CalEPA Department of Pesticide Regulation Environmental Monitoring Branch

Air Protection Program 1001 I Street

Sacramento California 95812

Environmental fate and ecotoxicology of paraquat a California perspective

Fabio Sartori and Edgar Vidrio

Department of Pesticide Regulation California Environmental Protection Agency Sacramento

California USA

Environmental fate and ecotoxicology of paraquat a California perspective

ABSTRACT

The herbicide paraquat belongs to the group of the bipyridylium salts In California it is used

primarily for control of broad-leaved grasses in fruit orchards and plantations as a cotton

defoliant and for inter-row control in many crop and non-crop areas In plants paraquat causes

the formation of reactive radicals leading to cell membrane damage and ultimately rapid

desiccation Soil clay minerals have a greater influence on paraquat adsorption and inactivation

compared with soil organic matter following an application Degradation mechanisms include

photolysis chemical and microbial degradation but these processes are generally extremely

slow In California during 2000ndash2014 paraquat was used primarily for the cultivation of

almonds cotton alfalfa and grapes median value for an application and annual mass applied

statewide were 053 kg ionha and 280 Mg respectively Paraquat was undetected in

groundwater as a non-point source pollutant Detections in surface waters (042ndash36 μgL) were

lt1 In earthworms and other invertebrates there is limited paraquat accumulation as toxic

effects are mitigated via soil inactivation Paraquat is among the most embryotoxic contaminants

for bird eggs but not to adult it causes toxic and teratogenic effects in amphibians and toxic

effects in honeybees fish and other aquatic species

KEYWORDS Paraquat California environmental fate toxicity soil deactivation

2

Contents

1 Introduction 4 2 Physical and chemical properties 5 3 Overview of paraquat use 6 31 Regulation 7

4 Use profile of paraquat in California 8 5 Plant resistance 9 6 Environmental fate 11 61 Soil 11 62 Water 15 63 Air 17

7 Environmental degradation 17 71 Microbial 18 72 Photochemical 19

8 Ecotoxicology 20 81 Mode of action paraquat redox chemistry 21 82 Plants 22 821 General physiological effects 22 822 Dose-response studies 25

83 Soil fauna and flora 26 831 Toxic effects 26 832 Teratogenic effects 28 833 Soil bacteria 29

84 Birds 29 841 Toxic and teratogenic effects 29 842 Reproductive effects 31

85 Honeybees 31 86 Amphibians 32 861 Toxic teratogenic and reproductive effects 33

87 Fish and other aquatic species 35 871 Toxic effects 35 872 Oxidative stress indicators 37 873 Behavioral analyses 38

9 Summary and Conclusions 39

3

1 Introduction

Paraquat (11-dimethyl-44-bipyridinium) was first synthesized by Weidel and Russo (1882)

and used as an herbicide in 1955 at the Jealottrsquos Hill Research Center (Brian et al 1958

Calderbank and Slade 1976) The first commercial paraquat formulation for agricultural use

became available in 1962 (USEPA 1987) Paraquat belongs to the ldquobipyridyliumrdquo (BP) or

ldquoviologenrdquo salts a group of compounds that has been known for their redox properties since

1933 (Michaelis and Hill 1933) The ability of BP salt compounds to accept or release an

electron in biochemical systems has long been considered the first step responsible for paraquat

toxicity in animals and plants (Homer et al 1960) Paraquat is a fast-acting non-selective contact

herbicide absorbed by foliage and is used in both agricultural and non-agricultural areas as well

as on both food and feed crops (Homer et al 1960 USEPA 1997) in reforestation programs

pine plantation establishment (USEPA 1997) and in some countries for the control of aquatic

weeds (Brooker and Edwards 1975 Peterson et al 1994)

In California it is used primarily for control of broad-leaved grasses in fruit orchards and

plantations (Wilson and Orloff 2008 Moretti et al 2015) as a cotton defoliant (Chester and

Ward 1984 Scarborough et al 1989) and for inter-row control in many crops (Kim and Hatzios

1993) such as alfalfa (Medicago sativa L) stands (Wilson and Orloff 2008) and non-crop areas

(CDPR 2016 Dennis et al 2016) It remains among the top five most used herbicides in

California with statewide applications averaging an annual mass of about 280 Mg ion during

2000ndash2014 on over 150 agricultural commodities (CDPR 2016)

Paraquat has revolutionized farming in the 1960s because it generally does not leave an

active residue in soil or plants to affect future or established crops does not leach into

groundwater and reduces or eliminate the need of ploughing the soil for vegetation control

4

2

acting as a ldquochemical ploughrdquo It is currently banned in 32 countries including the European

Union based primarily on human health concerns (The Court of First Instance 2007 USEPA

2018) however it is still registered and applied in over 90 countries (Fortenberry et al 2016)

Because of its toxicity to human health and potential impacts to the environment paraquat

remains one of the most controversial and studied herbicides of the last 50 years (Chester and

Ward 1984 USEPA 2018) Several past and recent reviews and analyses have already assessed

paraquat toxicology in mammals including humans (Autor and Schmitt 1977 Dinis-Oliveira et

al 2008 Lock and Wilks 2010 Xie et al 2016) and occupational and non-occupational exposure

(Hart 1987 Tsai 2013)

Because persistence and terminal residues play a major role in environmental regulation the

objectives of this review are to (1) summarize current knowledge of the physico-chemical

properties of paraquat (2) provide an historical overview of paraquat use focusing on California

agriculture and (3) critically review the most current and past knowledge on its environmental

fate degradation and ecotoxicology

Physical and chemical properties

This herbicide is suitable for many agricultural uses because of its physical and chemical

properties (Figure 1 and Table 1) (Florecircncio et al 2004) Its association with the chloride or

bromide anion to form a salt has no effect on the herbicidal properties of the paraquat cation

because cation and anion are dissociated when in aqueous solution (Calderbank and Slade 1976)

(Throughout this document we will refer to the paraquat cation as ldquoionrdquo or simply as ldquoparaquatrdquo

and its salt paraquat dichloride as ldquoPDrdquo) Michaelis and Hill (1933) first discovered the redox

properties of the BPs (γγrsquo-dipyridyl and dimethyl-dipyridylium chloride) in 1932 During a

titration with paraquat in an alkaline solution they used sodium hydrosulfite as a reducing agent

5

3

and measured an electrode potential E = ndash 0446 V which remained relatively stable at pH

ranging 84ndash13 Redox potential measurements of the BP compounds have been used to assess

their herbicidal properties considering that the more easily a BP compound is reduced the more

the toxic radicals that will be formed and vice versa (Homer et al 1960) These findings by

Homer and colleagues were instrumental and led to the commercialization of paraquat by the

Imperial Chemical Industries Limited London England who registered paraquat in that country

in 1962 and in the United States in 1964 (USEPA 1987) Reported Freundlich (Kf) soil-water

distribution coefficients are extremely variable because of the variability in soil type 68ndash50000

mLg (USEPA 1997) and 287 (sandy loam)ndash1419 (muck) mLg (Cheah et al 1997) However

this and other coefficients such as the organic carbon normalized partition coefficient (Koc) may

not be meaningful or applicable for certain soil types due to paraquat strong adsorption onto clay

minerals

Overview of paraquat use

In California all agricultural applicators are required to report information on the use of any

registered pesticide to the California Department of Pesticide Regulation (CDPR) The submitted

use information is recorded in the CDPRrsquos Pesticide Use Report (PUR) database (CDPR 2017)

During 2000ndash2014 there were 11 products registered that contained PD as an active ingredient

(ai) These six most common trade names of commercial paraquat herbicides are Gramoxone

Inteon Gramoxone Max Gramoxone Extra Herbicide Gramoxone SL 20 and Firestorm

representing more than 80 of reported paraquat uses (CDPR 2016) All these products are

formulated as aqueous solution with the ai concentration equal to or greater than 301

(USEPA 2016)

6

Paraquat is typically used as a nonselective herbicide to rapidly desiccate vegetation It is

defined as a ldquocontactrdquo herbicide because it quickly disrupts the plant tissues that are contacted by

the chemical due to its rapid desiccant action It is used as a ldquopost-emergencerdquo herbicide alone or

in combination with other herbicides (Moretti et al 2015) In California it is primarily used

individually or in combination with other herbicides in almond orchards (Connell et al 2001)

cotton plantations to cause cotton bolls to open or to control regrowth of late-season cotton

(Seiber and Woodrow 1981 Chester and Ward 1984 Wilhoit et al 1999) alfalfa stands (Wilson

and Orloff 2008) and grapes (UC IPM Program 2016) In most recent years paraquat has

become an important management tool to control glyphosate-resistant species (Moretti et al

2016) Paraquat is one of the few products registered for aerial application in alfalfa and is used

when fields are too wet to apply other herbicides by ground In grapes it is preferred to simazine

because simazine is a well-known groundwater contaminant in some parts of the state thereby

having more regulatory restrictions associated with its use (Wilhoit et al 1999)

31 Regulation

Paraquat was first registered in the United States as a contact herbicide and has been subject to

periodic reviews by the USEPA and other authorities Due to its acute toxicity to humans and

animals the USEPA classified paraquat as a restricted use material in 1978 and thereby ruled

that it can only be sold to and used by certified applicators (USEPA 1997) Additionally

paraquat is listed as a Restricted Use Pesticide (RUP) under Title 3 of the California Code of

Regulations (CCR) section 6400 in the production of an agricultural commodity The use of

both federally-restricted and California-restricted materials subjects paraquat to additional

restrictions and use limits Buying or using a California-restricted use pesticide requires a permit

7

4 Use profile of paraquat in California

from the County Agricultural Commissioner (CAC) which allows the CAC to make sure that

restricted pesticide users follow appropriate procedure to prevent harmful effects (CDPR 2017)

There was an upward trend from 2001 to 2006 (the overall maximum use year) followed by a

general decline with the lowest use occurring in 2013 (Figure 2 CDPR 2016) These trends may

be related primarily to the climatic conditions that affect the use of water-soluble herbicides As

found in a previous analysis on paraquat use from the PUR database by Wilhoit et al (1999)

paraquat use typically increases in several areas of the state in years of abundant rainfall which

promotes weed growth Similarly there was a weak (Spearmanrsquos ρ = 049) but significant

correlation (significance of both regression coefficients [p lt005] R2 = 03) between the annual

statewide precipitation data (2000ndash2014 series [NOAA 2017]) and one-year lagged annual use

supporting these previous findings (data not shown)

Many factors may affect paraquat use patterns changes in the area treated development of

plant resistance changes in the paraquat label requirements and in agricultural management

practices Paraquat can also be applied during wet years when fields are too wet to apply diuron

or hexazinone (Wilhoit et al 1999) When resistance occurs combinations of other herbicides are

typically used in alternative for example glufosinate and glyphosate (Moretti et al 2015) All of

these factors may ultimately affect the rate of application which remained relatively constant

during 2000ndash2014 as there were no major label changes (Figure 3[c]) although the median value

by year did show an increasing trend from 042 kg ionha in 2001 to 056 kgha in 2011 and 07

kgha in 2012 The average median value for an application (computed as average over 2000ndash

2014) was 053 kg ionha or 073 kg PDha (data not shown) In the years of greater paraquat use

and annual precipitation (765 cm and 600 cm for 2005 and 2006 respectively) the area of and

8

5 Plant resistance

mass applied per application were also greater (Figure 3[ab]) and vice versa in the years of

lower use and precipitation (201 and 505 cm for 2013 and 2014 respectively) (The six years of

lowest precipitation in California were 2011 2002 2008 2009 2007 and 2013 with annual

precipitation of 477 474 453 433 352 and 201 respectively [NOAA 2017])

The overall highest uses (2000ndash2014) were in Kern Fresno Kings and Tulare counties

Other counties with lower uses over the same period were Merced San Joaquin and Madera All

these counties are located in the San Joaquin Valley that is part of the Central Valley of

California (Figure 4) one of the most productive regions of agricultural crops in the world It is

historically an area of high pesticide use including the use of paraquat (Chester and Ward 1984

Weinbaum et al 1995 Carmichael et al 2014) Five commoditiesagricultural crops had a

corresponding 15-year cumulative paraquat use greater than 3300 Mg ion or in terms of

percentage greater than 77 of total use in California for that period (Figure 5) In descending

order of paraquat mass used they were almond (239 of total statewide use) cotton (204 )

alfalfa (152 ) grape wine (93 ) and grapes (88 ) During 2000ndash2014 PD was mainly

applied via ground application methods (801 of applications on a mass basis) whereas most

of the remaining mass (196 ) aerially (data not shown)

Increased resistance is among the important factors affecting paraquat application rates and the

development of new strategies for vegetation control (Wilhoit et al 1999) Resistance to

paraquat has been developing relatively slowly and is believed not to represent a serious

economic threat to agricultural production (Hawkes 2014) Researchers have thus far

documented 63 cases worldwide of species that have developed some sort of paraquat resistant

The reported cases in the United States were in eight separate locations two in California (hairy

9

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

Environmental fate and ecotoxicology of paraquat a California perspective

ABSTRACT

The herbicide paraquat belongs to the group of the bipyridylium salts In California it is used

primarily for control of broad-leaved grasses in fruit orchards and plantations as a cotton

defoliant and for inter-row control in many crop and non-crop areas In plants paraquat causes

the formation of reactive radicals leading to cell membrane damage and ultimately rapid

desiccation Soil clay minerals have a greater influence on paraquat adsorption and inactivation

compared with soil organic matter following an application Degradation mechanisms include

photolysis chemical and microbial degradation but these processes are generally extremely

slow In California during 2000ndash2014 paraquat was used primarily for the cultivation of

almonds cotton alfalfa and grapes median value for an application and annual mass applied

statewide were 053 kg ionha and 280 Mg respectively Paraquat was undetected in

groundwater as a non-point source pollutant Detections in surface waters (042ndash36 μgL) were

lt1 In earthworms and other invertebrates there is limited paraquat accumulation as toxic

effects are mitigated via soil inactivation Paraquat is among the most embryotoxic contaminants

for bird eggs but not to adult it causes toxic and teratogenic effects in amphibians and toxic

effects in honeybees fish and other aquatic species

KEYWORDS Paraquat California environmental fate toxicity soil deactivation

2

Contents

1 Introduction 4 2 Physical and chemical properties 5 3 Overview of paraquat use 6 31 Regulation 7

4 Use profile of paraquat in California 8 5 Plant resistance 9 6 Environmental fate 11 61 Soil 11 62 Water 15 63 Air 17

7 Environmental degradation 17 71 Microbial 18 72 Photochemical 19

8 Ecotoxicology 20 81 Mode of action paraquat redox chemistry 21 82 Plants 22 821 General physiological effects 22 822 Dose-response studies 25

83 Soil fauna and flora 26 831 Toxic effects 26 832 Teratogenic effects 28 833 Soil bacteria 29

84 Birds 29 841 Toxic and teratogenic effects 29 842 Reproductive effects 31

85 Honeybees 31 86 Amphibians 32 861 Toxic teratogenic and reproductive effects 33

87 Fish and other aquatic species 35 871 Toxic effects 35 872 Oxidative stress indicators 37 873 Behavioral analyses 38

9 Summary and Conclusions 39

3

1 Introduction

Paraquat (11-dimethyl-44-bipyridinium) was first synthesized by Weidel and Russo (1882)

and used as an herbicide in 1955 at the Jealottrsquos Hill Research Center (Brian et al 1958

Calderbank and Slade 1976) The first commercial paraquat formulation for agricultural use

became available in 1962 (USEPA 1987) Paraquat belongs to the ldquobipyridyliumrdquo (BP) or

ldquoviologenrdquo salts a group of compounds that has been known for their redox properties since

1933 (Michaelis and Hill 1933) The ability of BP salt compounds to accept or release an

electron in biochemical systems has long been considered the first step responsible for paraquat

toxicity in animals and plants (Homer et al 1960) Paraquat is a fast-acting non-selective contact

herbicide absorbed by foliage and is used in both agricultural and non-agricultural areas as well

as on both food and feed crops (Homer et al 1960 USEPA 1997) in reforestation programs

pine plantation establishment (USEPA 1997) and in some countries for the control of aquatic

weeds (Brooker and Edwards 1975 Peterson et al 1994)

In California it is used primarily for control of broad-leaved grasses in fruit orchards and

plantations (Wilson and Orloff 2008 Moretti et al 2015) as a cotton defoliant (Chester and

Ward 1984 Scarborough et al 1989) and for inter-row control in many crops (Kim and Hatzios

1993) such as alfalfa (Medicago sativa L) stands (Wilson and Orloff 2008) and non-crop areas

(CDPR 2016 Dennis et al 2016) It remains among the top five most used herbicides in

California with statewide applications averaging an annual mass of about 280 Mg ion during

2000ndash2014 on over 150 agricultural commodities (CDPR 2016)

Paraquat has revolutionized farming in the 1960s because it generally does not leave an

active residue in soil or plants to affect future or established crops does not leach into

groundwater and reduces or eliminate the need of ploughing the soil for vegetation control

4

2

acting as a ldquochemical ploughrdquo It is currently banned in 32 countries including the European

Union based primarily on human health concerns (The Court of First Instance 2007 USEPA

2018) however it is still registered and applied in over 90 countries (Fortenberry et al 2016)

Because of its toxicity to human health and potential impacts to the environment paraquat

remains one of the most controversial and studied herbicides of the last 50 years (Chester and

Ward 1984 USEPA 2018) Several past and recent reviews and analyses have already assessed

paraquat toxicology in mammals including humans (Autor and Schmitt 1977 Dinis-Oliveira et

al 2008 Lock and Wilks 2010 Xie et al 2016) and occupational and non-occupational exposure

(Hart 1987 Tsai 2013)

Because persistence and terminal residues play a major role in environmental regulation the

objectives of this review are to (1) summarize current knowledge of the physico-chemical

properties of paraquat (2) provide an historical overview of paraquat use focusing on California

agriculture and (3) critically review the most current and past knowledge on its environmental

fate degradation and ecotoxicology

Physical and chemical properties

This herbicide is suitable for many agricultural uses because of its physical and chemical

properties (Figure 1 and Table 1) (Florecircncio et al 2004) Its association with the chloride or

bromide anion to form a salt has no effect on the herbicidal properties of the paraquat cation

because cation and anion are dissociated when in aqueous solution (Calderbank and Slade 1976)

(Throughout this document we will refer to the paraquat cation as ldquoionrdquo or simply as ldquoparaquatrdquo

and its salt paraquat dichloride as ldquoPDrdquo) Michaelis and Hill (1933) first discovered the redox

properties of the BPs (γγrsquo-dipyridyl and dimethyl-dipyridylium chloride) in 1932 During a

titration with paraquat in an alkaline solution they used sodium hydrosulfite as a reducing agent

5

3

and measured an electrode potential E = ndash 0446 V which remained relatively stable at pH

ranging 84ndash13 Redox potential measurements of the BP compounds have been used to assess

their herbicidal properties considering that the more easily a BP compound is reduced the more

the toxic radicals that will be formed and vice versa (Homer et al 1960) These findings by

Homer and colleagues were instrumental and led to the commercialization of paraquat by the

Imperial Chemical Industries Limited London England who registered paraquat in that country

in 1962 and in the United States in 1964 (USEPA 1987) Reported Freundlich (Kf) soil-water

distribution coefficients are extremely variable because of the variability in soil type 68ndash50000

mLg (USEPA 1997) and 287 (sandy loam)ndash1419 (muck) mLg (Cheah et al 1997) However

this and other coefficients such as the organic carbon normalized partition coefficient (Koc) may

not be meaningful or applicable for certain soil types due to paraquat strong adsorption onto clay

minerals

Overview of paraquat use

In California all agricultural applicators are required to report information on the use of any

registered pesticide to the California Department of Pesticide Regulation (CDPR) The submitted

use information is recorded in the CDPRrsquos Pesticide Use Report (PUR) database (CDPR 2017)

During 2000ndash2014 there were 11 products registered that contained PD as an active ingredient

(ai) These six most common trade names of commercial paraquat herbicides are Gramoxone

Inteon Gramoxone Max Gramoxone Extra Herbicide Gramoxone SL 20 and Firestorm

representing more than 80 of reported paraquat uses (CDPR 2016) All these products are

formulated as aqueous solution with the ai concentration equal to or greater than 301

(USEPA 2016)

6

Paraquat is typically used as a nonselective herbicide to rapidly desiccate vegetation It is

defined as a ldquocontactrdquo herbicide because it quickly disrupts the plant tissues that are contacted by

the chemical due to its rapid desiccant action It is used as a ldquopost-emergencerdquo herbicide alone or

in combination with other herbicides (Moretti et al 2015) In California it is primarily used

individually or in combination with other herbicides in almond orchards (Connell et al 2001)

cotton plantations to cause cotton bolls to open or to control regrowth of late-season cotton

(Seiber and Woodrow 1981 Chester and Ward 1984 Wilhoit et al 1999) alfalfa stands (Wilson

and Orloff 2008) and grapes (UC IPM Program 2016) In most recent years paraquat has

become an important management tool to control glyphosate-resistant species (Moretti et al

2016) Paraquat is one of the few products registered for aerial application in alfalfa and is used

when fields are too wet to apply other herbicides by ground In grapes it is preferred to simazine

because simazine is a well-known groundwater contaminant in some parts of the state thereby

having more regulatory restrictions associated with its use (Wilhoit et al 1999)

31 Regulation

Paraquat was first registered in the United States as a contact herbicide and has been subject to

periodic reviews by the USEPA and other authorities Due to its acute toxicity to humans and

animals the USEPA classified paraquat as a restricted use material in 1978 and thereby ruled

that it can only be sold to and used by certified applicators (USEPA 1997) Additionally

paraquat is listed as a Restricted Use Pesticide (RUP) under Title 3 of the California Code of

Regulations (CCR) section 6400 in the production of an agricultural commodity The use of

both federally-restricted and California-restricted materials subjects paraquat to additional

restrictions and use limits Buying or using a California-restricted use pesticide requires a permit

7

4 Use profile of paraquat in California

from the County Agricultural Commissioner (CAC) which allows the CAC to make sure that

restricted pesticide users follow appropriate procedure to prevent harmful effects (CDPR 2017)

There was an upward trend from 2001 to 2006 (the overall maximum use year) followed by a

general decline with the lowest use occurring in 2013 (Figure 2 CDPR 2016) These trends may

be related primarily to the climatic conditions that affect the use of water-soluble herbicides As

found in a previous analysis on paraquat use from the PUR database by Wilhoit et al (1999)

paraquat use typically increases in several areas of the state in years of abundant rainfall which

promotes weed growth Similarly there was a weak (Spearmanrsquos ρ = 049) but significant

correlation (significance of both regression coefficients [p lt005] R2 = 03) between the annual

statewide precipitation data (2000ndash2014 series [NOAA 2017]) and one-year lagged annual use

supporting these previous findings (data not shown)

Many factors may affect paraquat use patterns changes in the area treated development of

plant resistance changes in the paraquat label requirements and in agricultural management

practices Paraquat can also be applied during wet years when fields are too wet to apply diuron

or hexazinone (Wilhoit et al 1999) When resistance occurs combinations of other herbicides are

typically used in alternative for example glufosinate and glyphosate (Moretti et al 2015) All of

these factors may ultimately affect the rate of application which remained relatively constant

during 2000ndash2014 as there were no major label changes (Figure 3[c]) although the median value

by year did show an increasing trend from 042 kg ionha in 2001 to 056 kgha in 2011 and 07

kgha in 2012 The average median value for an application (computed as average over 2000ndash

2014) was 053 kg ionha or 073 kg PDha (data not shown) In the years of greater paraquat use

and annual precipitation (765 cm and 600 cm for 2005 and 2006 respectively) the area of and

8

5 Plant resistance

mass applied per application were also greater (Figure 3[ab]) and vice versa in the years of

lower use and precipitation (201 and 505 cm for 2013 and 2014 respectively) (The six years of

lowest precipitation in California were 2011 2002 2008 2009 2007 and 2013 with annual

precipitation of 477 474 453 433 352 and 201 respectively [NOAA 2017])

The overall highest uses (2000ndash2014) were in Kern Fresno Kings and Tulare counties

Other counties with lower uses over the same period were Merced San Joaquin and Madera All

these counties are located in the San Joaquin Valley that is part of the Central Valley of

California (Figure 4) one of the most productive regions of agricultural crops in the world It is

historically an area of high pesticide use including the use of paraquat (Chester and Ward 1984

Weinbaum et al 1995 Carmichael et al 2014) Five commoditiesagricultural crops had a

corresponding 15-year cumulative paraquat use greater than 3300 Mg ion or in terms of

percentage greater than 77 of total use in California for that period (Figure 5) In descending

order of paraquat mass used they were almond (239 of total statewide use) cotton (204 )

alfalfa (152 ) grape wine (93 ) and grapes (88 ) During 2000ndash2014 PD was mainly

applied via ground application methods (801 of applications on a mass basis) whereas most

of the remaining mass (196 ) aerially (data not shown)

Increased resistance is among the important factors affecting paraquat application rates and the

development of new strategies for vegetation control (Wilhoit et al 1999) Resistance to

paraquat has been developing relatively slowly and is believed not to represent a serious

economic threat to agricultural production (Hawkes 2014) Researchers have thus far

documented 63 cases worldwide of species that have developed some sort of paraquat resistant

The reported cases in the United States were in eight separate locations two in California (hairy

9

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

Contents

1 Introduction 4 2 Physical and chemical properties 5 3 Overview of paraquat use 6 31 Regulation 7

4 Use profile of paraquat in California 8 5 Plant resistance 9 6 Environmental fate 11 61 Soil 11 62 Water 15 63 Air 17

7 Environmental degradation 17 71 Microbial 18 72 Photochemical 19

8 Ecotoxicology 20 81 Mode of action paraquat redox chemistry 21 82 Plants 22 821 General physiological effects 22 822 Dose-response studies 25

83 Soil fauna and flora 26 831 Toxic effects 26 832 Teratogenic effects 28 833 Soil bacteria 29

84 Birds 29 841 Toxic and teratogenic effects 29 842 Reproductive effects 31

85 Honeybees 31 86 Amphibians 32 861 Toxic teratogenic and reproductive effects 33

87 Fish and other aquatic species 35 871 Toxic effects 35 872 Oxidative stress indicators 37 873 Behavioral analyses 38

9 Summary and Conclusions 39

3

1 Introduction

Paraquat (11-dimethyl-44-bipyridinium) was first synthesized by Weidel and Russo (1882)

and used as an herbicide in 1955 at the Jealottrsquos Hill Research Center (Brian et al 1958

Calderbank and Slade 1976) The first commercial paraquat formulation for agricultural use

became available in 1962 (USEPA 1987) Paraquat belongs to the ldquobipyridyliumrdquo (BP) or

ldquoviologenrdquo salts a group of compounds that has been known for their redox properties since

1933 (Michaelis and Hill 1933) The ability of BP salt compounds to accept or release an

electron in biochemical systems has long been considered the first step responsible for paraquat

toxicity in animals and plants (Homer et al 1960) Paraquat is a fast-acting non-selective contact

herbicide absorbed by foliage and is used in both agricultural and non-agricultural areas as well

as on both food and feed crops (Homer et al 1960 USEPA 1997) in reforestation programs

pine plantation establishment (USEPA 1997) and in some countries for the control of aquatic

weeds (Brooker and Edwards 1975 Peterson et al 1994)

In California it is used primarily for control of broad-leaved grasses in fruit orchards and

plantations (Wilson and Orloff 2008 Moretti et al 2015) as a cotton defoliant (Chester and

Ward 1984 Scarborough et al 1989) and for inter-row control in many crops (Kim and Hatzios

1993) such as alfalfa (Medicago sativa L) stands (Wilson and Orloff 2008) and non-crop areas

(CDPR 2016 Dennis et al 2016) It remains among the top five most used herbicides in

California with statewide applications averaging an annual mass of about 280 Mg ion during

2000ndash2014 on over 150 agricultural commodities (CDPR 2016)

Paraquat has revolutionized farming in the 1960s because it generally does not leave an

active residue in soil or plants to affect future or established crops does not leach into

groundwater and reduces or eliminate the need of ploughing the soil for vegetation control

4

2

acting as a ldquochemical ploughrdquo It is currently banned in 32 countries including the European

Union based primarily on human health concerns (The Court of First Instance 2007 USEPA

2018) however it is still registered and applied in over 90 countries (Fortenberry et al 2016)

Because of its toxicity to human health and potential impacts to the environment paraquat

remains one of the most controversial and studied herbicides of the last 50 years (Chester and

Ward 1984 USEPA 2018) Several past and recent reviews and analyses have already assessed

paraquat toxicology in mammals including humans (Autor and Schmitt 1977 Dinis-Oliveira et

al 2008 Lock and Wilks 2010 Xie et al 2016) and occupational and non-occupational exposure

(Hart 1987 Tsai 2013)

Because persistence and terminal residues play a major role in environmental regulation the

objectives of this review are to (1) summarize current knowledge of the physico-chemical

properties of paraquat (2) provide an historical overview of paraquat use focusing on California

agriculture and (3) critically review the most current and past knowledge on its environmental

fate degradation and ecotoxicology

Physical and chemical properties

This herbicide is suitable for many agricultural uses because of its physical and chemical

properties (Figure 1 and Table 1) (Florecircncio et al 2004) Its association with the chloride or

bromide anion to form a salt has no effect on the herbicidal properties of the paraquat cation

because cation and anion are dissociated when in aqueous solution (Calderbank and Slade 1976)

(Throughout this document we will refer to the paraquat cation as ldquoionrdquo or simply as ldquoparaquatrdquo

and its salt paraquat dichloride as ldquoPDrdquo) Michaelis and Hill (1933) first discovered the redox

properties of the BPs (γγrsquo-dipyridyl and dimethyl-dipyridylium chloride) in 1932 During a

titration with paraquat in an alkaline solution they used sodium hydrosulfite as a reducing agent

5

3

and measured an electrode potential E = ndash 0446 V which remained relatively stable at pH

ranging 84ndash13 Redox potential measurements of the BP compounds have been used to assess

their herbicidal properties considering that the more easily a BP compound is reduced the more

the toxic radicals that will be formed and vice versa (Homer et al 1960) These findings by

Homer and colleagues were instrumental and led to the commercialization of paraquat by the

Imperial Chemical Industries Limited London England who registered paraquat in that country

in 1962 and in the United States in 1964 (USEPA 1987) Reported Freundlich (Kf) soil-water

distribution coefficients are extremely variable because of the variability in soil type 68ndash50000

mLg (USEPA 1997) and 287 (sandy loam)ndash1419 (muck) mLg (Cheah et al 1997) However

this and other coefficients such as the organic carbon normalized partition coefficient (Koc) may

not be meaningful or applicable for certain soil types due to paraquat strong adsorption onto clay

minerals

Overview of paraquat use

In California all agricultural applicators are required to report information on the use of any

registered pesticide to the California Department of Pesticide Regulation (CDPR) The submitted

use information is recorded in the CDPRrsquos Pesticide Use Report (PUR) database (CDPR 2017)

During 2000ndash2014 there were 11 products registered that contained PD as an active ingredient

(ai) These six most common trade names of commercial paraquat herbicides are Gramoxone

Inteon Gramoxone Max Gramoxone Extra Herbicide Gramoxone SL 20 and Firestorm

representing more than 80 of reported paraquat uses (CDPR 2016) All these products are

formulated as aqueous solution with the ai concentration equal to or greater than 301

(USEPA 2016)

6

Paraquat is typically used as a nonselective herbicide to rapidly desiccate vegetation It is

defined as a ldquocontactrdquo herbicide because it quickly disrupts the plant tissues that are contacted by

the chemical due to its rapid desiccant action It is used as a ldquopost-emergencerdquo herbicide alone or

in combination with other herbicides (Moretti et al 2015) In California it is primarily used

individually or in combination with other herbicides in almond orchards (Connell et al 2001)

cotton plantations to cause cotton bolls to open or to control regrowth of late-season cotton

(Seiber and Woodrow 1981 Chester and Ward 1984 Wilhoit et al 1999) alfalfa stands (Wilson

and Orloff 2008) and grapes (UC IPM Program 2016) In most recent years paraquat has

become an important management tool to control glyphosate-resistant species (Moretti et al

2016) Paraquat is one of the few products registered for aerial application in alfalfa and is used

when fields are too wet to apply other herbicides by ground In grapes it is preferred to simazine

because simazine is a well-known groundwater contaminant in some parts of the state thereby

having more regulatory restrictions associated with its use (Wilhoit et al 1999)

31 Regulation

Paraquat was first registered in the United States as a contact herbicide and has been subject to

periodic reviews by the USEPA and other authorities Due to its acute toxicity to humans and

animals the USEPA classified paraquat as a restricted use material in 1978 and thereby ruled

that it can only be sold to and used by certified applicators (USEPA 1997) Additionally

paraquat is listed as a Restricted Use Pesticide (RUP) under Title 3 of the California Code of

Regulations (CCR) section 6400 in the production of an agricultural commodity The use of

both federally-restricted and California-restricted materials subjects paraquat to additional

restrictions and use limits Buying or using a California-restricted use pesticide requires a permit

7

4 Use profile of paraquat in California

from the County Agricultural Commissioner (CAC) which allows the CAC to make sure that

restricted pesticide users follow appropriate procedure to prevent harmful effects (CDPR 2017)

There was an upward trend from 2001 to 2006 (the overall maximum use year) followed by a

general decline with the lowest use occurring in 2013 (Figure 2 CDPR 2016) These trends may

be related primarily to the climatic conditions that affect the use of water-soluble herbicides As

found in a previous analysis on paraquat use from the PUR database by Wilhoit et al (1999)

paraquat use typically increases in several areas of the state in years of abundant rainfall which

promotes weed growth Similarly there was a weak (Spearmanrsquos ρ = 049) but significant

correlation (significance of both regression coefficients [p lt005] R2 = 03) between the annual

statewide precipitation data (2000ndash2014 series [NOAA 2017]) and one-year lagged annual use

supporting these previous findings (data not shown)

Many factors may affect paraquat use patterns changes in the area treated development of

plant resistance changes in the paraquat label requirements and in agricultural management

practices Paraquat can also be applied during wet years when fields are too wet to apply diuron

or hexazinone (Wilhoit et al 1999) When resistance occurs combinations of other herbicides are

typically used in alternative for example glufosinate and glyphosate (Moretti et al 2015) All of

these factors may ultimately affect the rate of application which remained relatively constant

during 2000ndash2014 as there were no major label changes (Figure 3[c]) although the median value

by year did show an increasing trend from 042 kg ionha in 2001 to 056 kgha in 2011 and 07

kgha in 2012 The average median value for an application (computed as average over 2000ndash

2014) was 053 kg ionha or 073 kg PDha (data not shown) In the years of greater paraquat use

and annual precipitation (765 cm and 600 cm for 2005 and 2006 respectively) the area of and

8

5 Plant resistance

mass applied per application were also greater (Figure 3[ab]) and vice versa in the years of

lower use and precipitation (201 and 505 cm for 2013 and 2014 respectively) (The six years of

lowest precipitation in California were 2011 2002 2008 2009 2007 and 2013 with annual

precipitation of 477 474 453 433 352 and 201 respectively [NOAA 2017])

The overall highest uses (2000ndash2014) were in Kern Fresno Kings and Tulare counties

Other counties with lower uses over the same period were Merced San Joaquin and Madera All

these counties are located in the San Joaquin Valley that is part of the Central Valley of

California (Figure 4) one of the most productive regions of agricultural crops in the world It is

historically an area of high pesticide use including the use of paraquat (Chester and Ward 1984

Weinbaum et al 1995 Carmichael et al 2014) Five commoditiesagricultural crops had a

corresponding 15-year cumulative paraquat use greater than 3300 Mg ion or in terms of

percentage greater than 77 of total use in California for that period (Figure 5) In descending

order of paraquat mass used they were almond (239 of total statewide use) cotton (204 )

alfalfa (152 ) grape wine (93 ) and grapes (88 ) During 2000ndash2014 PD was mainly

applied via ground application methods (801 of applications on a mass basis) whereas most

of the remaining mass (196 ) aerially (data not shown)

Increased resistance is among the important factors affecting paraquat application rates and the

development of new strategies for vegetation control (Wilhoit et al 1999) Resistance to

paraquat has been developing relatively slowly and is believed not to represent a serious

economic threat to agricultural production (Hawkes 2014) Researchers have thus far

documented 63 cases worldwide of species that have developed some sort of paraquat resistant

The reported cases in the United States were in eight separate locations two in California (hairy

9

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

1 Introduction

Paraquat (11-dimethyl-44-bipyridinium) was first synthesized by Weidel and Russo (1882)

and used as an herbicide in 1955 at the Jealottrsquos Hill Research Center (Brian et al 1958

Calderbank and Slade 1976) The first commercial paraquat formulation for agricultural use

became available in 1962 (USEPA 1987) Paraquat belongs to the ldquobipyridyliumrdquo (BP) or

ldquoviologenrdquo salts a group of compounds that has been known for their redox properties since

1933 (Michaelis and Hill 1933) The ability of BP salt compounds to accept or release an

electron in biochemical systems has long been considered the first step responsible for paraquat

toxicity in animals and plants (Homer et al 1960) Paraquat is a fast-acting non-selective contact

herbicide absorbed by foliage and is used in both agricultural and non-agricultural areas as well

as on both food and feed crops (Homer et al 1960 USEPA 1997) in reforestation programs

pine plantation establishment (USEPA 1997) and in some countries for the control of aquatic

weeds (Brooker and Edwards 1975 Peterson et al 1994)

In California it is used primarily for control of broad-leaved grasses in fruit orchards and

plantations (Wilson and Orloff 2008 Moretti et al 2015) as a cotton defoliant (Chester and

Ward 1984 Scarborough et al 1989) and for inter-row control in many crops (Kim and Hatzios

1993) such as alfalfa (Medicago sativa L) stands (Wilson and Orloff 2008) and non-crop areas

(CDPR 2016 Dennis et al 2016) It remains among the top five most used herbicides in

California with statewide applications averaging an annual mass of about 280 Mg ion during

2000ndash2014 on over 150 agricultural commodities (CDPR 2016)

Paraquat has revolutionized farming in the 1960s because it generally does not leave an

active residue in soil or plants to affect future or established crops does not leach into

groundwater and reduces or eliminate the need of ploughing the soil for vegetation control

4

2

acting as a ldquochemical ploughrdquo It is currently banned in 32 countries including the European

Union based primarily on human health concerns (The Court of First Instance 2007 USEPA

2018) however it is still registered and applied in over 90 countries (Fortenberry et al 2016)

Because of its toxicity to human health and potential impacts to the environment paraquat

remains one of the most controversial and studied herbicides of the last 50 years (Chester and

Ward 1984 USEPA 2018) Several past and recent reviews and analyses have already assessed

paraquat toxicology in mammals including humans (Autor and Schmitt 1977 Dinis-Oliveira et

al 2008 Lock and Wilks 2010 Xie et al 2016) and occupational and non-occupational exposure

(Hart 1987 Tsai 2013)

Because persistence and terminal residues play a major role in environmental regulation the

objectives of this review are to (1) summarize current knowledge of the physico-chemical

properties of paraquat (2) provide an historical overview of paraquat use focusing on California

agriculture and (3) critically review the most current and past knowledge on its environmental

fate degradation and ecotoxicology

Physical and chemical properties

This herbicide is suitable for many agricultural uses because of its physical and chemical

properties (Figure 1 and Table 1) (Florecircncio et al 2004) Its association with the chloride or

bromide anion to form a salt has no effect on the herbicidal properties of the paraquat cation

because cation and anion are dissociated when in aqueous solution (Calderbank and Slade 1976)

(Throughout this document we will refer to the paraquat cation as ldquoionrdquo or simply as ldquoparaquatrdquo

and its salt paraquat dichloride as ldquoPDrdquo) Michaelis and Hill (1933) first discovered the redox

properties of the BPs (γγrsquo-dipyridyl and dimethyl-dipyridylium chloride) in 1932 During a

titration with paraquat in an alkaline solution they used sodium hydrosulfite as a reducing agent

5

3

and measured an electrode potential E = ndash 0446 V which remained relatively stable at pH

ranging 84ndash13 Redox potential measurements of the BP compounds have been used to assess

their herbicidal properties considering that the more easily a BP compound is reduced the more

the toxic radicals that will be formed and vice versa (Homer et al 1960) These findings by

Homer and colleagues were instrumental and led to the commercialization of paraquat by the

Imperial Chemical Industries Limited London England who registered paraquat in that country

in 1962 and in the United States in 1964 (USEPA 1987) Reported Freundlich (Kf) soil-water

distribution coefficients are extremely variable because of the variability in soil type 68ndash50000

mLg (USEPA 1997) and 287 (sandy loam)ndash1419 (muck) mLg (Cheah et al 1997) However

this and other coefficients such as the organic carbon normalized partition coefficient (Koc) may

not be meaningful or applicable for certain soil types due to paraquat strong adsorption onto clay

minerals

Overview of paraquat use

In California all agricultural applicators are required to report information on the use of any

registered pesticide to the California Department of Pesticide Regulation (CDPR) The submitted

use information is recorded in the CDPRrsquos Pesticide Use Report (PUR) database (CDPR 2017)

During 2000ndash2014 there were 11 products registered that contained PD as an active ingredient

(ai) These six most common trade names of commercial paraquat herbicides are Gramoxone

Inteon Gramoxone Max Gramoxone Extra Herbicide Gramoxone SL 20 and Firestorm

representing more than 80 of reported paraquat uses (CDPR 2016) All these products are

formulated as aqueous solution with the ai concentration equal to or greater than 301

(USEPA 2016)

6

Paraquat is typically used as a nonselective herbicide to rapidly desiccate vegetation It is

defined as a ldquocontactrdquo herbicide because it quickly disrupts the plant tissues that are contacted by

the chemical due to its rapid desiccant action It is used as a ldquopost-emergencerdquo herbicide alone or

in combination with other herbicides (Moretti et al 2015) In California it is primarily used

individually or in combination with other herbicides in almond orchards (Connell et al 2001)

cotton plantations to cause cotton bolls to open or to control regrowth of late-season cotton

(Seiber and Woodrow 1981 Chester and Ward 1984 Wilhoit et al 1999) alfalfa stands (Wilson

and Orloff 2008) and grapes (UC IPM Program 2016) In most recent years paraquat has

become an important management tool to control glyphosate-resistant species (Moretti et al

2016) Paraquat is one of the few products registered for aerial application in alfalfa and is used

when fields are too wet to apply other herbicides by ground In grapes it is preferred to simazine

because simazine is a well-known groundwater contaminant in some parts of the state thereby

having more regulatory restrictions associated with its use (Wilhoit et al 1999)

31 Regulation

Paraquat was first registered in the United States as a contact herbicide and has been subject to

periodic reviews by the USEPA and other authorities Due to its acute toxicity to humans and

animals the USEPA classified paraquat as a restricted use material in 1978 and thereby ruled

that it can only be sold to and used by certified applicators (USEPA 1997) Additionally

paraquat is listed as a Restricted Use Pesticide (RUP) under Title 3 of the California Code of

Regulations (CCR) section 6400 in the production of an agricultural commodity The use of

both federally-restricted and California-restricted materials subjects paraquat to additional

restrictions and use limits Buying or using a California-restricted use pesticide requires a permit

7

4 Use profile of paraquat in California

from the County Agricultural Commissioner (CAC) which allows the CAC to make sure that

restricted pesticide users follow appropriate procedure to prevent harmful effects (CDPR 2017)

There was an upward trend from 2001 to 2006 (the overall maximum use year) followed by a

general decline with the lowest use occurring in 2013 (Figure 2 CDPR 2016) These trends may

be related primarily to the climatic conditions that affect the use of water-soluble herbicides As

found in a previous analysis on paraquat use from the PUR database by Wilhoit et al (1999)

paraquat use typically increases in several areas of the state in years of abundant rainfall which

promotes weed growth Similarly there was a weak (Spearmanrsquos ρ = 049) but significant

correlation (significance of both regression coefficients [p lt005] R2 = 03) between the annual

statewide precipitation data (2000ndash2014 series [NOAA 2017]) and one-year lagged annual use

supporting these previous findings (data not shown)

Many factors may affect paraquat use patterns changes in the area treated development of

plant resistance changes in the paraquat label requirements and in agricultural management

practices Paraquat can also be applied during wet years when fields are too wet to apply diuron

or hexazinone (Wilhoit et al 1999) When resistance occurs combinations of other herbicides are

typically used in alternative for example glufosinate and glyphosate (Moretti et al 2015) All of

these factors may ultimately affect the rate of application which remained relatively constant

during 2000ndash2014 as there were no major label changes (Figure 3[c]) although the median value

by year did show an increasing trend from 042 kg ionha in 2001 to 056 kgha in 2011 and 07

kgha in 2012 The average median value for an application (computed as average over 2000ndash

2014) was 053 kg ionha or 073 kg PDha (data not shown) In the years of greater paraquat use

and annual precipitation (765 cm and 600 cm for 2005 and 2006 respectively) the area of and

8

5 Plant resistance

mass applied per application were also greater (Figure 3[ab]) and vice versa in the years of

lower use and precipitation (201 and 505 cm for 2013 and 2014 respectively) (The six years of

lowest precipitation in California were 2011 2002 2008 2009 2007 and 2013 with annual

precipitation of 477 474 453 433 352 and 201 respectively [NOAA 2017])

The overall highest uses (2000ndash2014) were in Kern Fresno Kings and Tulare counties

Other counties with lower uses over the same period were Merced San Joaquin and Madera All

these counties are located in the San Joaquin Valley that is part of the Central Valley of

California (Figure 4) one of the most productive regions of agricultural crops in the world It is

historically an area of high pesticide use including the use of paraquat (Chester and Ward 1984

Weinbaum et al 1995 Carmichael et al 2014) Five commoditiesagricultural crops had a

corresponding 15-year cumulative paraquat use greater than 3300 Mg ion or in terms of

percentage greater than 77 of total use in California for that period (Figure 5) In descending

order of paraquat mass used they were almond (239 of total statewide use) cotton (204 )

alfalfa (152 ) grape wine (93 ) and grapes (88 ) During 2000ndash2014 PD was mainly

applied via ground application methods (801 of applications on a mass basis) whereas most

of the remaining mass (196 ) aerially (data not shown)

Increased resistance is among the important factors affecting paraquat application rates and the

development of new strategies for vegetation control (Wilhoit et al 1999) Resistance to

paraquat has been developing relatively slowly and is believed not to represent a serious

economic threat to agricultural production (Hawkes 2014) Researchers have thus far

documented 63 cases worldwide of species that have developed some sort of paraquat resistant

The reported cases in the United States were in eight separate locations two in California (hairy

9

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

2

acting as a ldquochemical ploughrdquo It is currently banned in 32 countries including the European

Union based primarily on human health concerns (The Court of First Instance 2007 USEPA

2018) however it is still registered and applied in over 90 countries (Fortenberry et al 2016)

Because of its toxicity to human health and potential impacts to the environment paraquat

remains one of the most controversial and studied herbicides of the last 50 years (Chester and

Ward 1984 USEPA 2018) Several past and recent reviews and analyses have already assessed

paraquat toxicology in mammals including humans (Autor and Schmitt 1977 Dinis-Oliveira et

al 2008 Lock and Wilks 2010 Xie et al 2016) and occupational and non-occupational exposure

(Hart 1987 Tsai 2013)

Because persistence and terminal residues play a major role in environmental regulation the

objectives of this review are to (1) summarize current knowledge of the physico-chemical

properties of paraquat (2) provide an historical overview of paraquat use focusing on California

agriculture and (3) critically review the most current and past knowledge on its environmental

fate degradation and ecotoxicology

Physical and chemical properties

This herbicide is suitable for many agricultural uses because of its physical and chemical

properties (Figure 1 and Table 1) (Florecircncio et al 2004) Its association with the chloride or

bromide anion to form a salt has no effect on the herbicidal properties of the paraquat cation

because cation and anion are dissociated when in aqueous solution (Calderbank and Slade 1976)

(Throughout this document we will refer to the paraquat cation as ldquoionrdquo or simply as ldquoparaquatrdquo

and its salt paraquat dichloride as ldquoPDrdquo) Michaelis and Hill (1933) first discovered the redox

properties of the BPs (γγrsquo-dipyridyl and dimethyl-dipyridylium chloride) in 1932 During a

titration with paraquat in an alkaline solution they used sodium hydrosulfite as a reducing agent

5

3

and measured an electrode potential E = ndash 0446 V which remained relatively stable at pH

ranging 84ndash13 Redox potential measurements of the BP compounds have been used to assess

their herbicidal properties considering that the more easily a BP compound is reduced the more

the toxic radicals that will be formed and vice versa (Homer et al 1960) These findings by

Homer and colleagues were instrumental and led to the commercialization of paraquat by the

Imperial Chemical Industries Limited London England who registered paraquat in that country

in 1962 and in the United States in 1964 (USEPA 1987) Reported Freundlich (Kf) soil-water

distribution coefficients are extremely variable because of the variability in soil type 68ndash50000

mLg (USEPA 1997) and 287 (sandy loam)ndash1419 (muck) mLg (Cheah et al 1997) However

this and other coefficients such as the organic carbon normalized partition coefficient (Koc) may

not be meaningful or applicable for certain soil types due to paraquat strong adsorption onto clay

minerals

Overview of paraquat use

In California all agricultural applicators are required to report information on the use of any

registered pesticide to the California Department of Pesticide Regulation (CDPR) The submitted

use information is recorded in the CDPRrsquos Pesticide Use Report (PUR) database (CDPR 2017)

During 2000ndash2014 there were 11 products registered that contained PD as an active ingredient

(ai) These six most common trade names of commercial paraquat herbicides are Gramoxone

Inteon Gramoxone Max Gramoxone Extra Herbicide Gramoxone SL 20 and Firestorm

representing more than 80 of reported paraquat uses (CDPR 2016) All these products are

formulated as aqueous solution with the ai concentration equal to or greater than 301

(USEPA 2016)

6

Paraquat is typically used as a nonselective herbicide to rapidly desiccate vegetation It is

defined as a ldquocontactrdquo herbicide because it quickly disrupts the plant tissues that are contacted by

the chemical due to its rapid desiccant action It is used as a ldquopost-emergencerdquo herbicide alone or

in combination with other herbicides (Moretti et al 2015) In California it is primarily used

individually or in combination with other herbicides in almond orchards (Connell et al 2001)

cotton plantations to cause cotton bolls to open or to control regrowth of late-season cotton

(Seiber and Woodrow 1981 Chester and Ward 1984 Wilhoit et al 1999) alfalfa stands (Wilson

and Orloff 2008) and grapes (UC IPM Program 2016) In most recent years paraquat has

become an important management tool to control glyphosate-resistant species (Moretti et al

2016) Paraquat is one of the few products registered for aerial application in alfalfa and is used

when fields are too wet to apply other herbicides by ground In grapes it is preferred to simazine

because simazine is a well-known groundwater contaminant in some parts of the state thereby

having more regulatory restrictions associated with its use (Wilhoit et al 1999)

31 Regulation

Paraquat was first registered in the United States as a contact herbicide and has been subject to

periodic reviews by the USEPA and other authorities Due to its acute toxicity to humans and

animals the USEPA classified paraquat as a restricted use material in 1978 and thereby ruled

that it can only be sold to and used by certified applicators (USEPA 1997) Additionally

paraquat is listed as a Restricted Use Pesticide (RUP) under Title 3 of the California Code of

Regulations (CCR) section 6400 in the production of an agricultural commodity The use of

both federally-restricted and California-restricted materials subjects paraquat to additional

restrictions and use limits Buying or using a California-restricted use pesticide requires a permit

7

4 Use profile of paraquat in California

from the County Agricultural Commissioner (CAC) which allows the CAC to make sure that

restricted pesticide users follow appropriate procedure to prevent harmful effects (CDPR 2017)

There was an upward trend from 2001 to 2006 (the overall maximum use year) followed by a

general decline with the lowest use occurring in 2013 (Figure 2 CDPR 2016) These trends may

be related primarily to the climatic conditions that affect the use of water-soluble herbicides As

found in a previous analysis on paraquat use from the PUR database by Wilhoit et al (1999)

paraquat use typically increases in several areas of the state in years of abundant rainfall which

promotes weed growth Similarly there was a weak (Spearmanrsquos ρ = 049) but significant

correlation (significance of both regression coefficients [p lt005] R2 = 03) between the annual

statewide precipitation data (2000ndash2014 series [NOAA 2017]) and one-year lagged annual use

supporting these previous findings (data not shown)

Many factors may affect paraquat use patterns changes in the area treated development of

plant resistance changes in the paraquat label requirements and in agricultural management

practices Paraquat can also be applied during wet years when fields are too wet to apply diuron

or hexazinone (Wilhoit et al 1999) When resistance occurs combinations of other herbicides are

typically used in alternative for example glufosinate and glyphosate (Moretti et al 2015) All of

these factors may ultimately affect the rate of application which remained relatively constant

during 2000ndash2014 as there were no major label changes (Figure 3[c]) although the median value

by year did show an increasing trend from 042 kg ionha in 2001 to 056 kgha in 2011 and 07

kgha in 2012 The average median value for an application (computed as average over 2000ndash

2014) was 053 kg ionha or 073 kg PDha (data not shown) In the years of greater paraquat use

and annual precipitation (765 cm and 600 cm for 2005 and 2006 respectively) the area of and

8

5 Plant resistance

mass applied per application were also greater (Figure 3[ab]) and vice versa in the years of

lower use and precipitation (201 and 505 cm for 2013 and 2014 respectively) (The six years of

lowest precipitation in California were 2011 2002 2008 2009 2007 and 2013 with annual

precipitation of 477 474 453 433 352 and 201 respectively [NOAA 2017])

The overall highest uses (2000ndash2014) were in Kern Fresno Kings and Tulare counties

Other counties with lower uses over the same period were Merced San Joaquin and Madera All

these counties are located in the San Joaquin Valley that is part of the Central Valley of

California (Figure 4) one of the most productive regions of agricultural crops in the world It is

historically an area of high pesticide use including the use of paraquat (Chester and Ward 1984

Weinbaum et al 1995 Carmichael et al 2014) Five commoditiesagricultural crops had a

corresponding 15-year cumulative paraquat use greater than 3300 Mg ion or in terms of

percentage greater than 77 of total use in California for that period (Figure 5) In descending

order of paraquat mass used they were almond (239 of total statewide use) cotton (204 )

alfalfa (152 ) grape wine (93 ) and grapes (88 ) During 2000ndash2014 PD was mainly

applied via ground application methods (801 of applications on a mass basis) whereas most

of the remaining mass (196 ) aerially (data not shown)

Increased resistance is among the important factors affecting paraquat application rates and the

development of new strategies for vegetation control (Wilhoit et al 1999) Resistance to

paraquat has been developing relatively slowly and is believed not to represent a serious

economic threat to agricultural production (Hawkes 2014) Researchers have thus far

documented 63 cases worldwide of species that have developed some sort of paraquat resistant

The reported cases in the United States were in eight separate locations two in California (hairy

9

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

3

and measured an electrode potential E = ndash 0446 V which remained relatively stable at pH

ranging 84ndash13 Redox potential measurements of the BP compounds have been used to assess

their herbicidal properties considering that the more easily a BP compound is reduced the more

the toxic radicals that will be formed and vice versa (Homer et al 1960) These findings by

Homer and colleagues were instrumental and led to the commercialization of paraquat by the

Imperial Chemical Industries Limited London England who registered paraquat in that country

in 1962 and in the United States in 1964 (USEPA 1987) Reported Freundlich (Kf) soil-water

distribution coefficients are extremely variable because of the variability in soil type 68ndash50000

mLg (USEPA 1997) and 287 (sandy loam)ndash1419 (muck) mLg (Cheah et al 1997) However

this and other coefficients such as the organic carbon normalized partition coefficient (Koc) may

not be meaningful or applicable for certain soil types due to paraquat strong adsorption onto clay

minerals

Overview of paraquat use

In California all agricultural applicators are required to report information on the use of any

registered pesticide to the California Department of Pesticide Regulation (CDPR) The submitted

use information is recorded in the CDPRrsquos Pesticide Use Report (PUR) database (CDPR 2017)

During 2000ndash2014 there were 11 products registered that contained PD as an active ingredient

(ai) These six most common trade names of commercial paraquat herbicides are Gramoxone

Inteon Gramoxone Max Gramoxone Extra Herbicide Gramoxone SL 20 and Firestorm

representing more than 80 of reported paraquat uses (CDPR 2016) All these products are

formulated as aqueous solution with the ai concentration equal to or greater than 301

(USEPA 2016)

6

Paraquat is typically used as a nonselective herbicide to rapidly desiccate vegetation It is

defined as a ldquocontactrdquo herbicide because it quickly disrupts the plant tissues that are contacted by

the chemical due to its rapid desiccant action It is used as a ldquopost-emergencerdquo herbicide alone or

in combination with other herbicides (Moretti et al 2015) In California it is primarily used

individually or in combination with other herbicides in almond orchards (Connell et al 2001)

cotton plantations to cause cotton bolls to open or to control regrowth of late-season cotton

(Seiber and Woodrow 1981 Chester and Ward 1984 Wilhoit et al 1999) alfalfa stands (Wilson

and Orloff 2008) and grapes (UC IPM Program 2016) In most recent years paraquat has

become an important management tool to control glyphosate-resistant species (Moretti et al

2016) Paraquat is one of the few products registered for aerial application in alfalfa and is used

when fields are too wet to apply other herbicides by ground In grapes it is preferred to simazine

because simazine is a well-known groundwater contaminant in some parts of the state thereby

having more regulatory restrictions associated with its use (Wilhoit et al 1999)

31 Regulation

Paraquat was first registered in the United States as a contact herbicide and has been subject to

periodic reviews by the USEPA and other authorities Due to its acute toxicity to humans and

animals the USEPA classified paraquat as a restricted use material in 1978 and thereby ruled

that it can only be sold to and used by certified applicators (USEPA 1997) Additionally

paraquat is listed as a Restricted Use Pesticide (RUP) under Title 3 of the California Code of

Regulations (CCR) section 6400 in the production of an agricultural commodity The use of

both federally-restricted and California-restricted materials subjects paraquat to additional

restrictions and use limits Buying or using a California-restricted use pesticide requires a permit

7

4 Use profile of paraquat in California

from the County Agricultural Commissioner (CAC) which allows the CAC to make sure that

restricted pesticide users follow appropriate procedure to prevent harmful effects (CDPR 2017)

There was an upward trend from 2001 to 2006 (the overall maximum use year) followed by a

general decline with the lowest use occurring in 2013 (Figure 2 CDPR 2016) These trends may

be related primarily to the climatic conditions that affect the use of water-soluble herbicides As

found in a previous analysis on paraquat use from the PUR database by Wilhoit et al (1999)

paraquat use typically increases in several areas of the state in years of abundant rainfall which

promotes weed growth Similarly there was a weak (Spearmanrsquos ρ = 049) but significant

correlation (significance of both regression coefficients [p lt005] R2 = 03) between the annual

statewide precipitation data (2000ndash2014 series [NOAA 2017]) and one-year lagged annual use

supporting these previous findings (data not shown)

Many factors may affect paraquat use patterns changes in the area treated development of

plant resistance changes in the paraquat label requirements and in agricultural management

practices Paraquat can also be applied during wet years when fields are too wet to apply diuron

or hexazinone (Wilhoit et al 1999) When resistance occurs combinations of other herbicides are

typically used in alternative for example glufosinate and glyphosate (Moretti et al 2015) All of

these factors may ultimately affect the rate of application which remained relatively constant

during 2000ndash2014 as there were no major label changes (Figure 3[c]) although the median value

by year did show an increasing trend from 042 kg ionha in 2001 to 056 kgha in 2011 and 07

kgha in 2012 The average median value for an application (computed as average over 2000ndash

2014) was 053 kg ionha or 073 kg PDha (data not shown) In the years of greater paraquat use

and annual precipitation (765 cm and 600 cm for 2005 and 2006 respectively) the area of and

8

5 Plant resistance

mass applied per application were also greater (Figure 3[ab]) and vice versa in the years of

lower use and precipitation (201 and 505 cm for 2013 and 2014 respectively) (The six years of

lowest precipitation in California were 2011 2002 2008 2009 2007 and 2013 with annual

precipitation of 477 474 453 433 352 and 201 respectively [NOAA 2017])

The overall highest uses (2000ndash2014) were in Kern Fresno Kings and Tulare counties

Other counties with lower uses over the same period were Merced San Joaquin and Madera All

these counties are located in the San Joaquin Valley that is part of the Central Valley of

California (Figure 4) one of the most productive regions of agricultural crops in the world It is

historically an area of high pesticide use including the use of paraquat (Chester and Ward 1984

Weinbaum et al 1995 Carmichael et al 2014) Five commoditiesagricultural crops had a

corresponding 15-year cumulative paraquat use greater than 3300 Mg ion or in terms of

percentage greater than 77 of total use in California for that period (Figure 5) In descending

order of paraquat mass used they were almond (239 of total statewide use) cotton (204 )

alfalfa (152 ) grape wine (93 ) and grapes (88 ) During 2000ndash2014 PD was mainly

applied via ground application methods (801 of applications on a mass basis) whereas most

of the remaining mass (196 ) aerially (data not shown)

Increased resistance is among the important factors affecting paraquat application rates and the

development of new strategies for vegetation control (Wilhoit et al 1999) Resistance to

paraquat has been developing relatively slowly and is believed not to represent a serious

economic threat to agricultural production (Hawkes 2014) Researchers have thus far

documented 63 cases worldwide of species that have developed some sort of paraquat resistant

The reported cases in the United States were in eight separate locations two in California (hairy

9

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

Paraquat is typically used as a nonselective herbicide to rapidly desiccate vegetation It is

defined as a ldquocontactrdquo herbicide because it quickly disrupts the plant tissues that are contacted by

the chemical due to its rapid desiccant action It is used as a ldquopost-emergencerdquo herbicide alone or

in combination with other herbicides (Moretti et al 2015) In California it is primarily used

individually or in combination with other herbicides in almond orchards (Connell et al 2001)

cotton plantations to cause cotton bolls to open or to control regrowth of late-season cotton

(Seiber and Woodrow 1981 Chester and Ward 1984 Wilhoit et al 1999) alfalfa stands (Wilson

and Orloff 2008) and grapes (UC IPM Program 2016) In most recent years paraquat has

become an important management tool to control glyphosate-resistant species (Moretti et al

2016) Paraquat is one of the few products registered for aerial application in alfalfa and is used

when fields are too wet to apply other herbicides by ground In grapes it is preferred to simazine

because simazine is a well-known groundwater contaminant in some parts of the state thereby

having more regulatory restrictions associated with its use (Wilhoit et al 1999)

31 Regulation

Paraquat was first registered in the United States as a contact herbicide and has been subject to

periodic reviews by the USEPA and other authorities Due to its acute toxicity to humans and

animals the USEPA classified paraquat as a restricted use material in 1978 and thereby ruled

that it can only be sold to and used by certified applicators (USEPA 1997) Additionally

paraquat is listed as a Restricted Use Pesticide (RUP) under Title 3 of the California Code of

Regulations (CCR) section 6400 in the production of an agricultural commodity The use of

both federally-restricted and California-restricted materials subjects paraquat to additional

restrictions and use limits Buying or using a California-restricted use pesticide requires a permit

7

4 Use profile of paraquat in California

from the County Agricultural Commissioner (CAC) which allows the CAC to make sure that

restricted pesticide users follow appropriate procedure to prevent harmful effects (CDPR 2017)

There was an upward trend from 2001 to 2006 (the overall maximum use year) followed by a

general decline with the lowest use occurring in 2013 (Figure 2 CDPR 2016) These trends may

be related primarily to the climatic conditions that affect the use of water-soluble herbicides As

found in a previous analysis on paraquat use from the PUR database by Wilhoit et al (1999)

paraquat use typically increases in several areas of the state in years of abundant rainfall which

promotes weed growth Similarly there was a weak (Spearmanrsquos ρ = 049) but significant

correlation (significance of both regression coefficients [p lt005] R2 = 03) between the annual

statewide precipitation data (2000ndash2014 series [NOAA 2017]) and one-year lagged annual use

supporting these previous findings (data not shown)

Many factors may affect paraquat use patterns changes in the area treated development of

plant resistance changes in the paraquat label requirements and in agricultural management

practices Paraquat can also be applied during wet years when fields are too wet to apply diuron

or hexazinone (Wilhoit et al 1999) When resistance occurs combinations of other herbicides are

typically used in alternative for example glufosinate and glyphosate (Moretti et al 2015) All of

these factors may ultimately affect the rate of application which remained relatively constant

during 2000ndash2014 as there were no major label changes (Figure 3[c]) although the median value

by year did show an increasing trend from 042 kg ionha in 2001 to 056 kgha in 2011 and 07

kgha in 2012 The average median value for an application (computed as average over 2000ndash

2014) was 053 kg ionha or 073 kg PDha (data not shown) In the years of greater paraquat use

and annual precipitation (765 cm and 600 cm for 2005 and 2006 respectively) the area of and

8

5 Plant resistance

mass applied per application were also greater (Figure 3[ab]) and vice versa in the years of

lower use and precipitation (201 and 505 cm for 2013 and 2014 respectively) (The six years of

lowest precipitation in California were 2011 2002 2008 2009 2007 and 2013 with annual

precipitation of 477 474 453 433 352 and 201 respectively [NOAA 2017])

The overall highest uses (2000ndash2014) were in Kern Fresno Kings and Tulare counties

Other counties with lower uses over the same period were Merced San Joaquin and Madera All

these counties are located in the San Joaquin Valley that is part of the Central Valley of

California (Figure 4) one of the most productive regions of agricultural crops in the world It is

historically an area of high pesticide use including the use of paraquat (Chester and Ward 1984

Weinbaum et al 1995 Carmichael et al 2014) Five commoditiesagricultural crops had a

corresponding 15-year cumulative paraquat use greater than 3300 Mg ion or in terms of

percentage greater than 77 of total use in California for that period (Figure 5) In descending

order of paraquat mass used they were almond (239 of total statewide use) cotton (204 )

alfalfa (152 ) grape wine (93 ) and grapes (88 ) During 2000ndash2014 PD was mainly

applied via ground application methods (801 of applications on a mass basis) whereas most

of the remaining mass (196 ) aerially (data not shown)

Increased resistance is among the important factors affecting paraquat application rates and the

development of new strategies for vegetation control (Wilhoit et al 1999) Resistance to

paraquat has been developing relatively slowly and is believed not to represent a serious

economic threat to agricultural production (Hawkes 2014) Researchers have thus far

documented 63 cases worldwide of species that have developed some sort of paraquat resistant

The reported cases in the United States were in eight separate locations two in California (hairy

9

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

4 Use profile of paraquat in California

from the County Agricultural Commissioner (CAC) which allows the CAC to make sure that

restricted pesticide users follow appropriate procedure to prevent harmful effects (CDPR 2017)

There was an upward trend from 2001 to 2006 (the overall maximum use year) followed by a

general decline with the lowest use occurring in 2013 (Figure 2 CDPR 2016) These trends may

be related primarily to the climatic conditions that affect the use of water-soluble herbicides As

found in a previous analysis on paraquat use from the PUR database by Wilhoit et al (1999)

paraquat use typically increases in several areas of the state in years of abundant rainfall which

promotes weed growth Similarly there was a weak (Spearmanrsquos ρ = 049) but significant

correlation (significance of both regression coefficients [p lt005] R2 = 03) between the annual

statewide precipitation data (2000ndash2014 series [NOAA 2017]) and one-year lagged annual use

supporting these previous findings (data not shown)

Many factors may affect paraquat use patterns changes in the area treated development of

plant resistance changes in the paraquat label requirements and in agricultural management

practices Paraquat can also be applied during wet years when fields are too wet to apply diuron

or hexazinone (Wilhoit et al 1999) When resistance occurs combinations of other herbicides are

typically used in alternative for example glufosinate and glyphosate (Moretti et al 2015) All of

these factors may ultimately affect the rate of application which remained relatively constant

during 2000ndash2014 as there were no major label changes (Figure 3[c]) although the median value

by year did show an increasing trend from 042 kg ionha in 2001 to 056 kgha in 2011 and 07

kgha in 2012 The average median value for an application (computed as average over 2000ndash

2014) was 053 kg ionha or 073 kg PDha (data not shown) In the years of greater paraquat use

and annual precipitation (765 cm and 600 cm for 2005 and 2006 respectively) the area of and

8

5 Plant resistance

mass applied per application were also greater (Figure 3[ab]) and vice versa in the years of

lower use and precipitation (201 and 505 cm for 2013 and 2014 respectively) (The six years of

lowest precipitation in California were 2011 2002 2008 2009 2007 and 2013 with annual

precipitation of 477 474 453 433 352 and 201 respectively [NOAA 2017])

The overall highest uses (2000ndash2014) were in Kern Fresno Kings and Tulare counties

Other counties with lower uses over the same period were Merced San Joaquin and Madera All

these counties are located in the San Joaquin Valley that is part of the Central Valley of

California (Figure 4) one of the most productive regions of agricultural crops in the world It is

historically an area of high pesticide use including the use of paraquat (Chester and Ward 1984

Weinbaum et al 1995 Carmichael et al 2014) Five commoditiesagricultural crops had a

corresponding 15-year cumulative paraquat use greater than 3300 Mg ion or in terms of

percentage greater than 77 of total use in California for that period (Figure 5) In descending

order of paraquat mass used they were almond (239 of total statewide use) cotton (204 )

alfalfa (152 ) grape wine (93 ) and grapes (88 ) During 2000ndash2014 PD was mainly

applied via ground application methods (801 of applications on a mass basis) whereas most

of the remaining mass (196 ) aerially (data not shown)

Increased resistance is among the important factors affecting paraquat application rates and the

development of new strategies for vegetation control (Wilhoit et al 1999) Resistance to

paraquat has been developing relatively slowly and is believed not to represent a serious

economic threat to agricultural production (Hawkes 2014) Researchers have thus far

documented 63 cases worldwide of species that have developed some sort of paraquat resistant

The reported cases in the United States were in eight separate locations two in California (hairy

9

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

5 Plant resistance

mass applied per application were also greater (Figure 3[ab]) and vice versa in the years of

lower use and precipitation (201 and 505 cm for 2013 and 2014 respectively) (The six years of

lowest precipitation in California were 2011 2002 2008 2009 2007 and 2013 with annual

precipitation of 477 474 453 433 352 and 201 respectively [NOAA 2017])

The overall highest uses (2000ndash2014) were in Kern Fresno Kings and Tulare counties

Other counties with lower uses over the same period were Merced San Joaquin and Madera All

these counties are located in the San Joaquin Valley that is part of the Central Valley of

California (Figure 4) one of the most productive regions of agricultural crops in the world It is

historically an area of high pesticide use including the use of paraquat (Chester and Ward 1984

Weinbaum et al 1995 Carmichael et al 2014) Five commoditiesagricultural crops had a

corresponding 15-year cumulative paraquat use greater than 3300 Mg ion or in terms of

percentage greater than 77 of total use in California for that period (Figure 5) In descending

order of paraquat mass used they were almond (239 of total statewide use) cotton (204 )

alfalfa (152 ) grape wine (93 ) and grapes (88 ) During 2000ndash2014 PD was mainly

applied via ground application methods (801 of applications on a mass basis) whereas most

of the remaining mass (196 ) aerially (data not shown)

Increased resistance is among the important factors affecting paraquat application rates and the

development of new strategies for vegetation control (Wilhoit et al 1999) Resistance to

paraquat has been developing relatively slowly and is believed not to represent a serious

economic threat to agricultural production (Hawkes 2014) Researchers have thus far

documented 63 cases worldwide of species that have developed some sort of paraquat resistant

The reported cases in the United States were in eight separate locations two in California (hairy

9

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

fleabane Conyza bonariensis L [Cronq] and horseweed Conyza canadensis L [Cronq]

Moretti et al 2016) three in Florida (Solanum americanum Eleusine indica and Landoltia

punctate) two in Mississippi (Conyza canadensis) one in Delaware involving horseweed

(Conyza canadensis) Conyza bonariensis and Landoltia punctate (Heap 2016)

Plant resistance to paraquat has generally developed following prolonged selection pressure

from many applications of the herbicide (Preston et al 1994) An herbicide must be present for

prolonged times to foster the high selection pressure required to obtain resistant biotypes

particularly for paraquat that completely lacks active persistence In recent years hairy fleabane

and horseweed have become well adapted in tree nut and vineyard plantations of the Central

Valley of California thereby becoming one of the most problematic species to control despite

herbicidal control treatments The combined glyphosate-paraquat treatment has been shown to be

one of the least effective herbicide treatment combinations due to the development of herbicide

resistant in Conyza sp populations (Moretti et al 2015 Moretti et al 2016) Those authors were

the first to confirm the presence of populations of hairy fleabane that are resistant to both

glyphosate and paraquat in the California Central Valley The hypothesis of independent

mechanisms of resistance for the two herbicides is supported by the concurrent presence of a

multiple resistant population as well as glyphosate-resistantmdashbut paraquat-susceptiblemdash

populations of hairy fleabane in California (Moretti et al 2013)

Most recently such evolved resistance has prompted new research efforts in identifying

environmental conditions and new herbicides such as saflufenacil (Dennis et al 2016) for the

control of glyphosate-paraquat-resistant biotypes of Conyza bonariensis [L] Conquist in the

Central Valley of California similarly to other researchers in other US regions for example

combined paraquat-metrobuzin combination (Eubank et al 2012)

10

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

6 Environmental fate

Under current US regulations paraquat cannot be applied directly to aquatic environments

(Dial and Bauer 1984 USEPA 1997) therefore the focus of this review is on paraquat use in

terrestrial ecosystems Its environmental fate in aquatic environments was previously reviewed

by Calderbank and Slade (1976) Following a terrestrial application the primary targets are

biological materials and soils Because of its low vapor pressure (Table 1) the main pathway of

paraquat dissipation is sorption onto soil particles or plant residue materials The research

conducted in the 1960s and 1970s was the foundation to understand the fate and transport of

paraquat in the environment (Calderbank and Slade 1976) That key work showed three possible

pathways of paraquat degradation in soil (1) photolysis under ultraviolet (UV) or solar radiation

(Slade 1965) (2) chemical (Hance 1967) and (3) microbial (Funderburk and Bozarth 1967)

However other researchers further confirmed that if these degradation processes do occur they

are typically extremely slow or non-existent and cannot be considered a viable option for the

degradation of polluted soils or waters as paraquat does not hydrolyze under neutral or acidic

conditions (Staiff et al 1981 USEPA 1997)

61 Soil

The rapid sorption and consequent rapid deactivation of the BP cations in soil is among the

important reasons the BP herbicides diquat and paraquat have been important crop management

tools worldwide in the agriculture and forestry sector for over 40 years (Roberts et al 2002)

Together with glyphosate and glufosinate paraquat is one of the three non-selective and soil-

inactivated herbicides (Hawkes 2014) Such inactivation generally allows the replanting or

sowing of new crops almost immediately in treated soil without the risk of phytotoxicity

(Calderbank and Slade 1976 Bromilow 2004) although in sandy soils inactivation of paraquat

11

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

occurs slowly and occasionally some toxic effects to the new crops can occur following an

application (Khan et al 1975)

A large body of research has shown that the main mechanism in the adsorption of BP

cations to soil particles is cation exchange due to electrostatic (coulombic) forces by the soilrsquos

negatively charged sites at mineral and organic surfaces of the soil particles (Hayes et al 1975)

and it is speculated that to a lesser degree other forces are also responsible (Weber et al 1965

Burns et al 1973) These include charge transfer hydrogen bonding and van der Waals forces

(Knight and Tomlinson 1967 Burns et al 1973 Cheah et al 1997 Gondar et al 2012)

Paraquat sorption and desorption onto soils depend on the relative quantity and quality of

the clay minerals and organic matter (Knight and Tomlinson 1967) Those authors showed that

the main driving variable controlling paraquat adsorption to soil is the presence of clay minerals

rather than soil organic matter (SOM) although in certain soil types SOM may also be relevant

in controlling paraquat adsorption and transport (Khan et al 1975 Senesi et al 1995)

Adsorption behavior onto the expandable 21 type clay minerals such as montmorillonite that

allow for isomorphic substitutions in interlayer positions greatly differs from that of non-

expandable 11 types such as kaolinite (Knight and Tomlinson 1967 Kookana and Aylmore

1993) and is controlled by the soil pH-dependent charge (Khan 1974 Calderbank and Slade

1976) In the case of kaolinite adsorption occurs on the edges or faces of the clay particles rather

than in the interlayer positions (Weber et al 1965) Other variables that may affect paraquat

adsorptiondesorption with a relatively weaker influence are oxide minerals such as iron oxides

(Weber et al 1965 Amondham et al 2006) Weber et al (1965) also showed that temperature

and time of exposure did not significantly affect paraquat sorption onto kaolinite or

montmorillonite (Weber and Weed 1968)

12

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

In clay-rich soils the presence of SOM may not necessarily increase paraquat adsorption

The work by Pateiro-Moure et al (2009) showed that adsorption of BPs by agricultural soils

clearly increased when SOM was removed by treating the soil with H2O2 Their interpretation

was that in the presence of SOM-clay mineral complexes SOM occludes some of the potential

sites afforded by the clay minerals for binding paraquat cations In contrast to paraquat

adsorption onto clay particles paraquat adsorption onto SOM-rich charcoal was found to be

strongly temperature and time dependent relative to the adsorption of other herbicides (Weber et

al 1965)

The study of paraquat adsorptiondesorption tofrom soils has important implications for

paraquat fate into ground and surface waters Key investigations that elucidated the underpinning

mechanisms of paraquat fate and transport in soils with different SOM and clay contents were

conducted in the 1960s and confirmed further in the 1970s by the work of Burns et al (1973) and

Khan et al (1975) Paraquat transport through a soilrsquos profile may occur when the herbicide is

adsorbed onto colloidal clays or as paraquat-laden SOM (Burns et al 1973 Khan et al 1975

Senesi et al 1995) although paraquat transport in soil is generally very limited (Vinteacuten et al

1983 Cheah et al 1997) The soil column experiments conducted by Vinteacuten et al (1983) showed

that a Li-montmorillonite suspension transported over 50 of the applied 14C-paraquat to a

depth of 12 cm in sandy loam soils and the experiments by Leonard et al (1979) in the Georgia

Piedmont demonstrated that surface erosion of paraquat-laden sediment was the only possible

mechanism of paraquat transport to surface waters These authors used paraquat as a tracer to

study sediment runoff at a watershed level Their work represents one of the few publications

validating results from previous laboratory experiments showing that paraquat transport in clay-

rich soils occur mainly as runoff rather than as leaching through a soilrsquos profile

13

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

The determination of the total paraquat concentration in a typical agricultural soil requires

the ldquoextractionrdquo of paraquat through a soil digestion that is boiling the soil samples in 12 N

sulfuric acid for five h because paraquat is immobilizedprotected in soils The ion can withstand

such treatment because of its stability in acid solutions (Burns and Audus 1970 Hance et al

1980)

Researchers have also investigated paraquat release overtime from soilsmdashmainly as

leachingmdashdue to long-term safety concerns about its displacement by exchangeable cations

applied through agricultural management practices (eg fertilization) While Roberts et al

(2002) concluded that this is an unlikely occurrence based on their review of the literature other

authors believe that certain agronomic practices such as liming gypsum application and intense

fertilization could generate such high soil cation saturation and foster the release of soil-bound

paraquat particularly in coarse soils with low SOM or highly weathered soils rich in kaolinite

(ie with low CEC) (Kookana and Aylmore 1993) Similarly in their work about sorption onto

and desorption from clay minerals of the BP cations Weber and Weed (1968) found that using

the 1M BaCl2 extraction method only 5 of the total adsorbed cation mass could be extracted

from montmorillonite as opposed to 80 from kaolinite due to its weaker sorption capacity

In the presence of organic-rich soilsmaterialsmdashrather than clay particlesmdashthe desorption of

paraquat from soils can be expected to behave as the reverse process when ion exchange is the

dominant process controlling adsorption However the analysis of adsorption and desorption

isotherms by Burns et al (1973) using aqueous paraquat and five types of organic sorbent (soil

humic acid humin and two ion exchange resins) showed that paraquat desorption is not a

purely reversible phenomenon because the same mass action type of ion exchange isotherms

should have applied to both processes Those authors could extract only a small fraction of the

14

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

total paraquat adsorbed onto the organic materials in suspensions (through mixing and

centrifugation of the samples at varying HCl concentrations) indicating that other cooperative

mechanisms in addition to simple cation exchange were involved in paraquat adsorption

Several authors believe that when paraquat is applied following the typical ldquogood

agricultural practicesrdquo as specified by a productrsquos label it becomes strongly bound to soil

particles (gt99 of total mass) thereby extremely low paraquat concentrations are generally

present in the soil solution phase (Roberts et al 2002) Their proposed conceptual model was

that most paraquat (gt99 ) in soil is strongly bound to soil particles and in equilibrium with soil-

solution paraquat undergoing slow microbial degradationmineralization (Funderburk and

Bozarth 1967 Lee et al 1995 Ricketts 1999) (Table 2)

It is common belief that no microbial degradation generally occurs once

immobilizedprotected by clay minerals because it becomes unavailable to microbes (Burns and

Audus 1970 Fryer et al 1975 Kookana and Aylmore 1993 USEPA 1997) The work of Hance

(1967) showed that non-biological chemical processes do not play an important role in the loss

of paraquat from the soil In the absence of any microbial activities their estimated paraquat half-

life was gt9 years Most commonly reported half-life values range from about 7 years (Hance et

al 1980 Cheah et al 1998) up to 26 years when paraquat is applied at a rate corresponding to

five times the sorption capacity of the soil (Kookana and Aylmore 1993) (Table 3) Among the

most extreme values reported in the literature are those for paraquat applied in tropical soils of

Thailand with extremely short half-lives of 36ndash46 days (Amondham et al 2006)

62 Water

Adsorbed paraquat can potentially be found in surface water systems associated with soil

particles carried by erosion (Leonard et al 1979) Other possible mechanisms include paraquat

15

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

drift onto aquatic ecosystems when paraquat is applied near surface waters such as reservoirs

canals and rivers in countries with less stringent regulations (Wijeyaratne and Pathiratne 2006)

In the United States a monitoring program conducted by the USEPA found paraquat in 11

out of 971 water wells sampled between 1983 and 1990 having concentrations greater than 100

μgL in wells located in extremely permeable coarse-grained glacial soils whose saturated

hydraulic conductivities are about 20000 ftday (USEPA 1992) In a similar monitoring program

as part of the National Water-Quality Assessment Programrsquos first decade of water-quality

assessments by the USGS paraquat was never detected in water samples collected from 5047

groundwater wells distributed in 51 major hydrologic systems in the United States (Gilliom et al

2006)

In California paraquat has not been detected in groundwater as a non-point source pollutant

The CDPR Well Inventory Database lists three unconfirmed detections of paraquat in 1993 and

1997 all concentrations were below laboratory reporting limits Subsequent monitorings

conducted by the CDPR to confirm these detections resulted in no measurable concentrations of

paraquat (C Nordmark personal communication May 8 2017)

Few peer-review reports in the literature for California or the United States support findings

that paraquat can be detected in surface waters resulting from non-point source pollution A

search in the USGS database of the National Water-Quality Assessment Program which

includes data collected by over 400 state federal tribal and local agencies did not return any

detection (Gilliom et al 2006) Furthermore similar searches of the CDPR surface water

database and the California Environmental Data Exchange Network (CEDEN) database

maintained by the California State Water Resources Control Board returned seven detections

(042ndash36 μgL Irrigated Lands Regulatory Program) out of 1450 samples between 16 May and

16

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

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Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

2006 and 16 September 2014 (CSWRCB 2017) No additional detections were reported

nationwide by other agencies contributing to the water quality data of the National Water Quality

Monitoring Council (NWQMC 2017)

63 Air

The main pathways by which paraquat can travel into the air are through adhesion to particulate

matter that is transported in the air or from drift during application (Seiber and Woodrow 1981

Chester and Ward 1984 Ames et al 1993 Lee et al 2005) These pathways can lead to adverse

effects due to dermal and respiratory exposure particularly to field applicators or people residing

near an application (Weinbaum et al 1995)

Seiber and Woodrow (1981) monitored a field application in two mature cotton fields in

Kings County California using air samplers and installed additional paired samplers in two

enclosed-cab harvesters (inside and outside the operatorrsquos cabin) to monitor the airborne dust

generated during the harvesting operations Paraquat concentrations measured in the air

downwind regularly decreased from values ranging 431ndash107 μgm3 at 1 m from the downwind

border of the two fields to lt50 ngm3 at about 400 m downwind The post-application

concentrations decreased to 1ndash10 of the initial values twondashfour h since the end of the

application and to non-detectable values fivendashseven h afterwards Paraquat was also present in

the airborne particulate matter during harvesting operations at one of the sites (1245 and 506

ngm3 outside and inside the open cab respectively) They concluded that such values were well

below most acute and sub-acute LD50s recorded in animals

7 Environmental degradation

In surface soils or biological materials paraquat may undergo microbial degradation during the

time period from immediately after an application until it becomes inactivated and protected via

17

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

adsorption onto soil particles (Carr et al 1985) This bioavailable paraquat may also photo-

decompose over several weeks (Funderburk and Bozarth 1967 Roberts et al 2002) Degradation

on plant materials generally occurs much more rapidly than the degradation in soil (Lee et al

1995) When paraquat becomes protectedimmobilized in subsurface soils it may persist for

several years or longer and currently there is no effective degradation method to degrade

paraquat that is strongly bound in soil (Ye and Lemley 2008) Ultraviolet or solar radiation alone

are insufficient to ultimately mineralize paraquat and serve as a method for depolluting

contaminated waters or soils and microbiological processes are slow and require long incubation

times (Moctezuma et al 1999)

71 Microbial

A vast array of bacteria and fungi in a soilrsquos solution are capable of slowly degrading the

bioavailable paraquat that is believed in equilibrium with the paraquat strongly adsorbed onto the

soil mineral phase (Funderburk and Bozarth 1967 Ricketts 1999 Roberts et al 2002 Wu et al

2013) andor the paraquat that is weakly adsorbed onto SOM (Burns and Audus 1970) (Table 2)

Both laboratory and field incubation studies have confirmed that microbial degradation is

responsible for the generally slow paraquat degradation ifwhen paraquat becomes bioavailable

(Funderburk and Bozarth 1967 Burns and Audus 1970 Lee et al 1995 Murray et al 1997

Cheah et al 1998 Ricketts 1999 Ismail et al 2011) Paraquat-degrading microorganisms

include the bacteria Pseudomonas spp and Flavobacterium spp (Murray et al 1997) the fungi

Neocosmospora vasinfecta (Funderburk and Bozarth 1967) and Lipomyces starkeyi Lod and Rij

(Burns and Audus 1970 Carr et al 1985) and the patented microbial mat (consortium of

cyanobacteria [Oscillatoria sp] and bacteria) used by Murray et al (1997)

18

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

The bioavailable paraquat can be degraded by soil fauna when in presence of sufficient

carbon (C) and nitrogen (N) sources or only C sources because the herbicide can act as the sole

source of N to sustain microbial activities (Carr et al 1985) Degradation can occur both under

aerobic (Ismail et al 2011) and anaerobic conditions (Lee et al 1995) laboratory in vitro

experiments (Carr et al 1985) or in field dissipation studies (Amondham et al 2006) The

degradation rate has been shown to be higher for plant- than soil-associated paraquat under

aerobic than anaerobic conditions and directly proportional to a plantrsquos CN ratio (Lee et al

1995) it increases with the addition of C sources such as sucrose that enhance bacterial

degradation under aerobic (Ricketts 1999) and anaerobic conditions (Wu et al 2013)

Funderburk and Bozarth (1967) isolated paraquat-tolerant microorganisms from soil under

laboratory conditions They proposed that the pathway of paraquat degradation in soil by a non-

identified bacterial includes the de-methylating of the parent molecule splitting of one of the two

hetorocyclic rings followed by formation of a carboxilated 1-methylpyridnium ion (Figure 6) A

similar pathway with initial de-methylating of the parent molecule was reported also by Ricketts

(1999) In incubation experiments those authors showed that no paraquat remained in solution at

the end of each incubation and the analysis of the degradation products showed almost identical

metabolite profiles between the different microorganisms They could also identify 14C-oxalic

acid as the main degradation product (85 of total remaining radioactivity in the incubating

solution) and other non-identified products

72 Photochemical

Degradation due to UV radiation can be expected to act as an important mechanism on paraquat

bound to the surface of treated plants and paraquat-laden soil particles on the topsoil exposed to

solar radiation (Calderbank and Slade 1976) Using paper chromatography and other techniques

19

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

8

Slade (1965) found that paraquat under Ultraviolet (UV) irradiation is degraded to 1-methyl-4-

carboxypyridinium ion and methylamine hydrochloride (Figure 7) That author observed that

paraquat under solar light is degraded only when adsorbed to a surface but not when in aqueous

solution These findings on photo-degradation were later evaluated by other authors (Table 2)

who also identified additional products using the same or similar experimental conditions

(Funderburk and Bozarth 1967 Kearney et al 1985 Nguyen and Zahir 1999 Florecircncio et al

2004) Similarly the work by Kearney et al (1985) showed limited photodegradation of aqueous

paraquat under UV or solar radiation under normal aerobic conditions

Ecotoxicology

The main focus of this section will be on new toxicological data available for terrestrial

invertebrates because they are important indicators of ecosystem pollution for risk assessment

(Edwards and Bohlen 1992 Wang et al 2012 Givaudan et al 2014) and the model species

identified with a relatively high level of concern (LOC) on an acute basis by the USEPA

(USEPA 1997) In small mammals endangered species LOCs are exceeded for large herbivorous

and insectivorous As reported in the previous sections paraquat is used primarily in agricultural

and forestry settings including along rights-of-way fence lines pipelines These uses

particularly increase the risk of exposing wild birds and mammals to paraquat (Hoffman et al

1987 Berny 2007) Despite strong evidence in favor of paraquat deactivationimmobilization by

soil Eisler (1990) has also underscored that long-term fluxes of paraquat from the soil-adsorbed

phase into the food chain remain poorly understood

It is unlikely that paraquat may cause chronic effects in birds due to accumulation in the

food chain whereas the main concern for avian populations is that direct application to eggs may

cause birth defects The risk is the greatest immediately after an application and tails off

20

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

overtime due to wetting and drying cycles that may re-solubilize the paraquat cation Similarly

the risk for insects such as honey bees (Apis mellifera) would be greatest during an application

but paraquat is typically applied when honey bees are not active in the field (USEPA 1997)

From the time Eisler (1990) conducted his synoptic review of the literature available up until the

late 1980s new information has become available particularly on earthworms honeybees

amphibians and fish

81 Mode of action paraquat redox chemistry

The work by Farrington et al (1973) analyzed and elucidated the main chemical reactions

responsible for paraquat herbicidal action in plants These reactions occur because in aqueous

solution the paraquat cation PQ2+ acts as a terminal electron acceptor to form the monovalent

cation PQ∙+ that is stable in the absence of oxygen Researchers coined the word ldquoviologenrdquo

specifically for the BP salts because of their blue-violet color corresponding to the one-electron

reduction (Michaelis and Hill 1933 Ross and Krieger 1980) Assuming a continuous supply of

electrons to paraquat and aerobic conditions paraquat will cycle from reduced (PQ∙+) to oxidized

form (PQ2+) with continuous production of the superoxide anion free radical O2 ∙- (Michaelis and

Hill 1933 Smith 1985) When in contact with any biological tissues such as plant or animal

tissues the superoxide radical O2 ∙- reacts with any biological targets leading to the formation of

hydrogen peroxide (H2O2) and secondary hydroxyl radicals (OH∙) particularly superoxide

radicals OH- (Farrington et al 1973 Calderbank and Slade 1976 Beloqui and Cederbaum 1985

Hassett et al 1987) These reactive species are then reduced by any metal ions that are part of the

biological targetstissues acting as electron donors (Goldstein et al 2002)

21

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

82 Plants

It is more likely that paraquat may move offsite due to drift during ground or aerial application

rather than through volatilization (USEPA 1997) thereby affecting both target and non-target

plant species Main effects include wilting and general collapse in herbaceous plants Perennial

plants may regrow and the most resistant plants may be affected by temporary scorch (Eisler

1990)

821 General physiological effects

There is evidence that paraquat enters the plant through the leaf cuticle rather than through

stomata (Brian 1967) and depending on which kind of plant tissue is sprayed some amounts may

also be adsorbed onto cell walls before reaching the membranes (Funderburk and Lawrence

1964) When leaves are treated with paraquat the formation of extremely reactive free radicals

leads initially to damage due to the polymerization of the unsaturated lipids of the cell

membranes Thus cell membranes lose integrity which favors water loss and rapid desiccation

or in other words tissue necrosis (Dodge 1971 Farrington et al 1973) Because of the redox

processes involved and their strong tendency to act as an electron acceptors the BPs are also

referred as ldquoelectron divertersrdquo of the Photosystem I (PSI)

When paraquat is present in illuminated chloroplasts it acts as an electron acceptor that

traps all electrons from PSI at a diffusion-controlled rate (Farrington et al 1973) to form its

cation radicals generating O2- at a rapid rate via oxidation Thus ferridrin and NADP+ cannot be

photo-reduced This causes accumulation of H2O2 in the chloroplast Paraquat radical is formed

within the thylakoids of the chloroplast and then diffuses into the stroma where initially there

are aerobic conditions Given that the BPs have a redox potential more negative than NADP+

22

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

and ferrodoxin they interfere with NADP reduction in the chloroplast (Dodge 1971 Asada

1999)

The production of O2- and H2O2 may then lead to formation of more reactive oxygen

radicals which may be more toxic to the cell and initiate cell membrane disruption and

destruction of chlorophyll interfering with the activities of the chloroplasts These harmful

byproducts cause breaches in the integrity of the membranes surrounding cell organelles

resulting in uncontrolled electrolyte leakage and cell death (Calderbank and Slade 1976) The BP

herbicides damage plants mainly in the presence of light whereas in darkness damage develops

more slowly and only at high application rates as shown by Slade and Bell (1966) Based on the

findings of those authors paraquat movement into the plant was limited when the plant was

exposed to light due to tissue damage whereas in darkness the translocation of paraquat occurred

over greater distances into the xylem through undamaged tissue It is well established that

paraquat translocation throughout the main plant organs is the greatest under dark conditions

(Brian 1967) Under experimental conditions it has been shown that paraquat is normally

reduced in green tissues by energy derived from light even though such activity is also present

in the dark in a reduced form (Homer et al 1960) Paraquat may also be taken up by a plantrsquos

root systems although this is an uncommon occurrence given that paraquat mobilization under

illumination in a plant tissue is minimal due to the redox reactions leading to the destruction of

cell membranes and consequent desiccation of plant tissues (Hawkes 2014)

Since the 1970s researchers have investigated the possible mechanisms responsible for

plant resistance Two prevailing mechanisms have received most research attention since the

early 1980s (1) sequestration of paraquat away from its site of action in the chloroplast (Fuerst

et al 1985) and (2) an increase in the activity of oxygen radical-scavenging enzymes in resistant

23

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

(R) biotypes which foster tolerance against active oxygen species formed by paraquat (Hart and

Di Tomaso 1994) The first mechanism involves restricted rate of herbicide movement

throughout the plant (Preston et al 1994 Yu et al 2007 Powles and Yu 2010) and has not yet

been established unambiguously for any biotype (Hart and Di Tomaso 1994 Hawkes 2014)

Researchers have hypothesized that only limited amounts of paraquat may reach the site of

action in the chloroplasts (limited penetration has been detected in Hordeum glaucum Steud and

Conyza bonariensis (L) Cronq) and if paraquat does reach the chloroplasts it is rapidly

transferred and sequestered into a metabolic inactive compartment via a sequestration

mechanism (Hart and Di Tomaso 1994 Joacuteri et al 2007)

Most recent studies in Lolium rigidum suggest that resistance to paraquat may be associated

with a mechanism of the cell cytoplasm that causes a greater rate of vacuolar sequestration but

the precise biochemical and molecular basis of this sequestration mechanism remains unclear

(Busi and Powles 2011 Hawkes 2014) Other proposed mechanisms include (3) the lack of

penetration due to epicuticular wax (4) an alteration in the redox potential of the PSI primary

electron acceptor (5) adsorption of paraquat to lignified areas (6) the binding of paraquat onto

cell walls or (7) developed ability to prevent paraquat from entering the symplast To date there

is not enough empirical evidence in favor or against mechanisms 3ndash7 (Hart and Di Tomaso

1994) Since paraquat rapidly diverts electrons from the PSI acting as an electron acceptor in the

chloroplast there likely are no binding-site mutation-based mechanisms that foster paraquat

resistance Hawkes (2014) indicated that currently there is no report of paraquat resistance

dependent on site-specific mutations whose identification would have important implications to

develop paraquat resistant crops

24

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

822 Dose-response studies

The doses for lethal and sub-lethal effects vary depending on species and biotype In his review

of the literature Eisler (1990) reported that in sensitive species of terrestrial plants and soil

microflora adverse effects occur at 028ndash06 kgha This is in agreement with other findings by

Preston et al (1994) who reported that adverse effects start to occur in sensitive species at 02

kgha and that the difference in toxicity indices between susceptible (S) and R biotype varied 10ndash

50 times Those authors studied paraquat effects on capeweed (Arctotheca calendula [L]

Levyns) biotypes collected from an alfalfa (Medicago sativa L) field near Ararat Australia that

had received 24 consecutive annual applications of diquat and paraquat They estimated LD50 ~ 4

kg aiha and 04 kg aiha for the R and S biotype respectively Busi and Powles (2011) studied

paraquat resistance in Wimmera ryegrass (Lolium rigidum Gaud) R and S biotyptes that were

previously selected for glyphosate resistance at 015 025 or 035 kg glyphosateha In their

work glyphosate-selected and unselected plants were treated at 0 0006 0013 0025 and 0050

kg paraquat ionha both under laboratory and field conditions to estimate 15-day LD50s They

found that biotypes that were selected for glyphosate resistance showed also a concomitant

paraquat resistance and the LD50 of a three-time glyphosate-selected progeny (0044 kgha) was

fourfold greater than the unselected parent (0011 kgha)

Similar findings about concomitant resistance among biotypes were reported by Moretti et

al (2016) who studied accessions that were found resistant to both paraquat and glyphosate in

orchards and vineyards of the Central Valley in California Plants were collected from separate

genetic groups located at distances up to 160 km In a randomized complete block design

experiment they considered genetic group and application rate (0 0019 0056 0167 05 15

45 135 and 405 kg PDha) as the two main experimental factors to assess paraquat dose-

25

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

responses They estimated that the 28-day LD50 varied 50ndash393 times in hairy fleabane and 418

times in horseweed The highest and lowest overall LD50s were 001 and 155 kgha

respectively for hairy fleabane and 002 and 216 kgha respectively for horseweed

83 Soil fauna and flora

Earthworms are important indicators of ecosystem functioning and health as well as soil

contamination from agrochemicals (Edwards and Bohlen 1992 Givaudan et al 2014) Most

reports underscore that paraquat adsorbed through the gut of earthworms and other invertebrates

or microarthropods is rapidly excreted without any significant bioaccumulation in tissues

(Summers 1980) Past reviews comparing paraquat to other agrochemicals indicated that

paraquat is less toxic to earthworms than other herbicides fungicides and insecticidesmdashit is

ranked as one of the least toxic (Haque and Ebing 1983 Edwards and Bohlen 1992)

831 Toxic effects

Adverse effects on growth in soil start to occur at extremely high concentrations at about 1000

ion mgkg soil or 1500 kgha (assuming a soil bulk density of 1 gcm3 and depth of 15 cm) based

on Van Gestel et al (1992) Haque and Ebing (1983) estimated 14-day LC50 values of gt200 mg

ionkg soil substrate for Eisenia foetida and Lumbricus terrestris (assuming a soil bulk density as

before the corresponding content would be ~ 300 kg ionha)

Roberts et al (2002) revised the results from long-term research trials about monitoring

changes in soil properties following a single or annual paraquat applications (Table 2) Those

studies showed that an increase of paraquat in soil had caused temporary changes in soil fauna

composition within the first decade since cessation but levels were well below values that would

cause marked effects on crops or soil fauna indicating that repeated paraquat applications at

recommended application rates of about 1 kgha will not have any negative impact on the

26

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

number or activity of soil microorganisms Those authors concluded that the overall changes

were more the indirect result of eliminating the competing vegetation rather than direct action of

paraquat on soil fauna

The findings about low toxicity in earthworms reported by Roberts et al (2002) were

confirmed most recently by Papini et al (2006) who studied the influence of two substrates

representing a clayey and sandy soil type on paraquat bioaccumulation in the earthworm

(Eisenia foetida) under laboratory controlled conditions The two substrates were treated to

obtain a paraquat concentration of 12 12 and 120 μ 14C-paraquatg substrate Ten worms were

added to each glass vessels containing the substrate and maintained in dark at 20 plusmn1 degC for 90

days The bioassay was then dismantled At the end of the experiment they found that the

paraquat mass bio-accumulated was always less than 1 of total biomass No mortality due to

paraquat was detected supporting the general belief that paraquat is non-toxic to these worms at

commonly adopted field application rates

However when the action of paraquat is not buffered by soil inactivationsequestration

toxic effects do occur also in earthworms as shown by Muangphra et al (2014) through contact

toxicity tests Those authors conducted both acute and chronic toxicity studies on Pheretima

peguana the most common earthworm species found in agricultural fields of Thailand They

also evaluated the genotoxicity of glyphosate and paraquat as indicated by chromosomal

aberrations DNA damage and cytoskeleton damage in coelomocytes (immune cells in the

coelomic cavity) of P peguana Using probit analysis they estimated a 2-day LC50 of 039 kg

ionha and found that even the chronic dose of 00039 kg ionha induced genetic (clastogenic

and aneugenic) effects on earthworm coelomocytes

27

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

A comparison between LD50 estimates on filter paper and artificial soil is provided for the

earthworm Eisenia fetida by Wang et al (2012) Their estimated 2-day LD50 value in filter paper

was 235 kg ionha indicating moderate toxicity whereas the 7-day and 14-day LD50 values in

artificial soil were gt1000 mgkg (or 1500 kg ionha assuming a soil bulk density of 1 gcm3 and

depth of 15 cm) underscoring that paraquat is among the least toxic herbicides when applied to

soil as indicated by Edwards and Bohlen (1992)

832 Teratogenic effects

Van Gestel et al (1992) described both toxic and teratogenic effects in the earthworm Eisenia

foetida under laboratory controlled conditions They monitored cocoon production in

earthworms that were placed in artificial soil for an adaptation period without paraquat followed

by a three-week period of exposure to paraquat Dry PD and water were added to the soil (55

gravimetric water content) and earthworms were exposed for three weeks Cocoon production

diminished at 450 mgkg dry soil (or 675 kg ionha) and was significantly reduced at 1000 mg

ionkg dry soil Cocoons had abnormal shapes and the percentages of the small ones passing

through a 2-mm sieve were 07 45 and 108 for the 0 450 and 1000 mgkg (control)

rate respectively There was no paraquat effect on the number of juvenile fertile cocoon Instead

paraquat applied at 1000 mgkg significantly reduced the total number of offspring

(juvenilewormwk) The difference between the no observed effect concentration for

reproduction (NOEC = 56 mgkg dry soil) and the median lethal dose (LC50 = 1000 mgkg dry

soil) was a factor of gt100 Those authors concluded that their results were in agreement with

previous findings indicating that paraquat produces long-lasting and irreversible effects on

earthworms

28

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

833 Soil bacteria

Paraquat can cause negative impacts on organisms responsible for nutrient cycling Gadkari

(1988) studied the influence of atrazine and paraquat on a mixed culture of nitrifying bacteria in

a laboratory aqueous system Paraquat caused a complete inhibition of ammonium and nitrate

oxidation at 5 μgmL and 10 μgmL ammonium oxidation was completely inhibited for over 40

days nitrate oxidation was also completely inhibited at the same concentrations for 28 days

even at 1 μgmL ammonium and nitrate oxidation were inhibited for gt18 days

84 Birds

Paraquat is among the most embryotoxic environmental contaminants for bird eggs (LC50 ~ 17

kgha day 18) at the customary application rates as reported by Hoffman and Albers (1984) who

refers also to Lutz-Ostertag and Henou (1975) (chicken and Japanese quail [Coturnix japonica]

eggs) and Hoffman and Easting (1982) (mallard [Anas platyrhynchos] eggs) Bird nestlings also

appear sensitive as reported for the American kestrel (Falco sparverius) by Hoffman et al

(1985) and bobwhite quail (Colinus virginianus) Japanese quail ring-necked pheasant

(Phasianus colchicus) and mallard by Heath et al (1972) At the same time adult bobwhite quail

(Colinus virginiunus) could tolerate a diet containing paraquat at 100 ppm (mixture of water and

mash) for 60 days without showing signs of toxicity or impaired learning indicating that

paraquat is highly toxic to avian embryos but not to adults (Bunck et al 1986)

841 Toxic and teratogenic effects

Hoffman and Eastin (1982) conducted a series of experiments to investigate the effects of two

insecticides (lindane and toxaphene) and two herbicides (paraquat and 245-T) by treating

mallard eggs in paraquat formulations at actual field concentrations The eggs were immersed in

different mixtures even though paraquat is generally applied in aqueous emulsion containing

29

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

different herbicidal concentrations and different solvents Paraquat was the most toxic to the

embryos regardless of the type of mixture in agreement with previous findings (Lutz Ostertag

and Henou 1974) When treatment with aqueous paraquat emulsion was on three-day-old

mallard embryos the corresponding LC50 (day 18) was 15 lb aiacre (15 times the recommend

field application rate) Paraquat in aqueous emulsion caused impaired growth and was slightly

teratogenic at half the field level of application causing 23 mortality Brain defects

(anencephaly and exencephaly) occurred only at higher concentrations (15ndash3 times the field

level)

These results were confirmed by the work of Hoffman and Albers (1984) who conducted a

subsequent comparison of the effects on mallard eggs exposed to 42 pesticides including

herbicides on mallard They used the same methods as described by Hoffman and Eastin (1982)

Their results showed again that paraquat both in the aqueous emulsion and organic mixtures

was the most toxic The LC50 was about 15 lbacre in the aqueous emulsion causing grown

inhibitions and teratogenic effects They found that in comparison to the other herbicide studied

that is trifuralin paraquat was more teratogenetic causing extensive edema and brain defects

(anencephaly and exencephaly) as well as growth inhibition at treatment doses less than the

LC50 The LC50 of the eight considered pesticides were greater in the organic solvent than in the

aqueous emulsion likely due to enhanced penetration through the eggshell and its membranes

Similar effects on bird reproduction were reported by Dunachie and Fletcher (1967) who found

an extremely high rate of mortality in longhorn chickens (Gallus domesticus) from eggs

previously injected with paraquat at 015 ppm and a generally reduced hatchability

Predatory birds like kestrels feed on other species such as grasshoppers small rodents and

passerine birds which may come into contact with paraquat during agricultural spaying or by

30

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

ingestion (Hoffman et al 1985) Those authors studied the effects of paraquat oral administration

in distilled water to American kestrel nestlings by randomly assigning each hatchling to a

control 10 20 or 60 mgkg treatment level They found that 44 of the nestlings receiving the

highest dose died after four days and the growth rates of the paraquat-treated groups were

significantly lower than all controls These results indicated that American kestrel nestlings are

particularly sensitive to paraquat exposure Histopathological examinations showed liver

kidney brain and lung damages suggesting greater sensitivity to paraquat in altricial nestling

kestrels than in young or adult birds of precocial species

842 Reproductive effects

Northern bobwhite (Colinus virginianus) appeared less sensitive to paraquat use as reported by

Bauer (1985) who investigated the effects on reproduction and growth of a paraquat-

contaminated diet at levels equal or lower than those that could be found in paraquat-treated

fields (0 20 60 and 180 ppm for six weeks) The studied variables included egg production

fertility hatchability chick abnormalities and chick survival and weights up to age seven days

The parental (P) hens fed at 180 ppm laid fewer eggs than at lower rates and had lower ovary and

oviduct weights However there were no significant differences in P fertility chick

abnormalities or survival compared with controls First generation hens from paraquat treated

parents laid eggs with a one-week delay lower rate of egg production and 10-day delay in clutch

completion compared with controls

85 Honeybees

Habitat reduction has often led beekeepers to pasture their bees on insecticide-treated fields

(Johansen 1977) Paraquat caused extremely toxic effects to honey bees (Apis mellifera L) in

small cages that were exposed to paraquat at 4 lb aiacre as a water spray 90 of bees died

31

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

three days after exposure (Moffett et al 1972) Paraquat was extremely toxic in a sucrose syrup

diet at concentrations of 100 mgL (Morton et al 1972) In honey bee colonies placed in isolated

desert apiaries and fed exclusively with aqueous paraquat at 1000 ppm of ai large numbers of

bees died immediately and all were dead within five weeks (Morton et al 1974) Based on the

available information it had been concluded by the USEPA (USEPA 1997) that paraquat was

non-toxic to slightly toxic to adult bees

The most recent information available since that time supports instead the conclusions by

those previous authors (Moffett et al 1972 Morton et al 1972) The survival rate of paraquat-

injected queens was greater than that of paraquat-injected workers due to the presence of the

protein vitellogin acting as an antioxidant (Corona et al 2007) In a most recent work by Cousin

et al (2013) honeybee combs were transported to the laboratory and the newly hatched larvae

were exposed to paraquat (0 0001 001 01 and 1 μgkg of food) for two days These

exposures caused a reduction in embryonic cells that is oenocytes occurring at concentrations

as low as 1 ngkg Reduction in cell size was concentration dependent

86 Amphibians

Amphibians are generally more susceptible than mammals and birds to the negative effects of

agrochemicals because they have evolved adapting to both aquatic and terrestrial habitats and

their skin is highly permeable because it is involved in water gas and electrolyte exchange with

the environment In California there is concern that paraquat may affect the federally threatened

California red-legged frog (Rana aurora draytonii) whose habitat includes both coastal and

interior mountain ranges (USEPA 2009)

32

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

861 Toxic teratogenic and reproductive effects

Several researchers have investigated the sub-lethal and lethal effects of paraquat on

amphibians The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a well-established

screening method to identify the potential developmental toxicity of single or multiple chemicals

in amphibians and is considered less protective than using more traditional aquatic test species

because it is relatively insensitive It provides a tool not only to assess mortality but also

malformation and growth inhibition using a standardized method While there is general

agreement on the embryotoxic effects of paraquat in amphibians there is disagreement regarding

its teratogenic effects This is likely due to the challenges in explaining the number and true

cause of the observed malformations in tadpoles (Dial and Bauer 1984 Vismara et al 2000

Osano et al 2002)

Dial and Bauer (1984) investigated exposure to paraquat of northern leopard frog (Rana

pipiens) embryos at field application rates (0 01 05 20 and 10 mgL) They found that eggs

were very resistant to paraquat because development proceeded regularly to hatch up to day four

in all exposed groups However with the exception of the 01 mgL group there was an increase

in mortality of tadpoles starting three days post hatch in all other groups and survival rates to

day 12 were 755 691 55 0 and 0 for the 0 01 05 20 and 10 mgL rates respectively

Growth rates decreased in all groups and were inversely proportional to rate They interpreted

their data as a clear sign of teratogenic effects starting at as low as the rate of 05 mgL After

hatch on day four paraquat caused death retardation of growth multiple tail malformations

reduced head development and reduced motor ability

The same authors found tadpole survival is a function of age ie there are greater survival

rates in older tadpoles Dial and Dial (1987) compared developing embryos of the northern

33

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

leopard frog and 15-day-old tadpoles that were treated with diquat or paraquat Paraquat

treatments (at 05 and 2 mg ionL) were replicated and occurred when the eggs were at the early

gastrula phase for the first group and when the tadpoles were 15 days old for the second group

The treated eggs were found to be resistant to both herbicides because their development

proceeded normally to hatch (day four) in all groups However survival of the tadpoles in the

first group showed increased mortality On day 16 survival rate was 63 and 0 for the first

group receiving the 05 mgL and 20 mgL paraquat ion dose respectively for the same doses it

was 667 and 5 respectively for the 15-day-old tadpoles

Lajmanovich et al (1998) studied the acute tolerance to paraquat in Scinax nasica tadpoles

as well as conducting morphological analyses to detect alterations in the internal gill structure

Their LC50 estimates were 3896 2997 2495 and 2199 mgL for the 24 48 72 and 96 h

interval respectively The corresponding 120-h LC50 (~ 15 mgL) calculated via interpolation

was about 100 times higher than that computed by Vismara et al (2000) or by Linder et al

(1990) who reported a 96-h LC50 of 13 mgL in leopard frog embryos which would indicate

that adverse effects occur at much lower levels

Paraquat was found embryotoxic but not teratogenic on amphibian development based on

FETAX bioassay studies by Vismara et al (2000) Those authors used probit analysis on the

percentage of mortality and the percentage of malformed larvae to investigate toxicity and

teratogenic effects Tadpoles of Xenopus laevis were treated with aqueous paraquat at 00625

0125 018 025 and 05 mgL and were studied at specific developmental stages from 8 to 47

h and 120 h post fertilization (PF) They observed significant reductions in larvae growth that

were proportional to the treatment concentration Histological examinations of the larvae that

received the 0125 dose revealed that 29 of the treated larvae were affected by a specific

34

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

malformation identified as ventral tail flexure There were statistically significant differences in

larvae length between larvae treated at the lowest rate of 00625 mgL and the control However

they concluded that such differences were more the result of growth retardation rather than

teratogenic effects because no significant teratogenic effects were noticeable from the time of

hatching until day three at the onset of lethal effects Paraquat was only highly embryolethal with

a 120-h-PF LC50 of 0138 mgL and TC50 of 0267 mgL Mortality rates at 120 h PF were 124

419 835 888 and 966 for the rates of treatment reported above respectively

Osano et al (2002) studied the teratogenic effects of three pesticides including paraquat on

Xenopus laevis tadpoles using the FETAX bioassay They found a drastic increase in mortality

from 24 h to 96 h with corresponding 96-h LC50 estimate of 067 mgL Malformation in all

embryos occurred at concentrations greater than 02 mgL whereas growth reduction was

apparent at all test concentrations (01ndash5 mgL) Those authors concluded that paraquat should

be classified as teratogens as suggested also by the work of Quassinti et al (2009) showing that

paraquat interferes also in amphibian reproductive processes

87 Fish and other aquatic species

Paraquat can contaminate aquatic environments as water runoff due to its high aqueous

solubility and surface or marine waters in countries that allow its use for the control of aquatic

plant species (Tortorelli et al 1990 Ayanda et al 2015 Ling et al 2017) where fish mortality

may indirectly increase also due to oxygen consumption by dead plants (Summers 1980)

871 Toxic effects

The susceptibility of fish and other aquatic species in waters treated with paraquat was studied

by Fytizas (1980) who simulated under laboratory settings the accidental discharge of paraquat

in a water body and its effects on marine species They estimated the lethal time necessary to kill

35

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

50 of the fish exposed (LT50) in bony fish (Mugil cephalus) the small decapos crustaceous

(Pagurus sp) and the gastropod mollusc (Murex brandaris) treated with Gramoxone (20 ai)

through a bioassay that is in all-glass aquaria of 75 L for fish and 35 L for all other species

They found that the LT50 at 10 mgL for fish was 1 h whereas it was 36 h and 24 h for the

crustaceans and gasteropods respectively The sensitivity was inversed when considering low

concentrations instead LT50 values were 16 10 and 18 days for fish crustacean and

gasteropod respectively at the 1 mgL dose The fish exposed to the highest doses that were

killed within 24 h showed damaged gills covered in mucus hemorrhage and necrosis of the liver

and kidney and hemorrhagic ulcerations of the digestive tract

Paraquat action on fish is species dependent as shown by the early work of Earnest (1971)

who studied the effects of paraquat applied during 30 min to a pond at a concentration of 114

mgL (using a boat-mounted bar spreader) near the town of Morrison Colorado Acute effects

were more evident one and two days post application The most affected species was bluegill

(Lepomis macrochirus) and least affected was channel catfish (Ictalurus punctatus) Paraquat

caused distress in 50 bluegills three h following the application 89 dead bluegills and one dead

trout one day post treatment and at least 34 mortality of the bluegills within two days Most

common malformations were the presence of granuloma involving pancreatic cells

Toxicity also varies with fish development stages For example the Louisiana crayfish

(Procambarus clarkia) has also been often used as bioassay organism to study pesticide effects

due to its commercial role as food supply Leung et al (1980) studied adult and juvenile crayfish

individuals exposed to six different paraquat concentrations (adult 0 15 25 35 50 75 and 100

mgL juvenile 0 05 20 40 70 and 80 mgL) and used probit analysis to estimate LC50s for

different time intervals Differences in mortality between treated and untreated group started to

36

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

be detected 48 h post treatment at the lowest doses that is 15 mgL for the adult and 05 mgL

for the juvenile group Adult mortality was 10 in the 15 mgL-treated group and 09 in the

corresponding control juvenile mortality was 93 in the 05 mgL-treated group and 67 in

the control The 48-h LC50 estimates were 52 mgL and 39 mgL for juvenile and adult

respectively

The 48-h LC50 estimate for juveniles reported by Leung et al (1980) was the same (ie 52

mgL) as that reported by Tortorelli et al (1990) for catfish fry (Plecostomus commersoni) but

about 74 times higher than the 96-h LC50 of 007 mgL reported most recently by Ayanda et al

(2015) who studied the effects of paraquat and glyphosate acute exposure in African catfish

(Clarias gariepinus) juveniles Discrepancies in these estimates may vary also due to the effect

of temperature an important factor influencing the immune response in fish exposed to paraquat

as shown by Salazar-Lugo et al (2011)

The estimates for the toxicological indices reported above agree also with the very

comprehensive review conducted by Summers (1980) indicating that in general adverse sub-

chronic effects to adult fish start to occur at aquatic concentration of lt1 mg ionL for varying

durations and most recent work have indicated that acute effects occur at concentration gt10 mg

ionL (Figueiredo-Fernandes et al 2006 Parvez and Raisuddin 2006 Salazar-Lugo et al 2011

Ma et al 2014)

872 Oxidative stress indicators

In the 1980s the main research focus has shifted to also include the oxidative stress induced by

paraquat on fish species due to its redox properties following the early work of Bus et al (1976)

who hypothesized lipid peroxidation as one of the main mechanism of toxicity (Tortorelli et al

1990) Thus paraquat has been widely used as a model agent of oxidant injury (Gabryelak and

37

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

Klekot 1985) A large number of publications have focused on changes in biochemical and

histological parameters useful for monitoring environmental exposure of fish to contaminants

and laboratory and field studies (eg Parvez and Raisuddin 2006) Ayanda et al (2015) found

that paraquat also caused changes in the activities of aminotransferase aspartate

aminotransferase lactate dehydrogenase and alkaline phosphatase supporting a wealth of other

findings that it interferes with the normal functions in fish (Gabryelak and Klekot 1985)

Figueiredo-Fernandes et al (2006) monitored the effects of temperature and gender on selected

oxidative stress parameters (hepatic levels of superoxide dismutase[SOD] glutathione reductase

[GST] and glutathione S-transferase) in Nile tilapia (Oreochromis niloticus) exposed to 05 mg

ionL at 17 and 27 C for 45 days paraquat They found that the activities of SOD and GST were

sex dependent and were greater in males compared to females at both experimental

temperatures There was no temperature dependence

873 Behavioral analyses

Most recent research has been focused also on paraquat effects on zebrafish (Danio rerio) which

is currently considered as a promising experimental model to investigate neurological diseases or

for validation of psychopathological models (Bortolotto et al 2014 Nunes et al 2017) In their

work Bortolotto et al (2014) conducted behavioral analyses (locomotion social interaction and

Y-maze task) on adult zebrafish treated with paraquat at 10 mgkg and 20 mgkg (ie six

paraquat repeated injections one every 3 days during 16 days) compared to a control They

found that locomotion and distance travelled decreased after 24 h following each injection for

both doses but no significant differences were observed in non-motor behavior (ie anxiety-

related behavior and social interactions)

38

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

9

A similar study was conducted by Nunes et al (2017) on four- to six-month-old zebra fish

assigned to two experimental groups the treated (20 mgkg) and the untreated one following the

same injection procedure described in Bortolotto et al (2014) The two groups were monitored

for behavioral changes and biochemical parameters (tissue analysis mitochondrial viability

assay lipid peroxidation bioassay reactive oxygen species [ROS] levels antioxidant enzymes

non-protein thiols and protein determination) They found that paraquat caused an increase in

aggressive behavior alters non-motor patterns associated with defensive behaviors and changes

in redox parameters of the brain There were significant changes in most parameters associated

with the antioxidant defense system an unexpected decrease in lipid peroxidation and no change

in ROS concentration

Summary and Conclusions

Review of the literature reveals gaps considerations and recommendations for additional

research work These research inquiries will improve our understanding of paraquat

environmental fate and ultimately our ability to develop more accurate strategies for

environmental risk assessment

1 In California paraquat remains an important post-emergence herbicide that is used to

eliminate competing vegetation alone or in combination with other herbicides such as

glyphosate on the main cash crops cultivated in the state including almond alfalfa and

grapes The increasing number of reported paraquat-resistant species such as hairy

fleabane and horseweed is currently leading to the development of new strategies for

vegetation control

2 The available monitoring studies in California support findings that paraquat may

contaminate surface watersmdashin very limited casesmdashthrough runoff andor erosion due to

39

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

its high aqueous solubility or adsorption onto soil particle However conclusions about

the specific mechanism driving these detections remain speculative because available

experimental studies support the hypothesis that paraquat can be found in surface waters

due to soil erosion only as paraquat bound to soil or sediment particlesmdashnot in an

aqueous solution form The percentage of reported detections was lt1 of the total

sample size

3 Most recent studies have confirmed that aqueous paraquat can be degraded biologically

and photo-chemically but such degradation is extremely slow and cannot be considered

as an efficient and viable method to remediate paraquat-contaminated soils or waters

There are no existing technologies to reclaim contaminated soils containing strongly

bound paraquat that is considered environmentally unavailable Some transport may

occur due to erosion and runoff processes but these or similar fluxes from the soil into

terrestrial food chains remain poorly studied often due to the complexity in designing

long-term field studies

4 Paraquat is considered one of the least toxic pesticides to earthworms It is moderately

toxic to birds and moderately to highly toxic to many species of fish Most recent studies

have confirmed that paraquat causes both teratogenic and toxic effects in amphibians In

California the overall highest reported concentration (36 μgL) detected in surface waters

was about 17 times lower than the value (625 μgL) associated with adverse effects in

amphibians one of the most sensitive species and an important bio-indicator of pesticide

contamination Similarly this concentration was about 278 times lower than the reported

limit in the literature shown to cause adverse effects in fish ie 1 mgL

40

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

Acknowledgments

Funding for this work was provided by the California Department of Pesticide Regulation

(CDPR) California Environmental Protection Agency The statements and conclusions are those

of the authors and not necessarily those of the CDPR We also appreciate the valuable revisions

of Pamela Wofford and Madeline Brattesani during the initial phases of this work Any reference

to commercial products their source or their use in connection does not imply actual or implied

endorsement of such products

Disclosure statement

No potential conflict of interest was reported by the authors

41

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

References

Aeschbacher M SH Brunner RP Schwarzenbach and M Sander 2012 Assessing the

Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined

Electrochemical Reduction and Sorption Experiments Environmental Science and

Technology 46 (7) 3882ndash3890 doi 101021es204496d

Ames RG RA Howd and L Doherty 1993 Community Exposure to a Paraquat Drift

Archives of Environmental Health 48 (1) 47ndash52

Amondham W P Parkpian C Polprasert RD DeLaune and A Jugsujinda 2006 Paraquat

Adsorption Degradation and Remobilization in Tropical Soils of Thailand Journal of

Environmental Science and Health - Part B 41 (5) 485ndash507 doi

10108003601230600701635

Asada K 1999 The Water-Water Cycle in Chloroplasts Scavenging of Active Oxygens and

Dissipation of Excess Photons Annual Review of Plant Physiology and Plant Molecular

Biology 50 (1) 601ndash639 doi 101146annurevarplant501601

Autor AP and SL Schmitt 1977 Pulmonary Fibrosis and Paraquat Toxicity In

Biochemical Mechanisms of Paraquat Toxicity edited by AP Autor 175-186 New York

New York United States Academic Press doi httpdxdoiorg101016B978-0-12-

068850-050015-0

Ayanda OI SJ Oniye JA Auta VO Ajibola and OA Bello 2015 Responses of the

African Catfish Clarias Gariepinus to Long-Term Exposure to Glyphosate- and Paraquat-

Based Herbicides African Journal of Aquatic Science 40 (3) 261ndash267 doi

1029891608591420151074882

42

Bauer CA 1985 Effects of Paraquat on Reproduction and Growth in Northern Bobwhite The

Journal of Wildlife Management 49 (4) 1066ndash1073

Beloqui O and AI Cederbaum 1985 Microsomal Interactions between Iron Paraquat and

Menadione Effect on Hydroxyl Radical Production and Alcohol Oxidation Archives of

Biochemistry and Biophysics 242 (1) 187ndash196 doi 1010160003-9861(85)90492-8

Berny P 2007 Pesticides and the Intoxication of Wild Animals Journal of Veterinary

Pharmacology and Therapeutics 30 (2) 93ndash100 doi 101111j1365-2885200700836x

Bortolotto JW GP Cognato RR Christoff LN Roesler CE Leite LW Kist MR Bogo

MR Vianna and CD Bonan 2014 Long-Term Exposure to Paraquat Alters Behavioral

Parameters and Dopamine Levels in Adult Zebrafish (Danio Rerio) Zebrafish 11 (2) 142ndash

153 doi 101089zeb20130923

Brian RC 1967 Darkness and the Activity of Diquat and Paraquat on Tomato Broad Bean

and Sugar Beet Annals of Applied Biology 60 (1) 77ndash85 doi 101111j1744-

73481967tb05924x

Brian RC RF Homer J Stubbs and RL Jones 1958 A New Herbicide 1 1prime-Ethylene-2

2prime-Dipyridylium Dibromide Nature 181 446ndash447 doi 101038181446a0

Bromilow RH 2004 Paraquat and Sustainable Agriculture Pest Management Science 60 (4)

340ndash349 doi 101002ps823

Brooker MP and RW Edwards 1975 Aquatic Herbicides and the Control of Water Weeds

Water Research 9 (1) 1ndash15

Bunck CM TJ Bunck and L Sileo 1986 Discrimination Learning in Adult Bobwhite Quail

Fed Paraquat Environmental Toxicology and Chemistry 5 (3) 295ndash298 doi

101002etc5620050309

43

Burns IG MHB Hayes and M Stacey 1973 Some Physico-Chemical Interactions of

Paraquat with Soil Organic Materials and Model Compounds Weed Research 13 (1) 79ndash

90 doi 101111j1365-31801973tb01248x

Burns RG and LJ Audus 1970 Distribution and Breakdown of Paraquat in Soil Weed

Research 10 49ndash58 doi 101111j1365-31801970tb00922x

Bus J S S D Aust and J E Gibson (1976) Paraquat toxicity proposed mechanism of action

involving lipid peroxidation Environmental Health Perspectives 16 139-146

Busi R and SB Powles 2011 Reduced Sensitivity to Paraquat Evolves under Selection with

Low Glyphosate Doses in Lolium Rigidum Agronomy for Sustainable Development 31 (3)

525ndash531 doi 101007s13593-011-0012-6

Cairns JF and JR Case 1975 Manufacture of Bipyridylium Salts US Patent 3886172 filed

February 10 1972 and issued May 27 1975 Accessed 10 November 2016

httppatftusptogovnetahtmlPTOsrchnumhtm

Calderbank A and P Slade 1976 Chapter 10--Diquat and Paraquat In Herbicidesmdash

Chemistry Degradation and Mode of Action Volume 2 2nd Edition edited by PC Kearney

and DD Kaufman 501ndash540 New York United States Marcel Dekker Inc

Carmichael SL W Yang E Roberts SE Kegley AM Padula PB English EJ Lammer

and GM Shaw 2014 Residential Agricultural Pesticide Exposures and Risk of Selected

Congenital Heart Defects among Offspring in the San Joaquin Valley of California

Environmental Research 135 133ndash138 doi 101016jenvres201408030

Carr RJ RF Bilton and T Atkinson 1985 Mechanism of Biodegradation of Paraquat by

Lipomyces Starkeyi Applied and Environmental Microbiology 49 (5) 1290ndash1294

44

Burns IG MHB Hayes and M Stacey 1973 Some Physico-Chemical Interactions of

Paraquat with Soil Organic Materials and Model Compounds Weed Research 13 (1) 79ndash

90 doi 101111j1365-31801973tb01248x

Burns RG and LJ Audus 1970 Distribution and Breakdown of Paraquat in Soil Weed

Research 10 49ndash58 doi 101111j1365-31801970tb00922x

Bus J S S D Aust and J E Gibson (1976) Paraquat toxicity proposed mechanism of action

involving lipid peroxidation Environmental Health Perspectives 16 139-146

Busi R and SB Powles 2011 Reduced Sensitivity to Paraquat Evolves under Selection with

Low Glyphosate Doses in Lolium Rigidum Agronomy for Sustainable Development 31 (3)

525ndash531 doi 101007s13593-011-0012-6

Cairns JF and JR Case 1975 Manufacture of Bipyridylium Salts US Patent 3886172 filed

February 10 1972 and issued May 27 1975 Accessed 10 November 2016

httppatftusptogovnetahtmlPTOsrchnumhtm

Calderbank A and P Slade 1976 Chapter 10--Diquat and Paraquat In Herbicidesmdash

Chemistry Degradation and Mode of Action Volume 2 2nd Edition edited by PC Kearney

and DD Kaufman 501ndash540 New York United States Marcel Dekker Inc

Carmichael SL W Yang E Roberts SE Kegley AM Padula PB English EJ Lammer

and GM Shaw 2014 Residential Agricultural Pesticide Exposures and Risk of Selected

Congenital Heart Defects among Offspring in the San Joaquin Valley of California

Environmental Research 135 133ndash138 doi 101016jenvres201408030

Carr RJ RF Bilton and T Atkinson 1985 Mechanism of Biodegradation of Paraquat by

Lipomyces Starkeyi Applied and Environmental Microbiology 49 (5) 1290ndash1294

44

CDPR (California Department of Pesticide Regulation) 2016 Pesticide Use Reporting (PUR)

Sacramento CA California Environmental Protection Agency Department of Pesticide

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CDPR (California Department of Pesticide Regulation) 2017 A Guide to Pesticide Regulation

in California 2017 Update Sacramento California California Environmental Protection

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httpwwwcdprcagovdocspressrlsdprguidedprguidepdf

Cheah U-B RC Kirkwood and K-Y Lum 1997 Adsorption Desorption and Mobility of

Four Commonly Used Pesticides in Malaysian Agricultural Soils Pesticide Science 50 (1)

53ndash63 doi 101002(sici)1096-9063(199705)501lt53aid-ps558gt30co2-p

Cheah UB RC Kirkwood and KY Lum 1998 Degradation of Four Commonly Used

Pesticides in Malaysian Agricultural Soils Journal of Agricultural and Food Chemistry 46

(3) 1217ndash1223

Chester G and RJ Ward 1984 Occupational Exposure and Drift Hazard During Aerial

Application of Paraquat to Cotton Archives of Environmental Contamination and

Toxicology 13 (5) 551ndash563 doi 101007bf01056333

Connell JH F Colbert W Krueger D Cudney R Gast T Bettner and S Dallman 2001

Vegetation Management Options in Almond Orchards HortTechnology 11 (2) 254ndash257

Corona M RA Velarde S Remolina A Moran-Lauter Y Wang KA Hughes and GE

Robinson 2007 Vitellogenin Juvenile Hormone Insulin Signaling and Queen Honey Bee

Longevity Proceedings of the National Academy of Sciences 104 (17) 7128ndash7133

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Cousin M E Silva-Zacarin A Kretzschmar M El Maataoui J-L Brunet and L Belzunces

2013 Size Changes in Honey Bee Larvae Oenocytes Induced by Exposure to Paraquat at

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CSWRCB (California State Water Resources Control Board) 2017 ldquoCEDEN (California

Environmental Data Exchange Network)rdquo Sacramento California California State Water

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httpcedenwaterboardscagovAdvancedQueryTool

Dennis M KJ Hembree JT Bushoven and A Shrestha 2016 Growth Stage Temperature

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101016jcropro201512019

Dial NA and CA Bauer 1984 Teratogenic and Lethal Effects of Paraquat on Developing

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33 (1) 592ndash597 doi 101007bf01625589

Dial NA and CAB Dial 1987 Lethal Effects of Diquat and Paraquat on Developing Frog

Embryos and 15-Day-Old Tadpoles Rana pipiens Bulletin of Environmental

Contamination and Toxicology 38 (6) 1006ndash1011 doi 101007bf01609088

Dinis-Oliveira RJ JA Duarte A Saacutenchez-Navarro F Remiatildeo ML Bastos and F Carvalho

2008 Paraquat Poisonings Mechanisms of Lung Toxicity Clinical Features and

Treatment Critical Reviews in Toxicology 38 (1) 13ndash71 doi

10108010408440701669959

Dodge AD 1971 The Mode of Action of the Bipyridylium Herbicides Paraquat and Diquat

Endeavour 30 (111) 130ndash135

46

Dunachie JF and WW Fletcher 1967 Effect of Some Herbicides on the Hatching Rate of

Hens Eggs Nature 215 (5108) 1406ndash1407

Earnest RD 1971 The Effect of Paraquat on Fish in a Colorado Farm Pond The Progressive

Fish-Culturist 33 (1) 27-31 doi 1015771548-8640(1971)33[27teopof]20co2

Edwards CA and PJ Bohlen 1992 The Effects of Toxic Chemicals on Earthworms In

Reviews of Environmental Contamination and Toxicology edited by GW Ware 23ndash99

New York United States Springer-Verlag New York Inc doi 101007978-1-4612-2890-

5_2

Eisler R 1990 Paraquat Hazards to Fish Wildlife and Invertebrates A Synoptic Review

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httppubserusgsgovpublication5200090

Eubank WT KV Nandula HD Poston and RD Shaw 2012 Multiple Resistance of

Horseweed to Glyphosate and Paraquat and Its Control with Paraquat and Metribuzin

Combinations Agronomy 2 (4) 358ndash370 doi 103390agronomy2040358

Farrington JA M Ebert EJ Land and K Fletcher 1973 Bipyridylium Quaternary Salts and

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Oxygen Implications for the Mode of Action of Bipyridyl Herbicides Biochimica et

Biophysica Acta - Bioenergetics 314 (3) 372ndash381 doi 1010160005-2728(73)90121-7

Figueiredo-Fernandes A A Fontaiacutenhas-Fernandes E Rocha and MA Reis-Henriques 2006

The Effect of Paraquat on Hepatic Erod Activity Liver and Gonadal Histology in Males

and Females of Nile Tilapia Oreochromis niloticus Exposed at Different Temperatures

Archives of Environmental Contamination and Toxicology 51 (4) 626ndash632 doi

101007s00244-005-0208-3

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Florecircncio MH E Pires AL Castro MR Nunes C Borges and FM Costa 2004

Photodegradation of Diquat and Paraquat in Aqueous Solutions by Titanium Dioxide

Evolution of Degradation Reactions and Characterisation of Intermediates Chemosphere

55 (3) 345ndash355 doi 101016jchemosphere200311013

Fortenberry GZ J Beckman A Schwartz JB Prado LS Graham S Higgins M Lackovic

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in the US 1998ndash2013 Environmental Research 146 191ndash199 doi

101016jenvres201601003

Fryer JD RJ Hance and JW Ludwig 1975 Long-Term Persistence of Paraquat in a Sandy

Loam Soil Weed Research 15 (3) 189ndash194 doi 101111j1365-31801975tb01121x

Fuerst EP HY Nakatani AD Dodge D Penner and CJ Arntzen 1985 Paraquat

Resistance in Conyza Plant Physiology 77 (4) 984ndash989

Funderburk HHJ and GA Bozarth 1967 Review of the Metabolism and Decomposition of

Diquat and Paraquat Journal of Agricultural and Food Chemistry 15 (4) 563ndash567 doi

101021jf60152a011

Funderburk HHJr and JM Lawrence 1964 Mode of Action and Metabolism of Diquat and

Paraquat Weeds 12 (4) 259ndash264 doi 1023074040748

Fytizas R 1980 Toxicity of Paraquat to Three Marine Organisms Bulletin of Environmental

Contamination and Toxicology 25 (1) 283ndash288 doi 101007bf01985525

Gabryelak T and J Klekot 1985 The Effect of Paraquat on the Peroxide Metabolism

Enzymes in Erythrocytes of Freshwater Fish Species Comparative Biochemistry and

Physiology Part C Comparative Pharmacology 81 (2) 415ndash418 doi

httpdxdoiorg1010160742-8413(85)90030-1

48

Gadkari D 1988 Effects of Atrazine and Paraquat on Nitrifying Bacteria Archives of

Environmental Contamination and Toxicology 17 (4) 443ndash447 doi 101007bf01055509

Gilliom RJ JE Barbash CG Crawford PA Hamilton JD Martin N Nakagaki LH

Nowell et al 2006 The Quality of Our Nationrsquos WatersmdashPesticides in the Nationrsquos Streams

and Ground Water 1992ndash2001 Circular 1291 Reston Virginia US Department of

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httpspubsusgsgovcirc20051291

Givaudan N C Wiegand B Le Bot D Renault F Pallois S Llopis and F Binet 2014

Acclimation of Earthworms to Chemicals in Anthropogenic Landscapes Physiological

Mechanisms and Soil Ecological Implications Soil Biology and Biochemistry 73 49ndash58

doi httpdxdoiorg101016jsoilbio201401032

Goldstein S A Samuni Y Aronovitch D Godinger A Russo and JB Mitchell 2002

Kinetics of Paraquat and Copper Reactions with Nitroxides The Effects of Nitroxides on

the Aerobic and Anoxic Toxicity of Paraquat Chemical Research in Toxicology 15 (5)

686ndash691 doi 101021tx0155956

Gondar D R Loacutepez J Antelo S Fiol and F Arce 2012 Adsorption of Paraquat on Soil

Organic Matter Effect of Exchangeable Cations and Dissolved Organic Carbon Journal of

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Haley TJ 1979 Review of the Toxicology of Paraquat (1 1primeDimethyl-4 4primeBipyridinium

Chloride) Clinical Toxicology 14 (1) 1ndash46 doi 10310915563657909030112

Hance RJ 1967 Decomposition of Herbicides in the Soil by Non-Biological Chemical

Processes Journal of the Science of Food and Agriculture 18 (11) 544ndash547 doi

101002jsfa2740181111

49

Hance RJ TH Byast and PD Smith 1980 Apparent Decomposition of Paraquat in Soil

Soil Biology and Biochemistry 12 (4) 447ndash448

Haque A and W Ebing 1983 Toxicity Determination of Pesticides to Earthworms in the Soil

Substrate Journal of Plant Diseases and Protection 90 (4) 395ndash408

Hart TB 1987 Paraquatmdasha Review of Safety in Agricultural and Horticultural Use Human

toxicology 6 (1) 13ndash18

Hart JJ and JM Di Tomaso 1994 Sequestration and Oxygen Radical Detoxification as

Mechanisms of Paraquat Resistance Weed Science 42 (2) 277ndash284

Hassett DJ BE Britigan T Svendsen GM Rosen and MS Cohen 1987 Bacteria Form

Intracellular Free Radicals in Response to Paraquat and Streptonigrin Demonstration of the

Potency of Hydroxyl Radical The Journal of Biological Chemistry 262 (28) 13404ndash

13408

Hawkes TR 2014 Mechanisms of Resistance to Paraquat in Plants Pest Management

Science 70 (9) 1316ndash1323 doi 101002ps3699

Hayes MHB ME Pick and BA Toms 1975 Interactions between Clay Minerals and

Bipyridylium Herbicides In Residue Reviews Residues of Pesticides and Other

Contaminants in the Total Environment edited by FA Gunther 1ndash25 New York United

States Springer

Heap I 2016 The International Survey of Herbicide Resistant Weeds Accessed Thursday

May 19 2016 wwwweedscienceorg

Heath RG JW Spann EF Hill and JF Kreitzer 1972 Comparative Dietary Toxicities of

Pesticides to Birds Special Scientific Report -- Wildlife No 152 Accessed 11 April 2016

httppubserusgsgovpublication5230135

50

Hoffman DJ and PH Albers 1984 Evaluation of Potential Embryotoxicity and

Teratogenicity of 42 Herbicides Insecticides and Petroleum Contaminants to Mallard

Eggs Archives of Environmental Contamination and Toxicology 13 (1) 15ndash27 doi

101007bf01055642

Hoffman DJ and WC Eastin Jr 1982 Effects of Lindane Paraquat Toxaphene and 245-

Trichlorophenoxyacetic Acid on Mallard Embryo Development Arch Environ Contam

Toxicol 11 (1) 79ndash86

Hoffman DJ JC Franson OH Pattee and CM Bunck 1985 Survival Growth and

Histopathological Effects of Paraquat Ingestion in Nestling American Kestrels (Falco

sparverius) Archives of Environmental Contamination and Toxicology 14 (4) 495ndash500

doi 101007bf01055536

Hoffman DJ JC Franson OH Pattee CM Bunck and HC Murray 1987 Toxicity of

Paraquat in Nestling Birds Effects on Plasma and Tissue Biochemistry in American

Kestrels Archives of Environmental Contamination and Toxicology 16 (2) 177ndash183 doi

101007bf01055799

Homer RF GC Mees and TE Tomlinson 1960 Mode of Action of Dipyridyl Quaternary

Salts as Herbicides Journal of the Science of Food and Agriculture 11 (6) 309ndash315 doi

101002jsfa2740110604

Ismail BS M Sameni and M Halimah 2011 Kinetics of the Microbial Degradation of 24-D

and 14C-Labeled Paraquat in Two Types of Tropical Agricultural Soil World Applied

Sciences Journal 14 (2) 324ndash333

Johansen CA 1977 Pesticides and Pollinators Annual Reviews of Entomology 22 177ndash192

51

Joacuteri B V Sooacutes D Szego E Paacuteldi Z Szigeti I Raacutecz and D Laacutesztity 2007 Role of

Transporters in Paraquat Resistance of Horseweed Conyza canadensis (L) Cronq

Pesticide Biochemistry and Physiology 88 (1) 57ndash65 doi 101016jpestbp200608013

Kearney PC JM Ruth Q Zeng and P Mazzocchi 1985 UV-Ozonation of Paraquat

Journal of Agricultural and Food Chemistry 33 (5) 953ndash957 doi 101021jf00065a044

Khan SU 1974 Adsorption of Bipyridylium Herbicides by Humic Acid Journal of

Environmental Quality 3 (3) 202ndash206

Khan SU PB Marriage and WJ Saidak 1975 Residues of Paraquat in an Orchard Soil

Canadian Journal of Soil Science 55 (1) 73ndash75

Kidd H and DR James 1991 The Agrochemicals Handbook 3rd Ed Cambridge United

Kingdom Royal Society of Chemistry Information Services

Kim S and KK Hatzios 1993 Differential Response of Two Soybean Cultivars to Paraquat

Zeitschrift fur Naturforschung - Section C Journal of Biosciences 48 (3ndash4) 379ndash384

Knight BAG and TE Tomlinson 1967 The Interaction of Paraquat (11prime-Dimethyl 44prime-

Dipyridylium Dichloride) with Mineral Soils Journal of Soil Science 18 (2) 233ndash243 doi

101111j1365-23891967tb01503x

Kookana RS and LAG Aylmore 1993 Retention and Release of Diquat and Paraquat

Herbicides in Soils Australian Journal of Soil Research 31 (1) 97ndash109

Lajmanovich RC MF Izaguirre and VH Casco 1998 Paraquat Tolerance and Alteration of

Internal Gill Structure of Scinax nasica Tadpoles (Anura Hylidae) Archives of

Environmental Contamination and Toxicology 34 (4) 364ndash369 doi

101007s002449900331

52

Lee SJ A Katayama and M Kimura 1995 Microbial Degradation of Paraquat Sorbed to

Plant Residues Journal of Agricultural and Food Chemistry 43 (5) 1343ndash1347 doi

101021jf00053a040

Lee EH CA Burdick and DM Olszyk 2005 GIS-Based Risk Assessment of Pesticide Drift

Case Study Fresno County California Washington DC Environmental Protection

Agency Office of Pesticide Programs and Office of Research and Development

Leonard RA GW Langdale and WG Fleming 1979 Herbicide Runoff from Upland

Piedmont WatershedsmdashData and Implications for Modeling Pesticide Transport Journal of

Environmental Quality 8 (2) 223ndash229

Leung T-S SM Naqvi and NZ Naqvi 1980 Paraquat Toxicity to Louisiana Crayfish

(Procambarus clarkii) Bulletin of Environmental Contamination and Toxicology 25 (1)

465ndash469

Linder G J Barbitta and T Kwaiser 1990 Short-Term Amphibian Toxicity Tests and

Paraquat Toxicity Assessment In Aquatic Toxicology and Risk Assessment Thirteenth

Volume ASTM STP 1096 edited by WG Landis and WH van der Schalie 189ndash198

Philadelphia Pennsylvania USA American Society for Testing and Materials

Ling L-B Y Chang C-W Liu P-L Lai and T Hsu 2017 Oxidative Stress Intensity-

Related Effects of Cadmium (Cd) and Paraquat (PQ) on UV-Damaged-DNA Binding and

Excision Repair Activities in Zebrafish (Danio rerio) Embryos Chemosphere 167 10ndash18

doi httpdxdoiorg101016jchemosphere201609068

Lock EA and MF Wilks 2010 Paraquat In Hayes Handbook of Pesticide Toxicology

edited by RI Krieger 1771ndash1827 New York United States Academic Press

53

Joacuteri B V Sooacutes D Szego E Paacuteldi Z Szigeti I Raacutecz and D Laacutesztity 2007 Role of

Transporters in Paraquat Resistance of Horseweed Conyza canadensis (L) Cronq

Pesticide Biochemistry and Physiology 88 (1) 57ndash65 doi 101016jpestbp200608013

Kearney PC JM Ruth Q Zeng and P Mazzocchi 1985 UV-Ozonation of Paraquat

Journal of Agricultural and Food Chemistry 33 (5) 953ndash957 doi 101021jf00065a044

Khan SU 1974 Adsorption of Bipyridylium Herbicides by Humic Acid Journal of

Environmental Quality 3 (3) 202ndash206

Khan SU PB Marriage and WJ Saidak 1975 Residues of Paraquat in an Orchard Soil

Canadian Journal of Soil Science 55 (1) 73ndash75

Kidd H and DR James 1991 The Agrochemicals Handbook 3rd Ed Cambridge United

Kingdom Royal Society of Chemistry Information Services

Kim S and KK Hatzios 1993 Differential Response of Two Soybean Cultivars to Paraquat

Zeitschrift fur Naturforschung - Section C Journal of Biosciences 48 (3ndash4) 379ndash384

Knight BAG and TE Tomlinson 1967 The Interaction of Paraquat (11prime-Dimethyl 44prime-

Dipyridylium Dichloride) with Mineral Soils Journal of Soil Science 18 (2) 233ndash243 doi

101111j1365-23891967tb01503x

Kookana RS and LAG Aylmore 1993 Retention and Release of Diquat and Paraquat

Herbicides in Soils Australian Journal of Soil Research 31 (1) 97ndash109

Lajmanovich RC MF Izaguirre and VH Casco 1998 Paraquat Tolerance and Alteration of

Internal Gill Structure of Scinax nasica Tadpoles (Anura Hylidae) Archives of

Environmental Contamination and Toxicology 34 (4) 364ndash369 doi

101007s002449900331

52

Lee SJ A Katayama and M Kimura 1995 Microbial Degradation of Paraquat Sorbed to

Plant Residues Journal of Agricultural and Food Chemistry 43 (5) 1343ndash1347 doi

101021jf00053a040

Lee EH CA Burdick and DM Olszyk 2005 GIS-Based Risk Assessment of Pesticide Drift

Case Study Fresno County California Washington DC Environmental Protection

Agency Office of Pesticide Programs and Office of Research and Development

Leonard RA GW Langdale and WG Fleming 1979 Herbicide Runoff from Upland

Piedmont WatershedsmdashData and Implications for Modeling Pesticide Transport Journal of

Environmental Quality 8 (2) 223ndash229

Leung T-S SM Naqvi and NZ Naqvi 1980 Paraquat Toxicity to Louisiana Crayfish

(Procambarus clarkii) Bulletin of Environmental Contamination and Toxicology 25 (1)

465ndash469

Linder G J Barbitta and T Kwaiser 1990 Short-Term Amphibian Toxicity Tests and

Paraquat Toxicity Assessment In Aquatic Toxicology and Risk Assessment Thirteenth

Volume ASTM STP 1096 edited by WG Landis and WH van der Schalie 189ndash198

Philadelphia Pennsylvania USA American Society for Testing and Materials

Ling L-B Y Chang C-W Liu P-L Lai and T Hsu 2017 Oxidative Stress Intensity-

Related Effects of Cadmium (Cd) and Paraquat (PQ) on UV-Damaged-DNA Binding and

Excision Repair Activities in Zebrafish (Danio rerio) Embryos Chemosphere 167 10ndash18

doi httpdxdoiorg101016jchemosphere201609068

Lock EA and MF Wilks 2010 Paraquat In Hayes Handbook of Pesticide Toxicology

edited by RI Krieger 1771ndash1827 New York United States Academic Press

53

Lee SJ A Katayama and M Kimura 1995 Microbial Degradation of Paraquat Sorbed to

Plant Residues Journal of Agricultural and Food Chemistry 43 (5) 1343ndash1347 doi

101021jf00053a040

Lee EH CA Burdick and DM Olszyk 2005 GIS-Based Risk Assessment of Pesticide Drift

Case Study Fresno County California Washington DC Environmental Protection

Agency Office of Pesticide Programs and Office of Research and Development

Leonard RA GW Langdale and WG Fleming 1979 Herbicide Runoff from Upland

Piedmont WatershedsmdashData and Implications for Modeling Pesticide Transport Journal of

Environmental Quality 8 (2) 223ndash229

Leung T-S SM Naqvi and NZ Naqvi 1980 Paraquat Toxicity to Louisiana Crayfish

(Procambarus clarkii) Bulletin of Environmental Contamination and Toxicology 25 (1)

465ndash469

Linder G J Barbitta and T Kwaiser 1990 Short-Term Amphibian Toxicity Tests and

Paraquat Toxicity Assessment In Aquatic Toxicology and Risk Assessment Thirteenth

Volume ASTM STP 1096 edited by WG Landis and WH van der Schalie 189ndash198

Philadelphia Pennsylvania USA American Society for Testing and Materials

Ling L-B Y Chang C-W Liu P-L Lai and T Hsu 2017 Oxidative Stress Intensity-

Related Effects of Cadmium (Cd) and Paraquat (PQ) on UV-Damaged-DNA Binding and

Excision Repair Activities in Zebrafish (Danio rerio) Embryos Chemosphere 167 10ndash18

doi httpdxdoiorg101016jchemosphere201609068

Lock EA and MF Wilks 2010 Paraquat In Hayes Handbook of Pesticide Toxicology

edited by RI Krieger 1771ndash1827 New York United States Academic Press

53

Lutz Ostertag Y and C Henou 1974 Action of Paraquat on the Urogenital Tract of the Chick

and Quail Embryo Comptes Rendus des Seances de la Societe de Biologie et de Ses

Filiales 168 (2ndash3) 304ndash307

Lutz-Ostertag Y and C Henou 1975 Paraquat Embryonic Mortality and Effects on

Pulmonary Apparatus of Chick and Quail Embryos Comptes Rendus Hebdomadaires Des

Seances De lAcademie Des Sciences Serie D 281 (5ndash8) 439ndash442

Ma J X Li Y Li and D Niu 2014 Toxic Effects of Paraquat on Cytokine Expression in

Common Carp Cyprinus carpio L Journal of Biochemical and Molecular Toxicology 28

(11) 501ndash509 doi 101002jbt21590

Michaelis L and ES Hill 1933 Potentiometric Studies on Semiquinones Journal of the

American Chemical Society 55 (4) 1481ndash1494 doi 101021ja01331a027

Moctezuma E E Leyva E Monreal N Villegas and D Infante 1999 Photocatalytic

Degradation of the Herbicide Paraquat Chemosphere 39 (3) 511ndash517

Moffett JO HL Morton and RH MacDonald 1972 Toxicity of Some Herbicidal Sprays to

Honey Bees Journal of Economic Entomology 65 (1) 32ndash36

Moretti ML BD Hanson KJ Hembree and A Shrestha 2013 Glyphosate Resistance Is

More Variable Than Paraquat Resistance in a Multiple-Resistant Hairy Fleabane (Conyza

bonariensis) Population Weed Science 61 (3) 396ndash402 doi 101614ws-d-12-002011

Moretti ML A Shrestha KJ Hembree and BD Hanson 2015 Postemergence Control of

GlyphosateParaquat-Resistant Hairy Fleabane (Conyza bonariensis) in Tree Nut Orchards

in the Central Valley of California Weed Technology 29 (3) 501ndash508 doi 101614wt-d-

14-001491

54

Moretti ML LM Sosnoskie A Shrestha SD Wright KJ Hembree M Jasieniuk and BD

Hanson 2016 Distribution of Conyza sp in Orchards of California and Response to

Glyphosate and Paraquat Weed Science 64 (2) 339ndash347 doi 101614ws-d-15-001741

Morton HL JO Moffett and RH Macdonald 1972 Toxicity of Herbicides to Newly

Emerged Honey Bees Environmental Entomology 1 (1) 102ndash104 doi 101093ee11102

Morton HL JO Moffett and RD Martin 1974 Influence of Water Treated Artificially with

Herbicides on Honey Bee Colonies Environmental Entomology 3 (5) 808ndash812 doi

101093ee35808

Muangphra P W Kwankua and R Gooneratne 2014 Genotoxic Effects of Glyphosate or

Paraquat on Earthworm Coelomocytes Environmental Toxicology 29 (6) 612ndash620 doi

101002tox21787

Murray R P Phillips and J Bender 1997 Degradation of Pesticides Applied to Banana Farm

Soil Comparison of Indigenous Bacteria and a Microbial Mat Environmental Toxicology

and Chemistry 16 (1) 84ndash90

Nguyen C and KO Zahir 1999 UV Induced Degradation of Herbicide Methyl Viologen

Kinetics and Mechanism and Effect of Ionic Media on Degradation Rates Journal of

Environmental Science and Health - Part B 34 (1) 1ndash16

NOAA National Centers for Environmental information 2017 Climate at a Glance US Time

Series Precipitation Published March 2017 Accessed 14 April 2017

httpwwwncdcnoaagovcag

Nunes M E T E Muumlller et al (2017) Chronic Treatment with Paraquat Induces Brain

Injury Changes in Antioxidant Defenses System and Modulates Behavioral Functions in

Zebrafish Molecular Neurobiology 54 (6) 3925-3934 doi 101007s12035-016-9919-x

55

Moretti ML LM Sosnoskie A Shrestha SD Wright KJ Hembree M Jasieniuk and BD

Hanson 2016 Distribution of Conyza sp in Orchards of California and Response to

Glyphosate and Paraquat Weed Science 64 (2) 339ndash347 doi 101614ws-d-15-001741

Morton HL JO Moffett and RH Macdonald 1972 Toxicity of Herbicides to Newly

Emerged Honey Bees Environmental Entomology 1 (1) 102ndash104 doi 101093ee11102

Morton HL JO Moffett and RD Martin 1974 Influence of Water Treated Artificially with

Herbicides on Honey Bee Colonies Environmental Entomology 3 (5) 808ndash812 doi

101093ee35808

Muangphra P W Kwankua and R Gooneratne 2014 Genotoxic Effects of Glyphosate or

Paraquat on Earthworm Coelomocytes Environmental Toxicology 29 (6) 612ndash620 doi

101002tox21787

Murray R P Phillips and J Bender 1997 Degradation of Pesticides Applied to Banana Farm

Soil Comparison of Indigenous Bacteria and a Microbial Mat Environmental Toxicology

and Chemistry 16 (1) 84ndash90

Nguyen C and KO Zahir 1999 UV Induced Degradation of Herbicide Methyl Viologen

Kinetics and Mechanism and Effect of Ionic Media on Degradation Rates Journal of

Environmental Science and Health - Part B 34 (1) 1ndash16

NOAA National Centers for Environmental information 2017 Climate at a Glance US Time

Series Precipitation Published March 2017 Accessed 14 April 2017

httpwwwncdcnoaagovcag

Nunes M E T E Muumlller et al (2017) Chronic Treatment with Paraquat Induces Brain

Injury Changes in Antioxidant Defenses System and Modulates Behavioral Functions in

Zebrafish Molecular Neurobiology 54 (6) 3925-3934 doi 101007s12035-016-9919-x

55

NWQMC (National Water Quality Monitoring Council) 2017 USEPA STORET Data

Accessed 20 April 2017 httpswwwwaterqualitydatausportal

Osano O AA Oladimeji MHS Kraak and W Admiraal 2002 Teratogenic Effects of

Amitraz 24-Dimethylaniline and Paraquat on Developing Frog (Xenopus) Embryos

Archives of Environmental Contamination and Toxicology 43 (1) 42ndash49 doi

101007s00244-002-1132-4

Papini S T Langenbach L C Luchini and M M De Andreacutea 2006 Influence of Substrate

on Bioaccumulation of 14C-Paraquat in Compost Worms Eisenia foetida Journal of

Environmental Science and Health - Part B 41 (5) 523ndash530 doi

10108003601230600701650

Parvez S and S Raisuddin 2006 Preexposure to Copper Modulates Nonenzymatic

Antioxidants in Liver of Channa punctata (Bloch) Exposed to the Herbicide Paraquat

Bulletin of Environmental Contamination and Toxicology 76 (3) 545ndash551 doi

101007s00128-006-0954-6

Pateiro-Moure M C Peacuterez-Novo M Arias-Esteacutevez R Rial-Otero and J Simal-Gaacutendara

2009 Effect of Organic Matter and Iron Oxides on Quaternary Herbicide Sorption-

Desorption in Vineyard-Devoted Soils Journal of Colloid and Interface Science 333 (2)

431ndash438 doi 101016jjcis200902019

Peterson HG C Boutin PA Martin KE Freemark NJ Ruecker and MJ Moody 1994

Aquatic Phyto-Toxicity of 23 Pesticides Applied at Expected Environmental

Concentrations Aquatic Toxicology 28 (3ndash4) 275ndash292

Powles SB and Q Yu 2010 Evolution in Action Plants Resistant to Herbicides Annual

Review of Plant Biology 61 (1) 317ndash347 doi 101146annurev-arplant-042809-112119

56

Preston C S Balachandran and SB Powles 1994 Investigations of Mechanisms of

Resistance to Bipyridyl Herbicides in Arctotheca calendula (L) Levyns Plant Cell amp

Environment 17 (10) 1113ndash1123 doi 101111j1365-30401994tb02009x

Quassinti L E Maccari O Murri and M Bramucci 2009 Effects of Paraquat and

Glyphosate on Steroidogenesis in Gonads of the Frog Rana Esculenta in Vitro Pesticide

Biochemistry and Physiology 93 (2) 91ndash95 doi 101016jpestbp200811006

Ricketts DC 1999 The Microbial Biodegradation of Paraquat in Soil Pesticide Science 55

(5) 596ndash598 doi 101002(sici)1096-9063(199905)555lt596aid-ps961gt30co2-s

Roberts TR JS Dyson and MCG Lane 2002 Deactivation of the Biological Activity of

Paraquat in the Soil Environment A Review of Long-Term Environmental Fate Journal of

Agricultural and Food Chemistry 50 (13) 3623ndash3631 doi 101021jf011323x

Ross JH and RI Krieger 1980 Synthesis and Properties of Paraquat (Methyl Viologen) and

Other Herbicidal Alkyl Homologs Journal of Agricultural and Food Chemistry 28 (5)

1026ndash1031 doi 101021jf60231a041

Salazar-Lugo R C Mata A Oliveros LM Rojas M Lemus and E Rojas-Villarroel 2011

Histopathological Changes in Gill Liver and Kidney of Neotropical Fish Colossoma

macropomum Exposed to Paraquat at Different Temperatures Environmental Toxicology

and Pharmacology 31 (3) 490-495 doi 101016jetap201102002

Scarborough ME RG Ames MJ Lipsett and RJ Jackson 1989 Acute Health Effects of

Community Exposure to Cotton Defoliants Archives of Environmental Health 44 (6) 355ndash

360 doi 1010800003989619899935906

57

Seiber JN and JE Woodrow 1981 Sampling and Analysis of Airborne Residues of Paraquat

in Treated Cotton Field Environments Archives of Environmental Contamination and

Toxicology 10 (2) 133ndash149 doi 101007bf01055616

Senesi N V DOrazio and TM Miano 1995 Adsorption Mechanisms of S-Triazine and

Bipyridylium Herbicides on Humic Acids from Hop Field Soils Geoderma 66 (3ndash4) 273ndash

283 doi 1010160016-7061(94)00083-m

Slade P 1965 Photochemical Degradation of Paraquat Nature 207 (4996) 515ndash516 doi

101038207515a0

Slade P and EG Bell 1966 The Movement of Paraquat in Plants Weed Research 6 (3)

267ndash274 doi 101111j1365-31801966tb00891x

Smith LL 1985 Paraquat Toxicity Philosophical Transactions of the Royal Society of

London Series B Biological Sciences 311 (1152) 647ndash657

Staiff DC LC Butler and JE Davis 1981 A Field Study of the Chemical Degradation of

Paraquat Dichloride Following Simulated Spillage on Soil Bulletin of Environmental

Contamination and Toxicology 26 (1) 16ndash21 doi 101007bf01622047

Summers LA 1980 The Bipyridinium Herbicides New York United States Academic Press

The Court of First Instance 2007 Judgment of the Court of First Instance Accessed 4 April

2016

httpcuriaeuropaeujurisshowPdfjsftext=ampdocid=62401amppageIndex=0ampdoclang=enamp

mode=lstampdir=ampocc=firstamppart=1ampcid=268231

58

Seiber JN and JE Woodrow 1981 Sampling and Analysis of Airborne Residues of Paraquat

in Treated Cotton Field Environments Archives of Environmental Contamination and

Toxicology 10 (2) 133ndash149 doi 101007bf01055616

Senesi N V DOrazio and TM Miano 1995 Adsorption Mechanisms of S-Triazine and

Bipyridylium Herbicides on Humic Acids from Hop Field Soils Geoderma 66 (3ndash4) 273ndash

283 doi 1010160016-7061(94)00083-m

Slade P 1965 Photochemical Degradation of Paraquat Nature 207 (4996) 515ndash516 doi

101038207515a0

Slade P and EG Bell 1966 The Movement of Paraquat in Plants Weed Research 6 (3)

267ndash274 doi 101111j1365-31801966tb00891x

Smith LL 1985 Paraquat Toxicity Philosophical Transactions of the Royal Society of

London Series B Biological Sciences 311 (1152) 647ndash657

Staiff DC LC Butler and JE Davis 1981 A Field Study of the Chemical Degradation of

Paraquat Dichloride Following Simulated Spillage on Soil Bulletin of Environmental

Contamination and Toxicology 26 (1) 16ndash21 doi 101007bf01622047

Summers LA 1980 The Bipyridinium Herbicides New York United States Academic Press

The Court of First Instance 2007 Judgment of the Court of First Instance Accessed 4 April

2016

httpcuriaeuropaeujurisshowPdfjsftext=ampdocid=62401amppageIndex=0ampdoclang=enamp

mode=lstampdir=ampocc=firstamppart=1ampcid=268231

58

Tortorelli MC DA Hernaacutendez GR Vaacutezquez and A Salibiaacuten 1990 Effects of Paraquat on

Mortality and Cardiorespiratory Function of Catfish Fry Plecostomus commersoni

Archives of Environmental Contamination and Toxicology 19 (4) 523ndash529 doi

101007bf01059071

Tsai WT 2013 A Review on Environmental Exposure and Health Risks of Herbicide

Paraquat Toxicological and Environmental Chemistry 95 (2) 197ndash206 doi

101080027722482012761999

UC IPM (University of California Integrated Pest Management) Program Staff 2016 UC

Integrated Pest Management Guidelines Grape Davis CA University of California

Agriculture and Natural Resources Accessed 15 September 2016

httpwwwgencowinemakerscomdocsPest20Management20Guidelines-Grapepdf

USEPA (US Environmental Protection Agency) 1987 Guidance for the Reregistration of

Pesticide Products Containing Paraquat Dichloride as the Active Ingredient Washington

DC Environmental Protection Agency Office of Pesticide Programs Accessed 12 May

2016 httpswwwepagovnscep

USEPA (US Environmental Protection Agency) 1992 Pesticides in Ground Water Database -

a Compilation of Monitoring Studies 1971ndash1991 National Summary Washington DC

USEPA Office of Pesticide Programs Accessed 12 May 2016 httpswwwepagovnscep

USEPA (US Environmental Protection Agency) 1997 Reregistration Eligibility Decision

(Red) -- Paraquat Dichloride Washington DC Environmental Protection Agency Office

of Pesticide Programs Accessed 11 May 2016 httpswwwepagovnscep

59

USEPA (US Environmental Protection Agency) 2009 Risks of Paraquat Use to Federally

Threatened California Red-Legged Frog (Rana aurora draytonii) Washington DC

Accessed 11 January 2017 httpswwwepagovnscepampSeekPage=xampZyPURL

USEPA (US Environmental Protection Agency) 2016 Pesticide Product and Label System

Washington DC United States US Protection Agency Accessed 12 May 2016

httpsiaspubepagovapexpesticidesfp=PPLS1

USEPA (US Environmental Protection Agency) 2018 Memorandum Dated February 6 2018

from Marianne Mannix to Yu-Ting Guilaran Amendment to Paraquat Dichloride Human

Health Mitigation Decision Washington DC US Environmental Protection Agency

Van Gestel CAM EM Dirven-Van Breemen R Baerselman HJB Emans JAM Janssen

R Postuma and PJM Van Vliet 1992 Comparison of Sublethal and Lethal Criteria for

Nine Different Chemicals in Standardized Toxicity Tests Using the Earthworm Eisenia

andrei Ecotoxicology and Environmental Safety 23 (2) 206ndash220 doi 1010160147-

6513(92)90059-c

Vinteacuten AJA B Yaron and PH Nye 1983 Vertical Transport of Pesticides into Soil When

Adsorbed on Suspended Particles Journal of Agricultural and Food Chemistry 31 (3)

662ndash664 doi 101021jf00117a048

Vismara C V Battista G Vailati and R Bacchetta 2000 Paraquat Induced Embryotoxicity

on Xenopus laevis Development Aquatic Toxicology 49 (3) 171ndash179 doi 101016s0166-

445x(99)00080-6

Wang Y S Wu L Chen C Wu R Yu Q Wang and X Zhao 2012 Toxicity Assessment

of 45 Pesticides to the Epigeic Earthworm Eisenia fetida Chemosphere 88 (4) 484ndash491

doi 101016jchemosphere201202086

60

Weber JB PW Perry and RP Upchurch 1965 The Influence of Temperature and Time on

the Adsorption of Paraquat Diquat 24-D and Prometone by Clays Charcoal and an

Anion-Exchange Resin Soil Science Society of America Journal 29 (6) 678ndash688 doi

102136sssaj196503615995002900060026x

Weber JB and SB Weed 1968 Adsorption and Desorption of Diquat Paraquat and

Prometone by Montmorillonitic and Kaolinitic Clay Minerals Soil Science Society of

America Journal 32 (4) doi 102136sssaj196803615995003200040020x

Weed Science Society of America 2014 Herbicide Handbook Weed Science Society of

America Tenth Edition Lawrence Kansas Weed Science Society of America

Weidel H and M Russo 1882 Studien Uumlber Das Pyridin Monatshefte fuumlr Chemie und

verwandte Teile anderer Wissenschaften 3 (1) 850ndash885 doi 101007bf01516855

Weinbaum Z MB Schenker and SJ Samuels 1995 Risk Factors for Occupational Illnesses

Associated with the Use of Paraquat (11prime-Dimethyl-44prime-Bipyridylium Dichloride) in

California Archives of Environmental Health 50 (5) 341ndash348 doi

1010800003989619959935965

Wijeyaratne WMDN and A Pathiratne 2006 Acetylcholinesterase Inhibition and Gill

Lesions in Rasbora caverii an Indigenous Fish Inhabiting Rice Field Associated

Waterbodies in Sri Lanka Ecotoxicology 15 (7) 609ndash619 doi 101007s10646-006-0101-

5

Wilhoit L N Davidson D Supkoff J Steggall A Braun S Simmons B Hobza et al 1999

Pesticide Use Analysis and Trends from 1991 to 1996 Pest Management Analysis and

Planning Program Sacramento California California Department of Pesticide Regulation

Accessed 10 November 2016 httpwwwcdprcagovdocspurpur97reppur_analpdf

61

Wilson RG and SB Orloff 2008 Winter Annual Weed Control with Herbicides in Alfalfa-

Orchardgrass Mixtures Weed Technology 22 (1) 30ndash33 doi 101614wt-07-0701

Wu CY JK Liu SS Chen X Deng and QF Li 2013 Isolation and Characterization of

Paraquat-Degrading Extracellular Humus-Reducing Bacteria from Vegetable Field

Advanced Materials Research 807ndash809 1026ndash1030 doi

104028wwwscientificnetAMR807-8091026

Xie L D Zhou J Xiong J You Y Zeng and L Peng 2016 Paraquat Induce Pulmonary

Epithelial-Mesenchymal Transition through Transforming Growth Factor-β1-Dependent

Mechanism Experimental and Toxicologic Pathology 68 (1) 69-76 doi

101016jetp201509010

Ye P and AT Lemley 2008 Adsorption Effect on the Degradation of Carbaryl Mecoprop

and Paraquat by Anodic Fenton Treatment in an Swy-2 Montmorillonite Clay Slurry

Journal of Agricultural and Food Chemistry 56 (21) 10200ndash10207 doi 101021jf801922r

Yu Q A Cairns and S Powles 2007 Glyphosate Paraquat and ACCase Multiple Herbicide

Resistance Evolved in a Lolium rigidum Biotype Planta 225 (2) 499ndash513 doi

101007s00425-006-0364-3

62

Table 1 Selected physical and chemical properties of paraquat dichloride (C12H14Cl2N2) (IUPAC name 11-dimethyl-44-bipyridinium dichloride CAS Registry number 1910-42-5)

Property Value Description Reference Molecular weight (gmol) 2572 Molecular weight of paraquat ion is 1863 Kidd and James (1991) Density (gcm3) at 20 degC 124ndash126 Kidd and James (1991) Solubility in water Almost completely dissociated in aqueous Haley 1979 Weed

solution as cation and anion Science Society of America 2014

Melting point Approximately 300 degC Paraquat dichloride Kidd and James (1991) Stability Degrades under UV light stable in neutral and Calderbank and Slade

acid media and rapidly hydrolyzes in alkaline (1976) solutions

Photochemical degradation 3 days 01 paraquat dichloride solution Slade (1965) in water

Vapor pressure lt10 times 10-7 (mm Hg at 25 Negligible at room temperature Practically Lock and Wilks (2010) oC) non-volatile

Octanol-water distribution - 45 (at 20 degC) Weed Science Society of coefficient log(Kow) America (2014)

Linear Kd 1520ndash2516 (Lkg) Texture loam to sandy loam (clay content 11 Pateiro-Moure et al ndash22 ) (2009)

Koc 1738ndash3467 and 13183ndash Batch equilibrium studies on adsorption of Aeschbacher et al (2012) 45709 paraquat onto humic acids Organic carbon

normalized distribution coefficients based on Freundlich model parameters

Kd 73 times 104 (mLg) Li-montmorillonite suspension Vinteacuten et al (1983) Redox system potential E = ndash0446 V Redox potential for one-electron reduction Michaelis and Hill (1933)

(blue reduced form) measured at pH ranging 84ndash13

63

Degradation typemedia Experimental Main findings by-products Source Microbial degradationsoil Laboratory incubations over 16 days Selected microorganisms The fungus Neocosmospora vasinfecta was capable Funderburk and

from paraquat-treated soil (Cahaba loamy fine sand) were grown of reducing paraquat to the colored free radical Bozarth (1967) on paraquat and thin-layer substrate Autoradiographs of thin-

layer electrophoresis plates to track 14C-methyl-labeled paraquat without any paraquat degradation A non-identified

bacteria degraded paraquat Identified product was l-methyl-44-dipyridinium ion

Microbialsoil Laboratory incubation A 14C-paraquat dichloride (PD)soil Slow transfer of 14C-paraquat from SOM onto clay Burns and Audus complex was added to a sucrose medium inoculated with the minerals Microbial decomposition occurs only (1970) soil fungus Lipomyces starkeyi Lod and Rij to monitor the when paraquat is weakly adsorbed (reversible

degradation of PD over 72 h Separation of organic and inorganic soil fractions Determination of 14C-PD in soil extracts

and emitted 14C-CO2 Two silt loam soils had 29 and 091

adsorption) onto SOM no degradation when paraquat is adsorbed onto mineral fraction

soil organic carbon (SOC) two sandy loam soils had 25 and 14 SOC

Microbialin vitro The soil yeast Lipomyces starkeyi was added to a salt solution free of any N sources except N contained in the 14C-paraquat

Paraquat degradation was associated with CO2 evolution Loss of integrity of the cell wall

Carr et al (1985)

that was added as the sole N source Removal of the wall resulted in complete loss of degradative capacity

Microbialsoil and plant residues

Laboratory incubation studies using aerobic and anaerobic soil microbes 14C-paraquat and non-labelled paraquat three plant

No degradation of paraquat adsorbed to sterilized plant residues Plant-associated microorganisms had

Lee et al (1995)

residues (rice straw (Oryza sativa L cv Aoinokaze) dropwort higher degradation rates than the soil-associated (Oenanthe jauanica DC) and Chinese milk vetch (Astragalus ones Higher paraquat degradation under aerobic

sinicus L) and paraquat treated and non-treated soil Four conditions and for plant residues with higher CN conditions were tested sterilized plants sterilized with a soil ratio Suppression when urea was added

suspension intact plant residue and intact plant with soil Degradation products from rice straw experiment suspension monopyridone (12-dihydro-llrsquo-dimethyl-2-oxo-

44-bipyridynium ion) and I4CO2 for rice straw spiked with 14C-paraquat

Microbialsoil and plant Soils from a banana farm near St Vincent (West Indies) Pseudomonas spp and Flavobacterium spp were Murray et al (1997) residues Laboratory incubation experiment of soils incubated with only identified as the pesticide-resistant soil bacteria

100 mg ionkg soil or combined with other two pesticides at 50 Rapid microbial degradation in both experiments In mgkg each Twenty-one day exposure to indigenous soil the experiments with indigenous soil

bacteria or microbial mat (consortium of cyanobacteria microorganisms paraquat recovery was 40 and [Oscillatoria sp] and bacteria) vs sterilized control under 12 h 897 species in non-sterile vs sterilized soil

dark-12 h light and 25 degC respectively in the paraquat only experiment it was 52 at day 21 in the three-pesticide experiments

Table 2 Selected studies on the microbial degradation of bioavailable paraquat photochemical degradation of aqueous paraquat and combined mechanisms by environmental media

64

Lowest overall degradation was for the microbial mat (ie 60 recovery)

Microbialsoil Laboratory incubation experiment over 60 days under controlled Extremely slow paraquat degradation due to strong Cheah et al (1998) conditions (temperature and moisture) using Malaysian agricultural soils (sandy loam and muck soils Malaysia with

adsorption Sixty days after treatment the 14CO2 evolution was 473 in the aerobic sandy

soil organic C of 13 and 30 respectively Rate 06ndash08 kg ionha with 14C-paraquat Comparison sterilized vs non-

loam 818 in the aerobic muck and 194 in the anaerobic muck Greater 14CO2 evolution in the

sterilized non-sterilized soils indicating a slow rate of microbial degradation

Microbialsoil Laboratory incubations in a mineral salts medium to foster microbial degradation of bioavailable 14C-paraquat using

Degradation of bioavailable paraquat is rapid 50 mass was mineralized to 14CO2 in three weeks No

Ricketts (1999)

microbial cultures (from two UK sandy loam agricultural soils) paraquat could be detected at the end of the under dark and aerobic conditions Sucrose was also added as a

C source The paraquat-soil-culture mixture was incubated for incubations and the main identified products were

14C-oxalic acid (85 ) and other non-identified 20ndash36 days with regular sampling of volatile products products

Microbial degradationsoil Paraquat applied well above the recommended application rates The detected paraquat residue indicated that Roberts et al (2002) Investigations on the as a one-time or annual application in long-term research trials dissipation was extremely slow Significant

deactivation and sorption in four countries The Frensham (UK) trials were the most decrease in the number of earthworms for the high capacity of the soil through intensively studied They received within the 0ndash15 cm soil rate treatments compared to control one year since

field and laboratory depth increment a one-time application at rates ranging 90ndash720 application a change in the composition of the experiments kg ionha The fate of paraquat residue was then monitored over earthworm population and no significant difference

the following 20 years during which fields were maintained in the overall number of earthworms compared to under cereal or grassland cultivation Quantification of control six years since application no paraquat

earthworm micro-arthropod (some Collembola and Gamasina sorption by earthworms a significant decrease in species) microbial population (number of microorganisms total the number of micro-arthropod at the highest rates

propagules algae bacteria fungi and actinomycetes one year after application no statistically significant population) and biomass (via ATP determination) differences in total microbial biomass and the

number of microorganisms (total propagules algae bacteria fungi actinomycetes and the yeast

Lipomyces starkeyi) or in the ATP concentration the population of L starkeyi significantly decrease

in the 720 kgha treatment compared to the control Microbialsoil Greenhouse experiment under controlled conditions (soil Faster paraquat degradation for the non-sterilized Ismail et al (2011)

temperature and moisture) over 60 days (clay loam and clay soils compared to the sterilized ones soils from Malaysian agricultural soils) Non-sterilized vs

sterilized soils Use of 14C-paraquat to estimate degradation Photochemical and biological Field soil cores monitored for 1 month (applic rate of 075 and Paraquat degradation was faster under field than Amondham et al

degradationsoil 1725 kgha) Incubated laboratory soil columns for 12 weeks under laboratory conditions High degradation rates (2006) (Application rate of 345 kgha twice normal use) Laboratory compared to other studies may depend on

65

soil column incubation experiments (applic rate of 345 kgha) photochemical and microbial degradation of tropical over 12 weeks Determination of total paraquat concentration regions

via spectrophotometric methods after soil digestion and exchangeable paraquat via leaching using a NH4Cl solution

Microbial degradation under Use of three facultative anaerobic bacteria (PQ-1 PQ-2 and About 100 paraquat degradation in four days in Wu et al (2013) anaerobic conditions using PQ-3) isolated from vegetable soil in Sanya city China the presence of AQDS plus sucrose About 20

anthraquinone-26- Laboratory batch equilibrium experiments to monitor degradation in the presence of AQDS without disulphonate (AQDS) (in degradation of paraquat anaerobically with AQDS as the sole sucrose Sucrose addition can significantly enhance

place of humic substances) to electron acceptor with and without sucrose addition (source of paraquat degradation by anaerobic bacteria under simulate no-tillage paddy energy) anaerobic conditions

fields conditions

aPhotochemical Laboratory experiment Paraquat in aqueous solution was UV light degrades paraquat in presence of O2 over Slade (1965) degradationaqueous solution irradiated with Ultraviolet (UV) light generated by a lamp or three days However decomposition of paraquat

under sunlight in the presence of oxygen Autoradiograph of thin-layer chromatograph of UV-irradiated 14C-methyl paraquat

under sunlight appears to occur only when adsorbed to a surface eg paraquat adsorbed on filter paper

or thin layer silica gel but not when in aqueous solution

Methyl quaternary pyridinium (ie 4-carboxy-1- methylpyridinium ion) and methylamine

hydrochloride Photochemical Laboratory experiment Paraquat in (1) solid (dry) and (2) (1) About half dry paraquat was degraded after two Funderburk and

degradationaqueous solution aqueous form under UV radiation days and frac34 after four days to volatile compounds Bozarth (1967) (2) Paraquat concentration decreased with increased

UV exposure Very little aqueous paraquat was present after two days 1-methyl-4- carboxypyridinium chloride and two un-identified

degradation products

a Paraquat has a strong UV signal at 257 nm wavelength corresponding to a π-π transition of the electrons in the pyridinium ring

66

Table 3 Selected degradation studies reporting half-life values for paraquat in soil

Overall objectives Experimental Half-life estimate Source

Combined soil field and laboratory Paraquat dichloride (PD) applied annually (1967ndash1972) on All paraquat applied over a period of six Fryer et al (1975) dissipation study and phytotoxicity study a sandy loam soil as one-time single dose of 448 kgha or years could be extracted in 1971 and 1973 by the Weed Research Organization four separate doses of 12 kgha in plots under Medicago No degradation took place over seven years

Oxford England sativa L Soil digestion using H2SO4 for paraquat No phytotoxic effects could be observed determination Laboratory incubation experiments despite paraquat build up in soil

Re-sampling of the study plot described by Re-analysis of the same plots under the same paraquat Based on the 1975ndash1978 extractions some Hance et al (1980) Fryer et al (1975) at the Weed Research dose treatments (see above) during 1975ndash1978 paraquat disappeared from plots ie 10

Organization Oxford England yearly loss regardless of when paraquat was applied Estimated half-life was 66 years

Determination of the rate and by-products Use of 14C-paraquat to track degradation Laboratory soil 8392ndash1072 days (26 years) 4051ndash6509 Cheah et al (1998) of degradation for paraquat (24-D incubations under aerobic conditions for sandy loam days (14 years) and 2289ndash3652 days (72 lindane and glyphosate) in two muck and anaerobic for muck (Tanjong Karang and years) respectively

agricultural soils (sandy loam and muck Cameron Highlands Malaysia) ie about 30 soil organic C)

Assess degradation mobility sorption Field incubation of soil cores in polyvinyl chloride Degradation was faster under field Amondham et al (2006) desorption of paraquat in agricultural soils collected at 1 4 8 and 12 weeks following application conditions than under laboratory conditions

of the Yom River basin Thailand Two application rates of 075 kgha and 17 kgha with half-life estimate of 36ndash46 days

Monitoring paraquat degradation and Use of 14C-paraquat to estimate degradation comparing Clay loam soil 187 days (non-sterilized) Ismail et al (2011) testing the effect of soil temperature and non-sterilized vs sterilized soils Application rate was 06ndash and 1386 days (sterilized) Clay soil 231 moisture on paraquat degradation in 1 kgha days (non-sterilized) 1733 days (sterilized)

Malaysian agricultural soils

67

List of Figures

Figure 1 Synthesis of the paraquat dichloride salt In the paraquat cation the number-4 carbon

atom joins the two pyridine rings and each nitrogen atom has a methyl group (Based on Cairns

and Case [1975])

Figure 2 Annual paraquat dichloride mass used in California during 2000ndash2014

Figure 3 Cumulative frequency plot of paraquat dichloride-treated area (a) and mass per

application (b) and rate of application vs percentile (c) by year (2000ndash2014) in California (p5

through p95 are the 5h through the 95th percentile respectively of the frequency distribution of

interest CDPR [2016])

Figure 4 Map of the average annual mass of paraquat applied primarily on agricultural

commodities in different California counties during 2000ndash2014 (CDPR 2016)

Figure 5 Total paraquat dichloride mass applied to the top 20 crops (based on use) in California

during 2000ndash2014 (CDPR 2016)

Figure 6 Proposed pathway of paraquat dichloride (1) degradation by soil microfauna leading to

the formation of a 4-carboxilated-1-methylpyridnium ion (2) followed by oxalic acid (3) and

other non-identified products (not shown) and ultimately to mineralization products (4)

(modified after Funderburk and Bozarth [1967] and Ricketts [1999])

68

Figure 7 Pathway of paraquat dichloride (1) degradation by ultraviolet light in presence of

oxygen Identified degradation compounds were amino-aldehyde (likely) (2) 4-carboxy-1-

methylpyridinium ion (3) and methylamine hydrochloride (4) Compounds (3) and (4) do not

form in the absence of oxygen (modified after Slade [1965])

69

NaNH3 lack of O2

44rsquo- Bipyridyl

2 CH3Cl

2 Clndash

Paraquat dichloride

The initial reaction is between the product resulting from the metal-pyridine interaction and an alkylene oxide in liquid ammonia at temperatures normally not exciding -33 degC In its usually oxidized form paraquat is ionized with two positive charges Thus paraquat is usually manufactured as a salt containing two chloride anions

70

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

Table 1 Selected physical and chemical properties of paraquat dichloride (C12H14Cl2N2) (IUPAC name 11-dimethyl-44-bipyridinium dichloride CAS Registry number 1910-42-5)

Property Value Description Reference Molecular weight (gmol) 2572 Molecular weight of paraquat ion is 1863 Kidd and James (1991) Density (gcm3) at 20 degC 124ndash126 Kidd and James (1991) Solubility in water Almost completely dissociated in aqueous Haley 1979 Weed

solution as cation and anion Science Society of America 2014

Melting point Approximately 300 degC Paraquat dichloride Kidd and James (1991) Stability Degrades under UV light stable in neutral and Calderbank and Slade

acid media and rapidly hydrolyzes in alkaline (1976) solutions

Photochemical degradation 3 days 01 paraquat dichloride solution Slade (1965) in water

Vapor pressure lt10 times 10-7 (mm Hg at 25 Negligible at room temperature Practically Lock and Wilks (2010) oC) non-volatile

Octanol-water distribution - 45 (at 20 degC) Weed Science Society of coefficient log(Kow) America (2014)

Linear Kd 1520ndash2516 (Lkg) Texture loam to sandy loam (clay content 11 Pateiro-Moure et al ndash22 ) (2009)

Koc 1738ndash3467 and 13183ndash Batch equilibrium studies on adsorption of Aeschbacher et al (2012) 45709 paraquat onto humic acids Organic carbon

normalized distribution coefficients based on Freundlich model parameters

Kd 73 times 104 (mLg) Li-montmorillonite suspension Vinteacuten et al (1983) Redox system potential E = ndash0446 V Redox potential for one-electron reduction Michaelis and Hill (1933)

(blue reduced form) measured at pH ranging 84ndash13

63

Degradation typemedia Experimental Main findings by-products Source Microbial degradationsoil Laboratory incubations over 16 days Selected microorganisms The fungus Neocosmospora vasinfecta was capable Funderburk and

from paraquat-treated soil (Cahaba loamy fine sand) were grown of reducing paraquat to the colored free radical Bozarth (1967) on paraquat and thin-layer substrate Autoradiographs of thin-

layer electrophoresis plates to track 14C-methyl-labeled paraquat without any paraquat degradation A non-identified

bacteria degraded paraquat Identified product was l-methyl-44-dipyridinium ion

Microbialsoil Laboratory incubation A 14C-paraquat dichloride (PD)soil Slow transfer of 14C-paraquat from SOM onto clay Burns and Audus complex was added to a sucrose medium inoculated with the minerals Microbial decomposition occurs only (1970) soil fungus Lipomyces starkeyi Lod and Rij to monitor the when paraquat is weakly adsorbed (reversible

degradation of PD over 72 h Separation of organic and inorganic soil fractions Determination of 14C-PD in soil extracts

and emitted 14C-CO2 Two silt loam soils had 29 and 091

adsorption) onto SOM no degradation when paraquat is adsorbed onto mineral fraction

soil organic carbon (SOC) two sandy loam soils had 25 and 14 SOC

Microbialin vitro The soil yeast Lipomyces starkeyi was added to a salt solution free of any N sources except N contained in the 14C-paraquat

Paraquat degradation was associated with CO2 evolution Loss of integrity of the cell wall

Carr et al (1985)

that was added as the sole N source Removal of the wall resulted in complete loss of degradative capacity

Microbialsoil and plant residues

Laboratory incubation studies using aerobic and anaerobic soil microbes 14C-paraquat and non-labelled paraquat three plant

No degradation of paraquat adsorbed to sterilized plant residues Plant-associated microorganisms had

Lee et al (1995)

residues (rice straw (Oryza sativa L cv Aoinokaze) dropwort higher degradation rates than the soil-associated (Oenanthe jauanica DC) and Chinese milk vetch (Astragalus ones Higher paraquat degradation under aerobic

sinicus L) and paraquat treated and non-treated soil Four conditions and for plant residues with higher CN conditions were tested sterilized plants sterilized with a soil ratio Suppression when urea was added

suspension intact plant residue and intact plant with soil Degradation products from rice straw experiment suspension monopyridone (12-dihydro-llrsquo-dimethyl-2-oxo-

44-bipyridynium ion) and I4CO2 for rice straw spiked with 14C-paraquat

Microbialsoil and plant Soils from a banana farm near St Vincent (West Indies) Pseudomonas spp and Flavobacterium spp were Murray et al (1997) residues Laboratory incubation experiment of soils incubated with only identified as the pesticide-resistant soil bacteria

100 mg ionkg soil or combined with other two pesticides at 50 Rapid microbial degradation in both experiments In mgkg each Twenty-one day exposure to indigenous soil the experiments with indigenous soil

bacteria or microbial mat (consortium of cyanobacteria microorganisms paraquat recovery was 40 and [Oscillatoria sp] and bacteria) vs sterilized control under 12 h 897 species in non-sterile vs sterilized soil

dark-12 h light and 25 degC respectively in the paraquat only experiment it was 52 at day 21 in the three-pesticide experiments

Table 2 Selected studies on the microbial degradation of bioavailable paraquat photochemical degradation of aqueous paraquat and combined mechanisms by environmental media

64

Lowest overall degradation was for the microbial mat (ie 60 recovery)

Microbialsoil Laboratory incubation experiment over 60 days under controlled Extremely slow paraquat degradation due to strong Cheah et al (1998) conditions (temperature and moisture) using Malaysian agricultural soils (sandy loam and muck soils Malaysia with

adsorption Sixty days after treatment the 14CO2 evolution was 473 in the aerobic sandy

soil organic C of 13 and 30 respectively Rate 06ndash08 kg ionha with 14C-paraquat Comparison sterilized vs non-

loam 818 in the aerobic muck and 194 in the anaerobic muck Greater 14CO2 evolution in the

sterilized non-sterilized soils indicating a slow rate of microbial degradation

Microbialsoil Laboratory incubations in a mineral salts medium to foster microbial degradation of bioavailable 14C-paraquat using

Degradation of bioavailable paraquat is rapid 50 mass was mineralized to 14CO2 in three weeks No

Ricketts (1999)

microbial cultures (from two UK sandy loam agricultural soils) paraquat could be detected at the end of the under dark and aerobic conditions Sucrose was also added as a

C source The paraquat-soil-culture mixture was incubated for incubations and the main identified products were

14C-oxalic acid (85 ) and other non-identified 20ndash36 days with regular sampling of volatile products products

Microbial degradationsoil Paraquat applied well above the recommended application rates The detected paraquat residue indicated that Roberts et al (2002) Investigations on the as a one-time or annual application in long-term research trials dissipation was extremely slow Significant

deactivation and sorption in four countries The Frensham (UK) trials were the most decrease in the number of earthworms for the high capacity of the soil through intensively studied They received within the 0ndash15 cm soil rate treatments compared to control one year since

field and laboratory depth increment a one-time application at rates ranging 90ndash720 application a change in the composition of the experiments kg ionha The fate of paraquat residue was then monitored over earthworm population and no significant difference

the following 20 years during which fields were maintained in the overall number of earthworms compared to under cereal or grassland cultivation Quantification of control six years since application no paraquat

earthworm micro-arthropod (some Collembola and Gamasina sorption by earthworms a significant decrease in species) microbial population (number of microorganisms total the number of micro-arthropod at the highest rates

propagules algae bacteria fungi and actinomycetes one year after application no statistically significant population) and biomass (via ATP determination) differences in total microbial biomass and the

number of microorganisms (total propagules algae bacteria fungi actinomycetes and the yeast

Lipomyces starkeyi) or in the ATP concentration the population of L starkeyi significantly decrease

in the 720 kgha treatment compared to the control Microbialsoil Greenhouse experiment under controlled conditions (soil Faster paraquat degradation for the non-sterilized Ismail et al (2011)

temperature and moisture) over 60 days (clay loam and clay soils compared to the sterilized ones soils from Malaysian agricultural soils) Non-sterilized vs

sterilized soils Use of 14C-paraquat to estimate degradation Photochemical and biological Field soil cores monitored for 1 month (applic rate of 075 and Paraquat degradation was faster under field than Amondham et al

degradationsoil 1725 kgha) Incubated laboratory soil columns for 12 weeks under laboratory conditions High degradation rates (2006) (Application rate of 345 kgha twice normal use) Laboratory compared to other studies may depend on

65

soil column incubation experiments (applic rate of 345 kgha) photochemical and microbial degradation of tropical over 12 weeks Determination of total paraquat concentration regions

via spectrophotometric methods after soil digestion and exchangeable paraquat via leaching using a NH4Cl solution

Microbial degradation under Use of three facultative anaerobic bacteria (PQ-1 PQ-2 and About 100 paraquat degradation in four days in Wu et al (2013) anaerobic conditions using PQ-3) isolated from vegetable soil in Sanya city China the presence of AQDS plus sucrose About 20

anthraquinone-26- Laboratory batch equilibrium experiments to monitor degradation in the presence of AQDS without disulphonate (AQDS) (in degradation of paraquat anaerobically with AQDS as the sole sucrose Sucrose addition can significantly enhance

place of humic substances) to electron acceptor with and without sucrose addition (source of paraquat degradation by anaerobic bacteria under simulate no-tillage paddy energy) anaerobic conditions

fields conditions

aPhotochemical Laboratory experiment Paraquat in aqueous solution was UV light degrades paraquat in presence of O2 over Slade (1965) degradationaqueous solution irradiated with Ultraviolet (UV) light generated by a lamp or three days However decomposition of paraquat

under sunlight in the presence of oxygen Autoradiograph of thin-layer chromatograph of UV-irradiated 14C-methyl paraquat

under sunlight appears to occur only when adsorbed to a surface eg paraquat adsorbed on filter paper

or thin layer silica gel but not when in aqueous solution

Methyl quaternary pyridinium (ie 4-carboxy-1- methylpyridinium ion) and methylamine

hydrochloride Photochemical Laboratory experiment Paraquat in (1) solid (dry) and (2) (1) About half dry paraquat was degraded after two Funderburk and

degradationaqueous solution aqueous form under UV radiation days and frac34 after four days to volatile compounds Bozarth (1967) (2) Paraquat concentration decreased with increased

UV exposure Very little aqueous paraquat was present after two days 1-methyl-4- carboxypyridinium chloride and two un-identified

degradation products

a Paraquat has a strong UV signal at 257 nm wavelength corresponding to a π-π transition of the electrons in the pyridinium ring

66

Table 3 Selected degradation studies reporting half-life values for paraquat in soil

Overall objectives Experimental Half-life estimate Source

Combined soil field and laboratory Paraquat dichloride (PD) applied annually (1967ndash1972) on All paraquat applied over a period of six Fryer et al (1975) dissipation study and phytotoxicity study a sandy loam soil as one-time single dose of 448 kgha or years could be extracted in 1971 and 1973 by the Weed Research Organization four separate doses of 12 kgha in plots under Medicago No degradation took place over seven years

Oxford England sativa L Soil digestion using H2SO4 for paraquat No phytotoxic effects could be observed determination Laboratory incubation experiments despite paraquat build up in soil

Re-sampling of the study plot described by Re-analysis of the same plots under the same paraquat Based on the 1975ndash1978 extractions some Hance et al (1980) Fryer et al (1975) at the Weed Research dose treatments (see above) during 1975ndash1978 paraquat disappeared from plots ie 10

Organization Oxford England yearly loss regardless of when paraquat was applied Estimated half-life was 66 years

Determination of the rate and by-products Use of 14C-paraquat to track degradation Laboratory soil 8392ndash1072 days (26 years) 4051ndash6509 Cheah et al (1998) of degradation for paraquat (24-D incubations under aerobic conditions for sandy loam days (14 years) and 2289ndash3652 days (72 lindane and glyphosate) in two muck and anaerobic for muck (Tanjong Karang and years) respectively

agricultural soils (sandy loam and muck Cameron Highlands Malaysia) ie about 30 soil organic C)

Assess degradation mobility sorption Field incubation of soil cores in polyvinyl chloride Degradation was faster under field Amondham et al (2006) desorption of paraquat in agricultural soils collected at 1 4 8 and 12 weeks following application conditions than under laboratory conditions

of the Yom River basin Thailand Two application rates of 075 kgha and 17 kgha with half-life estimate of 36ndash46 days

Monitoring paraquat degradation and Use of 14C-paraquat to estimate degradation comparing Clay loam soil 187 days (non-sterilized) Ismail et al (2011) testing the effect of soil temperature and non-sterilized vs sterilized soils Application rate was 06ndash and 1386 days (sterilized) Clay soil 231 moisture on paraquat degradation in 1 kgha days (non-sterilized) 1733 days (sterilized)

Malaysian agricultural soils

67

List of Figures

Figure 1 Synthesis of the paraquat dichloride salt In the paraquat cation the number-4 carbon

atom joins the two pyridine rings and each nitrogen atom has a methyl group (Based on Cairns

and Case [1975])

Figure 2 Annual paraquat dichloride mass used in California during 2000ndash2014

Figure 3 Cumulative frequency plot of paraquat dichloride-treated area (a) and mass per

application (b) and rate of application vs percentile (c) by year (2000ndash2014) in California (p5

through p95 are the 5h through the 95th percentile respectively of the frequency distribution of

interest CDPR [2016])

Figure 4 Map of the average annual mass of paraquat applied primarily on agricultural

commodities in different California counties during 2000ndash2014 (CDPR 2016)

Figure 5 Total paraquat dichloride mass applied to the top 20 crops (based on use) in California

during 2000ndash2014 (CDPR 2016)

Figure 6 Proposed pathway of paraquat dichloride (1) degradation by soil microfauna leading to

the formation of a 4-carboxilated-1-methylpyridnium ion (2) followed by oxalic acid (3) and

other non-identified products (not shown) and ultimately to mineralization products (4)

(modified after Funderburk and Bozarth [1967] and Ricketts [1999])

68

Figure 7 Pathway of paraquat dichloride (1) degradation by ultraviolet light in presence of

oxygen Identified degradation compounds were amino-aldehyde (likely) (2) 4-carboxy-1-

methylpyridinium ion (3) and methylamine hydrochloride (4) Compounds (3) and (4) do not

form in the absence of oxygen (modified after Slade [1965])

69

NaNH3 lack of O2

44rsquo- Bipyridyl

2 CH3Cl

2 Clndash

Paraquat dichloride

The initial reaction is between the product resulting from the metal-pyridine interaction and an alkylene oxide in liquid ammonia at temperatures normally not exciding -33 degC In its usually oxidized form paraquat is ionized with two positive charges Thus paraquat is usually manufactured as a salt containing two chloride anions

70

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

Degradation typemedia Experimental Main findings by-products Source Microbial degradationsoil Laboratory incubations over 16 days Selected microorganisms The fungus Neocosmospora vasinfecta was capable Funderburk and

from paraquat-treated soil (Cahaba loamy fine sand) were grown of reducing paraquat to the colored free radical Bozarth (1967) on paraquat and thin-layer substrate Autoradiographs of thin-

layer electrophoresis plates to track 14C-methyl-labeled paraquat without any paraquat degradation A non-identified

bacteria degraded paraquat Identified product was l-methyl-44-dipyridinium ion

Microbialsoil Laboratory incubation A 14C-paraquat dichloride (PD)soil Slow transfer of 14C-paraquat from SOM onto clay Burns and Audus complex was added to a sucrose medium inoculated with the minerals Microbial decomposition occurs only (1970) soil fungus Lipomyces starkeyi Lod and Rij to monitor the when paraquat is weakly adsorbed (reversible

degradation of PD over 72 h Separation of organic and inorganic soil fractions Determination of 14C-PD in soil extracts

and emitted 14C-CO2 Two silt loam soils had 29 and 091

adsorption) onto SOM no degradation when paraquat is adsorbed onto mineral fraction

soil organic carbon (SOC) two sandy loam soils had 25 and 14 SOC

Microbialin vitro The soil yeast Lipomyces starkeyi was added to a salt solution free of any N sources except N contained in the 14C-paraquat

Paraquat degradation was associated with CO2 evolution Loss of integrity of the cell wall

Carr et al (1985)

that was added as the sole N source Removal of the wall resulted in complete loss of degradative capacity

Microbialsoil and plant residues

Laboratory incubation studies using aerobic and anaerobic soil microbes 14C-paraquat and non-labelled paraquat three plant

No degradation of paraquat adsorbed to sterilized plant residues Plant-associated microorganisms had

Lee et al (1995)

residues (rice straw (Oryza sativa L cv Aoinokaze) dropwort higher degradation rates than the soil-associated (Oenanthe jauanica DC) and Chinese milk vetch (Astragalus ones Higher paraquat degradation under aerobic

sinicus L) and paraquat treated and non-treated soil Four conditions and for plant residues with higher CN conditions were tested sterilized plants sterilized with a soil ratio Suppression when urea was added

suspension intact plant residue and intact plant with soil Degradation products from rice straw experiment suspension monopyridone (12-dihydro-llrsquo-dimethyl-2-oxo-

44-bipyridynium ion) and I4CO2 for rice straw spiked with 14C-paraquat

Microbialsoil and plant Soils from a banana farm near St Vincent (West Indies) Pseudomonas spp and Flavobacterium spp were Murray et al (1997) residues Laboratory incubation experiment of soils incubated with only identified as the pesticide-resistant soil bacteria

100 mg ionkg soil or combined with other two pesticides at 50 Rapid microbial degradation in both experiments In mgkg each Twenty-one day exposure to indigenous soil the experiments with indigenous soil

bacteria or microbial mat (consortium of cyanobacteria microorganisms paraquat recovery was 40 and [Oscillatoria sp] and bacteria) vs sterilized control under 12 h 897 species in non-sterile vs sterilized soil

dark-12 h light and 25 degC respectively in the paraquat only experiment it was 52 at day 21 in the three-pesticide experiments

Table 2 Selected studies on the microbial degradation of bioavailable paraquat photochemical degradation of aqueous paraquat and combined mechanisms by environmental media

64

Lowest overall degradation was for the microbial mat (ie 60 recovery)

Microbialsoil Laboratory incubation experiment over 60 days under controlled Extremely slow paraquat degradation due to strong Cheah et al (1998) conditions (temperature and moisture) using Malaysian agricultural soils (sandy loam and muck soils Malaysia with

adsorption Sixty days after treatment the 14CO2 evolution was 473 in the aerobic sandy

soil organic C of 13 and 30 respectively Rate 06ndash08 kg ionha with 14C-paraquat Comparison sterilized vs non-

loam 818 in the aerobic muck and 194 in the anaerobic muck Greater 14CO2 evolution in the

sterilized non-sterilized soils indicating a slow rate of microbial degradation

Microbialsoil Laboratory incubations in a mineral salts medium to foster microbial degradation of bioavailable 14C-paraquat using

Degradation of bioavailable paraquat is rapid 50 mass was mineralized to 14CO2 in three weeks No

Ricketts (1999)

microbial cultures (from two UK sandy loam agricultural soils) paraquat could be detected at the end of the under dark and aerobic conditions Sucrose was also added as a

C source The paraquat-soil-culture mixture was incubated for incubations and the main identified products were

14C-oxalic acid (85 ) and other non-identified 20ndash36 days with regular sampling of volatile products products

Microbial degradationsoil Paraquat applied well above the recommended application rates The detected paraquat residue indicated that Roberts et al (2002) Investigations on the as a one-time or annual application in long-term research trials dissipation was extremely slow Significant

deactivation and sorption in four countries The Frensham (UK) trials were the most decrease in the number of earthworms for the high capacity of the soil through intensively studied They received within the 0ndash15 cm soil rate treatments compared to control one year since

field and laboratory depth increment a one-time application at rates ranging 90ndash720 application a change in the composition of the experiments kg ionha The fate of paraquat residue was then monitored over earthworm population and no significant difference

the following 20 years during which fields were maintained in the overall number of earthworms compared to under cereal or grassland cultivation Quantification of control six years since application no paraquat

earthworm micro-arthropod (some Collembola and Gamasina sorption by earthworms a significant decrease in species) microbial population (number of microorganisms total the number of micro-arthropod at the highest rates

propagules algae bacteria fungi and actinomycetes one year after application no statistically significant population) and biomass (via ATP determination) differences in total microbial biomass and the

number of microorganisms (total propagules algae bacteria fungi actinomycetes and the yeast

Lipomyces starkeyi) or in the ATP concentration the population of L starkeyi significantly decrease

in the 720 kgha treatment compared to the control Microbialsoil Greenhouse experiment under controlled conditions (soil Faster paraquat degradation for the non-sterilized Ismail et al (2011)

temperature and moisture) over 60 days (clay loam and clay soils compared to the sterilized ones soils from Malaysian agricultural soils) Non-sterilized vs

sterilized soils Use of 14C-paraquat to estimate degradation Photochemical and biological Field soil cores monitored for 1 month (applic rate of 075 and Paraquat degradation was faster under field than Amondham et al

degradationsoil 1725 kgha) Incubated laboratory soil columns for 12 weeks under laboratory conditions High degradation rates (2006) (Application rate of 345 kgha twice normal use) Laboratory compared to other studies may depend on

65

soil column incubation experiments (applic rate of 345 kgha) photochemical and microbial degradation of tropical over 12 weeks Determination of total paraquat concentration regions

via spectrophotometric methods after soil digestion and exchangeable paraquat via leaching using a NH4Cl solution

Microbial degradation under Use of three facultative anaerobic bacteria (PQ-1 PQ-2 and About 100 paraquat degradation in four days in Wu et al (2013) anaerobic conditions using PQ-3) isolated from vegetable soil in Sanya city China the presence of AQDS plus sucrose About 20

anthraquinone-26- Laboratory batch equilibrium experiments to monitor degradation in the presence of AQDS without disulphonate (AQDS) (in degradation of paraquat anaerobically with AQDS as the sole sucrose Sucrose addition can significantly enhance

place of humic substances) to electron acceptor with and without sucrose addition (source of paraquat degradation by anaerobic bacteria under simulate no-tillage paddy energy) anaerobic conditions

fields conditions

aPhotochemical Laboratory experiment Paraquat in aqueous solution was UV light degrades paraquat in presence of O2 over Slade (1965) degradationaqueous solution irradiated with Ultraviolet (UV) light generated by a lamp or three days However decomposition of paraquat

under sunlight in the presence of oxygen Autoradiograph of thin-layer chromatograph of UV-irradiated 14C-methyl paraquat

under sunlight appears to occur only when adsorbed to a surface eg paraquat adsorbed on filter paper

or thin layer silica gel but not when in aqueous solution

Methyl quaternary pyridinium (ie 4-carboxy-1- methylpyridinium ion) and methylamine

hydrochloride Photochemical Laboratory experiment Paraquat in (1) solid (dry) and (2) (1) About half dry paraquat was degraded after two Funderburk and

degradationaqueous solution aqueous form under UV radiation days and frac34 after four days to volatile compounds Bozarth (1967) (2) Paraquat concentration decreased with increased

UV exposure Very little aqueous paraquat was present after two days 1-methyl-4- carboxypyridinium chloride and two un-identified

degradation products

a Paraquat has a strong UV signal at 257 nm wavelength corresponding to a π-π transition of the electrons in the pyridinium ring

66

Table 3 Selected degradation studies reporting half-life values for paraquat in soil

Overall objectives Experimental Half-life estimate Source

Combined soil field and laboratory Paraquat dichloride (PD) applied annually (1967ndash1972) on All paraquat applied over a period of six Fryer et al (1975) dissipation study and phytotoxicity study a sandy loam soil as one-time single dose of 448 kgha or years could be extracted in 1971 and 1973 by the Weed Research Organization four separate doses of 12 kgha in plots under Medicago No degradation took place over seven years

Oxford England sativa L Soil digestion using H2SO4 for paraquat No phytotoxic effects could be observed determination Laboratory incubation experiments despite paraquat build up in soil

Re-sampling of the study plot described by Re-analysis of the same plots under the same paraquat Based on the 1975ndash1978 extractions some Hance et al (1980) Fryer et al (1975) at the Weed Research dose treatments (see above) during 1975ndash1978 paraquat disappeared from plots ie 10

Organization Oxford England yearly loss regardless of when paraquat was applied Estimated half-life was 66 years

Determination of the rate and by-products Use of 14C-paraquat to track degradation Laboratory soil 8392ndash1072 days (26 years) 4051ndash6509 Cheah et al (1998) of degradation for paraquat (24-D incubations under aerobic conditions for sandy loam days (14 years) and 2289ndash3652 days (72 lindane and glyphosate) in two muck and anaerobic for muck (Tanjong Karang and years) respectively

agricultural soils (sandy loam and muck Cameron Highlands Malaysia) ie about 30 soil organic C)

Assess degradation mobility sorption Field incubation of soil cores in polyvinyl chloride Degradation was faster under field Amondham et al (2006) desorption of paraquat in agricultural soils collected at 1 4 8 and 12 weeks following application conditions than under laboratory conditions

of the Yom River basin Thailand Two application rates of 075 kgha and 17 kgha with half-life estimate of 36ndash46 days

Monitoring paraquat degradation and Use of 14C-paraquat to estimate degradation comparing Clay loam soil 187 days (non-sterilized) Ismail et al (2011) testing the effect of soil temperature and non-sterilized vs sterilized soils Application rate was 06ndash and 1386 days (sterilized) Clay soil 231 moisture on paraquat degradation in 1 kgha days (non-sterilized) 1733 days (sterilized)

Malaysian agricultural soils

67

List of Figures

Figure 1 Synthesis of the paraquat dichloride salt In the paraquat cation the number-4 carbon

atom joins the two pyridine rings and each nitrogen atom has a methyl group (Based on Cairns

and Case [1975])

Figure 2 Annual paraquat dichloride mass used in California during 2000ndash2014

Figure 3 Cumulative frequency plot of paraquat dichloride-treated area (a) and mass per

application (b) and rate of application vs percentile (c) by year (2000ndash2014) in California (p5

through p95 are the 5h through the 95th percentile respectively of the frequency distribution of

interest CDPR [2016])

Figure 4 Map of the average annual mass of paraquat applied primarily on agricultural

commodities in different California counties during 2000ndash2014 (CDPR 2016)

Figure 5 Total paraquat dichloride mass applied to the top 20 crops (based on use) in California

during 2000ndash2014 (CDPR 2016)

Figure 6 Proposed pathway of paraquat dichloride (1) degradation by soil microfauna leading to

the formation of a 4-carboxilated-1-methylpyridnium ion (2) followed by oxalic acid (3) and

other non-identified products (not shown) and ultimately to mineralization products (4)

(modified after Funderburk and Bozarth [1967] and Ricketts [1999])

68

Figure 7 Pathway of paraquat dichloride (1) degradation by ultraviolet light in presence of

oxygen Identified degradation compounds were amino-aldehyde (likely) (2) 4-carboxy-1-

methylpyridinium ion (3) and methylamine hydrochloride (4) Compounds (3) and (4) do not

form in the absence of oxygen (modified after Slade [1965])

69

NaNH3 lack of O2

44rsquo- Bipyridyl

2 CH3Cl

2 Clndash

Paraquat dichloride

The initial reaction is between the product resulting from the metal-pyridine interaction and an alkylene oxide in liquid ammonia at temperatures normally not exciding -33 degC In its usually oxidized form paraquat is ionized with two positive charges Thus paraquat is usually manufactured as a salt containing two chloride anions

70

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

Lowest overall degradation was for the microbial mat (ie 60 recovery)

Microbialsoil Laboratory incubation experiment over 60 days under controlled Extremely slow paraquat degradation due to strong Cheah et al (1998) conditions (temperature and moisture) using Malaysian agricultural soils (sandy loam and muck soils Malaysia with

adsorption Sixty days after treatment the 14CO2 evolution was 473 in the aerobic sandy

soil organic C of 13 and 30 respectively Rate 06ndash08 kg ionha with 14C-paraquat Comparison sterilized vs non-

loam 818 in the aerobic muck and 194 in the anaerobic muck Greater 14CO2 evolution in the

sterilized non-sterilized soils indicating a slow rate of microbial degradation

Microbialsoil Laboratory incubations in a mineral salts medium to foster microbial degradation of bioavailable 14C-paraquat using

Degradation of bioavailable paraquat is rapid 50 mass was mineralized to 14CO2 in three weeks No

Ricketts (1999)

microbial cultures (from two UK sandy loam agricultural soils) paraquat could be detected at the end of the under dark and aerobic conditions Sucrose was also added as a

C source The paraquat-soil-culture mixture was incubated for incubations and the main identified products were

14C-oxalic acid (85 ) and other non-identified 20ndash36 days with regular sampling of volatile products products

Microbial degradationsoil Paraquat applied well above the recommended application rates The detected paraquat residue indicated that Roberts et al (2002) Investigations on the as a one-time or annual application in long-term research trials dissipation was extremely slow Significant

deactivation and sorption in four countries The Frensham (UK) trials were the most decrease in the number of earthworms for the high capacity of the soil through intensively studied They received within the 0ndash15 cm soil rate treatments compared to control one year since

field and laboratory depth increment a one-time application at rates ranging 90ndash720 application a change in the composition of the experiments kg ionha The fate of paraquat residue was then monitored over earthworm population and no significant difference

the following 20 years during which fields were maintained in the overall number of earthworms compared to under cereal or grassland cultivation Quantification of control six years since application no paraquat

earthworm micro-arthropod (some Collembola and Gamasina sorption by earthworms a significant decrease in species) microbial population (number of microorganisms total the number of micro-arthropod at the highest rates

propagules algae bacteria fungi and actinomycetes one year after application no statistically significant population) and biomass (via ATP determination) differences in total microbial biomass and the

number of microorganisms (total propagules algae bacteria fungi actinomycetes and the yeast

Lipomyces starkeyi) or in the ATP concentration the population of L starkeyi significantly decrease

in the 720 kgha treatment compared to the control Microbialsoil Greenhouse experiment under controlled conditions (soil Faster paraquat degradation for the non-sterilized Ismail et al (2011)

temperature and moisture) over 60 days (clay loam and clay soils compared to the sterilized ones soils from Malaysian agricultural soils) Non-sterilized vs

sterilized soils Use of 14C-paraquat to estimate degradation Photochemical and biological Field soil cores monitored for 1 month (applic rate of 075 and Paraquat degradation was faster under field than Amondham et al

degradationsoil 1725 kgha) Incubated laboratory soil columns for 12 weeks under laboratory conditions High degradation rates (2006) (Application rate of 345 kgha twice normal use) Laboratory compared to other studies may depend on

65

soil column incubation experiments (applic rate of 345 kgha) photochemical and microbial degradation of tropical over 12 weeks Determination of total paraquat concentration regions

via spectrophotometric methods after soil digestion and exchangeable paraquat via leaching using a NH4Cl solution

Microbial degradation under Use of three facultative anaerobic bacteria (PQ-1 PQ-2 and About 100 paraquat degradation in four days in Wu et al (2013) anaerobic conditions using PQ-3) isolated from vegetable soil in Sanya city China the presence of AQDS plus sucrose About 20

anthraquinone-26- Laboratory batch equilibrium experiments to monitor degradation in the presence of AQDS without disulphonate (AQDS) (in degradation of paraquat anaerobically with AQDS as the sole sucrose Sucrose addition can significantly enhance

place of humic substances) to electron acceptor with and without sucrose addition (source of paraquat degradation by anaerobic bacteria under simulate no-tillage paddy energy) anaerobic conditions

fields conditions

aPhotochemical Laboratory experiment Paraquat in aqueous solution was UV light degrades paraquat in presence of O2 over Slade (1965) degradationaqueous solution irradiated with Ultraviolet (UV) light generated by a lamp or three days However decomposition of paraquat

under sunlight in the presence of oxygen Autoradiograph of thin-layer chromatograph of UV-irradiated 14C-methyl paraquat

under sunlight appears to occur only when adsorbed to a surface eg paraquat adsorbed on filter paper

or thin layer silica gel but not when in aqueous solution

Methyl quaternary pyridinium (ie 4-carboxy-1- methylpyridinium ion) and methylamine

hydrochloride Photochemical Laboratory experiment Paraquat in (1) solid (dry) and (2) (1) About half dry paraquat was degraded after two Funderburk and

degradationaqueous solution aqueous form under UV radiation days and frac34 after four days to volatile compounds Bozarth (1967) (2) Paraquat concentration decreased with increased

UV exposure Very little aqueous paraquat was present after two days 1-methyl-4- carboxypyridinium chloride and two un-identified

degradation products

a Paraquat has a strong UV signal at 257 nm wavelength corresponding to a π-π transition of the electrons in the pyridinium ring

66

Table 3 Selected degradation studies reporting half-life values for paraquat in soil

Overall objectives Experimental Half-life estimate Source

Combined soil field and laboratory Paraquat dichloride (PD) applied annually (1967ndash1972) on All paraquat applied over a period of six Fryer et al (1975) dissipation study and phytotoxicity study a sandy loam soil as one-time single dose of 448 kgha or years could be extracted in 1971 and 1973 by the Weed Research Organization four separate doses of 12 kgha in plots under Medicago No degradation took place over seven years

Oxford England sativa L Soil digestion using H2SO4 for paraquat No phytotoxic effects could be observed determination Laboratory incubation experiments despite paraquat build up in soil

Re-sampling of the study plot described by Re-analysis of the same plots under the same paraquat Based on the 1975ndash1978 extractions some Hance et al (1980) Fryer et al (1975) at the Weed Research dose treatments (see above) during 1975ndash1978 paraquat disappeared from plots ie 10

Organization Oxford England yearly loss regardless of when paraquat was applied Estimated half-life was 66 years

Determination of the rate and by-products Use of 14C-paraquat to track degradation Laboratory soil 8392ndash1072 days (26 years) 4051ndash6509 Cheah et al (1998) of degradation for paraquat (24-D incubations under aerobic conditions for sandy loam days (14 years) and 2289ndash3652 days (72 lindane and glyphosate) in two muck and anaerobic for muck (Tanjong Karang and years) respectively

agricultural soils (sandy loam and muck Cameron Highlands Malaysia) ie about 30 soil organic C)

Assess degradation mobility sorption Field incubation of soil cores in polyvinyl chloride Degradation was faster under field Amondham et al (2006) desorption of paraquat in agricultural soils collected at 1 4 8 and 12 weeks following application conditions than under laboratory conditions

of the Yom River basin Thailand Two application rates of 075 kgha and 17 kgha with half-life estimate of 36ndash46 days

Monitoring paraquat degradation and Use of 14C-paraquat to estimate degradation comparing Clay loam soil 187 days (non-sterilized) Ismail et al (2011) testing the effect of soil temperature and non-sterilized vs sterilized soils Application rate was 06ndash and 1386 days (sterilized) Clay soil 231 moisture on paraquat degradation in 1 kgha days (non-sterilized) 1733 days (sterilized)

Malaysian agricultural soils

67

List of Figures

Figure 1 Synthesis of the paraquat dichloride salt In the paraquat cation the number-4 carbon

atom joins the two pyridine rings and each nitrogen atom has a methyl group (Based on Cairns

and Case [1975])

Figure 2 Annual paraquat dichloride mass used in California during 2000ndash2014

Figure 3 Cumulative frequency plot of paraquat dichloride-treated area (a) and mass per

application (b) and rate of application vs percentile (c) by year (2000ndash2014) in California (p5

through p95 are the 5h through the 95th percentile respectively of the frequency distribution of

interest CDPR [2016])

Figure 4 Map of the average annual mass of paraquat applied primarily on agricultural

commodities in different California counties during 2000ndash2014 (CDPR 2016)

Figure 5 Total paraquat dichloride mass applied to the top 20 crops (based on use) in California

during 2000ndash2014 (CDPR 2016)

Figure 6 Proposed pathway of paraquat dichloride (1) degradation by soil microfauna leading to

the formation of a 4-carboxilated-1-methylpyridnium ion (2) followed by oxalic acid (3) and

other non-identified products (not shown) and ultimately to mineralization products (4)

(modified after Funderburk and Bozarth [1967] and Ricketts [1999])

68

Figure 7 Pathway of paraquat dichloride (1) degradation by ultraviolet light in presence of

oxygen Identified degradation compounds were amino-aldehyde (likely) (2) 4-carboxy-1-

methylpyridinium ion (3) and methylamine hydrochloride (4) Compounds (3) and (4) do not

form in the absence of oxygen (modified after Slade [1965])

69

NaNH3 lack of O2

44rsquo- Bipyridyl

2 CH3Cl

2 Clndash

Paraquat dichloride

The initial reaction is between the product resulting from the metal-pyridine interaction and an alkylene oxide in liquid ammonia at temperatures normally not exciding -33 degC In its usually oxidized form paraquat is ionized with two positive charges Thus paraquat is usually manufactured as a salt containing two chloride anions

70

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

soil column incubation experiments (applic rate of 345 kgha) photochemical and microbial degradation of tropical over 12 weeks Determination of total paraquat concentration regions

via spectrophotometric methods after soil digestion and exchangeable paraquat via leaching using a NH4Cl solution

Microbial degradation under Use of three facultative anaerobic bacteria (PQ-1 PQ-2 and About 100 paraquat degradation in four days in Wu et al (2013) anaerobic conditions using PQ-3) isolated from vegetable soil in Sanya city China the presence of AQDS plus sucrose About 20

anthraquinone-26- Laboratory batch equilibrium experiments to monitor degradation in the presence of AQDS without disulphonate (AQDS) (in degradation of paraquat anaerobically with AQDS as the sole sucrose Sucrose addition can significantly enhance

place of humic substances) to electron acceptor with and without sucrose addition (source of paraquat degradation by anaerobic bacteria under simulate no-tillage paddy energy) anaerobic conditions

fields conditions

aPhotochemical Laboratory experiment Paraquat in aqueous solution was UV light degrades paraquat in presence of O2 over Slade (1965) degradationaqueous solution irradiated with Ultraviolet (UV) light generated by a lamp or three days However decomposition of paraquat

under sunlight in the presence of oxygen Autoradiograph of thin-layer chromatograph of UV-irradiated 14C-methyl paraquat

under sunlight appears to occur only when adsorbed to a surface eg paraquat adsorbed on filter paper

or thin layer silica gel but not when in aqueous solution

Methyl quaternary pyridinium (ie 4-carboxy-1- methylpyridinium ion) and methylamine

hydrochloride Photochemical Laboratory experiment Paraquat in (1) solid (dry) and (2) (1) About half dry paraquat was degraded after two Funderburk and

degradationaqueous solution aqueous form under UV radiation days and frac34 after four days to volatile compounds Bozarth (1967) (2) Paraquat concentration decreased with increased

UV exposure Very little aqueous paraquat was present after two days 1-methyl-4- carboxypyridinium chloride and two un-identified

degradation products

a Paraquat has a strong UV signal at 257 nm wavelength corresponding to a π-π transition of the electrons in the pyridinium ring

66

Table 3 Selected degradation studies reporting half-life values for paraquat in soil

Overall objectives Experimental Half-life estimate Source

Combined soil field and laboratory Paraquat dichloride (PD) applied annually (1967ndash1972) on All paraquat applied over a period of six Fryer et al (1975) dissipation study and phytotoxicity study a sandy loam soil as one-time single dose of 448 kgha or years could be extracted in 1971 and 1973 by the Weed Research Organization four separate doses of 12 kgha in plots under Medicago No degradation took place over seven years

Oxford England sativa L Soil digestion using H2SO4 for paraquat No phytotoxic effects could be observed determination Laboratory incubation experiments despite paraquat build up in soil

Re-sampling of the study plot described by Re-analysis of the same plots under the same paraquat Based on the 1975ndash1978 extractions some Hance et al (1980) Fryer et al (1975) at the Weed Research dose treatments (see above) during 1975ndash1978 paraquat disappeared from plots ie 10

Organization Oxford England yearly loss regardless of when paraquat was applied Estimated half-life was 66 years

Determination of the rate and by-products Use of 14C-paraquat to track degradation Laboratory soil 8392ndash1072 days (26 years) 4051ndash6509 Cheah et al (1998) of degradation for paraquat (24-D incubations under aerobic conditions for sandy loam days (14 years) and 2289ndash3652 days (72 lindane and glyphosate) in two muck and anaerobic for muck (Tanjong Karang and years) respectively

agricultural soils (sandy loam and muck Cameron Highlands Malaysia) ie about 30 soil organic C)

Assess degradation mobility sorption Field incubation of soil cores in polyvinyl chloride Degradation was faster under field Amondham et al (2006) desorption of paraquat in agricultural soils collected at 1 4 8 and 12 weeks following application conditions than under laboratory conditions

of the Yom River basin Thailand Two application rates of 075 kgha and 17 kgha with half-life estimate of 36ndash46 days

Monitoring paraquat degradation and Use of 14C-paraquat to estimate degradation comparing Clay loam soil 187 days (non-sterilized) Ismail et al (2011) testing the effect of soil temperature and non-sterilized vs sterilized soils Application rate was 06ndash and 1386 days (sterilized) Clay soil 231 moisture on paraquat degradation in 1 kgha days (non-sterilized) 1733 days (sterilized)

Malaysian agricultural soils

67

List of Figures

Figure 1 Synthesis of the paraquat dichloride salt In the paraquat cation the number-4 carbon

atom joins the two pyridine rings and each nitrogen atom has a methyl group (Based on Cairns

and Case [1975])

Figure 2 Annual paraquat dichloride mass used in California during 2000ndash2014

Figure 3 Cumulative frequency plot of paraquat dichloride-treated area (a) and mass per

application (b) and rate of application vs percentile (c) by year (2000ndash2014) in California (p5

through p95 are the 5h through the 95th percentile respectively of the frequency distribution of

interest CDPR [2016])

Figure 4 Map of the average annual mass of paraquat applied primarily on agricultural

commodities in different California counties during 2000ndash2014 (CDPR 2016)

Figure 5 Total paraquat dichloride mass applied to the top 20 crops (based on use) in California

during 2000ndash2014 (CDPR 2016)

Figure 6 Proposed pathway of paraquat dichloride (1) degradation by soil microfauna leading to

the formation of a 4-carboxilated-1-methylpyridnium ion (2) followed by oxalic acid (3) and

other non-identified products (not shown) and ultimately to mineralization products (4)

(modified after Funderburk and Bozarth [1967] and Ricketts [1999])

68

Figure 7 Pathway of paraquat dichloride (1) degradation by ultraviolet light in presence of

oxygen Identified degradation compounds were amino-aldehyde (likely) (2) 4-carboxy-1-

methylpyridinium ion (3) and methylamine hydrochloride (4) Compounds (3) and (4) do not

form in the absence of oxygen (modified after Slade [1965])

69

NaNH3 lack of O2

44rsquo- Bipyridyl

2 CH3Cl

2 Clndash

Paraquat dichloride

The initial reaction is between the product resulting from the metal-pyridine interaction and an alkylene oxide in liquid ammonia at temperatures normally not exciding -33 degC In its usually oxidized form paraquat is ionized with two positive charges Thus paraquat is usually manufactured as a salt containing two chloride anions

70

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

Table 3 Selected degradation studies reporting half-life values for paraquat in soil

Overall objectives Experimental Half-life estimate Source

Combined soil field and laboratory Paraquat dichloride (PD) applied annually (1967ndash1972) on All paraquat applied over a period of six Fryer et al (1975) dissipation study and phytotoxicity study a sandy loam soil as one-time single dose of 448 kgha or years could be extracted in 1971 and 1973 by the Weed Research Organization four separate doses of 12 kgha in plots under Medicago No degradation took place over seven years

Oxford England sativa L Soil digestion using H2SO4 for paraquat No phytotoxic effects could be observed determination Laboratory incubation experiments despite paraquat build up in soil

Re-sampling of the study plot described by Re-analysis of the same plots under the same paraquat Based on the 1975ndash1978 extractions some Hance et al (1980) Fryer et al (1975) at the Weed Research dose treatments (see above) during 1975ndash1978 paraquat disappeared from plots ie 10

Organization Oxford England yearly loss regardless of when paraquat was applied Estimated half-life was 66 years

Determination of the rate and by-products Use of 14C-paraquat to track degradation Laboratory soil 8392ndash1072 days (26 years) 4051ndash6509 Cheah et al (1998) of degradation for paraquat (24-D incubations under aerobic conditions for sandy loam days (14 years) and 2289ndash3652 days (72 lindane and glyphosate) in two muck and anaerobic for muck (Tanjong Karang and years) respectively

agricultural soils (sandy loam and muck Cameron Highlands Malaysia) ie about 30 soil organic C)

Assess degradation mobility sorption Field incubation of soil cores in polyvinyl chloride Degradation was faster under field Amondham et al (2006) desorption of paraquat in agricultural soils collected at 1 4 8 and 12 weeks following application conditions than under laboratory conditions

of the Yom River basin Thailand Two application rates of 075 kgha and 17 kgha with half-life estimate of 36ndash46 days

Monitoring paraquat degradation and Use of 14C-paraquat to estimate degradation comparing Clay loam soil 187 days (non-sterilized) Ismail et al (2011) testing the effect of soil temperature and non-sterilized vs sterilized soils Application rate was 06ndash and 1386 days (sterilized) Clay soil 231 moisture on paraquat degradation in 1 kgha days (non-sterilized) 1733 days (sterilized)

Malaysian agricultural soils

67

List of Figures

Figure 1 Synthesis of the paraquat dichloride salt In the paraquat cation the number-4 carbon

atom joins the two pyridine rings and each nitrogen atom has a methyl group (Based on Cairns

and Case [1975])

Figure 2 Annual paraquat dichloride mass used in California during 2000ndash2014

Figure 3 Cumulative frequency plot of paraquat dichloride-treated area (a) and mass per

application (b) and rate of application vs percentile (c) by year (2000ndash2014) in California (p5

through p95 are the 5h through the 95th percentile respectively of the frequency distribution of

interest CDPR [2016])

Figure 4 Map of the average annual mass of paraquat applied primarily on agricultural

commodities in different California counties during 2000ndash2014 (CDPR 2016)

Figure 5 Total paraquat dichloride mass applied to the top 20 crops (based on use) in California

during 2000ndash2014 (CDPR 2016)

Figure 6 Proposed pathway of paraquat dichloride (1) degradation by soil microfauna leading to

the formation of a 4-carboxilated-1-methylpyridnium ion (2) followed by oxalic acid (3) and

other non-identified products (not shown) and ultimately to mineralization products (4)

(modified after Funderburk and Bozarth [1967] and Ricketts [1999])

68

Figure 7 Pathway of paraquat dichloride (1) degradation by ultraviolet light in presence of

oxygen Identified degradation compounds were amino-aldehyde (likely) (2) 4-carboxy-1-

methylpyridinium ion (3) and methylamine hydrochloride (4) Compounds (3) and (4) do not

form in the absence of oxygen (modified after Slade [1965])

69

NaNH3 lack of O2

44rsquo- Bipyridyl

2 CH3Cl

2 Clndash

Paraquat dichloride

The initial reaction is between the product resulting from the metal-pyridine interaction and an alkylene oxide in liquid ammonia at temperatures normally not exciding -33 degC In its usually oxidized form paraquat is ionized with two positive charges Thus paraquat is usually manufactured as a salt containing two chloride anions

70

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

List of Figures

Figure 1 Synthesis of the paraquat dichloride salt In the paraquat cation the number-4 carbon

atom joins the two pyridine rings and each nitrogen atom has a methyl group (Based on Cairns

and Case [1975])

Figure 2 Annual paraquat dichloride mass used in California during 2000ndash2014

Figure 3 Cumulative frequency plot of paraquat dichloride-treated area (a) and mass per

application (b) and rate of application vs percentile (c) by year (2000ndash2014) in California (p5

through p95 are the 5h through the 95th percentile respectively of the frequency distribution of

interest CDPR [2016])

Figure 4 Map of the average annual mass of paraquat applied primarily on agricultural

commodities in different California counties during 2000ndash2014 (CDPR 2016)

Figure 5 Total paraquat dichloride mass applied to the top 20 crops (based on use) in California

during 2000ndash2014 (CDPR 2016)

Figure 6 Proposed pathway of paraquat dichloride (1) degradation by soil microfauna leading to

the formation of a 4-carboxilated-1-methylpyridnium ion (2) followed by oxalic acid (3) and

other non-identified products (not shown) and ultimately to mineralization products (4)

(modified after Funderburk and Bozarth [1967] and Ricketts [1999])

68

Figure 7 Pathway of paraquat dichloride (1) degradation by ultraviolet light in presence of

oxygen Identified degradation compounds were amino-aldehyde (likely) (2) 4-carboxy-1-

methylpyridinium ion (3) and methylamine hydrochloride (4) Compounds (3) and (4) do not

form in the absence of oxygen (modified after Slade [1965])

69

NaNH3 lack of O2

44rsquo- Bipyridyl

2 CH3Cl

2 Clndash

Paraquat dichloride

The initial reaction is between the product resulting from the metal-pyridine interaction and an alkylene oxide in liquid ammonia at temperatures normally not exciding -33 degC In its usually oxidized form paraquat is ionized with two positive charges Thus paraquat is usually manufactured as a salt containing two chloride anions

70

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

Figure 7 Pathway of paraquat dichloride (1) degradation by ultraviolet light in presence of

oxygen Identified degradation compounds were amino-aldehyde (likely) (2) 4-carboxy-1-

methylpyridinium ion (3) and methylamine hydrochloride (4) Compounds (3) and (4) do not

form in the absence of oxygen (modified after Slade [1965])

69

NaNH3 lack of O2

44rsquo- Bipyridyl

2 CH3Cl

2 Clndash

Paraquat dichloride

The initial reaction is between the product resulting from the metal-pyridine interaction and an alkylene oxide in liquid ammonia at temperatures normally not exciding -33 degC In its usually oxidized form paraquat is ionized with two positive charges Thus paraquat is usually manufactured as a salt containing two chloride anions

70

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

NaNH3 lack of O2

44rsquo- Bipyridyl

2 CH3Cl

2 Clndash

Paraquat dichloride

The initial reaction is between the product resulting from the metal-pyridine interaction and an alkylene oxide in liquid ammonia at temperatures normally not exciding -33 degC In its usually oxidized form paraquat is ionized with two positive charges Thus paraquat is usually manufactured as a salt containing two chloride anions

70

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

71

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

72

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

Sacramento Valley San Joaquin Valley

Average mass (kgyr)

73

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

74

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

H3CmdashN+ N+mdashCH3 2 Clndash(1)

H3CmdashN+ N+ Clndash

(2) H3CmdashN+ COOndash

O O (3)

OH OH

(4) NH3 + CO2 + H2O

75

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final

H3CmdashN+ N+mdashCH3 2 Clndash (1)

CHO

(2) H3CmdashN+ NHCH3 Clndash

COOndash(3) H3CmdashN+

HCl

(4) CH3NH2HCl+

76

• 2018_0531_SartoriampVidrio_textamptables_final
• • 1 Introduction
  • 2 Physical and chemical properties
  • 3 Overview of paraquat use
  • • 31 Regulation
    • • 4 Use profile of paraquat in California
      • 5 Plant resistance
      • 6 Environmental fate
      • • 61 Soil
        • 62 Water
        • 63 Air
        • • 7 Environmental degradation
          • • 71 Microbial
            • 72 Photochemical
            • • 8 Ecotoxicology
              • • 81 Mode of action paraquat redox chemistry
                • 82 Plants
                • • 821 General physiological effects
                  • 822 Dose-response studies
                  • • 83 Soil fauna and flora
                    • • 831 Toxic effects
                      • 832 Teratogenic effects
                      • 833 Soil bacteria
                      • • 84 Birds
                        • • 841 Toxic and teratogenic effects
                          • 842 Reproductive effects
                          • • 85 Honeybees
                            • 86 Amphibians
                            • • 861 Toxic teratogenic and reproductive effects
                              • • 87 Fish and other aquatic species
                                • • 871 Toxic effects
                                  • 872 Oxidative stress indicators
                                  • 873 Behavioral analyses
                                  • • 9 Summary and Conclusions
                                    • • 2018_0531_SartoriampVidrio_figures_final