Faculty of Sciences

Department of Biology

Research group Marine Biology

__________________________________________________________________________________

Academic year 2013/2014

Alternative methods for the assessment of pollution

in marine nematodes: behavioral analysis versus

developmental and reproductive assays.

Van Butsel Jana

Supervisor: Prof. Dr. Tom Moens

Tutors: Luana Monteiro Thesis submitted to obtain the degree of

Nele De Meester Master of Science in Biology

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To my parents and Niels,

For making my life in Ghent possible.

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Table of contents

I. Introduction .......................................................................................................................................... 6

II. Aims and hypotheses ........................................................................................................................ 10

III. Material and Methods ...................................................................................................................... 11

III. 1 - Research .................................................................................................................................. 11

III. 2 - Nematode cultures .................................................................................................................. 11

III. 3 - Test substances and concentrations ........................................................................................ 11

III. 4 - Preparation of test solutions, polluted agar and polluted food .............................................. 12

III. 5 - General taxis to food experiment (GTTF) - four species, no pollution ..................................... 12

III. 6 - Taxis to food after exposure to pollutants (TTF) ..................................................................... 13

III. 7 - Growth and reproduction experiments ................................................................................... 15

III. 8 - Data analysis ........................................................................................................................... 17

IV. Results .............................................................................................................................................. 19

IV. 1 - General taxis to food (GTTF) ................................................................................................... 19

IV. 2 - Taxis to food (TTF) after exposure ........................................................................................... 20

IV. 3 - Growth .................................................................................................................................... 22

IV. 4 - Reproduction ........................................................................................................................... 24

V. Discussion .......................................................................................................................................... 25

V. 1 - Selection of test species............................................................................................................ 25

V. 2 - Effects of heavy metals on nematode behaviour ..................................................................... 26

V. 3 - Effects of heavy metals on nematode growth and reproduction ............................................. 28

V. 4 - Differences between species and ecological implications ....................................................... 29

V. 5 - Suitability of our test species, endpoints and assays in toxicity testing ................................... 31

VI. Conclusion ........................................................................................................................................ 32

VII. Summary ......................................................................................................................................... 33

VII. 1 - Summary ................................................................................................................................ 33

VII. 2 - Samenvatting ......................................................................................................................... 35

VIII. Acknowledgements ........................................................................................................................ 38

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I. Introduction Before the London Convention 1972 (11 ILM 1294(1972)), now replaced by the 1996 Protocol (36

ILM 1 (1997)), was enforced, oceans were deliberately used as convenient dumping sites for all kinds

of toxic waste (Nauke and Holland (1992). Moreover, the forms of marine pollution are as diverse as

their effects on organisms and ecosystems. Molecular pollutants, for example, can enter the food

web through microbial decomposers and primary producers and may accumulate at higher trophic

levels (Bryan and Langston (1992)), whereas larger elements such as plastic waste can directly kill

organisms by ingestion (Moore (2008); Claessens et al. (2011)) or by acting as a transport medium for

toxic chemicals (Mato et al. (2001)). The modes of action of the various forms of pollution need to be

well studied and understood for every part of the food web, in order to gain insight into their effects

on interactions between and within different trophic levels (Borgmann et al. (1989); Koivisto et al.

(1997); Salminen et al. (2002); Fleeger et al. (2003); Evans-White and Lamberti (2009); De Laender et

al. (2011)). Furthermore, identifying sensitive (local) species can prove useful in ecological risk

assessment and in monitoring an environment's condition (OECD (1992), Abrantes et al. (2009)).

Chemical analyses alone are not sufficient to gain proper insight in how hazardous substances affect

organisms and communities (Long et al. (1995)), which is why reliable biological methods are needed

(Rand (1995)). Nematodes have proven to be excellent organisms for ecotoxicological research

(Bongers and Ferris (1999); Sochovà et al. (2006); Höss and Williams (2009)). They form a very

diverse group and reach high abundances in soil and other sediments in different terrestrial (Yeates

(1979); Bongers and Bongers (1998)) and aquatic environments (Heip et al. (1985); Traunspurger

(2000)). Nematodes are limited in their active and passive dispersal capacities, generally not covering

more than 2 metres per tidal cycle (Thomas and Lana (2011); Commito and Tita (2002)). This implies

that they are likely to get affected by contaminants in case of (sudden) local pollution, as was

demonstrated by the response of the benthic meiofauna community following a simulated oil spill by

Kang et al. (2014). In addition, a variety of feeding types and trophic levels– bacterial, fungal or plant

feeders, predators and omnivores – can be found within this phylum (Yeates et al. (1993); Moens and

Vincx (1997); Traunspurger et al. (1997); Moens et al. (2004)), which makes them ideal organisms for

comparative studies that focus on how feeding habits and trophic interactions influence sensitivity

towards toxicants (Kammenga et al. (1994)). The immense diversity of nematode communities is

reflected in the diversity of their responses towards disturbances in the environment (Schratzberger

and Warwick (1999)). It is therefore interesting to investigate the consequences of pollution on

different scales, ranging from entire communities to single-species individuals (De Laender et al.

(2009)). If the main interest lies in the ecological consequences of pollution, community assessments

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probably provide the most relevant results (Korthals et al. (1996); Austen and McEvoy (1997);

Schratzberger et al. (2002); Gyedu-Ababio and Baird (2006); Beyrem et al. (2007)). By contrast,

studying the exact responses of single species can be a helpful tool to identify the toxicity

mechanisms of pollutants (Houck and Kavlock (2008); Scott and Hodson (2008)), or to predict

ecologically safe maximum concentrations of substances (Versteeg et al. (1999)). Another advantage

of single-species assays performed in laboratories under controlled conditions is that they are less

complex and results are often easier to interpret (Höss and Williams (2009)).

The model organism Caenorhabditis elegans has been used in numerous ecotoxicological studies

(Williams and Dusenbery (1990); Sochovà et al. (2006)). Toxic effects of a wide range of substances in

different media have been elaborately tested and described for this soil nematode (Traunspurger et

al. (1997); Dhawan et al. (2000); Anderson et al. (2001); Boyd and Williams (2003); Anderson et al.

(2004)) which has lead to the development of a standardized toxicity test for this organism (ISO

10872:2010). Researchers are working to identify other nematode species for toxicity testing that

prove equally suitable as C. elegans. For example, Kammenga et al. (1996) used Plectus acuminatus

for (artificial) soil toxicity tests and the aquatic species Panagrellus redivivus was proven suitable by

Samoiloff et al. (1980) and Samoiloff (1990). In this study, the free-living, bacterivorous marine

nematode Litoditis marina (Sudhaus (2011)) will be used as test organism. This species belongs to the

same family as C. elegans (Rhabditidae) and was formerly also known as Rhabditis marina (Bastian

(1865)) or Pellioditis marina (Andrássy (1983)). It is a cosmopolitan species that can be found on

decaying macroalgae or in sediment in the littoral zone of coasts and estuaries (Inglis and Coles

(1961)). Litoditis marina is one of few marine species that can successfully be cultivated under

laboratory conditions (Moens and Vincx (1998)), and has been used in a variety of studies, focusing

for example on different life strategies (Moens et al. (1996)), on cadmium toxicity (Vranken et al.

(1985); Derycke et al. (2007); Lira et al. (2011)) and the relative influences of environmental

conditions and food availability (Moens and Vincx (2000a); dos Santos et al. (2009); De Meester et al.

(2011)), along with various studies on genetics (Derycke et al. 2005, 2007, 2008, 2012). This has lead

to the documentation of some important life-history traits and strategies. Generation time is very

short (down to 3 days or less under optimal conditions (Moens and Vincx (2000b)), reproduction is

obligately heterosexual, females can be oviparous or ovoviviparous (Derycke et al. (2008) with up to

600 eggs per female (Vranken and Heip (1983)). Juveniles moult four times (J1-J4) before reaching

adulthood. The species displays a natural tolerance of a broad salinity and temperature range

(Moens and Vincx (2000a); De Meester et al. (2011, 2012)). However, the alleged broad tolerance to

environmental conditions might be partly biased in view of the discovery of substantial cryptic

diversity within the L. marina morphospecies complex. This has lead to a number of population

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genetic and phylogeographic studies that attempt to shed light onto the micro-evolutionary

background of these coexisting cryptic species (Derycke et al. (2005, 2007, 2008, 2012)). In Derycke

et al. (2008) the existence of at least ten cryptic species was revealed, of which four (PmI, PmII, PmIII,

PmIV) commonly occur in multispecies complexes along the south-western coast and estuaries of

The Netherlands (Derycke et al. (2005)). Recent studies have focussed on identifying possible

differences in autecology (De Meester et al. (2011); Derycke et al. (2012)) and dispersal behaviour

(De Meester et al. (2012)) of sympatrically occurring cryptic species. Laboratory cultures of L. marina

are often isolated from natural populations that occur along the Westerschelde estuary (e.g. Derycke

et al. (2007)).

Heavy metals are globally occurring, often very slowly degrading pollutants (Höss and Williams

(2009); Akpor and Muchie (2010)). Elevated concentrations in soil and aquatic environments are

often linked to urbanization, industrialization, and the release of contaminated waste water to

receiving water bodies (Hussein et al. (2005); Hu et al. (2013)). Copper (Cu) is an essential trace-

metal, meaning that it is an indispensable element to most organisms as it is involved in the

functioning of a variety of enzymes and proteins (Henry (1996); Holm et al. (1996); Solomon et al.

(1996)). However, when exposed to doses that the body cannot equilibrate in time, it causes adverse

effects on living organisms (EPA (2009)). Copper sulphate is used in a wide array of biocides either as

a drying agent, or as an additive for fertilizers and food, as well as in numerous industrial applications

(Boone et al. (2012)). This makes it an extensively studied pollutant with severely adverse effects

observed in terrestrial and aquatic organisms (Boone et al. (2012)). Lead (Pb) is a non-essential trace-

metal that is used to make e.g. batteries, ammunition (bullets), pipes, roofing material and, in the

past, paint and gasoline (EPA (2009)). It has been a commonly known toxicant since the discovery of

childhood lead poisoning (Needleman (2004)). The bioavailability of these two heavy metals depends

on their speciation in the environment and is influenced by pH (Zirino and Yamamoto (1972)), salinity

and the presence of complexing ligands like particulate and dissolved organic matter (Kozelka et al.

(1997); Deruytter et al. (2014); Bi et al. (2013)). Sánchez-Marín et al. (2007) documented that the

presence of humic acids, important dissolved organic constituents of sea water (Coble (1996)),

increases uptake and toxicity of lead in excised gills of the mussel Mytilus edulis and in larvae of the

sea urchin Paracentrotus lividus. Earlier, equivalent studies by Lorenzo et al. (2002) and Lorenzo et al.

(2005) confirmed a decrease of copper toxicity and uptake upon complexation with humic acids in

ASW by these species. On another note, Howell (1982a) discovered that the mucus secreted by some

marine nematode species has metal-binding capacities and may thus play a role in the uptake and

loss of heavy metals. Yet, the toxicity of a substance appears to be extremely species specific (Lewis

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(1992); Libralato et al. (2010)) and depends on its bioavailability and the ecology, lifestyle and

feeding habits of the exposed organism (Bryan and Langston (1992)).

Lethality tests have been popular in toxicological research, which has lead to the development of

several standardized acute toxicity tests for different organisms (EPS 1/RM/9, EPS 1/RM/11, EPS

1/RM/35, ISO 7346-1:1996, ISO 14669:1999, ISO 14380:2011). Traditionally, environmental risk

managers have been basing their choice for threshold concentrations of harmful substances in

different environmental compartments on such mortality studies (Chapman et al. (1998)). In many

cases, however, it is more relevant to assess the effects of sublethal concentrations on organisms,

populations or communities, as even low concentrations may already impact relations within and

between each of these entities (Fleeger et al. (2003)). Choosing appropriate and meaningful

endpoints is crucial and should take into account the research objective as well as specific

characteristics of the studied organism(s) and their ecological relevance (EPA, 1996). In toxicity

testing with nematodes, the most frequently studied sublethal endpoints are growth, fertility and

reproduction on the one hand, and behavioural endpoints like feeding and movement on the other

hand (Höss and Williams (2009)). Endpoints can differ in their ecological relevance and the way they

respond to different types of toxicants: endocrine disruptors may influence reproduction (Höss and

Weltje (2007)), leading to changes in population size and age structure (EPA, 1994); interactions with

developmental pathways may cause reduced growth, leading to e.g. decreased reproduction, lower

fecundity, smaller size and increased susceptibility to predation (EPA, 1994). Neurotoxicity can be

assessed by measuring responses at the neuronal level (Leung et al. (2008), or by studying

behavioural endpoints like movement and feeding activity (Dhawan et al. (2000); Boyd et al. (2003);

Anderson et al. (2004)) or more recently, chemotaxis (Monteiro et al. (2014); Song et al. (2014)). It is

therefore interesting to assess different endpoints when studying the effects of a substance on an

organism.

For our model species L. marina, so far the toxicity of cadmium (Vranken et al. (1985)) and barium

(Lira et al. (2011)) have been tested on the survival, and individual and population development of

PmI. Also the influence of lead, zinc and nickel has been tested by Monteiro et al. (2010) on growth,

reproduction and taxis to food of PmI. This study is therefore the first to investigate whether cryptic

species differ in their sensitivity and/or response towards different toxicants. For this study, lead was

chosen for its neural toxicity (Williams and Dusenbery (1990)), while copper is a more general

toxicant known to interfere with osmoregulation (Brooks and Mills (2003)) and oxidative stress

pathways in several aquatic organisms (Lushchak (2011)). Furthermore, studying the effects of these

two heavy metals on two cryptic species is interesting from both an evolutionary and an ecological

point of view. It may reveal yet unknown autecological differences between them, it could form a

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link to more detailed studies on the mechanisms that allow these species to coexist, and – provided

these cryptic species show substantial functional overlap – it may also reveal to what extent cryptic

diversity provides an insurance against the consequences of species loss for ecosystem functioning

(Naeem and Li (1997)).

II. Aims and hypotheses The aim of this study was to assess the effects of copper and lead contamination on cryptic species of

L. marina using different endpoints. This would also help us to further assay the suitability and

reliability of behavioural versus developmental and reproductive tests for this model species. To

accomplish the above described goals, nematodes' taxis to food (behaviour), growth and

reproduction after exposure to different concentrations of copper and lead were tested. Thereby we

would address the following hypotheses:

I. The three endpoints will indicate the same order of toxicity for Cu and Pb;

II. Cryptic species of L. marina will show similar responses to toxicants (Cu and Pb);

III. Behavioural experiments are suitable assays for cryptic L. marina species and can be used to

gain complementary information missed by classical toxicity tests that use growth and

reproduction as endpoints.

All experiments were conducted under controlled laboratory conditions. At first, a pollution-free

‘taxis-to-food’ assay was performed with four different cryptic species to identify two suitable test

species for the actual toxicity assays. Then, the selected endpoints were investigated to assess the

effects of different concentrations of Cu and Pb on the two selected species.

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III. Material and Methods

III. 1 - Research

The study was performed in the research group of Marine Biology, part of the Department of Biology

at the Faculty of Sciences of the University of Ghent. All practical work was carried out in the

laboratories of Campus De Sterre, S8, in Ghent, Belgium.

III. 2 - Nematode cultures

Stock cultures of four cryptic species of Litoditis marina (Sudhaus (2011)), referred to as PmI, PmII,

PmIII and PmIV after Derycke et al. (2005, 2008), have been maintained under laboratory conditions

as proposed by Moens and Vincx (1998, 2000) at a constant temperature of 20°C on sloppy agar

plates with unidentified bacteria as food (1% agar; bacto and nutrient agar in a 4:1 ratio, prepared

with artificial sea water (ASW) with a salinity of 25 and a pH 7.5-8, buffered with TRIS-HCl at a final

concentration of 5 mM). Stock cultures were renewed approximately every eight to ten days. To

avoid food depletion and to stimulate growth and reproduction, a few drops (30-50 µL) of a

suspension of dead Escherichia coli (K-12 strain, 3 x 109 cells/mL, ASW, cholesterol 0.1%), were added

to the stock plates from which nematodes would be harvested for toxicity experiments.

III. 3 - Test substances and concentrations

Establishing detailed concentration-response curves of L. marina to copper and lead fell beyond the

scope of this study. Our choice of copper and lead concentrations to be tested was based on lethal

and sublethal aquatic toxicity studies performed on related soil nematode species like Caenorhabditis

elegans (Anderson et al. (2001, 2004); Boyd et al. (2003); Boyd and Williams (2003)), which belongs

to the same family as L. marina (Rhabditidae), or Panagrellus redivivus and Pristionchus pacificus,

who belong to the same order (Rhabditida) (Boyd and Williams (2003)).

Table 1 shows which heavy metal concentrations were used for the taxis to food (TTF) and the

growth and reproduction assays (G, R), respectively, expressed as both molar and metric

concentrations. To allow easier comparison of the toxicity of the different metals, the same molar

concentrations were used for both copper and lead. Even though the TTF experiments and the G and

R assays handle different exposure times (shorter for TTF, longer for G and R), it seemed interesting

to use (partly) overlapping test concentrations, allowing to compare the utility of behavioural versus

'traditional' assays. For the TTF experiment a higher concentration was included (240 µM) that was

not used in the G and R assays because of the extended exposure time; for the G and R assays, a

lower concentration was tested instead (10 µM).

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Table 1. Overview of pollutant concentrations used in the different experiments, expressed both in molar (µM) and metric (mg/L) quantities. TTF stands for Taxis to Food, G for growth, R for reproduction.

Assay Concentration Copper (Cu) Lead (Pb)

µM mg/L µM mg/L

TTF, G, R C0 = control 0 0 0 0

G, R C1 = C10 10 0.64 10 2.07

TTF, G, R C2 = C40 40 2.54 40 8.29

TTF, G, R C3 = C80 80 5.08 80 16.58

TTF C4 = C240 240 15.25 240 49.73

III. 4 - Preparation of test solutions, polluted agar and polluted food

Copper and lead stock solutions of 24 mM (Cstock) were prepared with distilled water and

copper(II)sulfate pentahydrate (CuSO4*5H2O) and lead(II)nitrate Pb(NO3)2 respectively, and stored in

100 mL volumetric flasks.

Polluted agar was prepared by adding an appropriate amount of undiluted, or 3x, 6x, 24x diluted

stock solution in ASW in a ratio of 1:100 to hot, liquid 1% bacto agar to obtain final C4, C3, C2 and C1

toxicant concentrations in the agar. For control plates, agar was prepared in the same way, but

adding ASW instead of toxicant solution. Experimental plates were poured immediately, sealed with

laboratory film and stored at 4°C until further use.

For polluted food, Cstock was first diluted 10x, 30x, 60x or 24x*10x in ASW and then added in a 1:10

ratio to an E. coli stock suspension of 3x1010 cells/mL to obtain final food suspensions of 3x109

cells/mL and C4, C3, C2 and C1 toxicant concentrations. For control plates, food was prepared by

adding ASW instead of contaminant solution. New food suspensions were always prepared before

starting an experiment from thawed and refrigerated, uncontaminated food stock suspensions.

III. 5 - General taxis to food experiment (GTTF) - four species, no pollution

This first set of experiments was carried out to identify differences in food finding behaviour

between the four cryptic species (PmI-IV) and to choose two suitable species for further analysis. The

choice would be based on the ease of handling of the species (low mortality, quick recovery from

washing and incubation) and the 'strength' of their attraction to food. The experimental set-up is

similar to the design developed by Weber and Traunspurger (2013) and was also successfully applied

in Monteiro et al. (2014). Petri dishes of 9 cm inner diameter were filled with 18 mL agar (1.5% bacto

agar, ASW, TRIS-HCl buffer 5 mM). A sterile test tube of 1.5 cm diameter was used to pierce six

circles into the solidified agar at equal distances from the plate centre. The agar discs were removed

with sterile forceps and the bottom of the wells was again covered with a thinner layer (100 µL) of

agar to prevent test solutions from dispersing underneath the agar. Wells were filled alternately with

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300 µL of a control solution (autoclaved ASW) or unpolluted food

suspension (dead E. coli mix of 3x109 cells/mL, ASW, cholesterol 0.1%)

as depicted in Fig. 1.

Nematodes were handpicked from stock plates with a fine needle and

placed into staining blocks containing autoclaved ASW for a few

minutes to minimize the amount of bacteria being co-transferred to

the experimental plates. This washing step also helped screening for

and selecting only vital, motile adults. No distinction was made

between males and females. For each stock plate that was used

(usually 3-5), a washing recipient was prepared to contain only nematodes obtained from the same

stock plate. To avoid culture-dependent bias, an equal number of nematodes from each washing

recipient was incubated per replicate. For ease of handling and to avoid damaging the agar, a small

droplet (5 µL) of autoclaved ASW was used to mark the centre of the plate where nematodes would

be placed. Per replicate 30 nematodes were transferred from different washing recipients directly to

the droplet in the centre of the experimental plates. After the last nematode was placed on a plate,

the droplet was carefully smeared out with the picking needle to not let surface tension trap the

nematodes. The exact time of incubation was noted and the plate was covered with a lid and placed

in the dark at 20°C. This procedure was repeated for each replicate, using 5 replicates per species.

Nematodes were left to disperse across the plates and after intervals of 1, 2½, 4, 6 and 24 hours, the

number of individuals in each spot (food and control) was counted under a dissecting microscope.

III. 6 - Taxis to food after exposure to pollutants (TTF)

Based on the results of the GTTF experiments, PmII and PmIV were chosen for further toxicity assays.

The idea behind this experiment is to expose nematodes to a certain concentration of a toxicant for

24 hours and then test their performance in a pollution-free, taxis-to-food assay. Exposure was done

in 6-well multiwell plates (35 mm inner diameter). Wells were filled with 2 mL polluted agar

containing pollutant at the concentration to be tested. Shortly before nematodes were incubated,

the agar was covered with a thin film of polluted food suspension with the same toxicant

concentration. This was done by evenly smearing out 50 µL of food suspension with the heat-bent tip

of a sterile glass Pasteur pipette. Before smearing, the bent tip was sterilized by rinsing it in ethanol

and passing it through a flame. Instead of using different washing recipients as described for the

GTTF experiment, this time the nematodes needed for one replicate were picked from different stock

plates and placed together in one washing recipient with ASW and transferred to the exposure wells.

Each well (replicate) contained 25 vital adult nematodes, and there were 4 replicates per pollutant

Figure 1. Experimental plate design used in the general taxis to food (GTTF) experiment. F: food spot, C: control spot.

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concentration. For each replicate the exact time of incubation was noted, and finally the multiwell

plate was sealed with parafilm and placed in the dark at 20°C for 24 hours.

A few adaptations were made to the experimental GTTF design (see further), making it more similar

to the set-up used by Moens et al. (1999). Petri dishes of 9 cm inner diameter were filled with 18 mL

unpolluted agar into which only two excavations were made at equal distance and opposing sides

from the centre of the plate (Fig. 2). Here, the wells were bottom-covered with a slightly thicker layer

of agar (250 µL) and subsequently filled with either 150 µL autoclaved ASW for the control spot or

unpolluted food suspension (dead E. coli mix of 3x109 cells/mL) for the food spot. This adaptation

mainly served to increase visibility of nematodes in the food spots, which were often very hard to see

in the deeper GTTF food wells.

After 24±½ hours, 20 (out of 25) nematodes from the exposure

plates were hand-picked and washed in ASW , one replicate at a

time, and incubated on the TTF plates. It proved necessary to

incubate a small excess (5) of nematodes in the exposure plates

to compensate for background mortality and 'hiding' or 'suicidal'

nematodes that crawl up the borders of the Petri dish and die

from dehydration. The procedure was the same as described for

the GTTF experiment. Time of incubation was noted and after 1,

2½, 4, and 6 hours nematodes on the plates were counted and

given a score according to their vicinity to the food or control

spots. The scoring scheme is shown in Fig. 2 and is similar to the

ones used by Grewal and Wright (1992) and Rodger et al. (2004), but adapted to the bipolar design

and round shape of the plates. Nematodes in the centre received a score of 0 because they were

considered not to have migrated after incubation, while those inside food (F) or control (C) spots get

the highest (+5) or lowest (-5) score, respectively. It is important to note that nematodes that

reached a food or control spot often stayed there as long as the spot had not dried out (this was

increasingly the case after more than 6 hours of incubation, which is why the 24h count was left out

from the data analysis). During each count the number of nematodes per section was noted and

multiplied by the corresponding section score. Then, the positive and the negative scores were

summed to obtain one score per plate. The larger the number, positive or negative, the more

nematodes were found in or close to the food or control spots, respectively.

This adapted design has three important advantages compared to the GTTF design. First, it minimizes

the chance of confounding random spot choice with actual taxis (attraction) by creating one clear

gradient and limiting interfering signals from juxtaposed food and control spots. Secondly it allows to

Figure 2. Scoring scheme used in the taxis to food (TTF) experiment. F: side of the food spot, C: side of the control spot.

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use a scoring system which includes all nematodes on the plate, not only the ones that have reached

a control or food spot. Moreover, unlike the abundances of nematodes inside multiple spots of one

GTTF plate, the single score per plate in this assay does not pose statistical issues related to the

(in)dependence of data (Sokal and Rohlf (1995)).

III. 7 - Growth and reproduction experiments

The two following assays serve to analyse how different treatments with copper and lead impact the

growth of juvenile nematodes and the reproduction of young adults. Growth and reproduction are

two commonly used endpoints to study the (toxic) effects of substances on nematodes (Höss and

Williams (2009)). For Caenorhabditis elegans a standardized toxicity test was developed (ISO

10872:2010) based on these endpoints. However, we could not copy this test design for L. marina

and had to make two distinct assays for growth and reproduction. This is mainly because L. marina is

not a hermaphroditic species like C. elegans and thus, at least for the reproduction assay, distinction

between males and females needs to be made, which is not possible when picking very small

juveniles (J1-J2) as needed for the growth assay. Moreover, the ISO test is performed in liquid K-

medium, a medium that is unsuitable for L. marina (Monteiro et al. (2010)). The initial plan was to

use a semi-fluid gel-like medium based on gellan gum (Gelrite-Schweizerhall Inc., South Plainfield,

NJ), which has been proven suitable for tests with a number of nematode species (Ferris et al. (1995);

Muschiol and Traunspurger (2007); Muschiol et al. (2009)) and for toxicity testing with C. elegans

(Brinke et al. (2011)). However, even though our pilot studies showed that L. marina was able to

survive in this medium for several days, it did not reproduce. Therefore the growth and reproduction

assays were performed on agar medium.

For both assays, Petri dishes of 35 mm inner diameter were filled with 1 mL of either unpolluted

(control) or polluted agar of C1, C2 or C3 concentration (Table 1). Keeping the layer of agar this thin

still allows the nematodes to burrow into the medium, but they remain easily visible which greatly

facilitates counting of juveniles in the reproduction assay. Shortly before incubating nematodes, a

thin film of polluted or unpolluted food was smeared over the surface of the agar as described for

the TTF pre-exposure plates.

Growth assay

Age synchronisation proved difficult due to variation in egg deposition (oviparous PmII) or juvenile

birth (ovoviviparous PmIV). Therefore, the juveniles used in this experiment were the smallest stage

that could be found on stock plates (probably J1 juveniles). Dealing with such small individuals

proved challenging, but an eye lash attached to the tip of a needle allowed to pick the juveniles and

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transfer them to small droplets (5 µL) of ASW on the surface of an empty Petri dish to wash them. By

doing this, juveniles did not get lost in the comparatively large washing recipients that are used in the

other assays with adult nematodes.

The 'initial size' was obtained by calculating the average body lengths of 30 J1 juveniles. For this

purpose, twice 15 living juveniles were transferred to a small droplet of ASW surrounded by a

paraffin ring on a glass microscope slide, covered with a cover glass and held above a flame for 2-3

seconds until the paraffin just melted. This method does not yield permanent slides, but is time

saving, kills nematodes quickly and efficiently and stretches their bodies, which facilitates

measurements. Due to time constraints 'initial size' was measured only once for each species,

instead of separately each time from those stock plates from which juveniles were collected for a

particular treatment. Small differences between cultures may therefore have lead to some bias in the

growth measurements. For some replicates (at the highest copper treatment) this resulted in

negative growth values, which were interpreted as 'no growth' and set to 0 (zero) instead.

For treatments, 15 juveniles per replicate were washed and transferred to experimental plates. Each

treatment was replicated four times. The exact time of incubation was noted, the Petri dishes sealed

with parafilm and stored at 20°C for two days (50±½ hours), which was the approximate time for

nematodes in a (pilot) control treatment to reach maturity but not yet start to reproduce. After the

incubation period, 10 nematodes were picked from each replicate and prepared for measuring as

described above. Again, it proved necessary to incubate a small excess (5) of juveniles to compensate

for background and accidental mortality.

Growth was quantified as the difference in body length between the 'initial size' and the average

body length after the incubation period of a certain treatment. Nematodes were measured using

Leica LAS 3.3 equipment and software.

Reproduction assay

In this assay, young adults at the beginning of their reproductive phase were used. Per replicate 10

females and 5 males were hand-picked, washed in ASW, and incubated on the experimental plates.

Each treatment was replicated four times. The exact time of incubation was noted, the experimental

plates sealed with parafilm and placed at 20°C for five days in total. The number of living juveniles

was counted after three days (74±½ hours) and after five days by placing the sealed experimental

plates on a transparent counting grid (squares of 5x5 mm) under a dissecting microscope. We chose

to count offspring on two separate days to find the moment where both species had produced

sufficient offspring to be used as a parameter for toxicity. Between day 3 and 5, the latter proved to

be the moment when even the youngest gravid females had had sufficient time to deposit their eggs

and/or give birth to their juveniles, and eggs that had been laid on day 3 or 4 had hatched by then

17

(data not shown). Checking reproduction of PmII and PmIV after 5 days of incubation of young adults

would therefore be more representative and reliable than day 3.

III. 8 - Data analysis

Statistical analyses were performed, and graphs created, with the software STATISTICA 7.0, Primer 6

& PERMANOVA+ and Microsoft Office Excel 2007.

For parametric analyses normal distribution of the data was confirmed with Kolmogorov-Smirnov

tests and homogeneity of variances was verified with Levene's tests. Where necessary, data was

transformed to conform to these terms. Significant factors were further investigated with post-hoc

Tukey-HSD tests.

The GTTF data was used first to test whether the nematode species show actual attraction to food

and secondly to test whether the behavioural patterns differ between species. This was done by

comparing the response of each species over time. Per plate, the number of nematodes in the three

spots of the same type was summed to obtain one number for food and one number for control

spots, ignoring within-replicate variability between spots of the same type. It is also important to

note that the numbers of nematodes in spots on a same plate (replicate) are not independent of one

another, because the number of nematodes that reaches one spot influences the number of

nematodes that can reach the other spots. To detect attraction, the amount of nematodes inside a

particular spot type was expressed as a proportion of the total amount of nematodes that had

moved to either spot type. Replicated G-tests were performed which test the goodness-of-fit to an

expected distribution, as was also done to analyse similar data in Moens et al. (1999) and Monteiro

et al. (2014). Under the null-hypothesis there is equal attraction to food versus control spots, or in

other words, no significant difference between the proportion of nematodes that have reached

either spot type. Any significant deviation from this 1:1 ratio will be interpreted as attraction to the

concerned type of spot. For all species, this analysis was performed only on the data of the time at

which the highest average number of nematodes had reached a spot.

By contrast, the average number of nematodes that have migrated to a particular type of spot can be

compared between species without violating the requirement for independence of data. Overall

migration of the different species was analysed by comparing the total number of nematodes that

had moved (i.e. reached a spot, regardless of its type). Similarly, food-finding of the different species

was analysed by comparing only the numbers of nematodes that had reached food spots. However,

the counts at the different time intervals were obtained from the same replicates, meaning that the

18

data are not independent with regard to the time factor. Therefore, in both cases, a one-way

repeated measures analysis of variances (ANOVA) was performed with species (PmI, PmII, PmIII,

PmIV) as between-group factor and time (1h, 2½h, 4h, 6h, 24h) as the repeated measures factor.

Data of the TTF assay was used to test for differences in taxis to food between and within the species

in response to the pollution treatments. Data for the highest copper concentration (Cu240) was not

available for PmIV. To account for this imbalance in the design, data was analysed with an

assumption-free permutation-based ANOVA (PERMANOVA). This test can be used as an alternative

for a repeated-measures design, but unlike a repeated measures ANOVA, PERMANOVA is not able to

identify when (in time) species differ from one another. The PERMANOVA design was two-factorial

with species (PmII, PmIV) and treatment (Control, Cu40, Cu80, Pb40, Pb80, Pb240) as between-group

factors, and time intervals (1h, 2½h, 4h, 6h) as dependent variables. Monte Carlo p-values (p (MC))

were calculated for pairwise comparisons between species and treatments due to the low number of

unique permutations possible. The variability of the data was also compared between species and

between treatments using the PERMDISP procedure, which is the multivariate equivalent of Levene’s

test for homogeneity of variance in ANOVA.

To investigate how the two species' growth is affected by the heavy metal treatments, growth data

was subjected to a regular two-way ANOVA with species and treatment as factors (as for TTF).

For reproduction, we decided to analyse and present data from day 5 only (and not day 3), because

that was the day where a sufficiently large, representative number of offspring was present in both

species. A two-way ANOVA with species and treatment as factors (as for TTF) was performed on

log(x+1)-transformed data to reveal how the species' reproductive output was affected by our

treatments.

19

IV. Results

IV. 1 - General taxis to food (GTTF)

Attraction to food

The time at which most nematodes had reached spots was different for each species. For PmI this

was after 1 hour (Fig. 3 A), for PmII after 4 hours (Fig. 3 B), for PmIII after 24 hours (Fig. 3 C) and for

PmIV after 2½ hours (Fig. 3 D). G-tests on the data of these time intervals confirmed highly significant

differences (all p-values < 0.001) between the numbers of nematodes in food spots compared to

control spots, clearly confirming their attraction towards food.

General migration

The four cryptic species do not present significant differences in the total number of migrating

individuals (df = 3, p > 0.1), but the amount of nematodes that had moved to spots changed over

time (df = 4, p < 0.001) with different migration patterns among the species (interaction of

time*species: df = 12, p < 0.001) (Figures 3 A-D: significant differences in general migration between

time intervals indicated with letters above the bars).

Figure 3. Average numbers of nematodes ±SE at food and control spots over time of A: PmI, B: PmII, C: PmIII and D: PmIV.

Letters above the bars indicate significant differences between the moments in time with regard to the total number of

nematodes in spots of any type.

20

Table 2. Summary of p-values associated with the indicated comparisons of motility between early and later time intervals,

given per species. Code: *p < 0.05; ** p < 0.01; *** p < 0.001.

Comparison p-values

Species Time interval 2½h 4h 6h 24h

PmI 1h n.s. n.s. n.s. n.s.

PmII 1h *** *** *** ***

PmIII 1h n.s. * ** ***

PmIV 2½h n.s. n.s. n.s. *

Table 2 summarizes the significance of the p-values of the most important observations (with regard

to migration patterns) for each species within the time*species interaction factor. While PmI showed

no significant change over time during the entire experiment (Fig. 3 A), PmII showed a significant

increase in taxis after the first hour compared to the consecutive hours (Fig. 3 B). In PmIII, migration

towards food also increased over time, especially up to the fourth hour after incubation, and

presenting a moderate, but not significant increase after that (Fig. 3 C). Most PmIV nematodes

reached the food spots after 2½h of incubation, and a significant decrease on that number was only

observed after 24 hours (Fig. 3 D).

Food-finding

Food-finding behaviour of the four species was analysed by comparing only the amount of

nematodes that had migrated to food spots. All factors, species (df = 3, p < 0.01), time (df = 4, p <

0.001) and the interaction factor time*species (df = 12, p < 0.001) are significant, indicating that, in

addition to change over time and differences in patterns between species, also the total amount of

nematodes that moved to food spots differs between species (Fig. 3 A-D). In general, less PmI

nematodes seem to have migrated towards food spots compared to PmII and PmIII, and to a lesser

extent PmIV.

IV. 2 - Taxis to food (TTF) after exposure

In this assay a scoring system was used to quantify the animals' taxis to food. High, positive scores

are equivalent to a large proportion of nematodes in or close to the food spots, whereas negative

values would indicate a stronger attraction to the control spots. Since the graphs in Figures 4 A-D

show that all scores are positive, it is safe to say that nematodes have not entirely lost their ability to

locate food in any of the treatments. It is interesting to note that for PmII (Fig. 4 A and B) the control

treatment indicates a very similar migration pattern as the one observed in the GTTF (Fig. 3 B). For

PmIV (Fig. 4 C and D) the attraction pattern of the control looks slightly different than in the GTTF

(Fig. 3 D) (probably due to the different design), but the main characteristic - little change over time -

is still present.

21

Figure 4. Average TTF-scores attained under the indicated treatments in function of incubation time, given per species and

type of metal. A: PmII after exposure to copper; B: PmII after exposure to lead; C: PmIV after exposure to copper; D: PmIV

after exposure to lead. Error bars are not shown to improve the clarity of the figures.

Statistical analysis revealed that there is a general difference between the scores attained by our test

species (species factor: df = 1, p < 0.001). Also, the treatments have a significant influence on the

scores (treatment factor: df = 6, p < 0.01) and both species are affected in a different way by these

treatments (interaction of species*treatment factor: df = 5, p < 0.001). Only the significant p-values

of the most important comparisons within the species*treatment interaction factor are summarized

in Table 3.

Table 3. Summary of significant p-values associated with the indicated comparisons of TTF-scores between species or

treatments. Code: Den. df = Denominator degrees of freedom; *p < 0.05; ** p < 0.01; *** p < 0.001.

Comparison Treatments Den. Df p (MC)

PmII-PmIV

Control 5 *

Cu40 6 *

Pb40 6 **

Pb80 5 ***

PmII

Control-Cu240 5 *

Cu40-Cu240 6 *

Pb40-Pb80 6 *

Pb40-Pb240 6 *

PmIV

Control-Pb80 5 *

Pb40-Pb80 5 **

Pb80-Pb240 5 **

22

The tests confirm that the scores attained by PmII and PmIV differ from each other in the control, the

lowest copper treatment, and the lowest and the second lead treatments (Table 3 and Fig. 4). This

also suggests that at the intermediate copper concentration Cu80 the differences between PmII and

PmIV have disappeared. However, PmII kept migrating until it had reached a similar score as in the

control by the end (Fig. 4 A), whereas PmIV did not catch up with the control (Fig. 4 C). Also note that

there is no data of PmIV for Cu240, preventing proper comparison of this treatment between

species.

PmII reached significantly lower scores in Cu240 than in the control and the lowest copper treatment

Cu40 (Fig. 4 A), meaning that Cu240 inhibited PmII's taxis to food. Yet, none of the lead treatments

caused significant differences from the control (Fig. 4 B). However, the lowest lead concentration

Pb40 stimulated PmII to attain significantly higher scores than in the two higher concentrations Pb80

and Pb240. Statistical analysis indicates that PmIV's taxis is not significantly affected by either of the

two copper concentrations we tested (Fig 4 C), but in Cu40 we observe a sudden decrease between

2½h and 4h, suggesting that nematodes migrated away from the food. The effect is however

amplified by a specific artefact that will be explained further in the discussion. Also in Cu80 the curve

indicates that nematodes were, on average, less attracted to the food than in the control situation.

PmIV, on the other hand, showed a strong response to the intermediate lead treatment Pb80 (Fig. 4

D). The scores attained in this treatment were significantly higher than in the control and both other

lead concentrations (Table 3), meaning that the nematodes were strongly attracted to the food. In

contrast to PmII, the lowest lead treatment Pb40 caused the lowest migration to food in PmIV,

instead of the highest. The differences between the scores of Pb40 versus the control and the two

higher lead treatments are, however, borderline non-significant.

IV. 3 - Growth

Growth analysis indicates that, in general, the juveniles of both our test species grew an equal

amount over two days (non-significance of the species factor: df = 1, p > 0.05). However, on first

sight, this seems to contradict what is observed in the graphs for the control treatments (Fig. 5).

There, PmIV (Fig. 5 right) seems to have grown some more than PmII (Fig. 5 left). Moreover, growth

is significantly affected by some of the treatments (treatment factor: df = 6, p < 0.001) and both

species react differently to the contaminant concentrations (interaction of treatment*species: df = 6,

p < 0.01). Table 4 summarizes the significant p-values of the most important comparisons.

23

Table 4. Summary of the significant p-values associated with the indicated comparisons of the test species' growth between

treatments. Code: *p(MC) < 0.05; ** p(MC) < 0.01; *** p(MC) < 0.001.

Comparison Treatments p (MC)

PmII

Control-Pb10 ***

Control-Pb40 ***

Control-Pb80 *

all Pb - all Cu **

PmIV

Control-Cu40 **

Control-Cu80 ***

Control-Pb10 **

Pb10-Pb40 ***

all Pb - all CuX ** X except Pb40:Cu10 (p = 0.96)

Figure 5. Average growth (µm) of PmII (left) and PmIV (right) in response to the indicated treatments.

Copper seems to reduce average growth with increasing concentrations, but the differences are not

statistically significant for PmII (Fig. 5 left). For PmIV (Fig. 5 right), the two higher copper

concentrations result in significantly reduced growth compared to the control. By contrast, all lead

treatments significantly incrase growth in PmII compared to its control and to each of the copper

treatments (Fig. 5 left). Also, the stimulating effect appears to become smaller with increasing

concentrations, but differences between the Pb treatments (for PmII) are not significant. For PmIV

(Fig. 5 right), only the lowest lead concentration causes a significant increase in growth compared to

the control and to the second highest lead concentration Pb40. However, all lead treatments differ

significantly from all copper treatments, except for Cu10:Pb40 (Table 4).

24

IV. 4 - Reproduction

Only living juveniles were counted and only data of day 5 is presented (Fig. 6). PmII and PmIV in

general produced different amounts of progeny (species factor: df = 1, p < 0.001). Figure 6 (left)

suggests that PmII had, on average, more offspring than PmIV (Fig. 6 right), especially in the controls

and at the lower pollution concentrations Cu10 and Pb10. Treatments also significantly affected the

amount of progeny a species produced (treatment factor: df = 6, p < 0.001). In general, but especially

for PmII, variation between replicates was large (Fig 6 left). This could explain why the interaction

factor is borderline non-significant (species*treatment factor: df = 6, p = 0.06), suggesting that both

species were affected roughly in the same way by the treatments. A post-hoc test revealed that only

the highest copper treatment caused both species to produce significantly less offspring than they

did in the control and in the lowest copper and lead treatments (all p-values < 0.01). The graphical

illustration (Fig. 6), on the other hand, suggests that also the intermediate copper concentration

reduced reproduction in both species compared to their controls. In addition, PmII (left) seems to be

more affected by the highest lead treatment than by the lower lead concentrations, whereas PmIV

(right) seems largely unaffected by lead.

Figure 6. Average number of offspring of PmII (left) and PmIV (right) after 5 days of exposure to the indicated treatments.

25

V. Discussion All four cryptic species of L. marina were attracted to the offered food, but exhibited different

migration patterns. Also, nematodes' taxis to food was affected by our test pollutants in a species-

specific way. Similar effects were observed for growth, and to a lesser extent for reproduction.

Consequently, not all endpoints have proven equally suitable to assess toxicity in L. marina cryptic

species.

V. 1 - Selection of test species

The results of the GTTF analyses clearly demonstrate that all four species are attracted to the offered

food source and that movement in Litoditis marina is not completely random, but to a large extent

based on selective choice (Fig. 3). These findings are in accordance with previous studies on PmI.

Monteiro et al. (2010) demonstrated taxis to a bacterial food suspension (Escherichia coli (OP50)) in

PmI in a very similar way as was done in our study. However, food choice is highly selective and

species-specific among bacterial feeding nematodes (Moens et al. (1999); Grewal and Wright (1992);

Weber and Traunspurger (2013)). Therefore it was crucial to investigate food-finding behaviour in all

four cryptic species before using them in our toxicity assays. For this study, the food attractant was a

suspension of dead E. coli (K-12) of 3x109 cells/mL, which was found to be close to optimal for

population development and body growth of PmI (dos Santos et al. (2008)). Nevertheless, our

experiments revealed different migration patterns towards food between the cryptic species, even

though the total amount of motile nematodes was roughly the same in all four species. PmI and

PmIV exhibited the quickest response (higher number of nematodes that have moved after one

hour) but also seem to enter control spots more readily compared to PmII and PmIII (Fig. 3 A - D).

Similarities in the responses of PmI and PmIV could be ascribed to them being the most closely

related groups among our four cryptic test species (Derycke et al. (2008)). A quick initial response

may be indicative of easy recovery from handling. On a same note, Dhawan et al. (2000) point out

the short adaptation period C. elegans needs (±30 min) compared to Daphnia magna (±2h) before

behavioural endpoints can reliably be assessed following incubation. A longer acclimatisation period

needed by PmII and PmIII compared to PmI and PmIV could explain the delay in migration towards

food of the first two species. Quick recovery also seems to be associated with a higher proportion of

nematodes ending up in control spots (Fig. 3 A and D). In addition to presenting a shorter adaptation

period, it is perfectly possible that PmI and PmIV are also less attracted to the food compared to PmII

and PmIII (Moens et al. (1999); Weber and Traunspurger (2013)). Decreases in numbers of

nematodes inside food spots in all species except PmIII can be explained by the animals trying to

26

move away from their feeding site in response to the decrease in attractant concentration after

feeding (Gaugler and Bilgrami (2004)). PmIII were often found alive but immobile in food spots,

probably leading to the observed steady increase of individuals reaching food (Fig. 3 C). The exact

reason for this is unclear, but worms in food spots were often found surrounded by dense mucus

sheaths clogged with food, likely hampering their normal movement (Monteiro et al. (2010)). Since

PmIII proved the most fragile of the four test species, this could explain its immobility in the food

spots.

The main goal of the GTTF assays was to identify two suited species for further toxicity testing.

According to the main selection criterion - a clear attraction towards food - one would be inclined to

choose PmII and PmIII (Fig. 3 B and D). However, besides food-finding behaviour, each species was

also evaluated according to its ease of handling. This was mainly based on personal experience, but

helped making a conscious choice. PmIII proved the most difficult in handling of all four species and

was therefore discarded. The choice of PmIV over PmI was based on both the clear, persisting

response to food of PmIV (at least over the first 6 hours, Fig. 3 D), its high survival throughout all

handling steps and the fact that it does not form dense clusters in the washing recipients, unlike the

other three species.

V. 2 - Effects of heavy metals on nematode behaviour

Our results demonstrate that after exposure to pollutants the nematodes' attraction to food was

modified in a species-specific and concentration-dependent way, but never disappeared completely.

The shape of the control curve for PmII (Fig. 4 A and B), suggests that nematodes that are able to

detect food properly are likely to reach the spot within 2½ hours. This general pattern is also visible

in the two lower copper treatments (Fig. 4 A), indicating that migration itself (not taxis to food) was

not so much affected. In addition, the sudden sign of 'recovery' observed midway the intermediate

copper concentration indicates that towards the end of the experiment nematodes were again fully

attracted to their food in that treatment. At the highest copper treatment, however, both migration

(early flattening of the curve) and taxis to food (lower scores) were clearly impaired in PmII (note

that low scores are not simply the result of nematodes remaining immobile after incubation, since

the vitality of each animal was checked during the washing step before the start of an experiment).

Similarly, the constant, steep rise of nematodes moving towards food in the two higher lead

treatments (Fig. 4 B), suggests that their overall migration is somewhat delayed compared to the

control. However, nematodes remain attracted to the food as indicated by the high scores attained

towards the end of the experiment. This implies that the effect of lead may have worn off by that

time or is just not detectable anymore. Judging by the first significantly negative effect on taxis (at

27

Cu240), the order of toxicity for PmII would be Cu > Pb, for this endpoint after 24 hours of exposure.

For PmIV, this is less clear, since the highest Cu concentration (Cu240) could not be tested (Fig. 4 C).

Comparison of the control treatments of both species confirms that PmIV is generally less attracted

to the food than PmII (p < 0.05) and the effects of the two other copper treatments (Cu80 and Cu40)

appear to decrease taxis in PmIV even more. The sudden decrease observed in Cu40 (Fig. 4 C)

between 2½h and 4h, is in part due to nematodes moving away from the food, but the effect is

amplified due to an artefact coming from mainly one of the replicates; raw data counts confirm a

lower total count (20-18-12-15 for 1h-2½h-4h-6h respectively of the concerned replicate), due to

nematodes having 'disappeared' from inside and close to food spots, compared to the preceding

time interval (data not presented). Notes on personal observations also mention that during the

Cu40 experiment nematodes tended to burrow into cracks in the medium around food spots where

they became practically invisible. This did however not happen on a regular basis, and certainly not

to such an extent in the entire TTF experiment, meaning that data is generally accurate. On the other

hand, this burying behaviour could be a result from the treatment itself. After all, by hiding in the

medium nematodes do move away from the centre of the food spot. The exact effect of Cu40 on

taxis of PmIV is however not entirely clear and should be interpreted with caution. On a different

note, taxis in response to the lowest lead concentration was remarkably, but borderline non-

significantly weaker than in the other treatments (Fig. 4 D). By contrast, the second lead

concentration Pb80 (unlike Cu80) stimulated nearly all PmIV nematodes to migrate towards the food

within the first hour of incubation. The relative constancy (little change over time after the first hour)

of all curves of PmIV suggests that its migration speed is not negatively affected: nematodes moved

soon after incubation, but not necessarily to food spots. However, without statistically significant

decreases in taxis for both metal types in PmIV, the order of toxicity cannot be determined with

certainty. Yet, the (nearly significant) lowest scores being attained in the lowest lead treatment, and

the significant (albeit positive) effect at the next-higher lead concentration would support a toxicity

ranking of Pb > Cu. Few other studies have analysed the effects of heavy metal exposure on

nematodes' chemotaxis abilities (Monteiro et al. (2010); Song et al. (2014)), and no known studies

have simultaneously tested the effects of Cu and Pb on taxis towards food. Monteiro et al. (2010)

found PmI's and C. elegans' migration towards food to be more negatively affected by zinc than by

lead and nickel when being incubated on polluted agar. In contrast to our study, however, worms

had not been pre-exposed to the test concentrations before the actual taxis-to-food experiment.

Song et al. (2014) found that chronic exposure of C. elegans to 2500 µM copper sulphate for 20

generations was able to partially change chemotaxis behaviour towards different substances.

However, for more commonly studied behavioural endpoints, such as movement and feeding ability

(Dhawan et al. (1999, 2000); Boyd et al. (2003); Anderson et al. (2001, 2004); Wang and Xing (2008);

28

Yu et al. (2012)), the most frequently found order of toxicity after 24h exposure for C. elegans is Cu >

Pb. An exception is the study ofDhawan et al. (2000), who found both movement and lethality in the

same species to be more sensitive to Pb than to Cu. Interestingly, the study of Anderson et al. (2001)

demonstrated that shorter exposure times (4h) reveal a higher sensitivity of movement to the

neurotoxicity of Pb than to Cu. As suggested by the same researchers, this could indicate that

different toxicity mechanisms act at short term versus long term exposure. That could explain why

neuro(sensory)-toxicity of Pb is not significantly more apparent in our behavioural assay than the

negative influence of Cu, except maybe in PmIV at the lowest lead concentration (Fig. 4 D).

Nevertheless, our findings support that, for this endpoint, the effects of the test substances are

concentration- and species-specific.

V. 3 - Effects of heavy metals on nematode growth and reproduction

We observed a persistently negative effect of copper on growth and reproduction, but the first

significantly 'noticeable' effects were obtained from low lead concentrations on growth (Table 4).

The first striking observation is that copper and lead had an opposite effect on the growth of our test

species (Fig. 5). Increasing copper concentrations seemed to reduce growth ever more strongly, so

that growth was almost completely inhibited at the highest concentration in both species. By

contrast, our lead treatments clearly stimulated growth in PmIV and especially in PmII. The positive

effect is largest at our lowest lead concentration (Pb10 ≈ 2 mg/L) and decreases with increasing

concentration. In terms of negative effects, however, our findings would support a toxicity ranking of

Cu > Pb for growth in L. marina. Comparable results for 24h exposure to copper were found by

Calafato et al. (2008) (exposure range 100-500µM ≥ Cu240) and Anderson et al. (2001) (exposure

range 0-8 mg/L (≈0-126 µM, which would include Cu10-Cu80: Table 1)) on the reduction of growth in

C. elegans, but the latter study's findings on lead do not match ours at all. They found lead (exposure

range 0-15 mg/L (≈ 0-72 µM or ≈ Pb10-Pb80)) to consistently reduce growth of C. elegans. One other

study on C. elegans by Monteiro et al. (2010) tested lead concentrations from 0.01 to 1 mg/L (≈0.05-

5 µM < Pb10) in a liquid assay and found a significant increase of growth at 0.05 mg/L (≈0.24 µM).

The lower test concentration range could explain why this stimulating effect was missed by Anderson

et al. (2001). Also Roh et al. (2006) found sublethal lead concentrations to slightly increase growth in

C. elegans. This particular response of growth to low concentrations of lead is definitely intriguing

and it seems that our test concentrations resulted in hormesis (Stebbing (1982)). Hormesis is the

phenomenon observed when low concentrations of toxicants elicit the opposite response as they

would in high concentrations. It is thought to be an over-compensatory adaptive reaction to low

levels of stress, resulting in a positive effect (Mattson (2008)). This has been documented for several

29

types of stressors like irradiation, heat, heavy metals, antibiotics, ethanol, pro-oxidants, exercise and

food restriction (Lopez-Diazguerrero et al. (2013)) in a variety of taxa (plants, invertebrates and

vertebrates) for diverse endpoints (Calabrese and Baldwin (2003)). With regard to aquatic organisms,

hormesis has, for example, been documented in response to low levels of lead for growth and

reproduction in aquatic snails (Lefcort et al. (2008)), and to copper for reproduction in polychaetes

(Reish and Carr (1978)). Interestingly, our reproduction assay showed no sign of hormesis towards

lead exposure, in spite of identical concentrations tested as in the growth assay (Table 1, Fig. 5 and

6). In fact, lead did not appear to affect reproduction of PmIV at all (Fig. 6 right), whereas the

graphical illustration (Fig. 6 left) suggests that higher Pb concentrations increasingly inhibited

reproduction in PmII. Such a concentration-dependent response is also observed in C. elegans by

Anderson et al. (2001) (exposure range Pb: 0-15 mg/L (≈Pb10-Pb80)). This would mean that the

reproductive capacity of PmIV is more resistant than that of PmII to lead. Also, in spite of the large

variations in the control treatment and at low contaminant levels, it seems that reproduction in both

species is affected more severely by the intermediate copper level Cu40 than by the lowest Cu10

(Fig. 6). This would again be in accordance with the concentration-dependent effect of copper on

reproduction of C. elegans (Anderson et al. (2001); Calafato et al. 2008). In Boyd and Williams (2003)

only EC50 values (being the concentration required to reduce a certain parameter by 50% relative to

controls) are given for reproduction of C. elegans and Pristionchus pacificus and is about 2 mgCu/L

for both species and 24h of exposure. This would approximate our Cu40 (Table 1), but the latter

species presents larger variation than C. elegans, comparable to our situation. Yet, in our study the

only statistically significant decrease compared to the control and Cu10 and Pb10 was indicated for

both species at the highest copper treatment, supporting a ranking of Cu > Pb for reproduction in

both species. Similarly, Reish and Carr 1978 reported the order of toxicity for polychaete

reproduction to be Cu > Pb, and findings on C. elegans follow the same trend (Anderson et al.

(2011)). Other reproductive defects have also been reported to occur in C. elegans upon increasing

copper concentrations like egg-retaining (Hunt et al. (2012)) or internal hatching Song et al. (2014).

V. 4 - Differences between species and ecological implications

Apart from the specific differences between species pointed out in the previous sections, we also

found some more general similarities and dissimilarities between endpoints and species. For

example, the hormetic growth response observed at low lead concentrations in PmIV and all

concentrations in PmII is not reflected in reproduction and not in the TTF assay either. In PmII no

significant increase in taxis is observed at any lead treatment (Fig. 4 B), whereas the peak attraction

of PmIV at Pb80 (Fig. 4 D) does not overlap with the peak stimulation of growth, which appears to lie

30

at Pb10 or even lower (Fig. 5 right). Clearly, these inconsistencies in PmIV's response to lead in both

the TTF and growth assays ask for further investigation of additional concentrations on both

endpoints to clarify these observations. By contrast, within a same endpoint, the responses of PmII

towards different lead concentrations appear more consistent. Moreover, no positive effects were

observed for copper in either species, at any concentration, in any of the endpoints. In fact, a severe

effect at the highest copper concentration Cu240 was observed for PmIV; nematodes were in such a

poor condition after 24h of exposure that most of them could not be used for the actual TTF-test,

which therefore had to be interrupted. This means that, at least for 24h of exposure, this

concentration is generally too high to use in (behavioural) assays for L. marina. In terms of overall

'health', copper therefore proved more toxic than lead to PmIV after 24h exposure to 240µM. The

same order of toxicity was also found in lethality tests with C. elegans, for example in Williams and

Dusenbery (1990) and Chu and Chow (2002). This higher sensitivity to copper than to lead in PmIV is

also visible in the growth assay, where growth was already significantly reduced at Cu40 in PmIV but

not in PmII (Fig 5). Also Fig. 6 suggests that copper affected reproduction of PmIV more severely than

that of PmII.

It is well possible that the differences in response and sensitivity of these cryptic species towards

food and toxicants are part of the mechanisms that allow their coexistence in the field (Leibold and

McPeek (2006)). Our GTTF assay clearly revealed that all four cryptic species differ in their migration

pattern and strength of attraction towards our offered food source (Fig. 2 A - D). Species-specific

food preferences have already been suggested to be one of the mechanisms that allow bacterivorous

nematode species to coexist without competitive exclusion (Weber and Traunspurger (2013)).

Furthermore, Martinez et al. (2012) found sublethal concentrations of toxicants (in this case

cadmium) to be able to alter interspecific interactions, such as competition and facilitation, between

two bacterivorous soil nematodes. Interspecific interactions between cryptic species of L. marina

have also been documented by De Meester et al. (2011) and were found to be modified by different

levels of salinity. These findings raise curiosity on which effects metals could have on the interactions

between our test species. Since very few studies exist on the effects of heavy metals on nematode

interactions (Martinez et al. (2012)), the outcome would be hard to predict. Detailed studies would

be needed to further investigate the effect of different tolerances towards toxicants on L. marina

multispecies complexes, and to find indications for ecological or functional redundancy (Naeem and

Li (1997)). In a next step it would also be interesting to investigate the mixed effects of copper and

lead, as they have already been found to have a synergistic effect on lethality of, for example C.

elegans (Chu and Chow (2002)) and Amphiascus tenuiremis, an estuarine meiobenthic copepod

(Hagopian-Schlekat et al. (2001)).

31

V. 5 - Suitability of our test species, endpoints and assays in toxicity testing

One of the crucial steps in (eco)toxicity testing is the choice of a suitable test species. To obtain

accurate, relevant results it is important to use reliable, representative test organisms. In general, we

find Litoditis marina to be a suitable marine test nematode; it can be cultured under laboratory

conditions with relatively little effort and proved robust enough when handled with some care. Our

results demonstrated that the cryptic species present specific similarities and differences between

each other, with regard to behavioural, developmental and life-strategy traits. This allows one to

select the best suited (combination of) cryptic species, fitting to the particular aim of a study.

However, their different reproductive strategies (Derycke et al. (2008)) made culture synchronisation

problematic, unavoidably causing some age variation between individuals used in experiments. This

issue, along with the presence of two sexes was also pointed out by Boyd and Williams (2003) to be a

disadvantage when using such species for toxicity testing.

Taxis to food proved to be a suitable endpoint in our study. It was able to indicate varying responses

(increases and decreases in taxis) towards different metals and concentrations, and provided us with

valuable information on how our test species differ in their attraction and migration towards food,

and in their sensitivity towards pollutants. This makes it a great endpoint if the aim is, for example, to

better understand the general functioning of a species or its response to a certain pollutant. Yet, the

experiments need quite some preparation time and have to be followed up at multiple time intervals

for at least six hours after incubation, making it difficult to run multiple experiments simultaneously.

Another shortcoming was that nematodes that shelter in the medium under the food spots or in

small cracks around it can be very hard or even impossible to see. Such cracks however only showed

up occasionally around the wells of only few experimental plates and probably originated from when

the bottom of the wells was covered with a thin layer of agar during preparation work (see Materials

and Methods); it could be the result of the agar not being hot enough to perfectly merge with the

walls of the wells up to the top - an issue that could easily be corrected in the future. However, in

every study involving chemotaxis, mate attraction between opposite sexes could partly mask true

attraction effects towards the test substance (White et al. (2007); Choe et al. (2012)). Therefore, for

really exact and routine toxicity studies, highly sensitive and high through-put computer technology

that is able to identify even the slightest changes in nematode head movement, as applied in e.g.

Wang and Xing (2008), may be better suited.

Besides taxis, growth also showed clear responses to our treatments. Variation remained

considerably large though, which is partly due to some remaining problems with the experimental

procedure. Since age-synchronisation proved difficult and separating size-classes by means of sieves

is too rough a procedure for these fragile nematodes (Monteiro, personal communication), juveniles

were hand-picked directly from stock cultures. Yet, due to the extremely small size of the juveniles,

32

this unavoidably introduced some bias into the experiment. An alternative could be to use computer

based imaging to measure the growth of the same group of juveniles at the start and at the end of an

experiment, as did Anderson et al. (2001).

Reproduction proved to be the least accurate endpoint in our test species. High variability in the

control treatments, especially of PmII, prevented us from detecting potentially subtle effects at the

different toxicant concentrations. This could be due to accidental culture-dependent bias, the effects

of which can become large when the number of test organisms per replicate is small. In addition, as

also pointed out by Boyd et al. (2010) counting offspring manually can be tedious and imprecise,

especially if the number of moving juveniles is large. We conclude therefore that, for this species, our

way of assessing reproduction was not reliable enough for toxicity testing.

VI. Conclusion The aim of this study was to investigate the effect of copper and lead on taxis to food, growth and

reproduction of cryptic species of the marine nematode Litoditis marina. We found that:

Migration time and strength of attraction towards our bacterial food source differed

between cryptic species;

Taxis in PmII was more severely affected by Cu than by Pb, whereas taxis in PmIV can be both

stimulated or inhibited by different Pb concentrations;

At the concentrations tested, Pb and Cu had opposite effects on growth in both species;

PmIV was more sensitive than PmII to the negative effects of Cu on growth, whereas PmII

was more sensitive to the growth-stimulating effect of Pb;

Reproduction was strongly decreased at high Cu levels, and Pb did not affect PmIV, but it

seemed to affect PmII in a concentration-dependent way

PmIV in general proved more sensitive than PmII to Cu;

Our behavioural endpoint was affected in a species-, toxicant- and concentration-dependent

way, which neither growth or reproduction assays could have predicted.

33

VII. Summary

VII. 1 - Summary

The topic of this master thesis is situated in the research field of marine ecotoxicology. In this study,

the toxic effects of two heavy metals, copper and lead, will be tested on the behaviour, growth and

reproduction of a marine roundworm species. The research was performed in the research group of

Marine Biology at the University of Ghent under supervision of Prof. Dr. Tom Moens and tutorship of

Luana Monteiro and Nele De Meester.

Our oceans and coastal areas have been severely polluted over the past few decades. Human

activities, such as fishery, sea transport and various kinds of recreational have been major factors in

this process. In combination with discharges of contaminated waste water from industries and

urbanized areas they cause an enormous variety of chemical substances to end up in our surface

waters and ultimately in our oceans. This has numerous negative consequences for all organisms that

depend on these ecosystems.

Heavy metals are a particular kind of pollutants that are known for being very persistent in the

environment. This means that they remain available for uptake by plants and animals for a long time

before they are degraded or stored permanently in sediments. Copper and lead are two common

heavy metals with known hazardous effects on many species. Lead is known to induce neurotoxicity,

resulting in various defects that involve the nervous system. Copper, on the other hand, is a more

general toxicant, known to interfere with enzymes and proteins at the cellular level. Aquatic

organisms often appear to be particularly sensitive to this kind of pollution. As chemical analyses

alone cannot accurately predict the effects of toxic substances on living organisms, populations or

communities, toxicological research depends on reliable biological assays.

Roundworms, or nematodes, are generally very small organisms that occur in high abundances in all

ecosystems across the world and can be either parasitic or free-living. Litoditis marina is a free-living

marine nematode species that lives on decaying algae in coastal areas. It is closely related to the

well-known model organism Caenorhabditis elegans, and is one of the very few marine nematode

species that can be cultivated under laboratory conditions. These characteristics make L. marina a

good candidate test organism for marine oriented experimental research.

In this study we used four very closely related L. marina species that naturally occur in multispecies

complexes. These so-called cryptic species were subjected to four different assays. In the first

experiment nematodes' food finding behaviour was investigated, and based on those results two

34

suitable species were selected for further toxicity testing. In the subsequent toxicity assays the

effects of three concentrations of copper and lead were tested on three different endpoints: food-

finding behaviour, growth and reproduction.

These experiments served to investigate whether the toxicity of copper and lead is reflected in a

similar way in the species' responses to the three chosen endpoints. In addition, the tests could

reveal important differences in sensitivity between the test species, and thereby shed more light

onto the mechanisms that allow them to coexist. Moreover, this study would help us to further

evaluate the suitability of our behavioural assay for L. marina, compared to the more traditional

growth and reproduction assays used in toxicity research.

Food-finding behaviour was tested by placing nematodes onto experimental dishes filled with agar

medium, where they had the choice between migrating towards wells filled with a bacterial food

suspension, or with sterile sea water. The experiment was run for twenty-four hours and the amount

of nematodes in each well was noted at several moments in time. The test revealed that all four

cryptic species were attracted to the offered food source, but that they differed in their specific

temporal migration patterns. Based on those results, two species were selected for the following

experiments.

The first toxicity assay served to investigate whether the nematodes' ability to detect food was

affected after having been exposed to certain concentrations of copper and lead for twenty-four

hours. We found that the effects of the heavy metals were species-specific and concentration-

dependent; one species was more severely affected by copper, whereas the other species showed a

clearer response to lead. Our results also demonstrate that attraction to food can be increased and

decreased by the same type of metal.

For the growth assay we measured the increase in body length of juveniles after they had been

exposed to the toxicant concentrations for two days. The test revealed that copper generally reduces

growth ever more severely at increasing concentrations, whereas low concentrations of lead

stimulated growth in both species. In addition, the species presented differences in their sensitivity

and strength of response towards the inhibiting or stimulating effects.

In the third assay we tested to what extent the nematodes' reproductive capacity was affected by

the toxicants. We counted the amount of living offspring that small groups of males and females had

produced after five days of exposure. We found that both species' reproduction is sensitive to

copper, while lead seemed to affect one species more than the other. However, due to high

variability between and within treatments, these results should be interpreted with caution.

Copper generally had a negative effect on each of the endpoints, whereas certain concentrations of

lead were found to cause positive responses in both food-finding behaviour and growth. Our

35

research demonstrated that the effect of a toxicant can be very species-specific. Even though each

of the endpoints was able to indicate toxicity effects, we were unable to identify the toxicity

mechanism by which the test pollutants acted. On the other hand, we revealed valuable information

on how these cryptic species differ from one another with regard to food-finding behaviour and

sensitivity towards copper and lead. In fact, the behavioural test proved particularly interesting in

our research because it revealed differences between species that could not have been predicted

from the results of the growth and reproduction assays.

VII. 2 - Samenvatting

Het onderwerp van deze masterscriptie kadert binnen de mariene ecotoxicologie. In deze studie

werden de toxische effecten van twee zware metalen, koper en lood, onderzocht op het gedrag, de

groei en het voorplantingsvermogen van een mariene rondwormsoort. Het volledige onderzoek werd

uitgevoerd binnen de vakgroep Mariene Biologie van de Universiteit Gent, onder promotorschap van

Prof. Dr. Tom Moens en begeleiding van Luana Monteiro en Nele De Meester.

Onze zeeën en kustgebieden hebben vaak onder zware vervuiling te lijden. Dit komt grotendeels

door de menselijke activiteiten die er plaats vinden, zoals visserij, scheepvaart, of allerlei recreatieve

bezigheden. Afvalwaterlozingen uit industriegebieden en steden dragen bij tot de enorme variatie

aan chemische stoffen die in oppervlaktewateren en uiteindelijk in de oceanen terecht komen met

gevolgen voor alle levensvormen die afhankelijk zijn van de getroffen ecosystemen.

Zware metalen vormen een bijzondere groep polluenten en staan gekend voor hun bijzonder lange

levensduur in het milieu. Dat betekent dat ze in de natuur lang beschikbaar blijven voor opname

door dieren en planten, voor ze afgebroken of permanent gebonden geraken in sediment. Koper en

lood zijn twee veelvoorkomende zware metalen met gekende negatieve gevolgen op een groot

aantal soorten. Van lood is geweten dat het een zenuwgif is, en kan leiden tot zeer uiteenlopende

gevolgen waar het zenuwstelsel bij betrokken is. Koper daarentegen zou een algemenere toxische

werking hebben, die niet bepaald zenuwen aantast, maar eerder de werking van essentiële enzymen

en proteïnen binnen een cel of organisme ontregelt. Aquatische organismen lijken vaak bijzonder

gevoelig aan dergelijke vervuiling. Chemische wateranalyses zijn onvoldoende om de effecten van

vervuiling op individuen, populaties of gemeenschappen te voorspellen. Daarom zijn betrouwbare

bio-tests, die gebruik maken van levende wezens, onmisbaar in toxicologisch onderzoek.

Rondwormen, of nematoden, zijn gewoonlijk zeer kleine organismen die overal ter wereld in enorme

aantallen voorkomen, en zowel parasitair als vrij-levend kunnen zijn. Litoditis marina, een mariene

36

nematode uit dezelfde familie als het bekende modelorganisme Caenorhabditis elegans, komt vrij-

levend op vervallend algenmateriaal in kustwateren voor. Het is een van de weinige mariene soorten

die onder laboratoriumomstandigheden kan gekweekt worden en biedt zich dus aan als geschikte

vertegenwoordiger voor zoutwatergerelateerd onderzoek.

In dit onderzoek werd gebruik gemaakt van vier zeer nauw aan elkaar verwante L. marina soorten,

die onder natuurlijke omstandigheden in verschillende soortencombinaties voorkomen. Deze

zogenaamde cryptische soorten werden aan vier reeksen experimenten onderworpen. Een eerste

diende om het voedselzoekgedrag van deze rondwormen te analyseren en op basis daarvan twee

geschikte soorten te selecteren voor de drie daaropvolgende toxiciteitsexperimenten. Daarin zouden

vervolgens de invloed van telkens drie verschillende concentraties koper of lood getest worden op

drie eigenschappen: het voedselzoekgedrag, de groei en het aantal nakomelingen dat deze wormen

voortbrengen.

Met deze experimenten wouden we nagaan of de toxiciteit van koper en lood op een gelijkaardige

manier zou weerspiegeld worden in de veranderingen van alle drie de eigenschappen die we

onderzochten. Daarnaast gingen we op zoek naar verschillen in de respons van onze cryptische

soorten, waarvan we aannamen dat ze door hun nauw verwantschap heel gelijkaardig zouden

reageren. Tot slot moest het onderzoek uitwijzen in hoeverre gedragsstudies met deze soort een

waardevolle aanvulling zijn op de andere, algemener gebruikte benaderingen zoals groei en

voortplanting.

Voedselzoekgedrag werd als volgt onderzocht. Wormen werden op agar-gevulde Petriplaatjes gezet

waar ze de keuze hadden om naar kuiltjes gevuld met voedsel of met puur zeewater te bewegen. De

wormen werden gedurende een volledige dag opgevolgd waarbij hun aantallen in de verschillende

types kuiltjes op meerdere tijdstippen genoteerd werd. Daaruit bleek dat alle vier soorten duidelijk

aangetrokken worden door het aangeboden voedsel, maar dat ze verschillen in aantallen en de tijd

die ze erover doen om tot het voedsel te geraken. Twee van deze vier cryptische soorten werden

geselecteerd voor de volgende experimenten.

In het eerste vergiftigingsexperiment werd nagegaan of de twee soorten de weg naar hun voedsel

nog konden vinden nadat ze een volledige dag blootgesteld waren geweest aan bepaalde

concentraties koper en lood. Daaruit bleek dat de effecten van deze zware metalen soort-specifiek

en concentratie-afhankelijk zijn; de ene soort werd het zwaarst beïnvloed door koper, de andere

door bepaalde concentraties lood. We zagen ook dat de aantrekking tot voedsel zowel versterkt als

verlaagd kon worden door éénzelfde metaal.

37

In het groei-experiment werd de toename in lichaamslengte van juvenielen na twee dagen

blootstelling aan vervuiling opgemeten. Hieruit bleek dat koper algemeen de groei afremt naarmate

de concentratie hoger wordt, maar dat lood in lage concentraties groeibevorderend werkt. We zagen

ook hier soort-specifieke verschillen in gevoeligheid en mate waarin de groei afgeremd of verhoogd

werd.

Het derde experiment diende om na te gaan in hoeverre de dieren hun voortplanting aangetast

werd. Hiertoe werd na vijf dagen geteld hoeveel nakomelingen onze kleine groepjes van vrouwtjes

en mannetjes voortgebracht hadden. Daaruit bleek dat terug koper het snelst een negatieve invloed

uitoefende op beide soorten. De invloed van lood was minder duidelijk, maar ook algemeen laten de

resultaten van dit onderzoek ruimte voor interpretatie door een hoge variabiliteit tussen en binnen

de verschillende behandelingsgroepen.

Koper had dus algemeen enkel negatieve uitwerkingen op alle drie de eigenschapen, waarbij

bepaalde concentraties lood ook positieve effecten hadden op zowel het voedselzoekgedrag als de

groei van de test organismen. Dit onderzoek heeft een aantal punten duidelijk naar voor gebracht.

Het effect van een gifstof kan zeer soort-specifiek zijn. Hoewel dus alle drie eigenschappen beïnvloed

werden door de zware metalen, lieten onze analyses niet toe om de precieze toxische

werkingsmechanismen van deze metalen op een onbetwistbare manier af te leiden. Langs de andere

kant hebben we op die manier een beter overzicht gekregen over hoe deze cryptische soorten

precies verschillen van elkaar. Daarnaast heeft het gedragsexperiment zich als bijzonder interessant

uitgewezen om complementaire informatie over soorten te verzamelen die niet uit de traditionelere

onderzoeken van groei of voortplanting zou kunnen gehaald worden.

38

VIII. Acknowledgements

I sincerely want to thank Prof. Dr. Tom Moens for his trust, guidance and encouragement throughout

the year and for giving me the opportunity to perform this research. My wholehearted thanks also go

to Luana Monteiro, for assisting me from the beginning to the end and whose readiness to help I

could always count on. Likewise, I thank Nele De Meester for her support and valuable input in this

study. Of the staff of the Marine Biology research group I particularly want to thank Dirk Van

Gansbeke, Guy de Smet and Annick Van Kenhove. Very special thanks also go to Jeroen De

Caesemaeker for restoring hope (and computers) at the most critical moments. Also, many thanks to

everyone at C32 for great company and help (Bartelijn, Thomas, ... ), and to my dearest friends, for

their amazing support during this year.

39

IX. References 11 ILM 1294 (1972) - Convention on the Dumping of Wastes at Sea, London, 13 November 1972 36 ILM 1 (1997) - 1996 Protocol to the London Convention 1972, London, 7 November 1996 Abrantes, N., R. Pereira, D. R. de Figueiredo, C. R. Marques, M. J. Pereira and F. Goncalves (2009). A

Whole Sample Toxicity Assessment to Evaluate the Sub-Lethal Toxicity of Water and Sediment Elutriates from a Lake Exposed to Diffuse Pollution. Environmental Toxicology 24(3): 259-270.

Akpor, O. B. and M. Muchie (2010). Remediation of heavy metals in drinking water and wastewater treatment systems: Processes and applications. International Journal of the Physical Sciences 5(12): 1807-1817.

Anderson, G. L., W. A. Boyd and P. L. Williams (2001). Assessment of sublethal endpoints for toxicity testing with the nematode Caenorhabditis elegans. Environmental Toxicology and Chemistry 20(4): 833-838.

Anderson, G. L., R. D. Cole and P. L. Williams (2004). Assessing behavioral toxicity with Caenorhabditis elegans. Environmental Toxicology and Chemistry 23(5): 1235-1240.

Andrássy, I. (1983). A taxonomic review of the suborder Rhabditina (Nematoda: Secernentia). Office de la Recherche Scientifique et Technique Outre-Mer (ORSTOM), Paris.

Austen, M. C. and A. J. McEvoy (1997). Experimental effects of tributyltin (TBT) contaminated sediment on a range of meiobenthic communities. Environmental Pollution 96(3): 435-444.

Bastian, H. C. (1865). Monograph of the Anguillulidae, or Free Nematoids, Marine, Land, and Freshwater; with Descriptions of 100 New Species. The Transactions of the Linnean Society of London XXV(II): 73-184.

Beyrem, H., E. Mahmoudi, N. Essid, A. Hedfi, F. Boufahja and P. Aissa (2007). Individual and combined effects of cadmium and diesel on a nematode community in a laboratory microcosm experiment. Ecotoxicology and Environmental Safety 68(3): 412-418.

Bi, Z. S., P. Salaun and C. M. G. van den Berg (2013). The speciation of lead in seawater by pseudopolarography using a vibrating silver amalgam microwire electrode. Marine Chemistry 151: 1-12.

Bongers, T. and M. Bongers (1998). Functional diversity of nematodes. Applied Soil Ecology 10(3): 239-251.

Bongers, T. and H. Ferris (1999). Nematode community structure as a bioindicator in environmental monitoring. Trends in Ecology & Evolution 14(6): 224-228.

Boone, C., G. Jervais, B. Luukinen, K. Buhl and D. Stone (2012). Copper Sulfate Technical Fact Sheet, Oregon State University Extension Services.

Borgmann, U., E. S. Millard and C. C. Charlton (1989). Effect of Cadmium on a Stable, Large Volume, Laboratory Ecosystem Containing Daphnia and Phytoplankton. Canadian Journal of Fisheries and Aquatic Sciences 46(3): 399-405.

Boyd, W. A., R. D. Cole, G. L. Anderson and P. L. Williams (2003). The effects of metals and food availability on the behavior of Caenorhabditis elegans. Environ Toxicol Chem 22(12): 3049-55.

Boyd, W. A., S. J. McBride, J. R. Rice, D. W. Snyder and J. H. Freedman (2010). A high-throughput method for assessing chemical toxicity using a Caenorhabditis elegans reproduction assay. Toxicology and Applied Pharmacology 245(2): 153-+.

Boyd, W. A. and P. L. Williams (2003). Comparison of the sensitivity of three nematode species to copper and their utility in aquatic and soil toxicity tests. Environmental Toxicology and Chemistry 22(11): 2768-2774.

Brinke, M., P. Heininger and W. Traunspurger (2011). A semi-fluid gellan gum medium improves nematode toxicity testing. Ecotoxicology and Environmental Safety 74(7): 1824-1831.

40

Brooks, S. J. and C. L. Mills (2003). The effect of copper on osmoregulation in the freshwater amphipod Gammarus pulex. Comp Biochem Physiol A Mol Integr Physiol 135(4): 527-37.

Bryan, G. W. and W. J. Langston (1992). Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries: a review. Environmental Pollution 76(2): 89-131.

Calabrese, E. J. and L. A. Baldwin (2003). Inorganics and hormesis. Critical Reviews in Toxicology 33(6): 215-304.

Calafato, S., S. Swain, S. Hughes, P. Kille and S. R. Sturzenbaum (2008). Knock down of Caenorhabditis elegans cutc-1 Exacerbates the Sensitivity Toward High Levels of Copper. Toxicological Sciences 106(2): 384-391.

Chapman, P. M., A. Fairbrother and D. Brown (1998). A critical evaluation of safety (uncertainty) factors for ecological risk assessment. Environmental Toxicology and Chemistry 17(1): 99-108.

Choe, A., T. Chuman, S. H. von Reuss, A. T. Dossey, J. J. Yim, R. Ajredini, A. A. Kolawa, F. Kaplan, H. T. Alborn, P. E. A. Teal, F. C. Schroeder, P. W. Sternberg and A. S. Edison (2012). Sex-specific mating pheromones in the nematode Panagrellus redivivus. Proceedings of the National Academy of Sciences of the United States of America 109(51): 20949-20954.

Chu, K. W. and K. L. Chow (2002). Synergistic toxicity of multiple heavy metals is revealed by a biological assay using a nematode and its transgenic derivative. Aquatic Toxicology 61(1-2): 53-64.

Claessens, M., S. De Meester, L. Van Landuyt, K. De Clerck and C. R. Janssen (2011). Occurrence and distribution of microplastics in marine sediments along the Belgian coast. Marine Pollution Bulletin 62(10): 2199-2204.

Coble, P. G. (1996). Characterization of marine and terrestrial DOM in seawater using excitation-emission matrix spectroscopy. Marine Chemistry 51(4): 325-346.

Commito, J. A. and G. Tita (2002). Differential dispersal rates in an intertidal meiofauna assemblage. Journal of Experimental Marine Biology and Ecology 268(2): 237-256.

De Laender, F., K. A. C. De Schamphelaere, P. A. Vanrolleghem and C. R. Janssen (2009). Comparing ecotoxicological effect concentrations of chemicals established in multi-species vs. single-species toxicity test systems. Ecotoxicology and Environmental Safety 72(2): 310-315.

De Laender, F., P. J. Van den Brink and C. R. Janssen (2011). Functional redundancy and food web functioning in linuron-exposed ecosystems. Environmental Pollution 159(10): 3009-3017.

De Meester, N., S. Derycke, D. Bonte and T. Moens (2011). Salinity effects on the coexistence of cryptic species: a case study on marine nematodes. Marine Biology 158(12): 2717-2726.

De Meester, N., S. Derycke and T. Moens (2012). Differences in Time until Dispersal between Cryptic Species of a Marine Nematode Species Complex. Plos One 7(8): 8.

Deruytter, D., J. Garrevoet, M. B. Vandegehuchte, E. Vergucht, B. De Samber, B. Vekemans, K. Appel, G. Falkenberg, K. Delbeke, R. Blust, K. A. C. De Schamphelaere, L. Vincze and C. R. Janssen (2014). The Combined Effect of Dissolved Organic Carbon and Salinity on the Bioaccumulation of Copper in Marine Mussel Larvae. Environmental Science & Technology 48(1): 698-705.

Derycke, S., F. Hendrickx, T. Backeljau, S. D'Hondt, L. Camphijn, M. Vincx and T. Moens (2007). Effects of sublethal abiotic stressors on population growth and genetic diversity of Pellioditis marina (Nematoda) from the Westerschelde estuary. Aquatic Toxicology 82(2): 110-119.

Derycke, S., T. Remerie, T. Backeljau, A. Vierstraete, J. Vanfleteren, M. Vincx and T. Moens (2008). Phylogeography of the Rhabditis (Pellioditis) marina species complex: evidence for long-distance dispersal, and for range expansions and restricted gene flow in the northeast Atlantic. Molecular Ecology 17(14): 3306-3322.

Derycke, S., T. Remerie, A. Vierstraete, T. Backeljau, J. Vanfleteren, M. Vincx and T. Moens (2005). Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marina. Marine Ecology Progress Series 300: 91-103.

41

Derycke, S., R. S. Tezerji, A. Rigaux and T. Moens (2012). Investigating the ecology and evolution of cryptic marine nematode species through quantitative real-time PCR of the ribosomal ITS region. Molecular Ecology Resources 12(4): 607-619.

Dhawan, R., D. B. Dusenbery and P. L. Williams (1999). Comparison of lethality, reproduction, and behavior as toxicological endpoints in the nematode Caenorhabditis elegans. Journal of Toxicology and Environmental Health-Part A 58(7): 451-462.

Dhawan, R., D. B. Dusenbery and P. L. Williams (2000). A comparison of metal-induced lethality and behavioral responses in the nematode Caenorhabditis elegans. Environmental Toxicology and Chemistry 19(12): 3061-3067.

dos Santos, G. A. P., S. Derycke, V. G. Fonseca-Genevois, L. Coelho, M. T. S. Correia and T. Moens (2008). Differential effects of food availability on population growth and fitness of three species of estuarine, bacterial-feeding nematodes. Journal of Experimental Marine Biology and Ecology 355(1): 27-40.

dos Santos, G. A. P., S. Derycke, V. G. F. Genevois, L. Coelho, M. T. S. Correia and T. Moens (2009). Interactions among bacterial-feeding nematode species at different levels of food availability. Marine Biology 156(4): 629-640.

EPA (1994) - Using Toxicity Tests in Ecological Risk Assessment (1994). EPA (1996) - Ecological Significance and Selection of Candidate Assessment Endpoints (1996). EPA (2009) "Fact Sheet Lead." from

http://www.epa.gov/osw/hazard/wastemin/minimize/factshts/lead.pdf. EPS 1/RM/9 (1990) - Biological Test Method - Acute Lethality Test Using Rainbow Trout. Environment Canada. EPS 1/RM/11 (1990) - Biological Test Method - Acute Lethality Test Using Daphnia spp. Environment Canada. EPS 1/RM/35 (1998) - Biological Test Method - Reference Method for Determining Acute Lethality of Sediment to Marine or Estuarine Amphipods. Environment Canada. Evans-White, M. A. and G. A. Lamberti (2009). Direct and indirect effects of a potential aquatic

contaminant on grazer-algae interactions. Environmental Toxicology and Chemistry 28(2): 418-426.

Ferris, H., S. Lau and R. Venette (1995). Population energetics of bacterial-feeding nematodes: Respiration and metabolic rates based on CO2 production. Soil Biology and Biochemistry 27(3): 319-330.

Fleeger, J. W., K. R. Carman and R. M. Nisbet (2003). Indirect effects of contaminants in aquatic ecosystems. Science of the Total Environment 317(1-3): 207-233.

Gaugler, R. and A. L. Bilgrami (2004). Nemaode behaviour. Oxfordshire, UK, CABI publishing. Grewal, P. and D. J. Wright (1992). Migration of Caenorhabditis elegans (NEmatoda: Rhabditidae)

larvae towards bacteria and the nature of bacterial stimulus. Fundamental and Applied Nematology 15: 159-166.

Gyedu-Ababio, T. K. and D. Baird (2006). Response of meiofauna and nematode communities to increased levels of contaminants in a laboratory microcosm experiment. Ecotoxicology and Environmental Safety 63(3): 443-450.

Hagopian-Schlekat, T., G. T. Chandler and T. J. Shaw (2001). Acute toxicity of five sediment-associated metals, individually and in a mixture, to the estuarine meiobenthic harpacticoid copepod Amphiascus tenuiremis. Mar Environ Res 51(3): 247-64.

Heip, C., M. Vincx and G. Vranken (1985). The Ecology of Marine Nematodes. Oceanography and Marine Biology 23: 399-489.

Henry, R. P. (1996). Multiple roles of carbonic anhydrase in cellular transport and metabolism. Annual Review of Physiology 58: 523-538.

Holm, R. H., P. Kennepohl and E. I. Solomon (1996). Structural and functional aspects of metal sites in biology. Chemical Reviews 96(7): 2239-2314.

Höss, S. and L. Weltje (2007). Endocrine disruption in nematodes: effects and mechanisms. Ecotoxicology 16(1): 15-28.

42

Höss, S. and P. L. Williams (2009). Ecotoxicity testing with nematodes. Nematodes as environmental indicators. M. J. Wilson, Kakouli-Duarte, T., Wallingford : CABI publishing, 2009: 208-224.

Houck, K. A. and R. J. Kavlock (2008). Understanding mechanisms of toxicity: Insights from drug discovery research. Toxicology and Applied Pharmacology 227(2): 163-178.

Howell, R. (1982a). The Secretion of Mucus By Marine Nematodes (Enoplus Spp.): a Possible Mechanism Influencing the Uptake and Loss of Heavy Metal Pollutants. Nematologica 28(1): 110-114.

Hu, Y. N., X. P. Liu, J. M. Bai, K. M. Shih, E. Y. Zeng and H. F. Cheng (2013). Assessing heavy metal pollution in the surface soils of a region that had undergone three decades of intense industrialization and urbanization. Environmental Science and Pollution Research 20(9): 6150-6159.

Hunt, P. R., N. Olejnik and R. L. Sprando (2012). Toxicity ranking of heavy metals with screening method using adult Caenorhabditis elegans and propidium iodide replicates toxicity ranking in rat. Food and Chemical Toxicology 50(9): 3280-3290.

Hussein, H., S. Farag, K. Kandil and H. Moawad (2005). Tolerance and uptake of heavy metals by Pseudomonads. Process Biochemistry 40(2): 955-961.

Inglis, W. and J. Coles (1961). The species of Rhabditis (Nematoda) found in rotting seaweed on British beaches. Bull Brit Mus(Natur Hist) 7: 320–333.

ISO 10872:2010 Water quality - Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) ISO 14380:2011 Water quality - Determination of the acute toxicity to Thamnocephalus platyurus (Crustacea, Anostraca) ISO 14669:1999 Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) ISO 7346-1:1996 Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] -- Part 1: Static method Kammenga, J. E., C. A. M. Vangestel and J. Bakker (1994). Patterns of sensitivity to cadmium and

pentachlorophenol among nematode species from different taxonomic and ecological groups. Archives of Environmental Contamination and Toxicology 27(1): 88-94.

Kammenga, J. E., P. H. G. VanKoert, J. A. G. Riksen, G. W. Korthals and J. Bakker (1996). A toxicity test in artificial soil based on the life-history strategy of the nematode Plectus acuminatus. Environmental Toxicology and Chemistry 15(5): 722-727.

Kang, T., W.-G. Min, H. S. Rho, H.-S. Park and D. Kim (2014). Differential responses of a benthic meiofaunal community to an artificial oil spill in the intertidal zone. Journal of the Marine Biological Association of the United Kingdom 94(02): 219-231.

Koivisto, S., M. Arner and N. Kautsky (1997). Does cadmium pollution change trophic interactions in rockpool food webs? Environmental Toxicology and Chemistry 16(6): 1330-1336.

Korthals, G. W., A. D. Alexiev, T. M. Lexmond, J. E. Kammenga and T. Bongers (1996). Long-term effects of copper and pH on the nematode community in an agroecosystem. Environmental Toxicology and Chemistry 15(6): 979-985.

Kozelka, P. B., S. Sanudo-Wilhelmy, A. R. Flegal and K. W. Bruland (1997). Physico-chemical speciation of lead in South San Francisco Bay. Estuarine Coastal and Shelf Science 44(6): 649-658.

Lefcort, H., Z. Freedman, S. House and M. Pendleton (2008). Hormetic effects of heavy metals in aquatic snails: Is a little bit of pollution good? Ecohealth 5(1): 10-17.

Leibold, M. A. and M. A. McPeek (2006). Coexistence of the niche and neutral perspectives in community ecology. Ecology 87(6): 1399-1410.

Leung, M. C. K., P. L. Williams, A. Benedetto, C. Au, K. J. Helmcke, M. Aschner and J. N. Meyer (2008). Caenorhabditis elegans: An emerging model in biomedical and environmental toxicology. Toxicological Sciences 106(1): 5-28.

Lewis, M. A. (1992). The effects of mixtures and other environmental modifying factors on the toxicities of surfactants to freshwater and marine life. Water Research 26(8): 1013-1023.

43

Libralato, G., V. Ghirardini Annamaria and A. Francesco (2010). How toxic is toxic? A proposal for wastewater toxicity hazard assessment. Ecotoxicology and Environmental Safety 73(7): 1602-1611.

Lira, V. F., G. A. P. dos Santos, S. Derycke, M. E. L. Larrazabal, V. G. Fonseca-Genevois and T. Moens (2011). Effects of barium and cadmium on the population development of the marine nematode Rhabditis (Pellioditis) marina. Marine Environmental Research 72(4): 151-159.

Long, E. R., D. D. Macdonald, S. L. Smith and F. D. Calder (1995). Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments. Environmental Management 19(1): 81-97.

Lopez-Diazguerrero, N. E., V. Y. G. Puertos, R. J. Hernandez-Bautista, A. Alarcon-Aguilar, A. Luna-Lopez and M. K. Fainstein (2013). Hormesis: What doesn't kill you makes you stronger. Gaceta Medica De Mexico 149(4): 438-447.

Lorenzo, J. I., R. Beiras, V. K. Mubiana and R. Blust (2005). Copper uptake by Mytilus edulis in the presence of humic acids. Environ Toxicol Chem 24(4): 973-80.

Lorenzo, J. I., O. Nieto and R. Beiras (2002). Effect of humic acids on speciation and toxicity of copper to Paracentrotus lividus larvae in seawater. Aquat Toxicol 58(1-2): 27-41.

Lushchak, V. I. (2011). Environmentally induced oxidative stress in aquatic animals. Aquat Toxicol 101(1): 13-30.

Martinez, J. G., G. dos Santos, S. Derycke and T. Moens (2012). Effects of cadmium on the fitness of, and interactions between, two bacterivorous nematode species. Applied Soil Ecology 56: 10-18.

Mato, Y., T. Isobe, H. Takada, H. Kanehiro, C. Ohtake and T. Kaminuma (2001). Plastic resin pellets as a transport medium for toxic chemicals in the marine environment. Environmental Science & Technology 35(2): 318-324.

Mattson, M. P. (2008). Hormesis defined. Ageing Research Reviews 7(1): 1-7. Moens, T., L. Verbeeck, A. de Maeyer, J. Swings and M. Vincx (1999). Selective attraction of marine

bacterivorous nematodes to their bacterial food. Marine Ecology Progress Series 176: 165-178.

Moens, T., A. Vierstraete and M. Vincx (1996). Life strategies in two bacterivorous marine nematodes: Preliminary results. Marine Ecology-Pubblicazioni Della Stazione Zoologica Di Napoli I 17(1-3): 509-518.

Moens, T. and M. Vincx (1997). Observations on the feeding ecology of estuarine nematodes. Journal of the Marine Biological Association of the United Kingdom 77(1): 211-227.

Moens, T. and M. Vincx (1998). On the cultivation of free-living marine and estuarine nematodes. Helgolander Meeresuntersuchungen 52(2): 115-139.

Moens, T. and M. Vincx (2000a). Temperature, salinity and food thresholds in two brackish-water bacterivorous nematode species: assessing niches from food absorption and respiration experiments. Journal of Experimental Marine Biology and Ecology 243(1): 137-154.

Moens, T. and M. Vincx (2000b). Temperature and salinity constraints on the life cycle of two brackish-water nematode species. Journal of Experimental Marine Biology and Ecology 243(1): 115-135.

Moens, T., G. W. Yeates and P. Ley (2004). Use of carbon and energy sources by nematodes. Proceeding of the Fourth International Congress of Nematology. Pa Leiden, E J Brill. 2: 529-545.

Monteiro, L., M. Brinke, G. dos Santos, W. Traunspurger and T. Moens (2014). Effects of heavy metals on free-living nematodes: A multifaceted approach using growth, reproduction and behavioural assays. European Journal of Soil Biology 62: 1-7.

Monteiro, L., W. Traunspurger and T. Moens (2010). Effects of heavy metals on free-living nematodes: A multifacteted approach using growth, reproduction and behavioural assays. M.Sc. Thesis. Universiteit Gent: Belgium.

Moore, C. J. (2008). Synthetic polymers in the marine environment: A rapidly increasing, long-term threat. Environmental Research 108(2): 131-139.

44

Muschiol, D., F. Schroeder and W. Traunspurger (2009). Life cycle and population growth rate of Caenorhabditis elegans studied by a new method. BMC Ecol 9: 14.

Muschiol, D. and W. Traunspurger (2007). Life cycle and calculation of the intrinsic rate of natural increase of two bacterivorous nematodes, Panagrolaimus sp. and Poikilolaimus sp. from chemoautotrophic Movile Cave, Romania. NEMATOLOGY 9(2): 271-284.

Naeem, S. and S. B. Li (1997). Biodiversity enhances ecosystem reliability. Nature 390(6659): 507-509. Nauke, M. and G. L. Holland (1992). The Role and Development of Global Marine Conventions: Two

Case Histories. Marine Pollution Bulletin 25(1-4): 74-79. Needleman, H. (2004). Lead poisoning. Annual Review of Medicine 55: 209-222. OECD (1992) - Organization for Economic Cooperation and Development. 1992. Report of the OECD workshop on the extrapolation of laboratory aquatic toxicity data to the real environment. Environmental Monograph 58. Paris, France. Rand, G. M. (1995). Fundamentals of aquatic toxicology: Effects, environmental fate, and risk

assessment. Washington, D.C, Taylor & Francis. Reish, D. J. and R. S. Carr (1978). The effect of heavy metals on the survival, reproduction,

development, and life cycles for two species of polychaetous annelids. Marine Pollution Bulletin 9(1): 24-27.

Rodger, S., B. S. Griffiths, J. W. McNicol, R. W. Wheatley and I. M. Young (2004). The impact of bacterial diet on the migration and navigation of Caenorhabditis elegans. Microb Ecol 48(3): 358-65.

Roh, J.-Y., J. Lee and J. Choi (2006). Assessment of stress-related gene expression in the heavy metal-exposed nematode Caenorhabditis elegans: A potential biomarker for metal-induced toxicity monitoring and environmental risk assessment. Environmental Toxicology and Chemistry 25(11): 2946-2956.

Salminen, J., T. Korkama and R. Strommer (2002). Interaction modification among decomposers impairs ecosystem processes in lead-polluted soil. Environmental Toxicology and Chemistry 21(11): 2301-2309.

Samoiloff, M. (1990). Tthe nematode toxicity assay using Panagrellus redivivus. Toxicity Assessment 5(3): 309-318.

Samoiloff, M. R., S. Schulz, Y. Jordan, K. Denich and E. Arnott (1980). A Rapid Simple Long-Term Toxicity Assay for Aquatic Contaminants Using the Nematode Panagrellus redivivus. Canadian Journal of Fisheries and Aquatic Sciences 37(7): 1167-1174.

Sánchez-Marín, P., J. I. Lorenzo, R. Blust and R. Beiras (2007). Humic acids increase dissolved lead Bioavailability for marine invertebrates. Environmental Science & Technology 41(16): 5679-5684.

Schratzberger, M., C. M. Wall, W. J. Reynolds, J. Reed and M. J. Waldock (2002). Effects of paint-derived tributyltin on structure of estuarine nematode assemblages in experimental microcosms. Journal of Experimental Marine Biology and Ecology 272(2): 217-235.

Schratzberger, M. and R. M. Warwick (1999). Differential effects of various types of disturbances on the structure of nematode assemblages: an experimental approach. Marine Ecology Progress Series 181: 227-236.

Scott, J. A. and P. V. Hodson (2008). Evidence for multiple mechanisms of toxicity in larval rainbow trout (Oncorhynchus mykiss) co-treated with retene and alpha-naphthoflavone. Aquatic Toxicology 88(3): 200-206.

Sochovà, I., J. Hofman and I. Holoubek (2006). Using nematodes in soil ecotoxicology. Environment International 32(3): 374-383.

Sokal, R. R. and F. J. Rohlf (1995). Biometry: the principles and practice of statistics in biological research. W. H. Freeman, New York.

Solomon, E. I., U. M. Sundaram and T. E. Machonkin (1996). Multicopper oxidases and oxygenases. Chemical Reviews 96(7): 2563-2605.

Song, S. J., Y. P. Guo, X. M. Zhang, X. Y. Zhang, J. Z. Zhang and E. B. Ma (2014). Changes to Cuticle Surface Ultrastructure and Some Biological Functions in the Nematode Caenorhabditis

45

Elegans Exposed to Excessive Copper. Archives of Environmental Contamination and Toxicology 66(3): 390-399.

Stebbing, A. R. D. (1982). Hormesis — The stimulation of growth by low levels of inhibitors. Science of the Total Environment 22(3): 213-234.

Sudhaus, W. (2011). Phylogenetic systematisation and catalogue of paraphyletic "Rhabditidae" (Secernentea, Nematoda). Journal of Nematode Morphology and Systematics 14(2): 113-178.

Thomas, M. C. and P. C. Lana (2011). A new look into the small-scale dispersal of free-living marine nematodes. Zoologia 28(4): 449-456.

Traunspurger, W. (2000). The biology and ecology of lotic nematodes. Freshwater Biology 44(1): 29-45.

Traunspurger, W., M. Haitzer, S. Hoss, S. Beier, W. Ahlf and C. Steinberg (1997). Ecotoxicological assessment of aquatic sediments with Caenorhabditis elegans (nematoda) - A method for testing liquid medium and whole-sediment samples. Environmental Toxicology and Chemistry 16(2): 245-250.

Versteeg, D. J., S. E. Belanger and G. J. Carr (1999). Understanding single-species and model ecosystem sensitivity: Data-based comparison. Environmental Toxicology and Chemistry 18(6): 1329-1346.

Vranken, G. and C. Heip (1983). Calculation of the intrinsic rate of natural increase, rm, with Rhabditis marina Bastian 1865 (Nematoda). Nematologica 29(4): 468-477.

Vranken, G., R. Vanderhaeghen and C. Heip (1985). Toxicity of cadmium to free-living marine and brackish water nematodes (Monhystera microphthalma, Monhystera disjuncta, Pellioditis marina). Diseases of Aquatic Organisms 1(1): 49-58.

Wang, D. Y. and X. J. Xing (2008). Assessment of locomotion behavioral defects induced by acute toxicity from heavy metal exposure in nematode Caenorhabditis elegans. Journal of Environmental Sciences-China 20(9): 1132-1137.

Weber, S. and W. Traunspurger (2013). Food choice of two bacteria-feeding nematode species dependent on food source, food density and interspecific competition. Nematology 15: 291-301.

White, J. Q., T. J. Nicholas, J. Gritton, L. Truong, E. R. Davidson and E. M. Jorgensen (2007). The sensory circuitry for sexual attraction in C. elegans males. Current Biology 17(21): 1847-1857.

Williams, P. L. and D. B. Dusenbery (1990). Aquatic toxicity testing using the nematode Caenorhabditis elegans. Environmental Toxicology and Chemistry 9(10): 1285-1290.

Yeates, G. W. (1979). Soil nematodes in terrestrial ecosystems. Journal of Nematology 11(3): 213-229.

Yeates, G. W., T. Bongers, R. G. M. Degoede, D. W. Freckman and S. S. Georgieva (1993). Feeding Habits in Soil Nematode Families and Genera—An Outline for Soil Ecologists. Journal of Nematology 25(3): 315-331.

Yu, Z. Y., J. Zhang and D. Q. Yin (2012). Toxic and recovery effects of copper on Caenorhabditis elegans by various food-borne and water-borne pathways. Chemosphere 87(11): 1361-1367.

Zirino, A. and S. Yamamoto (1972). A pH-Dependent Model for the Chemical Speciation of Copper, Zinc, Cadmium, and Lead in Seawater. Limnology and Oceanography 17(5): 661-671.