Acute toxicological response of Daphnia and Moina …...Acute toxicological response of Daphnia and...

Acute toxicological

response of Daphnia

and Moina to

hydrogen peroxide

for the improvement

of water quality in

stabilisation ponds

Leanne Zheng (20151494)

Supervised by Anas Ghadouani & Elke Reichwaldt

This dissertation is submitted in partial fulfillment for the degree of Bachelor of

Environmental Engineering (Water Resources) from the School of Environmental

Systems Engineering at the University of Western Australia, 2010.

Abstract

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Abstract

Cyanobacteria present in wastewater have fatal effects when exposed to humans and animals.

Its removal from wastewater is vital for protection of the community. Although there are

already current treatment methods in practice, harmful by-products are produced. As a result,

alternative treatment methods are being sought. Recently, hydrogen peroxide was found to

effectively induce cyanobacteria death and due to the environmentally benign biodegradation

products, it may potentially provide a more environmentally sensitive method for treating

wastewater. The Water Corporation has proposed to use hydrogen peroxide for long term

treatment of cyanobacteria in stabilisation ponds. Before a long term treatment scheme could

be implemented, the effects that hydrogen peroxide may have on the biological functioning of

stabilisation ponds needed to be analysed. Daphnia and Moina are filter feeding organisms

present in stabilisation ponds and are significant contributors to the biological processing of

wastewater. Commonly used in ecotoxicological studies, this makes them useful indicators

for identifying adverse effects resulting from application of hydrogen peroxide. An acute

toxicity test was performed on both Daphnia and Moina for a 48 hour period. Results showed

that Daphnia and Moina are highly sensitive to hydrogen peroxide. The NOAEC for Daphnia

was found to be 0.002 g/L with an LC50 of 0.007 g/L. The risk assessment parameters for

Moina were lower, with an NOAEC of 0.0015 g/L and LC50 of 0.002 g/L. Previous studies

found that the optimal concentration to induce death in cyanobacteria under controlled

laboratory conditions was 0.296 g/L and an optimal field dose of 0.04 g/L. Although

comparison shows that the recommended application dose would be lethal for both Daphnia

and Moina, conclusions cannot be drawn that hydrogen peroxide is unsuitable for long term

treatment of cyanobacteria. The toxicity study was performed under constant laboratory

conditions and does not account for changes to conditions in the environment occurring on

site, the dynamics of the pond or the relative volume differences between the volume used for

experimentation and the volume in the pond. Accounting for site conditions, the risk

assessment parameters are likely to have been under estimated. Application of hydrogen

peroxide to stabilisation ponds is unlikely to result in any significant adverse effects, although

on site testing would be required to ascertain the use of hydrogen peroxide for long term

treatment in stabilisation ponds.

Acknowledgements

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Acknowledgements

Many thanks go to my supervisors, Anas Ghadouani and Elke Reichwaldt for their support

over the year. I would like to thank Danielle Barrington for the trips she made with me to

Wundowie for collection of the test species and her advice during the course of my project.

Thanks to Shian Min Liau, for the help she has provided me during the seemingly endless

days working at the environmental research laboratory. I would also like to thank Som Cit

Sinang for the laboratory demonstrations and Brett Kerenyi from the Water Corporation for

the trips to the Wundowie stabilisation pond. Finally, I would like to thank Michael Smirk

and Darryl Roberts from FNAS for their assistance for POC calculations.

Table of Contents

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Table of Contents

Abstract ................................................................................................................................ I

Acknowledgements ............................................................................................................. II

Table Of Contents ............................................................................................................. III

Figures ............................................................................................................................... IV

Tables ................................................................................................................................. IV

1. Introduction ....................................................................................................... 1

2. Literature Review .............................................................................................. 3

2.1 Applications Of Hydrogen Peroxide ............................................................................. 3

2.2 Stabilisation Ponds ........................................................................................................ 4

2.3 Ecotoxicology Testing .................................................................................................... 6 2.3.1 Types Of Tests .......................................................................................................... 6

2.3.2 Choice Of Test Organisms ........................................................................................ 7

2.4 Daphnia And Moina ...................................................................................................... 8 2.4.1 Daphnia .................................................................................................................... 8

2.4.2 Moina ....................................................................................................................... 9

2.5 Case Studies Of Hydrogen Peroxide Tested On Animals .......................................... 11

3. Methodology ..................................................................................................... 13

3.1 Collection And Culturing Of Test Species .................................................................. 13

3.2 Experimental Design ................................................................................................... 16

3.3 Data Analysis ............................................................................................................... 19

4. Results............................................................................................................... 21

5. Discussion ......................................................................................................... 32

6. Recommendations ............................................................................................ 36

7. Conclusions ....................................................................................................... 37

8. References......................................................................................................... 39

Appendix A: Protocols ...................................................................................................... 42

Appendix B: Survival Data ............................................................................................... 44

Figures & Tables

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Figures

Figure 1: Photo of Daphnia under microscope (Zheng 2010) ................................................ 9

Figure 2: Photo of Moina under microscope (Zheng 2010). ................................................. 11

Figure 3: Plankton net used to sieve Daphnia and Moina from waterbodies and transferred

into corresponding containers (Zheng 2010). ....................................................................... 13

Figure 4: Desmodesmus cultures in the laboratory (Zheng 2010). ........................................ 15

Figure 5: Calibration curve for Desmodesmus, showing the volume required to achieve 1 mg

C. ........................................................................................................................................ 16

Figure 6: Experiment setup (Zheng 2010). .......................................................................... 18

Figure 7: Theoretical survival function. At timestep t=0, survival probability is 1. .............. 20

Figure 8: Survival response of Daphnia when different concentrations of hydrogen peroxide

were added. ......................................................................................................................... 22

Figure 9: Survival response of Moina when different concentrations of hydrogen peroxide

were added. ......................................................................................................................... 23

Figure 10: Survival curve of Daphnia and Moina for 0.002 g/L hydrogen peroxide tested. .. 25


Figure 12: Survival curve of Daphnia and Moina for 0.0125 g/L hydrogen peroxide tested. 27


Figure 14: Survival curve of Daphnia and Moina for 1.25 g/L hydrogen peroxide tested. .... 30

Tables

Table 1: Statistical analysis of Daphnia for 0.002 g/L hydrogen peroxide tested. ................. 25

Table 2: Statistical analysis of Moina for 0.002 g/L hydrogen peroxide tested. .................... 25

Table 3: Comparison of survival mean time, standard deviation and confidence intervals

between Daphnia and Moina for 0.002 g/L hydrogen peroxide tested. ................................ 26

Table 4: Statistical analysis of Daphnia for 0.005 g/L hydrogen peroxide tested. ................ 26




Table 7: Statistical analysis of Daphnia for 0.0125 g/L hydrogen peroxide tested. ............... 28

Table 8: Statistical analysis of Moina for 0.0125 g/L hydrogen peroxide tested. .................. 28

Table 9: Comparison of survival, standard deviation and confidence intervals between

Daphnia and Moina for 0.0125 g/L hydrogen peroxide tested. ............................................ 28

Table 10: Statistical analysis of Daphnia for 0.125 g/L hydrogen peroxide tested. ............... 29

Table 11: Statistical analysis of Moina for 0.125 g/L hydrogen peroxide tested. .................. 29



Table 13: Statistical analysis of Daphnia for 1.25 g/L hydrogen peroxide tested. ................ 30



between Daphnia and Moina for 1.25 g/L hydrogen peroxide tested. .................................. 31

Table 16: Survival data of Moina for hydrogen peroxide concentrations trialed. .................. 44

Table 17: Survival data of Daphnia for hydrogen peroxide concentrations trialed. .............. 47

Introduction

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1. Introduction

Cyanobacteria is commonly referred to as blue green algae, and it produces toxins which are

detrimental to the health of humans and animals (eds Huisman, Matthijs & Visser 2005).

They occur in many freshwater and marine ecosystems, and due to the high exposure to

sunlight, as well as the high nutrient loadings, cyanobacterial blooms are often observed in

stabilisation ponds (eds Chorus & Bartram 1999). Potential health risks are posed by the

presence of cyanobacteria and its removal from wastewater prior to releasing into the

environment is vital. Considerations for water reuse implications are not feasible until

cyanobacteria and the toxins it produces is removed from wastewater (Barrington &

Ghadouani 2008).

Recently, hydrogen peroxide has been identified to exhibit properties which effectively

induce death in cyanobacteria. Unlike current treatments methods used in practice to treat

cyanobacteria, hydrogen peroxide produces environmentally benign biodegradation products.

As a result, hydrogen peroxide may potentially provide a more environmentally sensitive

alternative for the removal of cyanobacteria from wastewater (Barrington & Ghadouani

2008) and has been proposed by the Water Corporation for long term use in the treatment of

stabilisation ponds.

Before a long term treatment program using hydrogen peroxide can be established, it is

important to identify whether hydrogen peroxide may affect the biological functions that

occur within the stabilisation ponds. Stabilisation ponds are able to self purify wastewater

through the natural biological processes that occur through the interaction of organisms in the

ponds (Spellman 1996). Daphnia and Moina form part of these complex ecological groups

present in stabilisation ponds. They contribute to the treatment of wastewater by feeding on

bacteria, organic matter and algae present (Gray 2004). Because Daphnia and Moina are

important for the natural processing of wastewater in stabilisation ponds, any adverse effects

hydrogen peroxide may pose on the two organisms would consequently affect the biological

functioning of the ponds. As a result, Daphnia and Moina are suitable bioindicators to

identify any adverse effects for using hydrogen peroxide. The suitability for using hydrogen

peroxide for long term treatment of cyanobacteria in stabilisation ponds can consequently be

assessed.

Introduction

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The main objectives of the project are as follows:

i. Determine the no observed adverse effect concentration (NOAEC) of hydrogen

peroxide on the bioindicators Daphnia and Moina.

ii. Estimate the hydrogen peroxide concentration lethal to 50% of Daphnia and

Moina, the LC50.

iii. Assess the suitability for using hydrogen peroxide for long term treatment in

stabilisation ponds.

This study effectively gives an indication of the sensitivity of Daphnia and Moina to

hydrogen peroxide. It provides a baseline for determining whether the use of hydrogen

peroxide would be a better solution for treating cyanobacteria in wastewater compared to

current methods in practice. Implications for finding a suitable dose to remove cyanobacterial

biomass from stabilisation ponds without influencing the biological functions of the ponds

will benefit water treatment facilities and provide an environmentally sensitive method for

future treatment. The safe threshold concentration obtained may also be used for future

reference for studies involving aquatic toxicity of hydrogen peroxide.

Literature Review

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2. Literature Review

2.1 Applications of hydrogen peroxide

Hydrogen peroxide is a strong oxidizing agent occurring in the form of a clear and colourless

liquid (Jones 1999). It is widely used for environmental applications, including water

treatment and disinfection (Drabkova et al. 2007). The presence of an active oxygen

component in hydrogen peroxide enables it to eradicate pollutants through an oxidation

reaction (Jones 1999). It is particularly suitable for environmental practice due to its

efficiency and safe biodegradation products, oxygen gas and water (Antoniou et al. 2005).

This is shown in the following chemical equation:

H2O2(l) � H2O(l) + O2(g)

The rate of degradation of hydrogen peroxide is affected by many factors, including changes

in temperature, the presence of metal contaminants, contact with active surfaces, and the pH.

Other applications include chemical purification and paper bleaching (Jones 1999).

The toxicity of hydrogen peroxide is increased when combined with a metal catalyst, UV

irradiation or ozonation. The oxidative power becomes much stronger and these reactions are

known as advanced oxidation processes (AOPs) (Jones 1999). Reaction with a reduced form

of a transition metal causes the production of the hydroxyl radical, which is able to react with

any molecule to produce further molecules. These free radicals produced can result in

damage to cells through lipid peroxidation, DNA damage and protein oxidation (Forman

2008). AOP through the transition metal ferrous iron has been found to effectively increase

degradation rates of pollutants in wastewater (Kallel 2009).

Hydrogen peroxide has also been used to treat sewers to inhibit the formation of harmful

hydrogen sulfide gases, (Jones 1999), control the growth of filamentous bacteria which cause

sludge bulking (Gray 2004), and manage algae biomass in waterways (Jones 1999). The use

of hydrogen peroxide as an algicide was also mentioned by Kay et al. (1984). Another

application for hydrogen peroxide is the formation of hypochlorous, hypobromous and

hypothiocyanous. These acids are able to assist in the defense against infection and have been

used to treat fungi infected fish in a study by Rach et al. (1997). However, due to the toxicity

Literature Review

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of the acids formed from hydrogen peroxide, if inflammation is already occurring, exposure

would result in tissue damage (Forman 2008).

More recently, hydrogen peroxide was tested for its effectiveness in the removal of

cyanobacteria commonly present in wastewater. Results showed that application of hydrogen

peroxide was effective to induce cell death in cyanobacteria (Barrington & Ghadouani 2008).

Other studies have shown that hydrogen peroxide can effectively remove toxins, such as

microcystin-RR when combined with UV irradiation (Qiao 2005). Application of hydrogen

peroxide for inducing death in cyanobacteria in wastewater is currently under consideration

for long term use by water industries (Barrington & Ghadouani 2008). Before this method

can be approved for regular use, it would be necessary to investigate the adverse effects, if

any, that hydrogen peroxide may pose on the dynamics of the stabilisation ponds used for

wastewater treatment. Considerations for the biological interactions involved would need to

be made.

2.2 Stabilisation ponds

Natural water systems are able to self-purify through micro-organisms living in the water

(Spellman 1996). Organic matter acts as a food source for the aquatic organisms present and

as a result, organic matter is able to be degraded. Similarly, wastewater can be treated

through the utilisation of the natural self-purification process in stabilisation ponds (Gray

2004). Though stabilisation ponds appear to be a simple treatment process, the ecological

systems within the ponds are very complex. The ponds contain communities of viruses, algae,

protozoa, rotifers, insects, fungi and crustaceans (Kehl 2009). Under controlled conditions

which optimize microbial activity, most of the organic matter can be degraded through the

interactions of these communities (Gray 2004).

The process of treating wastewater through stabilisation ponds involves pumping wastewater

to the primary pond, typically the anaerobic pond. It utilises anaerobic processes to remove

settled solids and decrease biological oxygen demand (BOD). The BOD is a measure of the

oxygen required by micro-organisms to decompose organic waste (Gloyna 1971).

Consequently, a low BOD indicates water quality of a high standard (Gray 2004).

Wastewater pre-treated by the primary pond is transferred to the secondary pond, usually the

Literature Review

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facultative pond. Aerobic and anaerobic processes act to further break down solids for

removal and decrease the BOD (Gloyna 1971). In the final step, the tertiary pond, also known

as the maturation pond, has the purpose of improving the quality of the treated wastewater

and removing pathogens present. The pond is typically shallow to allow for maximum light

exposure and is well aerated to kill pathogens (Gray 2004).

Factors including pH, temperature and light intensity affect the abundance and behavior of

micro-organisms. Combined, they can influence the biological performance of the pond

(Kayombo et al. 2002). Bacteria are mainly responsible for oxidation of organic matter in

wastewater systems, although many other organisms contribute to this process by

transforming matter to biomass for removal (Gray 2004). A large quantity of bacteria is

involved in early treatment due to the significant quantities of organic waste, which supplies

energy for bacterial growth. High quantities of organic load results in predominance of

anaerobic bacteria (Spellman 1996). Other organisms contributing to the purification process

of wastewater in stabilisation ponds include the zooplankton organisms Cladocera, which

occurs mainly in facultative ponds (Gray 2004).

Among the Cladocera are filter feeders, which feed through ingesting particles from the water

(Lampert 1987, p 145). Their role in stabilisation ponds is important as they primarily feed on

bacteria, suspended organic matter and algae, effectively reducing algal and bacterial

concentrations in the pond. They are also able to contribute through the formation of boluses

resulting from excess food. This is common in stabilisation ponds due to the high organic

loadings (Gray 2004). Boluses are compacted food prepared for digestion or expulsion by the

body (Gerristen, Porter & Strickler 1988), and excess food causes the Cladocera to reject the

boluses. Due to the high density, it rapidly settles and can be removed from the pond as

sludge (Gray 2004).

Although Cladocera contributes to the processing of material in the ponds, dense populations

are undesirable. When dense populations occur, as common in stabilisation ponds due to the

high organic loading, high levels of algae would be consumed. Reduction of algae present in

the ponds decreases photosynthetic activity, resulting in a reduction in the ability for the pond

to reaerate (Gray 2004). This is particularly important as dissolved oxygen is utilised from

the water by organisms to break down organic matter present in the ponds. Reduction in

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dissolved oxygen within the ponds would inhibit the natural process of breaking down the

waste (Gloyna 1971).

2.3 Ecotoxicology testing

Ecotoxicology assesses the extent of toxicity a substance may impose on a population within

the ecosystem. It integrates the study of lethal chemicals and its interactions with the natural

environment and ecological systems (Connell et al. 1999). It is now common practice to use

aquatic organisms to identify adverse effects resulting from the introduction of chemicals in

the ecosystem (Anderson-Carnahan 2004). Through the applications of ecotoxicological

testing, thresholds for chemical acceptability can be determined to provide guidelines for the

protection of the environment. It is useful to conduct tests at various concentrations to

quantify the effects of a chemical (Adams & Rowland 2003, p 22).

Aquatic toxicity tests are able to determine the endpoints of risk assessment parameters used

to determine the safe level of exposure. The no observed adverse effect concentration

(NOAEC) is the threshold concentration where the organism tested is not biologically

influenced by the compound and can function normally (U.S. EPA 1994). Another parameter

typically used is the LC50, which is the concentration by which there is only 50% survival. At

this concentration, aquatic life is not biologically influenced by the compound and can

function normally (Adams & Rowland 2003, p 29). In the study of interest, it would be useful

to conduct an aquatic toxicity test, since the objective is to find an acceptable concentration

where the biological functioning of the stabilisation ponds will not be affected from the

application of hydrogen peroxide.

2.3.1 Types of tests

There are two types of toxicity tests used in toxicology; acute and chronic. Acute toxicity

tests are used to determine the short term effects of aquatic species when exposed to toxins or

chemicals (U.S. EPA 2002). It typically evaluates survival response over 24 to 96 hours

(Adams & Rowland 2003, p 22). This test is suitable for assessing contaminants which move

quickly through a system or breaks down readily. If results show no significant effects in an

acute test, the chemical cannot be concluded as being non-toxic until a chronic toxicity test is

also performed (Anderson-Carnahan 2004).

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Chronic toxicity tests monitor a longer time period of an organism’s life cycle (Adams &

Rowland 2003, p 22). They are used to determine the long term effects on factors including

reproduction, mortality (U.S. EPA 1994), behavior and physiological interference (Adams &

Rowland 2003, p 22). Chronic tests are particularly suitable for testing natural aquatic

systems to ensure a thorough ecosystem exposure risk assessment is made (Anderson-

Carnahan 2004). Observations of long term factors are not possible with an acute toxicity test

(Meinzertz 2008). A chronic toxicity test can also provide a more precise estimate of the

NOAEC, particularly if no observed effect was found after conducting an acute test. A 7 day

cladoceran partial-life cycle test may also be used to monitor a longer period of exposure.

The early stage of cladoceran has been found to be most sensitive and is suitable for toxicity

testing (U.S. EPA 1994).

2.3.2 Choice of test organisms

Daphnia and Moina are useful bioindicators for toxicity testing. A wide range of organisms

have been found to be useful for the purposes of toxicity testing, but Daphnia is a particularly

common choice for ecotoxicological studies. These organisms have been used in such tests

for an extensive period already (Baudo 1987, p 462). Daphnia is a popular choice for various

reasons. It has a relatively high sensitivity to toxins, and is simple to culture and maintain in

the laboratory (Movahedian, Bina & Asghari 2005). They have a relatively short lifecycle,

which makes them suitable for testing long term effects which take course over a lifetime or

part of the lifecycle. Aside from this, they are also highly productive. As a result, Daphnia

are able to produce mass cultures within a short period of time (Ebert 2005). Due to the

cloning ability of Daphnia, they are also particularly ideal for genetic studies as a population

of Daphnia can be created initially from a single organism (Ebert 2005). The presence of

Daphnia in stabilisation ponds makes it particularly useful as a test organism. Also, since

Daphnia contributes to the biological processing of stabilisation ponds (Jones 2005), adverse

effects posed by hydrogen peroxide on Daphnia will be a reasonable reflection of how well

the ponds function.

There is less understanding of the genus Moina and the literature available is limited

(Anderson-Carnahan 1994). Hundreds of species branch from the genus Daphnia but there is

much less species branching from Moina (Goulden 1968). Daphnia is found in a larger range

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of water bodies, is easier to handle due to their larger size and has slower movements (Ebert

2005). There is also a better biological understanding of the genus. However, Moina are

closely related to Daphnia and exhibit many of the same properties. Moina have also been

used before in toxicity studies and are present in stabilization ponds. For this reason, both

Daphnia and Moina are suitable organisms for toxicity tests.

Communities within the ponds interact with each other during the self-purification process

(Gray 2004). Any negative response posed on Daphnia and Moina from application of

hydrogen peroxide would indicate the self-purification process of wastewater may be

adversely affected. Water samples may also be processed through laboratory analysis to

monitor any adverse changes after application. However, the process may take a significantly

long period of time before results are returned and is also costly. The use of bioindicators like

Daphnia and Moina is not only a more cost effective solution for testing toxicity, they are

also able to test for toxicity below instrument detectable limits and produces results more

efficiently.

2.4 Daphnia and Moina

2.4.1 Daphnia

Extensive research has been performed on the freshwater zooplankton, Daphnia (Ankley et

al. 2002). Daphnia are classified as Cladocera and are filter feeders (figure 1). Filter feeders

remove small particles suspended in the water for consumption using a filtering apparatus

(Ebert 2005). It commonly feeds on planktonic algae, particularly favouring green algae, but

bacteria is also collected from the water (Lampert 1987, p 176). They are found in most

waterbodies, including wastewater treatment ponds and contribute to the biorecycling process

through consumption of algae, protozoa, bacteria and organic matter present (Shiny et al.

2005).

Daphnia begins to reach sexual maturity between 5 to 10 days and reproduces every 3 to 4

days in a lifetime. Under optimal conditions, they may live up to 2 months (Ebert 2005).

Habitation within temporary ponds is likely to result in the production of resting eggs.

Resting eggs sink to the bottom of the ponds and will reproduce asexually when conditions

become ideal for growth again (Zaffagnini 1987, p 245). Ideally, the optimum water quality

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is between pH of 7.2 and 8.5, although pH levels between 6.5 and 9.5 is still within habitable

limits. Low salinity levels are also ideal. The behaviour of Daphnia is influenced by

predation. It moves away from the sunlight during the day and surfaces during the night.

Daphnia can be found in many different water bodies, from temporary ponds to large lakes.

They are often predated by fish and play an important role in the food chain (Threlkeld 1987,

p 379).

Figure 1: Photo of Daphnia under microscope (Zheng 2010)

2.4.2 Moina

Like Daphnia, Moina are Cladocera and have similar biological characteristics. There is

limited literature based on the genus Moina (Anderson-Carnahan 1994), although Moina has

been previously selected for use in toxicity studies. Due to the close relation between genera,

Moina is sometimes referred to as Daphnia and are collectively referred to as water fleas due

to the jerky swimming movements in water (Rottman et al. 2003). However, there are notable

differences between Daphnia and Moina.

Moina are typically smaller in length, with adult males occurring between 0.6 to 0.9 mm,

while adult females are between 1.0 and 1.5 mm (figure 2) (eds Lavens & Sorgeloos 1996).

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They take between 4 to 5 days to reach sexual maturity and at that point, female adults carry

two eggs within the ephippium located near its back. It takes an approximate 2 days for the

production of each brood, with a total of 2 to 6 broods being produced during the course of

their lifetime.

Unlike Daphnia where the brood pouch is completely enclosed, the brood pouch for Moina is

open (Rottman et al. 2003). Commonly, both Daphnia and Moina populations are dominated

by females and reproduce asexually under optimal conditions for growth. When

environmental conditions become harsh due to a shortage of food, both organisms begin to

reproduce sexually (eds Lavens & Sorgeloos 1996). Unlike Daphnia where reproduction

decreases when population densities are increased, Moina is able to maintain reproduction.

However, Moina are typically summer inhabitors and only reappears during the warmer

months from resting eggs (Anderson-Carnahan 1994).

Moina is primarily found in temporary ponds, ditches, swamps, lakes and reservoirs with

high levels of organic material, such as in stabilisation ponds (Goulden 1968). They have a

significant role in stabilisation ponds as they contribute to the decomposition process of

wastewater. Due to the high quantities of food in stabilisation ponds, large populations are

usually present (Gray 2004). These organisms are resilient and are able to tolerate poor water

quality. Ponds with pH ranging from 6.5 to 9.8 were found to contain Moina (Anderson-

Carnahan 1994). They are capable of withstanding extreme low and high levels of dissolved

oxygen, as well as extremes in water temperature ranging between 5 to 31° Celcius. Ideally,

temperatures ranging between 24 to 31° Celcius are optimal for growth and reproduction

(Rottman et al. 2003).

Under conditions where there is abundant food present, population blooms are common. This

is applicable to stabilisation ponds, where there are high organic loads (Gray 2004). Moina

feed on bacteria, phytoplankton, bacteria and organic matter, although higher levels of

consumption occur for bacteria and fungi (Rottman et al. 2003). This is due to the filtering

size of the setae (Gray 2004). They are also capable of consuming the cyanobacteria,

Microcystis aeruginosa (eds Lavens & Sorgeloos 1996). However, Moina are weaker

competitors in the presence of Daphnia, and their grazing rate is reduced by up to 3 times

under cohabitation (Anderson-Carnahan 1994). The filtering setae of Daphnia and Moina are

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also comparative, with Daphnia having larger setae. This makes Moina a more efficient

bacteria feeder compared to Daphnia, which primarily feeds on algae (Gray 2004).

Although Moina are resilient to some extremes in water conditions, they are particularly

sensitive to toxic materials including pesticides, metals, detergents and bleaches (Rottman et

al. 2003). As hydrogen peroxide is commonly used for bleaching, using Moina for toxicity

testing would give an indication of the level of sensitivity. Due to the habitation of Moina in

stabilisation ponds, the effects of hydrogen peroxide on Moina is required. In a study made

by Anderson-Carnahan (1994), it was found that the M. australiensis have similar sensitivity

to other cladocerans and are suitable for toxicity tests. Other toxicity studies have also been

performed using other species of Moina.

Figure 2: Photo of Moina under microscope (Zheng 2010).

2.5 Case studies of hydrogen peroxide tested on animals

Previous studies have been performed on the toxicity of hydrogen peroxide exposed to

animals. Of particular relevance is a flow-through chronic toxicity study performed by

Meinertz et al. (2007), which involves the continuous pumping of hydrogen peroxide into the

system (U.S. EPA 2002). A specially formulated hydrogen peroxide product was proposed

for use by the U.S. aquaculture to treat infectious fungal organisms but approval for

environmental safety towards aquatic organisms was required before it could be utilised.

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Daphnia magna was used as a test organism to assess the risks posed to aquatic invertebrates.

The effects on survival, growth, production and gender ratio were examined. The

concentrations tested ranged from 0.32 to 5.0 mg/L and were tested on juvenile Daphnia.

Results showed that at concentrations lower than 1.25 mg/L, mortality was not affected.

Concentrations below 0.63 mg/L did not affect the brood production and concentrations

lower than 0.32 mg/L did not affect the growth.

In another study made by Rach et al. (1997), the toxicity of hydrogen peroxide to fish was

tested. Hydrogen peroxide was found to exhibit the properties to treat fungi infected fish and

fish eggs, but the effects of hydrogen peroxide on other fish present were unknown. An acute

toxicity test was performed on three fish species and concentrations of hydrogen peroxide

ranging between 100 to 5000 µL/L. Results showed that different species of fish had varied

levels of tolerance to the toxicity of hydrogen peroxide. Larger fish were more sensitive to

hydrogen peroxide and toxicity increased with increased water temperature.

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3. Methodology

3.1 Collection and culturing of test species

Moina sp. was caught from the Wundowie stabilisation pond, while Daphnia sp. was caught

from the lake in Sir James Mitchell Park. This was achieved through the use of a 250 µm

plankton net, which was thrown into the water body and slowly pulled back to shore. Trapped

species and material was released into prepared containers and rinsed with distilled water for

transport, as shown in figure 3. The containers were transported with care to avoid premature

deaths through vigorous water movements. The Daphnia sp. and Moina sp. were immediately

separated from other organisms and transferred into filtered lake water prepared upon arrival

at the laboratory.

Figure 3: Plankton net used to sieve Daphnia and Moina from waterbodies and transferred into

corresponding containers (Zheng 2010).

Daphnia and Moina are capable of reproducing asexually through parthogenesis (Zaffagnini

1987, p 245), and because of this, a single Daphnia sp. and Moina sp. was selected for

cloning to ensure the genetic material of the offspring is the same. This was achieved by

placing individual organisms into separate jars for observation and the healthiest organism

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was chosen for mass culturing for experimentation. By creating a culture from the same

strain, the responses to toxins between the offspring are expected to be similar. Genetic

variation is prevalent for many traits in Cladocera and by using organisms containing the

same genetic makeup for testing, variation in results between tests would be less significant.

This is particularly important for testing chemicals at different concentrations (Ebert 2005).

There are many suitable mediums for culturing Daphnia and Moina. Filtered lake water was

primarily chosen because it mimics the natural water conditions that Daphnia and Moina are

accustomed to (Rottman et al. 2003). The cultures were kept under a constant temperature of

21 degrees Celsius and were transferred into larger containers once it became congested. To

keep the cultures growing comfortably, no more than 40 Daphnia and 50 Moina were kept

per litre of water.

Daphnia primarily feeds on algae (Lampert 1987, p 176), while Moina is a bacteria strainer

(Gray 2004). A variety of foods are recommended for feeding Cladocera, including yeast,

algae and fertilizer. Fertiliser is recommended for culturing Moina as it is rich in organic

matter and bacteria (Rottman et al. 2003). Besides requiring care when handling, Daphnia is

also not as adapted for consumption of bacteria. Since green algae is a suitable source of food

for both Daphnia and Moina, it was cultured in the laboratory as food supply for the duration

of the study.

Desmodesmus sp. (CSIRO strain CS-899) is a type of green algae commonly used as a food

source for water fleas. This was cultured in the laboratory under constant conditions of 21

degrees Celsius and daily exposure of 12 hours fluorescent lighting, as recommended by

Anderson (2005) (shown in figure 4). WC medium, adapted from Guillard and Lorenzen

(1972) as shown in appendix A, was added regularly under aseptic conditions to the algae

cultures for nutrient supply. The cultures were also aerated to promote growth (Anderson

2005).

Methodology

15 | P a g e

Figure 4: Desmodesmus cultures in the laboratory (Zheng 2010).

Daphnia has a filtering rate of 20ml per day, and has an optimal feeding concentration of 0.2

mg C/L (Lampert 1987, p 157). To predict the amount of carbon present in Desmodesmus, a

calibration curve as shown in figure 5 was developed (as shown in appendix A). This was

achieved by initially measuring the absorption of several Desmodesmus dilutions using the

spectrophotometer, filtering the dilutions made and processing it through the Elementar vario

MACRO. This is an instrument designed for the determination of carbon, nitrogen and

oxygen content. The results produced gave an indication of the relationship between light

absorption of Desmodesmus with respect to carbon content. From this, the volume of algae

required to achieve 1mg C could be found. Using the calibration curve, the absorption value

found from the spectrophotometer is able to provide an expected carbon content value. The

water fleas could then be fed accordingly.

Methodology

16 | P a g e

Figure 5: Calibration curve for Desmodesmus, showing the volume required to achieve 1 mg C.

Due to the high sensitivity of Daphnia and Moina to toxins, care was taken to not introduce

toxins to the cultures. Several cultures were created in case of contamination. Cultures were

maintained through regular feeding and occasional water change. Water changes refreshed

and aerated the cultures, which renewed the dissolved oxygen present. The cultures were also

shaded to avoid strong light penetration as intense light was unfavoured by Daphnia and

Moina. When the cultures grew to a substantial size, declines in growth and reproduction may

occur (Peters 1987, p. 491). As recommended by Rottman et al. (2003), new cultures were

created in a fresh container to prevent overcrowding.

3.2 Experimental design

An acute toxicity test was adapted to observe the effects of Daphnia sp. and Moina sp. when

both species were exposed to a range of hydrogen peroxide concentrations. The focus of the

test was to assess the survivorship of Daphnia and Moina after exposure to hydrogen

peroxide. Due to the ability of hydrogen peroxide to rapidly break down into oxygen and

water (Jones 1999), the effects in the long term are not as apparent. Hence, a chronic toxicity

test is not required. An acute toxicity test not only requires a shorter period of time to run, it

is also a more cost effective approach for the purposes of this study. Results obtained would

y = 5.4024x-1.031

R² = 0.9873

0

50

100

150

200

250

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Vo

lum

e r

eq

uir

ed

fo

r 1

mg

C (

ml)

Absorption

Methodology

17 | P a g e

give an immediate indication of the degree that hydrogen peroxide affects the organisms

tested and allow for estimation of the risk assessment parameters. Consequently, adverse

effects observed would indicate that the biological functioning of stabilisation ponds may be

affected through the application of hydrogen peroxide.

A static non-renewal procedure was adapted to observe the initial response of Daphnia and

Moina. Static non-renewal tests expose test organisms only once to the chemical being tested

for the entire length of the test, while static renewal tests are exposed to a new dose of the

chemical at regular intervals (U.S. EPA 1994). Flow through tests exposes test organisms to a

chemical continuously (Meinertz 2008). Choice depends on the nature of the chemical used

for testing. In practice, reapplication of hydrogen peroxide is required to continuously treat

the stabilisation ponds but since hydrogen peroxide breaks down readily, a static non-renewal

procedure is sufficient.

A definitive procedure was used to assess the extent of toxicity in a particular sample through

serial dilutions. A dose-response relationship can be observed through this test and it is

particularly useful for determining toxic thresholds for regulation purposes (Anderson-

Carnahan 2004). This is typically used in acute toxicity tests (U.S. EPA 2002). The U.S. EPA

(2002) recommends that a toxicity range finding test be used as a first step, which tests

concentrations of increasing order of magnitude to determine a range of concentrations where

the aquatic species tested begins to show a response. For this test, 5 widely spaced dilutions

are required over a period of 8 to 24 hours.

The rate of degradation of hydrogen peroxide is affected by factors including temperature,

sunlight, pH and impurities (Jones 1999). To keep the results constant, all experiment sets

were performed under the same conditions. The temperature was maintained at 21° Celcius,

the optimal temperature for water fleas, and shadowed fluorescent lighting. At least 2 days

before experimentation, a known volume of filtered lake water was prepared for individual

test chambers where 20 adult species were transferred to, as recommended by Adams &

Rowland (2003, p 22). Any test species which have died prior to experimentation were

replaced. In each chamber, 1mg C of Desmodesmus was injected daily.

Methodology

18 | P a g e

In the study by Barrington & Ghadouani (2008), the lowest hydrogen peroxide dose to cause

significant exponential decay for phytoplankton groups was found to be 3.0×10-3

g hydrogen

peroxide/µg phytoplankton chlorophyll-a. The initial chlorophyll concentration was 99.8

µg/L (Ms D Barrington 2009 pers. comm., 9 November) and after conversion, it was found

that the optimum dose for inducing death in cyanobacteria is 0.296 g/L. Field scale trials

required only 0.04 g hydrogen peroxide/L water to create the same effect due to AOP from

solar radiation (Ms D Barrington 2009 pers. comm., 9 November). Another study has shown

that continuous exposure of hydrogen peroxide in a flow through experiment did not result in

increased mortality through a chronic toxicity test at 0.125 g/L (Meinertz et al. 2008).

Since Meinertz et al. (2008) found 0.125 g/L of hydrogen peroxide concentrations did not

result in increased mortalities in a flow through test, although flow through tests typically

produce higher concentration endpoints than acute based tests. The initial concentrations used

for the range finding test were 0.0125 g/L, 0.125 g/L, 1.25 g/L, 12.5 g/L and 125 g/L. For

each concentration tested, there were three replicates and a control to ensure resulting

mortality is due to exposure to hydrogen peroxide concentrations alone. The volume of

hydrogen peroxide required for application was calculated through serial dilution calculations

and applied using automatic pipettes. The corresponding volumes measured were injected

into each chamber and slowly mixed. The experiment set up is as shown in figure 6.

Figure 6: Experiment setup (Zheng 2010).

Methodology

19 | P a g e

Each experiment set was run for 48 hours as a result of the short term nature of the test. As

the expected effects after application are immediate, observations for survival were made

every hour for the first 6 hours, at least every 3 hours for the first 12 hours and finally at 24

and 48 hours. After conducting the range finding test, subsequent concentrations could then

be applied to further refine the range and obtain a value for the NOAEC and LC50. This

procedure was applied to both Daphnia and Moina.

3.3 Data analysis

In toxicology, the estimation of the risk assessment parameters would help determine the

level of toxicity of a compound. The NOAEC is a useful estimate showing the concentration

by which the chemical does not affect the organism in any unfavourable way. In this study,

the adverse effects observed were denoted by changes to survival. The NOAEC was found by

continuous application of concentrations to create a range where no adverse effects were

observed to a concentration where mortality rates increased.

Another risk assessment parameter used to indicate toxicity is the LC50, the concentration

where 50% of deaths occur after dosage. This was also determined through graphical

prediction after testing numerous concentrations. Although there are several other methods

which can be used to predict the LC50, prediction through graphical means is the simplest,

particularly with a thorough data set from testing numerous concentrations. When the

NOAEC and LC50 are compared, the difference in concentration would give an indication of

the significance that increased exposure to the chemical may have.

In survival analysis, the Kaplan-Meier method is commonly used to analyse survival data and

is particularly useful when censored data is included. The variables considered include the

survival time, the event of failure and censored data. Survival time refers to the time until

death occurs and the event of failure refers to the occurrence of death in this particular case.

Censoring refers to data where the true survival time is unknown due to survivors being

present at the time of conclusion of the test. The use of the Kaplan Meier methods predicts

the survival function (figure 7), which estimates the probability that an organism’s survival

time exceeds that of a specified time. In theory, at timestep t=0, the survival probability is 1

but as the timestep reaches infinity, the survival function would decrease towards survival

Methodology

20 | P a g e

probability of 0. In practice, the survival function predicted through data collected is shown

in step functions. Survival curves show a statistical viewpoint of the probability of survival

with respect to time (Kleinbaum & Klein 2005).

Figure 7: Theoretical survival function. At timestep t=0, survival probability is 1.

JMP is a powerful statistical software which has previously been used by Oberhaus et al.

(2007) in the analysis of survivorship. This statistical tool has the capability of performing

survival analysis and through the use of this program, the survival curves for each

concentration of hydrogen peroxide exposed to Daphnia and Moina were plotted for analysis

and interpretation.

Results

21 | P a g e

4. Results

Results from the range finding test showed that both Daphnia sp. and Moina sp. were highly

sensitive to hydrogen peroxide. After 48 hours from the initial hydrogen peroxide dosing, it

was found that there were no survivors for 0.0125 g/L, the lowest concentration tested, for

either species. Further doses of lower concentrations were applied to both species to predict

the NOAEC and LC50 through graphical methods.

In figure 8, all of the concentrations trialed for Daphnia sp. are as shown. It can be observed

that most of the higher concentrations resulted in rapid mortality. Concentrations as high as

125 g/L resulted in 100% mortality within the first hour. As the strength of hydrogen

peroxide applied decreased, a longer period of time was taken before complete mortality was

reached. When concentrations of 0.008 g/L and lower were tested, a positive survival

response was obtained for the time period tested. The LC50 was predicted by matching the

50% survival response to the 48 hour time period when the test ended. From this, an LC50 of

0.007 g/L at the corresponding time period was estimated for Daphnia. At concentrations of

0.002 g/L and lower, it was found to have a 100% survival response. This concentration can

then be deduced as the NOAEC.

Figure 9 shows the concentrations tested on Moina sp. All concentrations tested that were

higher than 0.002 g/L resulted in rapid mortality, particularly concentrations above 0.003 g/L.

At 0.002 g/L, the rate of survival ranged between 40% to 60%. It can then be deduced that

the LC50 for Moina is 0.002 g/L in a 48 hour period. The NOAEC can be predicted to be

0.0015 g/L as the survivorship did not change from the application of hydrogen peroxide. The

difference in concentration between the LC50 and the NOAEC is only slight, indicating that

Moina is highly sensitive to the concentration of hydrogen peroxide applied. These values

can be confirmed through survival analysis.

Comparing figures 8 and 9, it is clear that Daphnia has a higher resilience in comparison to

Moina. In figure 8, it can be observed that a small decrease in strength of concentration

would increase the survival response. Compared to figure 9, most of the concentrations tested

on Moina resulted in complete mortality within 12 hours of applying the dose. The survival

response time for Daphnia improved gradually with lower concentrations applied, whereas

Results

22 | P a g e

for Moina, the survival response time did not show drastic improvements when lower

concentrations were applied. Comparing the NOAEC value with the LC50, the difference for

Moina is very small whereas for Daphnia, there is a larger difference in concentration. This

shows that Moina is highly sensitive to hydrogen peroxide and at the point where any effect

is found, high mortality rates are already observed. Daphnia however, is not as strongly

affected as Moina.

Figure 8: Survival response of Daphnia when different concentrations of hydrogen peroxide

were added.

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40 45

Su

rviv

al

(%)

Time (hr)

0.001 g/L Trial 1 0.001 g/L Trial 2 0.001 g/L Trial 3 0.002 g/L Trial 1











125 g/L Trial 1 125 g/L Trial 2 125 g/L Trial 3

Results

23 | P a g e

Figure 9: Survival response of Moina when different concentrations of hydrogen peroxide were

added.

As the results obtained showed a surprisingly low NOAEC for both species, the experiment

was repeated for certain concentrations. Bottles used in the tests were labeled and randomized

before application to ensure that the results are not influenced by any unknown factors.

Yielded results showed no significant changes between original results obtained.

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40 45

Su

rviv

al

(%)

Time (hr)

0.001 g/L Trial 1 0.001 g/L Trial 2 0.001 g/L Trial 3 0.0015 g/L Trial 10.0015 g/L Trial 2 0.0015 g/LTrial 3 0.002 g/L Trial 1 0.002 g/L Trial 20.002 g/L Trial 3 0.003 g/L Trial 1 0.003 g/L Trial 2 0.003 g/L Trial 30.004 g/L Trial 1 0.004 g/L Trial 2 0.004 g/L Trial 3 0.005 g/L Trial 10.005 g/L Trial 2 0.005 g/L Trial 3 0.0075 g/L Trial 1 0.0075 g/L Trial 20.0075 g/L Trial 3 0.01 g/L Trial 1 0.01 g/L Trial 2 0.01 g/L Trial 30.0125 g/L Trial 1 0.0125 g/L Trial 2 0.0125 g/L Trial 3 0.06875 g/L Trial 10.06875 g/L Trial 2 0.06875 g/L Trial 3 0.125 g/L Trial 1 0.125 g/L Trial 20.125 g/L Trial 3 12.5 g/L Trial 1 12.5 g/L Trial 2 12.5 g/L Trial 3125 g/L Trial 1 125 g/L Trial 2 125 g/L Trial 3

Results

24 | P a g e

Using the statistical tool JMP, survival curves were obtained for selective concentrations

which were of significance. The concentrations of hydrogen peroxide analysed include 0.002

g/L, 0.005 g/L, 0.0125 g/L, 0.125 g/L and 1.25 g/L. The survival curves for these

concentrations are shown in figures 10 to 15 and are estimates of the true distribution.

Because surviving organisms beyond the duration of the test is considered during analysis,

the predicted curves are particularly useful.

Tables 1, 2, 4, 5, 7, 8, 10, 11, 13 and 14 shows the statistical analysis of the data obtained for

Daphnia and Moina. The survival probability indicates the chance of survival at the given

time and the survival standard error shows the uncertainty of survival. It is a measure of the

distribution error that a given value varies from the actual value. Comparing the standard

error for all the concentrations for both Daphnia and Moina, the values did not vary much

and were relatively low. Since the sample size is large, the distribution can be assumed

normal and the confidence interval is calculated as follows:

[Sample mean – (1.96 x Standard error), Sample mean + (1.96 x Standard error)]

The confidence interval shows the range that 95% of the true population lies (Petrie & Sabin

2009). Using the final survival standard error value in the statistical analysis tables, the

confidence interval can be calculated. The time by which mortality is predicted to occur for

each concentration is predicted to have a 95% confidence within the confidence interval

range.

For the concentration 0.002 g/L tested (figure 10), the probability of survival is evident. For

Daphnia sp., over 95% survival is evident past the 48 hour period of testing. Between 24 to

48 hours, the survival probability was relatively constant. This is an indication that at 0.002

g/L, hydrogen peroxide does not affect Daphnia survival in any significant way. The

NOAEC can therefore be predicted as 0.002 g/L. Comparing to Moina sp., only 50% remain

surviving at the conclusion of the testing period. A significant decrease in survival can be

observed at the 24 hours. The LC50 for Moina can be predicted as 0.002 g/L.

Results

25 | P a g e

Figure 10: Survival curve of Daphnia and Moina for 0.002 g/L hydrogen peroxide tested.

Statistical analysis at 0.002 g/L is summarized in table 1 and 2. Mortality is not evident until

24 hours for Daphnia, whereas Moina showed failure by 12 hours into the experiment. The

censored data represents the number of organisms still surviving after the conclusion of the

test. From the tables, it is can be seen that 57 Daphnia survived whereas only 30 Moina

survived past the test.

Table 1: Statistical analysis of Daphnia for 0.002 g/L hydrogen peroxide tested.

Time

(hr)

Survival

probability

Failure

probability

Survival

standard error

N

Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

24 0.983 0.017 0.017 1 0 60

48 0.950 0.050 0.028 2 57 59

Table 2: Statistical analysis of Moina for 0.002 g/L hydrogen peroxide tested.

Time

(hr)

Survival

probability

Failure

probability

Survival

standard error

N

Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

12 0.983 0.017 0.017 1 0 60

24 0.800 0.200 0.052 11 0 59

48 0.500 0.500 0.065 18 30 48

Results

26 | P a g e


between Daphnia and Moina for 0.002 g/L hydrogen peroxide tested.

Mean Standard deviation Confidence interval

Daphnia 47.9 0.486 [47.845, 47.955]

Moina 45.3 0.725 [45.173, 45.437]

There is a more distinct change in survival rate when 0.005 g/L was tested (figure 11).

Daphnia survival was over 50% at the end of the test but Moina survival rapidly decreased,

particularly during the 9 hour time period. The LC50 for Daphnia can be predicted to be over

0.005 g/L through this step curve.



Time

(hr)

Survival

probability

Failure

probability

Survival

standard error N Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

10 0.867 0.133 0.044 8 0 60

12 0.667 0.333 0.061 12 0 52

24 0.567 0.433 0.064 6 0 40

48 0.533 0.467 0.064 2 32 34

Results

27 | P a g e


Time

(hr)

Survival

probability

Failure

probability

Survival

standard error

N

Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

4 0.800 0.200 0.052 12 0 60

5 0.617 0.383 0.063 11 0 48

6 0.550 0.450 0.064 4 0 37

9 0.100 0.900 0.039 27 0 33

12 0 1 0 6 0 6




Daphnia 33.333 2.255 [33.205, 33.455]

Moina 7.367 0.339 [7.291, 7.443]

At 0.0125 g/L, both Daphnia and Moina had no survivors by the end of the test period (figure

12). The survival curve for 0.005 g/L and 0.0125 g/L were very similar for Moina. This

indicates that Moina survival is not improved with decreased concentration until the point

where no adverse effects are observed at all. Daphnia however, showed a consistent decrease

in survival with increasing concentration.


Results

28 | P a g e


Time

(hr)

Survival

probability

Failure

probability

Survival

standard error

N

Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

3 0.983 0.017 0.017 1 0 60

4 0.883 0.117 0.041 6 0 59

5 0.850 0.150 0.046 2 0 53

6 0.800 0.200 0.052 3 0 51

9 0.667 0.333 0.061 8 0 48

12 0.567 0.433 0.064 6 0 40

24 0.217 0.783 0.053 21 0 34

48 0 1 0 13 0 13


Time

(hr)

Survival

probability

Failure

probability

Survival

standard error

N

Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

3 0.950 0.050 0.028 3 0 60

4 0.850 0.150 0.046 6 0 57

5 0.467 0.533 0.064 23 0 51

6 0.217 0.783 0.053 15 0 28

9 0 1 0 13 0 13

Table 9: Comparison of survival, standard deviation and confidence intervals between Daphnia

and Moina for 0.0125 g/L hydrogen peroxide tested.

The survival curve for 0.125 g/L (figure 10) shows that complete mortality occurred within

12 hours of the test. Rapid decreases to survival occurred early within the testing period.


Daphnia 22.117 2.022 [21.992, 22.242]

Moina 5.917 0.232 [5.792,6.042]

Results

29 | P a g e



Time

(hr)

Survival

probability

Failure

probability

Survival

standard error

N

Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

1 0.933 0.067 0.032 4 0 60

2 0.917 0.083 0.036 1 0 56

3 0.833 0.167 0.048 5 0 55

4 0.717 0.283 0.058 7 0 50

5 0.700 0.300 0.059 1 0 43

6 0.533 0.467 0.064 10 0 42

9 0.183 0.817 0.050 21 0 32

12 0 1 0 11 0 11


Time

(hr)

Survival

probability

Failure

probability

Survival

standard error

N

Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

1 0.850 0.150 0.046 9 0 60

2 0.483 0.517 0.065 22 0 51

3 0.117 0.883 0.041 22 0 29

4 0.017 0.983 0.017 6 0 7

5 0 1 0 1 0 1

Results

30 | P a g e



At 1.25 g/L, the concentration is highly toxic for Daphnia and Moina. Complete mortality for

Moina occurred within 2 hours of testing but Daphnia showed higher resilience to hydrogen

peroxide and lasted a further 3 hours before complete mortality occurred.



Time

(hr)

Survival

probability

Failure

probability

Survival

standard error

N

Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

1 0.733 0.267 0.057 16 0 60

2 0.683 0.317 0.060 3 0 44

3 0.433 0.567 0.064 15 0 41

4 0.083 0.917 0.036 21 0 26

5 0 1 0 5 0 5


Daphnia 7.25 0.439 [7.125, 7.375]

Moina 2.467 0.120 [2.340, 2.594]

Results

31 | P a g e


Time

(hr)

Survival

probability

Failure

probability

Survival

standard error

N

Failed

N

Censored

At

Risk

0 1 0 0 0 0 60

1 0.15 0.85 0.046 51 0 60

2 0 1 0 9 0 9




Daphnia 7.25 0.439 [7.125, 7.375]

Moina 2.467 0.120 [2.377, 2.557]

Discussion

32 | P a g e

5. Discussion

Hydrogen peroxide is highly toxic, particularly in AOPs. Mortality may have resulted

through various reasons. Hydroxyl radicals produced from hydrogen peroxide may induce

cell damage, and acids produced may result in tissue damage due to its toxicity. Excess

oxygen gas produced after the hydrogen peroxide is ingested and degrades may also cause

embolisation, the blocking of a vein (Forman 2008). The high toxicity of hydrogen peroxide

is evident from the results obtained in this study. All concentrations tested on Moina with a

hydrogen peroxide concentration greater than 0.002 g/L resulted in the complete mortality

within the first 12 hours of testing. Further increases in strength of hydrogen peroxide did not

change the time of survival significantly. Unlike Moina, Daphnia survival response was

found to show a greater response to changes in concentration. Increased strength of hydrogen

peroxide dosed resulted in a gradual decreased time before reaching complete mortality.

Moina is much more sensitive than Daphnia, in that a range of concentrations higher than the

NOAEC would result in a similar effect. This is evident through the small difference between

the NOAEC and the LC50. Complete mortality within the same short period of time would

occur for concentrations as low as 0.005 g/L and as high as 0.125 g/L.

The differences causing the varied sensitivity between Daphnia and Moina could be due to a

number of factors. Although both Daphnia and Moina are closely related, they are of a

different genus. The biological differences may have an influence on the sensitivity.

Compared to Daphnia, Moina are much smaller in size so it may be that the free radicals

produced from hydrogen peroxide are able to cause tissue damage in a shorter period of time.

It has been mentioned by Rottman et al. (2003) that Moina are particularly sensitive to toxic

materials such as bleaches. Hydrogen peroxide is often used for bleaching, and as evident

from results, Moina’s sensitivity towards toxic substances has been confirmed.

Under laboratory conditions, 0.299 g/L hydrogen peroxide is the optimal dosage

recommended to effectively induce cyanobacteria death (Barrington & Ghadouani 2008). The

ideal field dosage was recommended to be 0.04 g/L hydrogen peroxide (Ms D Barrington

2009 pers. comm., 9 November). The results show that if Daphnia and Moina are exposed to

the laboratory dosage of 0.299 g/L, complete mortality is certain. However, this does not

mean that hydrogen peroxide is not suitable for treatment of stabilisation ponds. The risk

Discussion

33 | P a g e

assessment parameters obtained in this study are likely to have been underestimated for use

on site.

Field conditions are different from laboratory conditions, and the data obtained through the

toxicity test cannot be applied directly on site. In the laboratory, the tests were performed in

strictly constant conditions to ensure the results obtained are due to the application of

hydrogen peroxide only. In the field, mixing in the water due to wind and stratification has

not been accounted for in the laboratory experiments. Stratification occurs from variation in

the vertical profiles of factors including water temperature, dissolved oxygen and pH are all

contributing factors to movement of wastewater in the stabilisation ponds. There is also the

direct inflow and outflow of the treated wastewater (Gu 1995). Because stabilisation ponds

are also quite big, the natural response for Daphnia and Moina is to move away from the

hydrogen peroxide. In the laboratory experiments conducted, the Daphnia and Moina were

confined to a small volume and escape from the plume of hydrogen peroxide is not possible.

Due to the rapid degradation time of hydrogen peroxide, it is likely that the survival for

Daphnia and Moina may have been underestimated.

Other factors that may have influenced the toxicity of hydrogen peroxide must also be

considered however. The presence of metals in wastewater can cause hydroxyl radicals to be

produced, as well as UV light (Forman 2008). The toxicity of hydrogen peroxide is

significantly increased, and although this may result in higher mortality rates when tested

under laboratory conditions, in the field there are many environmental factors involved and

further field based tests would need to be conducted. Hydrogen peroxide has been found by

Jones (1999) to contribute to treatment of sewers and controlling the levels of bacteria

present. Ferrous iron reacted with hydrogen peroxide has also been found to effectively

increase degradation rates of pollutants in wastewater (Kallel 2009). Even if Daphnia and

Moina were shown to be affected adversely in the field, overpopulations are common in

stabilisation ponds. High population densities can also affect the functioning of stabilisation

ponds in an adverse way (Gray 2004).

Although filter feeders contribute to the biodegradation process of bacteria, suspended matter

and algae in stabilisation ponds, large populations within the ponds can result in adverse

effects due to the excessive feeding on algae. High levels of grazing results in reduction in

Discussion

34 | P a g e

algal photosynthesis, and consequently decreases the reaeration of the stabilisation ponds

(Gray 2004). Aerobic bacteria and other organisms existing in stabilisation ponds require

dissolved oxygen to break down waste and are crucial to the natural renewal process of

wastewater (Gloyna 1971). In the case of low dissolved oxygen levels, the productivity of the

natural purification process is adversely affected. There have been instances where control

measures, including the application of lime and the introduction of fish to feed on the

Daphnids were required to be implemented to control the population size (Gray 2004).

Besides treating cyanobacteria, hydrogen peroxide may potentially act as a measure for

controlling Cladocera populations present to increase efficiency of the stabilisation pond.

As this study was performed using an acute toxicity test, the long term effects were not

considered. In an acute test, the risk assessment parameters are only estimates due to the short

length of the test. It has been observed that by extending the length of time for the toxicity

test, the LC50 decreases and the NOAEC increases for many chemicals. In a chronic test, long

term factors including growth, reproduction, physiology and behaviour can be observed. This

gives a better scope of any adverse effects observed, as the consideration is not limited to

mortality.

Although the accuracy of the NOAEC and LC50 is increased as a result, the simulation of life

cycle tests within the laboratory is under strictly controlled conditions (U.S. EPA 2002). As

external effects from the natural environment were also not accounted for, the simulation

created is not an estimate of the lethality in the natural environment but in controlled

conditions. Chronic toxicity tests also require a substantially longer period of time to conduct

and are not always cost effective to run. In this study, a chronic toxicity test is unnecessary

due to the rapid degradation of hydrogen peroxide, although the long term effects may

indicate whether there may be decreased efficiency of the stabilisation ponds.

The use of the risk assessment parameters, NOAEC and the LC50 was criticized by

Laskowski (1995). These parameters were determined based on a sample used to represent

the population. Although the samples used for this study was large, it is not sufficient to

represent the population. Variations of effects occur for different species but certain species

may have higher resilience to toxins. The outcomes of the results may depend on the location

the species were obtained, as well as the type of species. The risk assessment parameters

Discussion

35 | P a g e

obtained are true for the particular organism tested in that environment but can only be used

as an indication. Values are only estimated, as these values cannot truly be determined. The

Kaplan-Meier method was used to analyse the data. Although this method allows

comparisons between groups, the test is limited to data involving deaths. For concentrations

where survival did not change, JMP was unable to analyse the data.

Due to the significantly low threshold concentrations obtained for both Daphnia and Moina,

the suitability for applying hydrogen peroxide to stabilisation ponds cannot be determined

until further testing. However, the study has found that Daphnia and Moina are highly

sensitive to hydrogen peroxide. Due to the wide range of applications for hydrogen peroxide,

its usage in aquatic systems in future is likely. This study provides a recommended safe

concentration for Daphnia and Moina exposure to hydrogen peroxide. This threshold can be

used as a guideline in prospective studies.

Recommendations

36 | P a g e

6. Recommendations

The results presented are based on the survivorship of the two types of water fleas but the

behaviour after being dosed with hydrogen peroxide was not taken into account. Observed

change in behaviour of the water fleas gives a more precise view of any adverse effects after

exposure to a toxin or chemical. A useful tool for detecting this is the bbe DaphTox II, which

is an instrument designed for the biomonitoring of Daphnia. The DaphTox is able to observe

daphnids under constant running water to detect any hazardous effects resulting through

chemicals or toxins present. The technology alerts when changes in behaviour occur and such

changes in behaviour are analysed through image analysis (Lechelt et al. 2000).

The immediate effects that hydrogen peroxide imposes on biological communities are

significant due to the rapid degradation of hydrogen peroxide. However, since the proposed

method of cyanobacteria treatment has long term implications, it may also be useful to

consider any long term adverse effects resulting from exposure to hydrogen peroxide. By

conducting a chronic test, factors including changes in growth, reproduction, behaviour, and

physiology would be considered. Although this may not necessarily inhibit the self-

purification ability of the stabilisation ponds completely, there may be decreased efficiency if

changes occurred to reproduction rates or food intake.

As discussed, the results produced from laboratory experiments cannot be applied directly in

the field due to the strictly controlled conditions by which the experiments were performed

under. Although the laboratory results show that the recommended dosage concentration is

toxic for Daphnia and Moina, this may not be the actual case in field conditions. To further

understand the adverse effects of hydrogen peroxide in stabilisation ponds, a field study

focusing on the biological communities is recommended. A quantitative assessment can be

performed to determine population changes and photosynthetic activity (Gray 2004). By

conducting a field based assessment, the environmental factors including temperature, wind,

and sunlight, the interaction of communities within the ponds, the scale of the pond and

mixing through stratification are accounted for. The ability of the stabilisation pond to

function without decreased efficiency the suitability for using hydrogen peroxide for

cyanobacteria treatment.

Conclusions

37 | P a g e

7. Conclusions

Exposure of hydrogen peroxide to Daphnia and Moina in an acute toxicity study was

necessary to assess any adverse effects that the use of hydrogen peroxide may have on the

biological functioning of stabilisation ponds. Through application of several concentrations

of hydrogen peroxide to the test organisms, it was found that the risk assessment parameters

estimated a concentration lower than the optimal concentration of hydrogen peroxide for

inducing cyanobacteria death under laboratory conditions for both Daphnia and Moina. The

NOAEC was found to be 0.002 g/L and 0.0015 g/L. Although this indicates the

recommended concentration of 0.269 g/L is lethal for Daphnia and Moina, conclusions

cannot be drawn that hydrogen peroxide is unsuitable for long term treatment of

cyanobacteria in stabilisation ponds.

As the study was laboratory based, the toxicity test was performed under constant conditions.

This gives an indication of the extent that Daphnia and Moina are solely affected by

hydrogen peroxide in stabilisation ponds, and other factors which may impose on the two

species were not considered. Combined environmental factors may result in an under

prediction of the risk assessment parameters. Field conditions typically vary through the day,

including temperature fluctuations, exposure to sunlight, wind and mixing. Natural response

for Daphnia and Moina is to escape from harmful substances present in the water but tests

were performed in a relatively small volume compared to the volume in stabilisation ponds.

In the stabilisation ponds, Daphnia and Moina are able to swim away from the hydrogen

peroxide. As a result, the survival response is likely to be much higher than that predicted

through laboratory experimentation.

Compared to other methods of treatment for cyanobacteria, hydrogen peroxide is evidently a

more environmentally sensitive choice due to its safe biodegradation products. Although

hydrogen peroxide is unlikely to affect the functioning of stabilisation ponds, it would be

necessary to undergo further testing on site to assess any adverse changes to water quality

when hydrogen peroxide is applied. Provided the natural purification of wastewater in

stabilisation ponds is not inhibited through the application of hydrogen peroxide, prospects

for long term use for cyanobacteria treatment in stabilisation ponds are likely. Also, Daphnia

and Moina are highly productive animals, and produce dense populations easily. High

Conclusions

38 | P a g e

populations of Cladocera are detrimental to the purification process of stabilisation ponds

(Gray 2004), and since hydrogen peroxide is lethal at low concentrations, hydrogen peroxide

may have implications for controlling Cladocera populations within stabilisation ponds.

Besides providing an indication for the suitability for using hydrogen peroxide for treatment

in stabilisation ponds, this study also provides a toxicity reference of aquatic species to

hydrogen peroxide. Due to the range of applications for using hydrogen peroxide in aquatic

systems, future plans involving hydrogen peroxide are likely. Assessment of the risks

hydrogen peroxide may have on aquatic species would be required. The sensitivity of the

aquatic bioindicators used in this study, Daphnia and Moina would contribute to decision

making for future use in aquatic systems.

References

39 | P a g e

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Appendix A

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Appendix A: Protocols

WC Medium

Adapted from Guillard and Lorenzen (1972)

Preparation of nutrient stock solution are as follows:

Macronutrients Stock solution (g/100ml)

CaCl2.H2O 3.68

MgSO4.7H2O 3.7

NaHCO3 1.26

K2HPO4.3H2O 1.14

NaNO3 8.5

Na2SiO3.9H2O 2.12

Micronutrients Stock solution (g/100ml)

CuSO4.5H2O 0.98

ZnSO4.7H2O 2.2

CoCl2.6H2O 1.0

MnCl2.4H2O 18

Na2MoO4.2H2O 0.63

H3BO3 0.1

The micronutrients working solution was prepared by adding:

i. 0.315 g FeCl3.6H2O

ii. 0.436 g Na2EDTA.2H2O

iii. 0.1 ml of each micronutrients

Vitamin stock solution was prepared in sterile 100ml bottles:

i. 0.1g/100ml Biotin

ii. 0.1g/100ml Vitamin B12

Vitamin working solution was prepared with 95 ml sterile distilled water, and dissolved with

0.02 g thiamine, 0.1 ml biotin and 0.1 ml vitamin B12 from stock solution.

WC medium is prepared by:

i. Adding 0.1 ml of all macronutrients stock solution and micronutrients working

solution to 95 ml of distilled water.

ii. Adding 0.0115 g TES buffer

iii. Bringing final volume to 99.9 ml by adding 4.2 ml distilled water

iv. Autoclave and cool to room temperature before adding 0.1 ml sterile vitamin working

solution.

Appendix A

43 | P a g e

Calibration curve for Desmodesmus sp.

Adapted from Elke Reichwaldt (2010)

Dilutions were made from original algal culture as shown:

Dilution Volume filtered

(original+water)

Volume of

original algae for

3 replicates

Volume of

water for

dilution of 3

replicates

original original 5 15 0

1:10 1 part original + 9 parts

water

50 (5+45) 15 135


water

500 (5+495) 15 1485


water

25 (5+20) 15 60


water

250 (5+245) 15 735


water

20 (4+16) 12 48


water

10 (5+5) 15 15

102 ml 2478 ml

Using the photometer, the extinction for each dilution was measured at 800nm for three

times. For each dilution (3 replicates),

i. Algae volumes were filtered through pre-combusted (550°C for 2h) and pre-weighted

GF/C filters, and rinsed with DI water.

ii. Filter valve is closed and 10ml of 1M hydrochloric acid was added.

iii. After 30 seconds, the valve was opened and rinsed with DI water.

iv. Filter paper was folded and frozen in wrapped aluminium foil

v. Filters were dried at 60°C for 24h

Samples were processed through the Elementar vario MACRO to measure the POC and plot

the calibration curve for Desmodesmus sp.

Appendix B

44 | P a g e

Appendix B: Survival data

Table 16: Survival data of Moina for hydrogen peroxide concentrations trialed.

Time (hr) 1 2 3 4 5 6 9 12 24 48

Concentration (g/L) Trial Survival (%)

0.001 1 100 100 100 100 100 100 100 100 100 100

2 100 100 100 100 100 100 100 100 100 100

3 100 100 100 100 100 100 100 100 100 100

0.0015 1 100 100 100 100 100 100 100 100 100 100

2 100 100 100 100 100 100 100 100 95 90

3 100 100 100 100 100 100 100 100 100 95

0.002 1 100 100 100 100 100 100 100 95 75 40

2 100 100 100 100 100 100 100 100 85 60

3 100 100 100 100 100 100 100 100 80 50

0.003 1 100 100 100 100 100 75 50 25 0

2 100 100 100 100 100 55 35 30 20 0

3 100 100 100 100 100 80 40 20 15 0

0.004 1 100 100 100 100 75 55 10 0

2 100 100 100 100 60 25 10 10 0

3 100 100 100 95 75 55 10 0

Appendix B

45 | P a g e

0.005 1 100 100 100 90 65 55 10 0

2 100 100 100 75 60 50 5 0

3 100 100 100 75 60 60 15 0

0.0075 1 100 100 100 95 85 40 5 0

2 100 100 100 90 65 35 5 0

3 100 100 100 95 75 40 10 0

0.01 1 100 100 100 85 75 20 10 0

2 100 100 90 85 80 25 10 0

3 100 100 95 90 70 25 15 0

0.0125 1 100 100 95 85 75 55 0

2 100 100 100 90 40 5 0

3 100 100 90 80 25 5 0

0.06875 1 100 100 75 25 10 0

2 100 95 65 10 0

3 100 100 100 100 5 0

0.125 1 85 50 10 0

2 75 35 5 0

3 95 60 20 5 0

1.25 1 25 0

Appendix B

46 | P a g e

2 10 0

3 10 0

12.5 1 0

2 0

3 0

125 1 0

2 0

3 0

Appendix B

47 | P a g e

Table 17: Survival data of Daphnia for hydrogen peroxide concentrations trialed.

Time 1 2 3 4 5 6 9 12 24 48

Concentration (g/L) Trial Survival (%)

0.001 1 100 100 100 100 100 100 100 100 100 95

2 100 100 100 100 100 100 100 100 100 100

3 100 100 100 100 100 100 100 100 100 100

0.002 1 100 100 100 100 100 100 100 100 100 95

2 100 100 100 100 100 100 100 100 95 90

3 100 100 100 100 100 100 100 100 100 100

0.003 1 100 100 100 100 100 100 100 100 100 90

2 100 100 100 100 100 100 100 100 95 80

3 100 100 100 100 100 100 100 100 100 75

0.004 1 100 100 100 100 100 100 90 75 65 55

2 100 100 100 100 100 100 100 80 75 60

3 100 100 100 100 100 100 100 75 75 55

0.005 1 100 100 100 100 100 100 90 70 55 55

2 100 100 100 100 100 100 75 65 55 55

3 100 100 100 100 100 100 95 65 60 50

0.006 1 100 100 100 95 90 85 80 80 60 50

2 100 100 100 100 100 100 75 75 50 25

Appendix B

48 | P a g e

3 100 100 100 100 100 95 60 60 55 50

0.007 1 100 100 100 100 100 90 85 75 60 40

2 100 100 100 100 95 85 65 65 50 40

3 100 100 100 100 100 85 80 60 55 50

0.008 1 100 100 95 90 90 80 80 75 55 40

2 100 100 100 100 90 75 65 65 40 25

3 100 100 100 100 100 80 70 70 50 45

0.0125 1 100 100 95 85 85 75 65 60 25 0

2 100 100 100 90 85 85 65 55 20 0

3 100 100 100 90 85 80 70 55 20 0

0.02 1 95 95 90 85 85 85 45 30 10 0

2 100 100 95 90 85 85 50 35 15 0

3 95 90 90 85 80 80 35 25 20 0

0.03 1 90 80 80 70 65 55 10 5 5 0

2 80 70 70 60 50 35 5 5 0

3 90 80 80 75 75 70 30 20 10 0

0.04 1 95 95 95 90 80 75 25 20 0 2 100 95 90 90 70 70 25 15 0

3 95 80 80 70 60 50 20 15 0

0.125 1 95 95 90 75 70 50 15 0 2 90 90 85 75 75 60 20 0

Appendix B

49 | P a g e

3 95 90 75 65 65 50 20 0

1.25 1 70 65 40 5 0 2 70 65 45 10 0

3 80 75 45 10 0

12.5 1 0 2 0

3 0

125 1 0 2 0

3 0

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