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
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 LC 50 of 0.007 g/L. The risk assessment parameters for
Moina were lower, with an NOAEC of 0.0015 g/L and LC 50 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.
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Acknowledgements
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.
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Table of Contents
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
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Figures & Tables
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 11: Survival curve of Daphnia and Moina for 0.005 g/L hydrogen peroxide tested. .. 26 Figure 12: Survival curve of Daphnia and Moina for 0.0125 g/L hydrogen peroxide tested. 27 Figure 13: Survival curve of Daphnia and Moina for 0.125 g/L hydrogen peroxide tested. .. 29 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 5: Statistical analysis of Moina for 0.005 g/L hydrogen peroxide tested...... 27 Table 6: Comparison of survival mean time, standard deviation and confidence intervals between Daphnia and Moina for 0.005 g/L hydrogen peroxide tested...... 27 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 12: Comparison of survival mean time, standard deviation and confidence intervals between Daphnia and Moina for 0.125 g/L hydrogen peroxide tested...... 30 Table 13: Statistical analysis of Daphnia for 1.25 g/L hydrogen peroxide tested...... 30 Table 14: Statistical analysis of Moina for 1.25 g/L hydrogen peroxide tested...... 31 Table 15: Comparison of survival mean time, standard deviation and confidence intervals 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
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Introduction
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.
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Introduction
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 LC 50 . 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.
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Literature Review
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) + O 2(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
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Literature Review
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
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Literature Review
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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Literature Review
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 LC 50 , 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). 6 | P a g e
Literature Review
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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Literature Review
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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Literature Review
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). 9 | P a g e
Literature Review
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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Literature Review
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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Literature Review
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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Methodology
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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Methodology
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).
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Methodology
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.
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Methodology
250 y = 5.4024x -1.031 R² = 0.9873 200
150
100
50 Volume required for 1mg C (ml) C 1mgfor required Volume
0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 Absorption
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
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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.
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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). 18 | P a g e
Methodology
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 LC 50 . 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 LC 50 , 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 LC 50 , prediction through graphical means is the simplest, particularly with a thorough data set from testing numerous concentrations. When the
NOAEC and LC 50 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
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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.
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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 LC 50 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 LC 50 was predicted by matching the
50% survival response to the 48 hour time period when the test ended. From this, an LC 50 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 LC 50 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 LC 50 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
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for Moina , the survival response time did not show drastic improvements when lower concentrations were applied. Comparing the NOAEC value with the LC 50 , 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 .
100
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0 0 5 10 15 20 25 30 35 40 45 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 0.002 g/L Trial 2 0.003 g/L Trial 1 0.003 g/L Trial 2 0.003 g/L Trial 3 0.004 g/L Trial 1 0.004 g/L Trial 2 0.004 g/L Trial 3 0.005 g/L Trial 1 0.005 g/L Trial 2 0.005 g/L Trial 3 0.006 g/L Trial 1 0.006 g/L Trial 2 0.006 g/L Trial 3 0.007 g/L Trial 1 0.007 g/L Trial 2 0.007 g/L Trial 3 0.008 g/L Trial 1 0.008 g/L Trial 2 0.008 g/L Trial 3 0.0125 g/L Trial 1 0.0125 g/L Trial 2 0.0125 g/L Trial 3 0.02 g/L Trial 1 0.02 g/L Trial 2 0.02 g/L Trial 3 0.03 g/L Trial 1 0.03 g/L Trial 2 0.03 g/L Trial 3 0.04 g/L Trial 1 0.04 g/L Trial 2 0.04 g/L Trial 3 0.125 g/L Trial 1 0.125 g/L Trial 2 0.125 g/L Trial 3 1.25 g/L Trial 1 1.25 g/L Trial 2 1.25 g/L Trial 3 12.5 g/L Trial 1 12.5 g/L Trial 2 12.5 g/L Trial 3 125 g/L Trial 1 125 g/L Trial 2 125 g/L Trial 3
Figure 8: Survival response of Daphnia when different concentrations of hydrogen peroxide were added.
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100
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Survival Survival (%) 40
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0 0 5 10 15 20 25 30 35 40 45 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 1 0.0015 g/L Trial 2 0.0015 g/LTrial 3 0.002 g/L Trial 1 0.002 g/L Trial 2 0.002 g/L Trial 3 0.003 g/L Trial 1 0.003 g/L Trial 2 0.003 g/L Trial 3 0.004 g/L Trial 1 0.004 g/L Trial 2 0.004 g/L Trial 3 0.005 g/L Trial 1 0.005 g/L Trial 2 0.005 g/L Trial 3 0.0075 g/L Trial 1 0.0075 g/L Trial 2 0.0075 g/L Trial 3 0.01 g/L Trial 1 0.01 g/L Trial 2 0.01 g/L Trial 3 0.0125 g/L Trial 1 0.0125 g/L Trial 2 0.0125 g/L Trial 3 0.06875 g/L Trial 1 0.06875 g/L Trial 2 0.06875 g/L Trial 3 0.125 g/L Trial 1 0.125 g/L Trial 2 0.125 g/L Trial 3 12.5 g/L Trial 1 12.5 g/L Trial 2 12.5 g/L Trial 3 125 g/L Trial 1 125 g/L Trial 2 125 g/L Trial 3
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.
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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 LC 50 for Moina can be predicted as 0.002 g/L.
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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 Survival Failure Survival N N At (hr) probability probability standard error Failed Censored 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 Survival Failure Survival N N At (hr) probability probability standard error Failed Censored 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
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Table 3: Comparison of survival mean time, standard deviation and confidence intervals 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 LC 50 for Daphnia can be predicted to be over 0.005 g/L through this step curve.
Figure 11: Survival curve of Daphnia and Moina for 0.005 g/L hydrogen peroxide tested.
Table 4: Statistical analysis of Daphnia for 0.005 g/L hydrogen peroxide tested.
Time Survival Failure Survival N At (hr) probability probability standard error N Failed Censored 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
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Table 5: Statistical analysis of Moina for 0.005 g/L hydrogen peroxide tested.
Time Survival Failure Survival N N At (hr) probability probability standard error Failed Censored 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
Table 6: Comparison of survival mean time, standard deviation and confidence intervals between Daphnia and Moina for 0.005 g/L hydrogen peroxide tested.
Mean Standard deviation Confidence interval 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.
Figure 12: Survival curve of Daphnia and Moina for 0.0125 g/L hydrogen peroxide tested.
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Table 7: Statistical analysis of Daphnia for 0.0125 g/L hydrogen peroxide tested.
Time Survival Failure Survival N N At (hr) probability probability standard error Failed Censored 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
Table 8: Statistical analysis of Moina for 0.0125 g/L hydrogen peroxide tested.
Time Survival Failure Survival N N At (hr) probability probability standard error Failed Censored 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.
Mean Standard deviation Confidence interval Daphnia 22.117 2.022 [21.992, 22.242] Moina 5.917 0.232 [5.792,6.042]
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.
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Figure 13: Survival curve of Daphnia and Moina for 0.125 g/L hydrogen peroxide tested.
Table 10: Statistical analysis of Daphnia for 0.125 g/L hydrogen peroxide tested.
Time Survival Failure Survival N N At (hr) probability probability standard error Failed Censored 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
Table 11: Statistical analysis of Moina for 0.125 g/L hydrogen peroxide tested.
Time Survival Failure Survival N N At (hr) probability probability standard error Failed Censored 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
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Table 12: Comparison of survival mean time, standard deviation and confidence intervals between Daphnia and Moina for 0.125 g/L hydrogen peroxide tested.
Mean Standard deviation Confidence interval Daphnia 7.25 0.439 [7.125, 7.375] Moina 2.467 0.120 [2.340, 2.594]
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.
Figure 14: Survival curve of Daphnia and Moina for 1.25 g/L hydrogen peroxide tested.
Table 13: Statistical analysis of Daphnia for 1.25 g/L hydrogen peroxide tested. Time Survival Failure Survival N N At (hr) probability probability standard error Failed Censored 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
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Table 14: Statistical analysis of Moina for 1.25 g/L hydrogen peroxide tested.
Time Survival Failure Survival N N At (hr) probability probability standard error Failed Censored 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
Table 15: Comparison of survival mean time, standard deviation and confidence intervals between Daphnia and Moina for 1.25 g/L hydrogen peroxide tested.
Mean Standard deviation Confidence interval Daphnia 7.25 0.439 [7.125, 7.375] Moina 2.467 0.120 [2.377, 2.557]
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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 LC 50 . 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
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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
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Discussion
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 LC 50 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 LC 50 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 LC 50 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
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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.
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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.
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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
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Conclusions
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.
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References
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Appendix A
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 1.1 4 NaNO3 8.5 Na2SiO3.9H2O 2.12
Micronutrients Stock solution (g/100ml) CuSO4.5H2O 0.9 8 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.
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Appendix A
Calibration curve for Desmodesmus sp. Adapted from Elke Reichwaldt (2010) Dilutions were made from original algal culture as shown: Dilution Volume filtered Volume of Volume of (original+water) original algae for water for 3 replicates dilution of 3 replicates original original 5 15 0 1:10 1 part original + 9 parts 50 (5+45) 15 135 water 1:100 1 part original + 99 parts 500 (5+495) 15 1485 water 1:5 1 part original + 4 parts 25 (5+20) 15 60 water 1:50 1 part original + 49 parts 250 (5+245) 15 735 water 1:4 1 part original + 3 parts 20 (4+16) 12 48 water 1:2 1 part original + 1 parts 10 (5+5) 15 15 water 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.
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Appendix B
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
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Appendix B
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
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Appendix B
2 10 0 3 10 0
12.5 1 0 2 0 3 0
125 1 0 2 0 3 0
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Appendix B
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
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Appendix B
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
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Appendix B
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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