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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
i | P a g e
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
ii | P a g e
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
iii | P a g e
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
iv | P a g e
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
Introduction
1 | P a g e
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
2 | P a g e
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
3 | P a g e
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
4 | P a g e
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
5 | P a g e
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
Literature Review
6 | P a g e
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).
Literature Review
7 | P a g e
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
Literature Review
8 | P a g e
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
Literature Review
9 | P a g e
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).
Literature Review
10 | P a g e
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
Literature Review
11 | P a g e
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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12 | P a g e
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.
Methodology
13 | P a g e
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
Methodology
14 | P a g e
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
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
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
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 LC50 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
(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
Table 5: Statistical analysis of Moina for 0.005 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
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.
Results
28 | P a g e
Table 7: Statistical analysis of Daphnia for 0.0125 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
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
(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.
Mean Standard deviation Confidence interval
Daphnia 22.117 2.022 [21.992, 22.242]
Moina 5.917 0.232 [5.792,6.042]
Results
29 | P a g e
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
(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
Table 11: Statistical analysis of Moina for 0.125 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
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
Table 12: Comparison of survival mean time, standard deviation and confidence intervals
between Daphnia and Moina for 0.125 g/L hydrogen peroxide tested.
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
(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
Mean Standard deviation Confidence interval
Daphnia 7.25 0.439 [7.125, 7.375]
Moina 2.467 0.120 [2.340, 2.594]
Results
31 | P a g e
Table 14: Statistical analysis of Moina for 1.25 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
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]
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
42 | P a g e
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
1:100 1 part original + 99 parts
water
500 (5+495) 15 1485
1:5 1 part original + 4 parts
water
25 (5+20) 15 60
1:50 1 part original + 49 parts
water
250 (5+245) 15 735
1:4 1 part original + 3 parts
water
20 (4+16) 12 48
1:2 1 part original + 1 parts
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