Hydrogen Peroxide treatments of cyanobacterial blooms

Hydrogen Peroxide treatments of cyanobacterial blooms

A laboratory study on the effect of hydrogen peroxide

treatments on Microcystis and Dolichospermum at different light

intensities

Phaedra Hernández, Future Planet Studies Bsc, Biology major, University of Amsterdam, The Netherlands

Phaedra Hernández Bachelors thesis

Institute for Biodiversity and Ecosystem Dynamics University of Amsterdam

The Netherlands

Abstract

Harmful cyanobacterial blooms are proliferating as a result of eutrophication and climate change, and they pose a threat to the surrounding ecosystem as well as freshwater resources used for drinking water and agricultural purposes. Hydrogen peroxide treatments are suggested as a selective and sustainable method for harmful algal bloom mitigation. In this research, a laboratory experiment is conducted to test the effect of hydrogen peroxide treatments on Microcystis

aeru-ginosa and Dolichospermum flos-aquae. The

effective-ness of hydrogen peroxide as a treatment for algal blooms is dependent on irradiance. Therefore,

Microcystis and Dolichospermum were treated with a

range of hydrogen peroxide concentrations in high and low light intensity. The optical density, the photo-synthetic yield and the degradation of hydrogen peroxide during the experiment was measured to present a minimum dosage of hydrogen peroxide needed for the treatments to be successful. In this experiment, Microcystis is more sensitive to hydrogen peroxide and need lower dosage compared to

Dolichospermum and high light intensities increase the

toxicity of hydrogen peroxide for both species. Supervisors: mw. dr. Petra Visser, Nikoletta Tsiarta University of Amsterdam, The Netherlands Examiner:

prof. dr. Jef Huisman University of Amsterdam, The Netherlands

Introduction

Rapid growth of cyanobacteria, caused by eutrophication of freshwater systems and climate change is a threat to water quality worldwide (Paerl & Huisman, 2009; (Paerl and Huisman, 2008). Not only are these cyanobacterial blooms potentially toxic to humans and other species in the surrounding ecosystem, but they also alter the food web by dominating the ecosystems they bloom in, increase water turbidity and suppress the growth of other phytoplankton that form the base of the food web (Scheffer 1998; Schuurmans et. al, 2018). It is suggested that cyanobacteria have a competitive advantage over other species due to their higher affinity uptake and storage capabilities for Nitrogen (N) and Phosphorus (P) which are usually limiting nutrients. Also, the ability of some species to fix nitrogen enhances their abilities to survive, even when Nitrogen levels in the water are low (Flores & Herrero, 2005). Microcystis aeruginosa is a species of cyanobacteria often associated with high levels of toxins and known to cause harmful algal blooms (Chorus, 2012). The filamentous Dolichospermum flos-aquae is an example of a Nitrogen-fixing species known to cause harmful algal blooms (Li et al., 2016). In addition to the potential health effects and food web alteration, the economic losses due to the degradation of water resources such as drinking water and agricultural resources caused by cyanobacteria proliferation, have pushed a need for sustainable, selective and cost effective mitigation options (Dodds et al., 2009).

At IBED, a new method using hydrogen peroxide (H2O2) has been developed that allows for selective killing of cyanobacteria in a natural phytoplankton population leaving other phytoplankton, zooplankton and higher life-forms largely undisturbed (Drábková et al., 2007; Matthijs et. al., 2012). H2O2 can be found naturally in aquatic environments as a by-product of oxygenic photosynthesis, and many organisms possess enzymes to break down this component (Häkkinen et al. 2004; Quimby et. al, 1988). However, there are indications that cyanobacteria are far more susceptible to H2O2 than other species because they do not are less capable of breaking down H2O2 (Drábková, et. al., 2007).

An advantage of this method is the fact that H2O2 is not expected to lead to chemical residual accumulation because of its rapid natural decomposition. An example of this is the enzymatic degradation of H2O2 to O2 and H2O accompanied

by the production of hydroxyl radicals under light exposure, which are the cause for algal cell mortality (Yang et. al, 2018). Therefore, the effectiveness of H2O2 as a treatment for cyanobacterial blooms is dependent on irradiance, among other things. According to Drábková, Admiraal & Maršálek (2007), high light intensities increase the toxicity of H2O2, because more hydroxyl radicals are produced with higher irradiance.

In this research project, a laboratory experiment was conducted to answer the following research question: “What is the effect of hydrogen peroxide treatments on Microcystis and Dolichospermum at different light intensities?” Weenink et. al. (2015), argues that for hydrogen peroxide treatments of cyanobacteria, the dose should be as low as possible, in order to ensure selective killing of cyanobacteria without affecting other non-target species.

The goal of this experiment was to find the minimal dose of H2O2 concentration needed to kill two harmful bloom causing species of cyanobacteria in low and high light densities by testing a range of H2O2 concentrations as treatments for Microcystis and Dolichospermum blooms. Changes in cell density was analysed using the optical density and the photosynthetic yield was measured using the mini-PAM to test photosynthetic vitality of both species when treated with hydrogen peroxide. A hydrogen peroxide degradation assay was also conducted.

II. Materials and methods

II.a Growth conditions

All experiments were carried out with axenic cultures of Microcystis aeruginosa PCC7806 and Dolichospermum flos-aquae PCC7839 (aka Anabaena cylindrica). The cultures were obtained from Pasteur Culture Collection (Pasteur Institute, Paris) and grown in chemostats with BG-11 medium in which the nutrients correspond with the conditions in hypereutrophic aquatic systems. The average light intensity in chemostats was approximately 30µmol photons m–2 _s–1_{, with a CO2}

flow of 1000 ppm.

After approximately 2 weeks of growing, the cultures were measured with the CASY counter (table 1) and viewed under the microscope (figure 1).

Table 1: Average of two Casy counter measurements for Microcystis and Dolichospermum.

Figure 1: Pictures of microscopic view of unicellular Microcystis (left) and filamentous Dolichospermum

(right). Microcystis Counts 5504 Cells/µL 1,082E+07 Biovolume [fL] 3,928E+08 Diameter [μm] 3,69 Dolichospermum Counts 1357 Cells/µL 2,668E+06 Biovolume [fL] 5,680E+08 Diameter [μm] 4,47

II.b Laboratory experiment

Before the start of the experiment, the samples taken from the chemostats were diluted to approximately the same starting biovolume [femtoliter] and transferred to 100ml flasks. The dilution was done with BG-11, ensuring that nutrients are not a limiting factor to the growth of Microcystis and Dolichospermum. For Microcystis the starting biovolume was 1,088 * 10^8 fL and for Dolichospermum the starting biovolume was 1,196 * 10^8 fL.

Preliminary tests exposing both cultures to a large range of hydrogen peroxide (hereafter HP) concentrations from 0mg/L to 100 mg/L resulted in the selection of the following range of HP concentrations to be used as treatments for this experiment: 0 (control), 1, 2, 3, 4, 5, 10 and 15 mg/L HP. Each treatment was done in triplicates.

All treatments were done in two different light intensities, namely, 15 and 30 µmol photons m–2 _s–1_{. This was done by keeping treatments for both species in two}

different incubators during the course of the experiment. All incubators had the temperature set to 20 °C, 100 rounds per minute and no oxygen flow. In figure 2 below, the experimental setup is illustrated.

Figure 2: A schematic overview of the experiment.

Optical density Photosynthetic yield and fluorescence HP concentration

Measurements were taken before HP addition (time 0), and 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours after HP addition.

The exposure time was 4 hours, meaning that after taking samples for time point 4, catalase was added to all flasks to stop the treatment by breaking down all HP that was left.

II.c Hydrogen peroxide concentrations

The hydrogen peroxide dilutions were made from a 3% hydrogen peroxide drug store bottle. These dilutions were used for the different treatments, as well as making a calibration curve from which the HP concentrations in the samples could be calculated. After adding the HP dilutions to the corresponding treatments, the development of the HP concentrations was measured by filtering the samples from each treatment through a Millipore filtration unit, and placing 100µL of the filtrate into 96 well plates with 100 µL of p-nitrophenyl boronic acid reagent, as was done by Lu et al. (2011). The plates were then measured at 404 nm using a photospectometer (SpectostarNano by BMG Labtech).

II.d Optical density

The optical density was used in order to track changes in the cell density in the HP treatments during the experiment. The optical density was measured by sampling 200µL from each treatment and placing them into 96 well plates at the set time points after HP addition. Measurements were done with the photospectometer (SpectostarNano by BMG Labtech) at 750 nm for every time point. The values for the absorbance at 750 nm were corrected for the BG-11 medium, and the average absorbance of each treatment was calculated for every time point and plotted to view the changes in cell density of Microcystis and Dolichospermum treatments over time.

II.e Photosynthetic yield

The photosynthetic yield was measured using the mini-PAM. This was done to determine the changes in photosynthetic vitality of the cells in all HP treatments over time. First, a 5mL sample of each treatment was filtered through a Millipore filtration unit over 1,2µm filters (Whatman glass fibre filters, GF/C, CAT no. 1822-047) at every time point. The samples were then kept in the dark for 10 minutes by placing rubber plugs on the filtration unit before taking measurements. Averages of the yield (fv/fm) were calculated for each treatment and plotted over time as percentages of the controls. The filtrates were used to measure HP concentrations and the filters were saved to visually examine any changes in the cell density of Microcystis and Dolichospermum.

III. Results

V

conditions (Dolichospermum-15), the filters for the 10 and 15 mg/L HP treatments are lighter than the other treatments, indicating a decrease in cell density. Dolichospermum kept in 30 µmol photons m–2 _s–1 _{light conditions}

(Dolicho-spermum-30) show a decrease in cell density starting from the 4 mg/L HP treatment. Visually, Microcystis treatments in both light intensities show no significant changes in cell densities for any treatment, or a significant difference between light intensities.

Figure 3: filters after time 4 measurements show Microcystis exposed to 15 µmol photons m–2 _s–1_{(top left),}

Microcystis exposed to 30 µmol photons m–2 _s–1 _{(bottom left), Dolichospermum exposed to 15 µmol photons}

III.a Optical density

Optical density measurements confirm the observation made from the filters. As can be seen in figure 3a, Dolichospermum-30 shows a significant decline of the optical density in the 4 to 15 mg/L HP treatments at time 4, while Doli-chospermum-15 shows a decline of the optical density in the 10 to 15 mg/L HP treatments at time 4. At time 8, the optical density of Dolichospermum-15 treatments 10 and 15 mg/L HP continue to decline and the optical density for these treatments remains between 0 and 0,02 up to time 72, while the optical densities of treatments 0 to 5 mg/L show growth starting at time 8 up to time 72. For Dolichospermum-30, the optical density of treatments 0 to 3 mg/L HP show growth starting at time 8 up to time 72, while treatments 5 to 15 mg/L HP remain between 0 and 0,02 up to time 72.

Figure 3a: The effect of 8 different concentrations of hydrogen peroxide (0,1,2,3,4,5,10 and 15 mg/L) on the

optical density for Dolichospermum kept in 15 µmol photons m–2 _s–1 _{light conditions (A) and Dolichospermum}

kept in 30 µmol photons m–2 _s–1 _{light conditions (B).} 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0 6 12 18 24 30 36 42 48 54 60 66 72

Optical density (A750)

time after HP addition (hours)

Dolichospermum - 30 Optical density

0 1 2 3 4 5 10 15 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0 6 12 18 24 30 36 42 48 54 60 66 72

Optical density (A750)

time after HP addition (hours)

Dolichospermum - 15 Optical density

0 1 2 3 4 5 10 15 A B

For both Microcystis-15 and Microcystis-30, the optical density at time 72 shows growth for treatments 0 to 3 mg/L, while treatments 4 to 15 mg/L stay between 0.00 and 0.02.

Figure 3b: The effect of 8 different concentrations of hydrogen peroxide (0,1,2,3,4,5,10 and 15 mg/L) on the optical density for Microcystis kept in 15 µmol photons m–2 _s–1 _{light conditions(A) and Microcystis kept in 30}

µmol photons m–2 _s–1 _{light conditions (B).} 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0 6 12 18 24 30 36 42 48 54 60 66 72

Optical density (A750)

time after HP addition (hours)

Microcystis - 30 Optical density

0 1 2 3 4 5 10 15 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0 6 12 18 24 30 36 42 48 54 60 66 72 Op tic al d en si ty (A7 50 )

time after HP addition (hours)

Microcystis - 15 Optical density

0 1 2 3 4 5 10 15 A B

III.b Photosynthetic yield

Dolichospermum-15 and Dolichospermum-30 show a difference in sensitivity to the HP treatments (figure 5a). For example, in the 5mg/L HP treatment of Dolicho-spermum-15, the photosynthetic yield (as a percentage of the control) at time 4 decreases to approximately 70% and increases at later time points while for Dolichospermum-30, the photosynthetic yield for the 5mg/L HP treatment decreases to approximately 20% at time 4. At time 24, the photosynthetic yield of treatments 5 to 15 mg/L reach 0 for Dolichospermum-30. Treatments 10 and 15 mg/ L HP of Dolichospermum-15 reach 0% at time 48.

Figure 5a: The effect of hydrogen peroxide treatments on the photosynthetic yield of Dolichospermum kept in 15 µmol photons m–2 _s–1 _{light conditions (A) and 30 µmol photons m}–2 _s–1 _{light conditions (B).}

0 20 40 60 80 100 120 140 0 6 12 18 24 30 36 42 48 54 60 66 72 phot os yn the tic vit ality as % of c on tr ol time (hours)

Dolichospermum-30 Photosynthetic yield

0 1 2 3 4 5 10 15 0 20 40 60 80 100 120 140 0 6 12 18 24 30 36 42 48 54 60 66 72 phot os yn the tic vit ality as % of c on tr ol time (hours)

Dolichospermum-15 Photosynthetic yield

0 1 2 3 4 5 10 15 A B

Similar to Dolichospermum, Microcystis-15 and Microcystis-30 show a difference in sensitivity to the HP treatments (figure 5b). In the 5 mg/L HP treatment of Microcystis-15, the photosynthetic yield at time 4 decreases to approximately 55% and decreases further to reach 0% at time 72, while for Microcystis-30, the photosynthetic yield for the 5 mg/L HP treatment decreases to approximately 35% at time 4 and decreases further to reach 0% at time 24. For Microcystis-30, the 3 mg/L HP treatment decreases gradually to 0% at time 48 while for Microcystis-15, the 3 mg/L HP treatment decreases to approximately 80% at time 8 and then increases back to approximately 100% at time 48. At time 24, 10 and 15 mg/L HP treatments of Microcystis-15 reach 0%. At time 48, Microcystis-30 treatments of 3 to 15 mg/L HP of are 0% and stay 0% at time 72.

Figure 5b: The effect of hydrogen peroxide treatments on the photosynthetic yield for Microcystis exposed to 15 µmol photons m–2 _s–1_{(A) and Microcystis exposed to 30 µmol photons m}–2 _s–1 _(B)

0 20 40 60 80 100 120 140 0 6 12 18 24 30 36 42 48 54 60 66 72 phot os yn the tic vit ality as % of c on tr ol time (hours)

Microcystis-30 Photosynthetic yield

0 1 2 3 4 5 10 15 0 20 40 60 80 100 120 140 0 6 12 18 24 30 36 42 48 54 60 66 72 phot os yn the tic vit ality as % of c on tr ol time (hours)

Microcystis-15 Photosynthetic yield

0 1 2 3 4 5 10 15 A B

III.c Hydrogen Peroxide concentrations

The rate of HP degradation for all treatments is shown in figure 6. It seems that the HP concentrations degrade faster when exposed to 30 µmol photons m–2 _s–1

compared to 15 µmol photons m–2 _s–1_{. For example, at time 2, Dolichospermum-15}

treatments of 0 to 3 mg/L HP degrade to 0mg/L while Dolichospermum-30 treatments 0 to 4 mg/L HP degrade to 0 mg/L HP at time 2. At time 4, Dolichospermum-30 treatments 5 mg/L HP also degrades to 0 mg/L HP.

At time 4, the 0 to 5 mg/L HP treatments of Microcystis-15 reach 0 mg/L, while Microcystis-30 treatments 0 to 10 mg/L reach 0 mg/L at time 4.

Figure 6: HP degradation throughout the experiment

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 HP concentration (mg/L)

time after HP addition (hours)

Microcystis -15 HP concentration 0 1 2 3 4 5 10 15 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 HP concentrati on (mg/ L)

time after HP addition (hours) Microcystis - 30 HP concentration 0 1 2 3 4 5 10 15 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 HP concentration (mg/L)

time after HP addition (hours)

Dolichospermum - 30 HP concentration 0 1 2 3 4 5 10 15 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 HP concentration (mg/L)

time after HP addition (hours)

Dolichospermum - 15 HP concentration 0 1 2 3 4 5 10 15

Discussion

Hydrogen peroxide seems to be an effective method for treating cyanobacterial blooms. Table 2 shows an overview of the results for the photosynthetic yield and clarifies. It is clear that the toxicity of HP is dependent of irradiance. Dolichospermum and Microcystis kept in high light intensity conditions show a higher sensitivity to HP compared to Dolichospermum and Microcystis kept in low light intensity conditions. It is also clear that the sensitivity to HP is also species dependent, considering Microcystis and Dolichospermum show different results for the same treatments.

Table 2: overview of the HP treatments in which the photosynthetic yield reaches 0%

Initially, Dolichospermum showed a decrease in photosynthetic yield as well as the optical density as early as time 4 for the highest HP concentrations treatments, which was a lot earlier than Microcystis. However, Microcystis reached a photosynthetic yield of 0 at later time points for lower HP concentration treatments than Dolichospermum needed to reach a photosynthetic yield of 0.

Moreover, from table 2 we can conclude that the minimal dosage of HP needed for the photosynthetic yield to decrease to 0 is 4 mg/L for Microcystis-15 (72 hours after HP addition), 10 mg/L for Dolichospermum-15 (48 hours after HP addition), 3mg/L for Microcystis-30 (after 48 hours) and 4 mg/L for Dolicho-spermum-30 (after 48 hours).

Weenink et. al. (2015) advices to use minimally 2,3 mg/L HP as the treatment for cyanobacteria and not to exceed this dosage in order to prevent the killing of non-target species. However, in this experiment, the use of 2,3 mg/L HP would not be sufficient to kill the cyanobacteria (table 2). This might be because the experiments done by Weenink et. al. (2015) were done with lake samples and did not include the addition of catalase to stop the treatments.

3 4 5 10 15 30 µmol Time 24 X X X photons Time 48 X X X X X m–2 _s–1 _{Time 72 X X X X X} HP Treatments reaching 0% [mg/L] HP Treatments reaching 0% [mg/L] 3 4 5 10 15 15 µmol Time 24 X X photons Time 48 X X m–2 _s–1 _{Time 72 X X X X} 3 4 5 10 15 15 µmol Time 24 photons Time 48 X X m–2 _s–1 _{Time 72 X X} 3 4 5 10 15 30 µmol Time 24 X X X X photons Time 48 X X X X m–2 _s–1 _{Time 72 X X X X} Microcystis Dolichospermum

Drábková, Admiraal & Maršálek (2007) suggests that high light intensities increase the toxicity of HP because more hydroxyl radicals are produced with higher irradiance. This experiment shows a clear increase of sensitivity of the cyanobacteria to HP with higher light intensities and is thus in correspond with Drábková, Admiraal & Maršálek (2007). In order to take the step from a laboratory experiment such as this one, to actual lake treatments the effect of irradiance on the HP treatments needs to be further researched.

Before the start of this experiment a number of preliminary tests were done. First, both species were treated with a large range of hydrogen peroxide (0 to 100 mg/L). These treatments were then repeated for different exposure times (0 to 8 hours) and the optical density as well as the photosynthetic yield were measured up to 72 hours after HP addition. Results for time 72 were used to design the experimental setup used in the laboratory experiment. Since the goal of this experiment was to present a minimum dosage for the killing of two cyanobacteria, we concluded that time 4 was an interesting time point for catalase addition because the HP concentrations needed to kill the cyanobacteria were the lowest for the shortest exposure time.

In this study, the mini-PAM was used to measure to photosynthetic yield in order to draw conclusions about changes in the vitality of the algal cells for the different HP treatments in different light intensities. It might be interesting for future research to also measure the fluorescence and further investigate damage to the photosynthesis apparatus as a result of HP addition. Previous experiments by Drábková, Admiraal & Maršálek (2007), also suggest that in the case of cyanobacteria, the H2O2 causes damage to the photosynthesis apparatus. This was concluded because the Pulse Amplitude Modulated (PAM) fluorometry showed an increase in the fluorescence, and a decrease in the yield in an experiment done with Microcystis aeruginosa. Furthermore, the difference in sensitivity between species was interesting suggests that it would be meaningful to find minimum HP concentrations and exposure times for more harmful bloom causing species such as for example Planktothrix and Cilindrospermopsis.

Acknowledgements

This report would not have been possible without the help, patience and guidance of my supervisors Petra Visser and Nikoletta Tsiarta. Therefore, I would hereby like to express my deepest gratitude and appreciation to them for all the time and work put into helping me learn as much as possible during this internship and for counselling and inspiring me to continue in this field.

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