Effects of Hydrogen Peroxide on Anabaena and Scenedesmus in a Freshwater Environment

Waynick, K., Kielhofer, D., Humfeld, N., Brink, S. Many species of cyanobacteria can be a threat to water quality in lakes and ponds, our goal in this study was to find an environmentally safe way to reduce the overgrowth of the Anabaena in a local pond while maintaining the abundance of Scenedesmus, which is the primary food source to some of the organisms in the pond. Previous studies suggest hydrogen peroxide (H2O2) would be an effective, and targeted, control of cyanobacteria. The prokaryotic cyanobacteria lacks the beneficial peroxisome organelle found in green algae like Scenedesmus, therefore we hypothesized that the Anabaena would be adversely affected by H2O2 while it would have little or no effect on the Scenedesmus. Using water from McDermott Lake, we ran a controlled, replicated laboratory experiment testing the effect of various H2O2 concentrations (0.0mg/L, 0.5mg/L, 1.0mg/L, 2.0mg/L, 3.0mg/L) on Anabaena and Scenedesmus under typical late-summer conditions. Algal growth responded as predicted for both species, particularly at the highest concentration of 3.0mg/L when independently tested. We did not see the same significant differences in the combined treatment, and we also saw negative effects on an unexpected addition to our study, diatoms. For this reason, while our study supports the negative impact of H2O2 on Anabaena, we cannot conclude that H2O2 is safe for all beneficial algal species in the pond.

Introduction Freshwater ponds are home to a variety of organisms from microbes and fish, to birds and other terrestrial life. The ecosystems provided by these small bodies of water play a key role in the environmental balance and offer valuable resources to a wide range of organisms. The balance of life in freshwater ponds is delicate, and a slight increase or decrease in any key feature or resource can drastically change the pond from an ecological standpoint. A common issue is the algal growth and the potentially harmful effects that come with it. The over growth of blue green algae, such as cyanobacteria, can have a large detrimental effect on a ponds ecology. This is one of the many reasons the past use of community lakes and ponds has become a growing issue. McDermott Lake, found in Lafayette County, WI., was created for use as an agricultural pond. Surrounded by what used to be cultivated land for crops and livestock, the lake was subject to large amounts of runoff, which was full of nutrients and fertilizers (Doyle-Morin et al., unpublished). These runoff issues have led to the buildup of organic material, and are being noticed in many man-made ponds and small lakes due to the fact that they are shallow and require little time to acquire the buildup on the bottom. This results in a more concentrated level of cyanobacteria toxins due to the lower volume of water. By rapidly spreading, the cyanobacteria have an advantage over most other plants and assorted photosynthetic life under the pond surface (Dupuis, 2009). When the cyanobacteria bloom, toxins are released into the water harming other organisms, such as animals, that use the pond as a water source or habitat. The toxins can then be transferred to humans using the organisms in the pond as a food source (Stewart, 1998). Daphnia is a common invertebrate zooplankton in freshwater ecosystems that is known to show decreased growth and reproduction rates during the peak of cyanobacteria blooms. They are also not a great food source for other organisms in the pond, providing

minimal nutrients (Repka, 1996). In addition to the invertebrates being negatively affected by the abundance of cyanobacteria, larger organisms like dogs and cattle can also be affected by drinking the water (Rose, 2005). Our primary goal in this study is to find a cost effective way of maintaining and controlling the cyanobacteria growth to minimize the release of toxins in the ecosystem, while also preserving the species of algae that are beneficial to the ecosystem. We focused on two main algal types in this pond: harmful cyanobacteria Anabaena, and beneficial green algae Scenedesmus. Anabaena is a type of freshwater cyanobacteria which belongs to the bacteria kingdom and is prokaryotic (Rose, 2005). Cyanobacteria usually accumulate on the surface of the water, which is commonly referred to as pond scum and can cause low dissolved oxygen (DO) levels, toxins, and foul tastes (Hellweger, 2008). They often cause problems with aquatic life and the water supply as well as make the water look unpleasant. They bloom in a short period of time and thrive in temperatures around 25C (Rhee, 2008). Scenedesmus, a eukaryote from the plant kingdom, is a type of green algae commonly seen as a component of freshwater plankton (Rose, 2005; Chalifour, 2011). Scenedesmus are a major food source for the zooplankton, making them important to the survival of many other species in the pond (Chalifour, 2011). Maintaining their population is important to the survival of the ecosystem as a whole, which is why our aim is to find a treatment that has little to no effect on their population in the pond. Scenedesmus growth may be very dense in nutrient rich waters, but is rarely detrimental. The optimal temperature range for Scenedesmus to grow in the pond is 20-25C usually occurring during the mid-summer months at McDermott Lake, when the Anabaena is also in full bloom (Rhee, 2008; Doyle-Morin, et al., unpublished).

Methods for controlling cyanobacteria range from physically removing it from the water, to using chemical treatments to kill it. Chemicals such as copper sulfate and endothall amine salts are very effective; however they pose serious and sometimes lethal effects on the fish (Lynch, 2009; Fodorpataki, 2009). Less invasive methods of algal control include the use of barley straw, which releases H2O2 when decomposing and has a negative effect on the cyanobacteria (Rajabi, 2010). The barley straw has less detrimental effects on beneficial aquatic life than the chemical treatments, making it a safer, more natural method (Rajaniemi et al., 2005). Cost can become an issue with the use of the barley straw in larger bodies of water, as well as increasing the amount of biomass and nutrient levels in the pond, giving the algae more nutrients once it has decomposed (Doyle-Morin et al., unpublished). We want to determine if using H2O2 alone can be a cost effective way to control cyanobacteria and not kill off the beneficial algae without increasing biomass in the lake or using harsh chemicals. H2O2 will affect photosynthetic organisms by targeting specific organelles in the cell structure (Drbkov et al., 2009). However, some eukaryotic organisms have peroxisomes, which help them utilize the H2O2 (Rose, 2005). For this reason, we expected that the prokaryotic Anabaena population would decrease with the increasing levels of H2O2 as it did in the previous study, due to the lack of the H2O2 processing cellular component, the peroxisome (Rose, 2005; Colln, 1995). H2O2 is a byproduct of cellular metabolic processes in most green algae species, which are eukaryotic, meaning that their photosynthetic sites are better protected and therefore better able to survive in the presence of H2O2 (Drbkov et al.,2009). Doyle-Morin et al., unpublished showed that a concentration of 1.95mg/L of H2O2 or higher would decrease the abundance of Anabaena. However, in the middle of their study the incubators unexpectedly malfunctioned, which could have caused some of the Anabaena to die

due to unfavorable temperatures rather than the effect of the H2O2 being present (Doyle-Morin et al., unpublished). For our study we intended to replicate their methods in order to verify their results, focusing on concentrations around the threshold value of 1.95mg/L (0.5mg/L, 1.0mg/L, 2.0mg/L, and 3.0mg/L) to try to better pinpoint the lowest effective threshold level. Thus, we expect the H2O2 to have a negative impact on the nuisance algae, Anabaena, and to have no impact on the beneficial algae, Scenedesmus. We looked at the effects of H2O2 on Anabaena to see if it will have a negative effect on nuisance organisms, but were also interested in whether it would negatively affect other beneficial species of algae in the pond, like Scenedesmus. H2O2 is expected to have little to no effect on the Scenedesmus, due to the presence of the peroxisome (Rose, 2005). Methods This study was done in the University of Wisconsin Platteville Biology lab. Water samples were collected from an area of McDermott Lake, near Belmont, WI, that was approximately 3 feet deep, in the late afternoon on February 19, 2013. The water was taken from 8 inch holes in 18 inch thick ice, made by a motorized auger. The pond water was filtered using a KNF filter pump using 9cm Whatman/Double Rings qualitative medium speed filter paper and Ahlstrom quantitative fast speed filter paper. This filtering was for the purpose of eliminating dirt and other organic material while retaining the nutrients in the water. The filtered water was stored in a refrigerator at 8 Celsius. The Anabaena was densely clumped in the shipping container from NASCO Biological Supply, so clumps were manually broken into smaller colonies, and the remaining larger pieces were filtered out using a fine mesh net prior to addition. Upon looking at the Anabaena under a compound light microscope, we discovered two similar unknown species of the genus and

diatoms. The Scenedesmus were evenly distributed in the shipping container, along with other organic filaments and diatoms as well. To replicate the environmental conditions of the lake at the peak of algal bloom, the test beakers were stored in an incubator at 26 Celsius. We set the duration of the experiment to 21 days to match the life cycle of Anabaena (Rhee, 2008). Each 250mL beaker was filled to a total volume of 200 mL with filtered water, supplemented with algae: 10mL of each species for the separate treatments, and 5mL of each in the combined treatments. We tested four different concentrations of H2O2 (0.5mg/L, 1.0mg/L, 2.0mg/L, and 3.0mg/L) and a control with no H2O2 added in each of the three treatments. H2O2 was added following the addition of each species of algae to the beakers. Because the lake is spring-fed and artificially aerated, we stirred the beakers daily to replicate the constant motion of the water. They were also rotated every 4 days in the incubator to create even exposure to the light and a more even temperature distribution among samples. H2O2 and pond water were added twice a week to restore the water volume lost due to evaporation and replace the H2O2 degraded by natural processes (Mathjs at al., 2011). Milwaukee smart DO and Fischer Scientific Accumet pH meters were used and calibrated prior to use. DO measurements were taken after the beakers were filled with their treatments each week, then we refilled them with pond water and stirred them in an X formation. At the conclusion of the experiment, a final DO, pH and temperature measurements were taken. We filled the beakers back up with pond water to have a consistent volume among the samples, stirred them, and placed 1mL from each sample onto a Sedgewick Rafter slide, counting three random 1 uL grid cells using a compound light microscope. For Anabaena we counted the number of filaments and cells in each filament, giving us our total number of cells.

The Scenedesmus were counted in colonies of two and four, as well as free floating cells; combining these gave us the total number of cells. We also recorded diatom numbers even though it was not part of our intended design due to their potential impact on our test algae growth results. Statistical Analysis The analysis of the data was completed using Microsoft Excel version 2010. Each of the separate treatment sets was analyzed using one-way ANOVA. A two-tailed, unequal variance ttest was then used to determine what specific treatments presented significant data. All error estimates for the counts were recorded as () standard error. The DO content, pH, and temperature for each of the treatments were also compared using the one-way ANOVA, only using the data from the final day. All estimated errors for environmental factors were reported in () standard deviation. All t-tests and ANOVAs were run using an alpha significance value of 0.05.

Results Experimental Conditions: Water Chemistry The temperatures, DO, and pH varied slightly between treatments from data collected on the final day. ANOVA tests confirmed there were no significant differences between treatments with the exception of temperature, specifically in the Anabaena and Scenedesmus alone treatments (Table 1, ANOVA: p>0.10 for all DO and pH comparisons; p<0.001 for temperature comparisons). Among the Anabaena treatments, there was significant increase between 0.05mg/L and each of the other concentrations (p<0.05 for all comparisons). Among the

Scenedesmus treatments, there was significant decrease from the control to all of the different concentrations (p=<0.05 for all comparisons). There was no significance found among the other treatments. Table 1: Average DO, pH, and temperature on the final day for all treatments ( standard deviation, n=30). DO (mg/L) Anabaena Scenedesmus 6.780.51 6.190.62 pH 9.120.14 9.220.14 9.140.18 Temp(C) 21.910.30 21.570.39 22.320.59

Scenedesmus+Anabaena 6.520.89

Treatment Effects Anabaena: Treatments with only Anabaena showed significant difference in algal growth. The 3mg/L treatments were all significantly lower than the other treatments and control (Figure 1, t-test p=0.0055, 0.0048, 0.0024, 0.034). Anabaena in combined treatments showed no significant difference in growth (Figure 4, ANOVA p=0.0042).
80000 Abundance (# cells/mL) 70000 60000 50000 40000 30000 20000 10000 0 0 0.5 1.0 2.0 3.0 H2O2concentration (mg/L)

Figure 1: Effect of H2O2 Concentration on Anabaena growth, in Anabaena alone treatment. Error bars represent standard error (n=6). (ANOVA: df=4, p=0.0043).

25000 Abundance (# cell/mL) 20000 15000 10000 5000 0 0 0.5 1.0 2.0 3.0 H2O2 Concentration (mg/L)

Figure 2: Effect of H2O2 Concentration on Anabaena growth, in Scenedesmus and Anabaena mixed treatment. Error bars represent standard error (n=6). (ANOVA: df=4, p=0.10).

Scenedesmus The Scenedesmus individual treatments showed no significant difference in any of the treatments except for an increase between the control and the 3.0mg/L concentration. (Figure 3, t-test p=0.028). While Figure 4 suggests a similar pattern of increase, there were no significant differences between treatments when mixed with Anabaena (Figure 4, ANOVA p=0.39).

140000 Abundance (# cells/mL) 120000 100000 80000 60000 40000 20000 0 0 0.5 1.0 2.0 3.0 H2O2 Concentration (mg/L)

Figure 3: Effect of H2O2 Concentration on Scenedesmus growth, in Scenedesmus alone treatment. Error bars represent standard error (n=6). (ANOVA: df=4, p=0.21).

120000 Abundance (#cells/mL) 100000 80000 60000 40000 20000 0 0 0.5 1.0 2.0 3.0 H2O2 Concentration (mg/L)

Figure 4: Effect of H2O2 Concentration on Scenedesmus growth, in Scenedesmus and Anabaena mixed treatment. Error bars represent standard error (n=6). (ANOVA: df=4, p=0.39).

The diatoms decreased with the addition of stronger concentrations of H2O2. Figure 5 shows the results of all replicates of Scenedesmus, Anabaena, and the Scenedesmus/Anabaena mixture. Each of these showed a significant decrease in diatom counts (ANOVA p<0.05 for all tests).

30 25 Abundance (#cells/mL) 20 15 10 5 0 Control 0.5 1.0 2.0 3.0 H2O2 Concentration (mg/L) Scenedesmus Anabaena Scenedesmus All

Figure 5: Effect of H2O2 on diatoms in all concentrations. Error bars represent standard error (n=6). (ANOVA: df=4, p=0.00019, 0.0024, 0.015 respectively).

Discussion Our research objective was to test the effects of H2O2 on population growth of Anabaena and Scenedesmus. We conditionally accept our hypothesis that H2O2 has a negative effect on Anabaena. Throughout our data sets there was a clear decrease in the population of Anabaena on its own as the concentrations of H2O2 increased. We also, however, tentatively reject our hypothesis that H2O2 will have little to no effect on the Scenedesmus population, since we saw no effect in the combined treatment. There was a clear increase in the Scenedesmus population visually, and statistical results showed a significant increase in population from 0mg/L to 3.0mg/L concentrations of H2O2. The pH and DO measurements at the end of the study did not show significant differences among treatments (Table 1), suggesting no effect on algal growth. However, we did find significant difference in the temperature, which we do not think drove the differences in algal growth that we saw. The differences in temperature were likely due to the order that the temperature was taken in among samples, and could therefore have resulted in warmer temperatures for those measured first as opposed to cooler temperatures for those that sat out of the incubator for a longer period of time, as we saw within some of the 3.0mg/L test beakers. If the temperature had been the cause for the rise in Scenedesmus abundance or the decrease in Anabaena abundance, we would expect to see a significant increase in temperature at the same time we noticed an increase in the Scenedesmus, and a significant decrease in temperature with the decrease of Anabaena. Instead, for Scenedesmus we saw a significant decrease in temperature from the control to all other concentrations, but saw a significant increase in abundance between the control and the 3.0mg/L samples. In the Anabaena, the concentration of algae showed the most significant decrease from all treatments with the addition of 3.0mg/L,

while the temperature was significantly lower in the 0.5mg/L treatments than in the 3.0mg/L. Because the significant differences in temperature did not correspond to the significant differences in algal growth, we believe that this had no effect on the final algae abundance. However, t-test results do show marginally significant increases in Scenedesmus and significant decreases in Anabaena at the 1.0mg/L concentration. While we saw significant decreases in Anabaena when alone, but when both the Anabaena and Scenedesmus were added together in the same treatments, there appeared to be a significant increase in the Scenedesmus concentration, while the Anabaena abundance decreased in comparison to the control. This relationship could be due to the Scenedesmus being able to use the dead diatoms, or possibly Anabaena, as a nutrient source to help them grow and increase their abundance, along with decreased competition from Anabaena allowing the Scenedesmus to thrive in our designed environment (Asada, 2006). Because cyanobacteria do not have an encased photosynthetic system (prokaryotes have a membrane bound organelle) they are more sensitive to the H2O2 being added to the environment (Drbkov, 2009). The H2O2 restricts the Anabaena from sufficient energy production, therefore decreasing its ability to survive. Scenedesmus, on the other hand, naturally produces H2O2 and has evolved metabolic processes to dispose of it (Apel and Hirt, 2004; Asada 2006). Our results seem to follow this pattern, indicating that the H2O2 may be disrupting the Anabaenas ability to photosynthesize properly, resulting in death. Diatoms were present in the samples for the duration of the experiment, which was not originally accounted for in the experimental design, and could have potentially played a part in driving the results we found. The results showed a significant decrease in diatom abundance with an increase in hydrogen peroxide concentration. While this is not ideal because diatoms are

also considered to be beneficial algae, this could explain the increase in Scenedesmus. As the number of diatoms decreased, the abundance of Scenedesmus increased, possibly due to them using the nutrients given off by the decaying diatoms, and the reduction in competition. Although the diatoms have a trend matching the Anabaena in terms of H2O2 response, they are functionally similar to Scenedesmus because a major food source to other organisms in the pond. Diatoms are also big players in carbon fixation, which contributes to the health of the ecosystem (Reichwaldt, 2007). There are a few factors that could have limited our experiment or could have caused some issues along the way. One way to improve on our design would be to run the experiment for a longer period of time, seeing if any of the variances from the results turn up with more time. Doing more replicates would help to obtain more consistent data and limit the effect of statistical outliers. During our counts of Anabaena, we had one sample that had 83 total cells counted in a single grid cell, followed by 8 in the next, and 3 in the last cell counted. These counts alone create a standard error over 25, which is over 80% of the average, reducing our chance of getting significant differences between treatments. This experiment took place in a lab environment where mid to late summer conditions were replicated through use of incubators set at 25C, utilizing a day-night cycle of 16 hours of light exposure and 8 hours of darkness. Some factors not effectively replicated in the lab setting were the temperature variations between day and night and the spring fed movement of the pond that would be present in the natural environment of the pond. The variation of light intensity that would be present in the natural environment cannot be replicated in a lab setting, which could have an effect on the range of optimal survival conditions of the algal species studied (Dupuis and Hann, 2009). The potential for increased levels of DO due to dispersion from the natural

motion on the pond surface, as well as the presence of other micro fauna and zooplankton, can present a large experimental limitation. Based on this, we believe that not only could our overall DO values be significantly less than would be found in the actual pond environment, this may have also have limited the potential for growth in some cases. Another way these factors may have caused limitations in our results is that in the experiment, we replicated only the ideal conditions. In the natural pond environment the variables are constantly changing, and it never stays perfectly optimal for algal growth. With this, it is unknown if the temperature, light exposure, DO content, or several other factors, may cause a change in our results. Originally our study was to include only one species of Anabaena and Scenedesmus, but when our samples arrived we observed that our Anabaena culture contained several species of this genus. Both the Scenedesmus and Anabaena culture samples also contained diatoms. These extra species may have been a factor in the decrease in Anabaena concentration or in the increased abundance of the Scenedesmus. Future studies could be done to test how the absence of these species affects the population growth of Scenedesmus and Anabaena. Studies could also be done that include these species to support the results found in this study. We also had to account for the presence of other strains of diatoms since they are present in the ecosystems. These were not included in our original design, but are naturally present with the Anabaena and Scenedesmus in the pond during the peak season, making them an unexpected but useful addition. This study was done to determine a safe and cost effective concentration of H2O2 to be used as a treatment for the harmful algal blooms in McDermott Lake. The majority of our results, like the significant decrease in Anabaena abundance when alone and the rise in Scenedesmus numbers when mixed with Anabaena, support our hypothesis. However, due to a lack of

significant difference among all samples, further research must be done to gain a better idea of the effects. Based on our results, we have seen that H2O2 may be used as a safe treatment to lower the amount of harmful algae in a small pond without having a harmful effect on the Scenedesmus in the ecosystem. We did, however, see that the H2O2 had a negative effect on diatoms, which was not accounted for in our original experimental design, but is still an important result. This should be considered when conducting further research on H2O2 treatment as tool to the restoration of many small lakes and ponds throughout the world. If the elimination of negative effects on other beneficial algal species was determined by future research, our study suggests that H2O2 is a promising treatment. Small bodies of water, such as McDermott Lake, could be cleaned up and restored to their original health levels with the addition of a naturally produced compound that breaks down in the water.

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