EFFECTS OF TEMPERATURE AND POLLUTION ON ALGAE GROWTH

EFFECTS OF FRESHWATER POLLUTION AND TEMPERATURE ON

THE GROWTH OF GREEN ALGAE (​Chlamydomonas​)

Jenna Mazur
Leo Peters

Loras College, Department of Biology

1450 Alta Vista Street
Dubuque, IA 52001

ABSTRACT

Green algae is an important component of freshwater aquatic life. ​Chlamydomonas​ is a

microscopic unicellular algae commonly found in freshwater bodies. It is known for its ability to quickly
reproduce by undergoing cell division daily if in favorable conditions. This research project aimed to test
the boundaries of the ​Chlamydomonas​’ favorable conditions by culturing it in different levels of water
quality and temperature in attempts to simulate the effects of global warming and common water
pollution through agricultural runoff and city pollution. We compared ​Chlamydomonas​ cell density
count between two different locations differing in water quality and among three different temperatures:
17 C, 25 C, 30 C. Cell density counts were taken weekly for four weeks. At the end of the experiment,
we found that cell density differed significantly by decreasing in density through the consecutive weeks,
but cell count did not differ significantly between sampling sites or temperatures. The differences in
water quality and temperatures did not have a significant enough impact on the change of cell density
over the course of the four weeks; the lack of significance within the test between subjects could suggest
that we did not have a broad enough range of temperatures and water qualities. From running a paired
samples t-test, the only chemical differences of the five tested chemical concentrations (pH, chloride,
phosphate, oxygen and nitrate) between the retention basin and Catfish creek were in chloride and
phosphate concentrations. Therefore we conclude that ​Chlamydomonas​ is tolerant to our tested chemical
variations and temperatures.
 EFFECTS OF TEMPERATURE AND POLLUTION ON ALGAE GROWTH
2
INTRODUCTION
Freshwater aquatic ecosystems are an essential component of Earth’s biosphere. Rivers, lakes
and ponds are not only used for recreational activities, but also contribute to ecological productivity and
biological diversity (Poff ​et al​, 2002 ). However, aquatic ecosystems are at risk of increased pollution
levels and declining health due to global warming. Global warming is a widely-known phenomenon that
refers to the increase of Earth’s surface temperature. It is caused by air pollutants and greenhouse gasses
in the atmosphere trapping heat in the form of sunlight and solar radiation (MacMillan, 2016 ).

Due to climate change, more frequent and severe weather is likely to occur, including increased
precipitation (MacMillan, 2016 ). An increase in precipitation increases surface runoff, which causes a
rise in pollution levels in bodies of freshwater (Kundzewicz ​et al,​ 2009). The mix of pollutants washed
into bodies of water is known as urban runoff (Taebi and Droste, 2004). As the name suggests, it is
common in urban, populated areas. High amounts of Chloride can also be found in bodies of water that
experience urban runoff. Chloride in excess can hinder an organism's ability to osmoregulate (Hunt ​et al,
2012 ). However, water pollution can result from agricultural areas as well. Agriculturists typically use
fertilizers loaded with excessive amounts of nitrogen (Good and Beatty, 2011). In excess, nitrogen
contributes to the eutrophication of a body of water which causes heavy plant growth and lack of
oxygen, harming the aquatic ecosystem (Carpenter ​et al,​ 1998). Along with increased pollution,
increasing temperature can lead to a decrease in algae growth (Butterwick ​et al, ​2004).
Algae are the photosynthetic organisms that serve as the base of the food chain and play an
important role in the health of an aquatic ecosystem. However, abnormal growth of green algae could be
detrimental. If there was a lack of growth, the food chain would slowly begin to collapse. In excess, algal
blooms can alter the level of pH in a body of water (Ghorbani ​et al​, 2014). This is due to an increased
amount of carbon dioxide produced by photosynthesis(Ghorbani ​et al​, 2014).
We designed a study to investigate whether green algae, specifically ​Chlamydomonas,​ can thrive
in several different temperatures (21⁰C, 25⁰C and 30⁰C) in polluted and pristine waters. We
hypothesized that when ​Chlamydomonas​ is subjected to pollution and increased temperature, there will
be a decrease in reproduction.We predicted that the water sample collected at Catfish Creek will be more
pristine than the sample from the retention basin. We are addressing the issues of climate change and
pollution levels on algae growth as a way of determining if and how global warming will have an effect
on our freshwater ecosystems.

METHODS
Twenty 250 mL samples were obtained from two bodies of freshwater (ten samples per site); the
th​
16​ Street retention basin, located in Dubuque, Iowa, and Catfish Creek at Swiss Valley Nature Preserve
in Peosta, Iowa. Catfish Creek hosts Brown Trout, which are known to be sensitive to polluted waters.
The retention basin is used to ​manage stormwater runoff to prevent flooding and downstream erosion
and to also improve water quality of adjacent rivers/lakes, meaning that they are somewhat
‘contaminated water’ sinks.​ ​After collecting the samples, tests were performed to assess water quality.
Each sample was subjected to the following tests: pH, nitrate concentration, phosphate concentration and
oxygen level. The pH and nitrate levels of each sample were tested using the Aquachek Water Quality
strips. Chloride was measured using the Aquachek titrators. Oxygen levels and phosphate concentration
were tested using the CHEMets Kits we received from Dr. Davis. ​After initial testing, the samples were
 EFFECTS OF TEMPERATURE AND POLLUTION ON ALGAE GROWTH
3
autoclaved to kill all living organisms. The green algae, ​Chlamydomonas​, was added in 200 microliter
increments to each sample.​ ​The samples were then divided up to into 3 groups,​ ​21⁰C, 25⁰C and 30⁰C.
Each individual sample was stored in a clear container with a lid. The lids were loosely screwed on the
tubes.
In order to determine if the algae were reproducing, a sample of 10 microliters was taken from
each sample to count the number of living cells. This was done using a Hemocytometer. The corner
square of the Hemocytometer H slide was used when counting the cells. Once this was determined, the
formula in equation 1 was used to determine cell concentration. To determine the amount of cells in the
entire sample, the number calculated in equation 1 was multiplied by the total sample volume​. Cell
counts were performed weekly to determine algae growth.
In order to determine if there was a significant difference in chemical concentrations between
Catfish Creek and the retention basin, an independent samples t-test was performed. In terms of cell
density, a factorial ANOVA repeated measurements analysis and tukey post-hoc test were performed in
order to compare and test variance within the temperature and water type groups. Both tests were
performed using SPSS.

Equation 1​: The equation to calculate cell concentration of algae when using an Hemocytometer H
slide.

RESULTS
Comparing the water quality in both the retention basin and Catfish Creek, chloride(t​8​ = -30.358,
p<0.001) and phosphate(t​8​ =17.441, p<0.001) were the only water quality values of the five that were
significantly different in the two locations. Referring to figure 1, the retention basin had a higher mean
chloride concentration at 3.12 compared to the mean of 1.16 found in Catfish Creek. Whereas Catfish
Creek had a higher mean phosphate concentration at 0.96 and the retention basin had a lower mean of
0.18. The error bars in figure 1 also show that nitrate(t​8​=1.633, p=0.141) did not differ between sample
locations due to the large standard error within the retention basin. Oxygen and pH did not have any
variation within the water samples and were therefore unable to be tested for any significant differences.

Fig 1​: Initial water quality averages of

the five samples collected from the
Dubuque retention basin and Catfish
Creek at Swiss Valley Nature Center.
 EFFECTS OF TEMPERATURE AND POLLUTION ON ALGAE GROWTH
4

A repeated measures analysis of variance was performed on the four weeks of cell density
counts for retention basin and Catfish Creek water samples. The multivariate tests suggested that cell
density count changed significantly over time by decreasing gradually in cell density count (F3=23.880,
p=0.005) whereas interactions between time and water type were insignificant (F3=2.922, p=0.164) as
well as time and temperature (F6=1.939, p=0.190).
The tests of between subjects suggested that the cell density counts between water samples
(retention basin, catfish creek) were not significantly different from each other (F2=0.102, p=0.905).
Figure 3 shows a similarity between growth patterns of algae, the retention basin samples had lower cell
density counts after week 1, but not low enough to be significantly different from the cell densities found
in the Catfish creek samples. Similarly to the water samples, cell density counts between temperatures
(17C, 25C, 30C) were also not significantly different enough from each other (F1=0.110, p=0.751). In
reference to figure 2, the 30C group is seen to have affected cell density counts in the most negative way,
but not in a way significantly different from the other two temperature groups. It is clear that
temperatures 17C and 25C exhibited the same level of ​Chlamydomonas​ growth through similar cell
density counts.

Fig 2​: plot of ​Chlamydomonas​ cell

density counts over 4 weeks in
various temperatures (17C, 25C,
30C).

Fig 3​: plot of ​Chlamydomonas​ cell

density counts over 4 weeks within
Catfish creek, Swiss Valley and
retention basin water samples.
 EFFECTS OF TEMPERATURE AND POLLUTION ON ALGAE GROWTH
5

DISCUSSION
The results did support our predictions that there would be a difference in water quality between
the two sites. The retention basin had a significantly higher amount of chloride and a significantly lower
amount of phosphate. This is logical because with the retention basin being used to manage urban runoff,
which is known to include higher amounts of chloride. High levels of phosphate would be more common
in areas which collect agricultural runoff due to the fact that phosphorus is a common nutrient in
fertilizers. With the results not displaying any significant cell density variation between the water
samples, it can be said that these chemical differences were not significantly large enough to show any
statistical changes in the growth of ​Chlamydomonas​. Overall, in terms of temperature variations and
water quality, the results did not support our hypothesis that ​Chlamydomonas​ would have decreased
success in reproduction due to the changes of temperature as it increased and introduction of supposed
pollution through the collection of water samples within the retention basin.
In a similar study, planktonic algae were subjected to varying temperatures and still exhibited
growth. However, they also found significant differences in the rate at which the algae were growing at
low (<10C) and high (>20C) temperatures (Butterwick ​et al, ​2004). One reason why we did not find
significant differences in cell density could be that the temperatures were not in a large enough range;
ranging from 17C to 25C, an 8C difference. Another experiment determining the effects of temperature
on algae growth proved that when the scientists stirred their samples during experimentation, it lead to
increased algae growth (Ahlgren, 1987) which could have been useful in our experiment to increase cell
density counts. In terms of algae growth in pollution, a study found that excess nitrogen and phosphorous
are two of the biggest contributors to the eutrophication of freshwater bodies (Carpenter ​et al​, 1998).
Based on the study and our results not leading to an increase in algal growth, we can conclude that our
water samples did not have a significant amount of nitrogen or phosphorous.
Researchers should continue to test the effects of climate change and pollution on the overall
health of freshwater ecosystems. In our experiment, we have found that the presence of climate change
could potentially not have an influence on the growth of Chlamydomonas based on the temperatures in
which we tested. Future studies should look further into other consequences of global warming, such as
severe weather changes, which could potentially have an effect on aquatic ecosystems.

ACKNOWLEDGEMENTS

We would like to thank Dr. Shealer for the collection of the Catfish creek and retention basin
water samples. We would also like to thank Mary Bowman for her efforts in providing the supplies
needed to successfully conduct our research. Finally, we would like to acknowledge the Loras College
Science Department for making it possible to do our research by providing the funding necessary for this
project.
 EFFECTS OF TEMPERATURE AND POLLUTION ON ALGAE GROWTH
6

LITERATURE CITED

Ahlgren, G. (1987, June). Temperature Functions in Biology and Their Application to Algal Growth
Constants. ​Oikos,​ ​49(​ 2), 177. doi:10.2307/3566025

Carpenter, S. R., Caraco, N. F., Correll, D. L., Howarth, R. W., Sharpley, A. N., & Smith, V. H. (1998).
Nonpoint Pollution of Surface Waters with Phosphorus and Nitrogen. ​Ecological Applications,
8(​ 3), 559. doi:10.2307/2641247

Butterwick, C., Heaney, S. I., & Talling, J. F. (2004). Diversity in the influence of temperature on the
growth rates of freshwater algae, and its ecological relevance. ​Freshwater Biology,​ ​50(​ 2),
291-300. doi:10.1111/j.1365-2427.2004.01317.x

Ghorbani, M., Mirbagheri, S., Hasani, A., Nouri, J., & Monavari, S. (2014). Algal Bloom in Aquatic
Ecosystems - An Overview. ​Current World Environment Journal,​ ​9​(1), 105-108.
doi:10.12944/cwe.9.1.15

Good, A. G., & Beatty, P. H. (2011). Fertilizing Nature: A Tragedy of Excess in the Commons. ​PLoS
Biology PLoS Biol,​ ​9​(8). doi:10.1371/journal.pbio.1001124

Hunt, M., Herron, E., & Green, L. (2012, March 4). Chlorides in Fresh Water. ​The University of Rhode
Island College of Environment and Life Sciences​. Retrieved from
http://cels.uri.edu/docslink/ww/water-quality-factsheets/Chlorides.pdf

Kundzewicz, Z. W., Mata, L. J., Arnell, N. W., Döll, P., Jimenez, B., Miller, K., . . . Shiklomanov, I.
(2008). The implications of projected climate change for freshwater resources and their
management. ​Hydrological Sciences Journal THSJ,​ ​53(​ 1), 3-10. doi:10.1623/hysj.53.1.3

MacMillan, A. (2016, March 11). Global Warming 101. Retrieved November 19, 2016, from
https://www.nrdc.org/stories/global-warming-101

Poff, N. L., Brinson, M. M., & Day, J. W. (2002, January). Aquatic Ecosystems and Global Climate
Change. Retrieved from http://www.sysecol2.ethz.ch/AR4_Ch04/Ch4-GreyLit/Po59.pdf

Taebi, A., & Droste, R. L. (2004). Pollution loads in urban runoff and sanitary wastewater. ​Science of
The Total Environment,​ ​327(​ 1-3), 175-184. doi:10.1016/j.scitotenv.2003.11.015