Introduction

Phytoplankton are components of the plankton community and an integral part of ocean and freshwater ecosystems. They provide over 50% of our annual oxygen supply. Not only that, they also provide the foundation for marine life. They are the base of every food-web and food chain. Additionally their primary function is to absorb CO2 on the water’s surface and use it to perform photosynthesis through a process called carbon cycling in which they are able to utilize atmospheric carbon to generate chlorophyll while releasing oxygen into the atmosphere. However, due to an increase in the output of CO2 through activities such as the burning of fossil fuels like coal, natural gas, oil as well as deforestation, the temperature, mineral concentrations, as well as pH of ocean water has been steadily decreasing.

Also, the production and increasing use of copper has widely risen in the past century. Copper is essential for industry, however, the disposal or leaching of copper in oceans makes it a highly toxic pollutant which is detrimental to marine life, especially phytoplankton. That alongside the decreasing pH are two major factors that in combination interfere with the process of photosynthesis. Primarily because they affect the utilization of energy in marine phytoplankton by limiting cell divisions and inhibiting binding sites for other essential elements such as nitrate, silicate, carbonate, copper, and iron. As a result, phytoplankton may consume additional amounts copper and limit uptake of other essential nutrients required for cellular growth.

Currently, the pH of the ocean is around 8.05-8.10. However, the pH level of the ocean is expected to fall to 7.67 by 2100 due to increased amounts of atmospheric CO2. How does copper get into the ocean? Through minerals in the soil, biological particles ,volcanic activity, anthropogenic contributions, and precipitation (Blossom). Copper content in the world’s oceans is about 0.25 ug/L (0.000393 ). This is supposed to rise to 0.575 ug/L (0.000904 ) in the next century, a staggering 115% increase. As a result, we devised an experiment to establish the copper concentration that reduces phytoplankton cell count in an acidic environment. Due to the exposure to a number of environmental and physical stressors, we hypothesize that an acidic pH as well as high copper concentrations will minimize intake of nutrients in T. Weiss, thus hindering their growth.

Methods

In order to test the effects of pH and copper on phytoplankton, we used the species Thalassiosira Weissflogii. We obtained a small sample of this species that had been replicating to start a new culture. We used a 500mL container to prepare our cultures. 8mL of sea salt was added in first and the container was shaken vigorously in order to homogenize the mixture. Other ingredients such as nitrate, minimal amounts of copper, vitamin solution, iron, etc . This same method was utilized to develop all 3 cultures. A new batch was made once every week for 3 weeks. In order to vary pH to 7.67 and 7.57, we made buffers using TRIS and TRIS-HCL. Their concentrations are displayed in Table 1.

Table 1- TRIS and TRIS-HCL concentrations

Table 2- Culture 1 copper concentrations

In order to determine the effect of copper alone on our cultures, no changes were made in regards to the pH. For the purposes of this experiment, a normal pH refers to pH 8.1. Copper concentrations ranged from 0.0393 to 39.3 . The recipe called for a copper concentration of 0.0393 for optimal growth since copper is one of many elements’ phytoplankton require to grow. Based on that concentration, we chose to dilute the base level of copper 100X with the highest being 39.3 . Culture 2 and 3 differed in pH and copper concentrations

Table 3- Culture 2 treatment groups

This culture was used in order to test the factorial experiment. pH and type of vials varied in order to determine whether or not they had a significant impact on cell growth.

Table 4- Culture 3 treatment groups

This culture tested the effects of a more acidified pH 7.57, as well as various copper concentrations.

Figure 2-Color scale to observe T. Weiss growth

Each culture was measured for color Monday, Wednesday, and Friday using the color scale below. The color scale is representative of growth. This means that a darker solution of phytoplankton will be predicted to have a greater number of cells. However, because the color scale is objective, we determined that there was a 7% uncertainty in the color readings. This would mean that solely graphing color vs days would not provide us with accurate information thus, a Beckman Coulter counter was utilized to determine cell count in each vial. The solution was prepared using 2mL of phytoplankton solution and 18mL of isoton (salt water solution). The sigma (s) values correspond to cell count.

Table 5: Key

Glass = +1

Polypropylene = -1

Normal pH 8.1 = +1

Acidic pH 7.67 = -1

Table 5- Set Up for The Factorial Experiment with Three Factors- pH, Environment, noise

Table 6. Determining the Influence Of Factors on T. Weiss Cell Count Using Culture 2 Cell Counts

The bolded numbers on the last row of Table 2 (SUM/4) shows the response factors and how significant each individual factor is on the growth of phytoplankton. For the noise, factor C, since the magnitude of the effect is the largest, in comparison to Factor A and B, we can be certain that the changes in cell growth for either conditions, pH or type of vial, is primarily due to random variation. In the future, in order to minimize the variation, we could add more replicates to the experiment.

Note- culture 2 growth did not grow as well in comparison to cultures 1 and 3.  Thus, the variation between Factors A and B seems to be too high.

Results and Discussion

Figure 3-

The control group (containing no additional copper) had the highest cell count compared to the rest of the treatment groups which contained additional copper. The 39.3 peaked at day 7, and then the cell growth started to plateau. This is a direct result of cell death due to high copper concentrations. Since the results for the highest copper level were so detrimental to phytoplankton growth, we eliminated that treatment and instead assigned 3.93 to be our highest level for cultures 2 and 3. This further proves that any amount additional copper prevents T. Weiss from reaching their optimal cell count.

Figure 4- Comparing the effects of high and low copper concentrations on the growth of Culture 3

The figure above compares the effects of copper on Culture 3 growth. In a normal pH, the control group which contains no additional copper has the highest cell count throughout. However, any additional amount of copper lowers phytoplankton growth.

Figure 5- Comparing the effects of pH 7.67 using Culture 2

In order to test the effects of pH 7.67, samples from culture 2 were compared. The blue line in the graph above represents a sample with no additional copper and a normal pH of 8.1. The orange line represents a sample with no additional copper as well, however, with an acidified pH. Although the factorial experiment determined that the difference in cell counts for acidic vs normal treatments groups was most likely due to random variation, we analyzed each culture separately. The graph above clearly displays that the more acidified treatment group had higher cell counts meaning T. Weiss in this culture did better in an acidified environment of 7.67.

Figure 6- Comparing the effects of pH 7.57 using Culture 3

In comparing the cell counts for a normal pH of 8.1 with no additional copper to a pH of 7.57, the results show that after day 8, cell counts for T. Weiss in solution of pH 7.57 plateau and eventually decrease. In contrast, the control group mostly follows a linear growth trajectory.

Overall, although there was no statistical significance that pH and copper concentrations affect the growth of phytoplankton, using data for individual cultures we observed that the hypothesis was supported. There is in fact a trend in terms of copper concentrations and pH which is- the greater the copper concentration, the lower the cell growth and the lower the pH, the lower the cell count. The aim of this experiment was to determine what concentration of copper coupled with a slightly acidic pH causes a more toxic effect in phytoplankton. Since we were not able to delve deeper into the interaction between copper and pH, unfortunately the relationship between copper and pH remains unanswered.

It is clear, however, that any amount of additional copper does affect growth in phytoplankton. It is more pronounced in a copper concentration of 39.3 because the cells started to die off within a couple days. Additionally, a pH of 7.67 is in fact suitable for phytoplankton growth, however, a pH of 7.57 makes it extremely difficult for phytoplankton to reach their optimal cell counts. In order to prevent further decline of phytoplankton as a result of increase copper and acidified pH, there needs to be immense changes like reducing the copper used in boats, yacht, as well as ships since their bottom surfaces add additional copper ions into the ocean. Another solution is to have stricter regulations for any waste disposed into the oceans since it may contain harmful chemicals that interfere with phytoplankton growth. Lastly, climate change is a global issue. Although we may not consider phytoplankton to be essential in our daily lives, their extinction would eventually deplete oxygen available to humans which in turn, would eradicate humans as well.

References

Barton, A., Irwin, A., Finkel, Z., & Stock, C. (2016, March 15). Anthropogenic climate change drives shift and shuffle in North Atlantic phytoplankton communities. Retrieved March 23, 2021, from https://www.pnas.org/content/113/11/2964

Blossom, Neal. “Copper in the Ocean Environment.” American Chemet Corporation, 2001, pp. 1–8. Chemet.com, www.chemet.com/assets/1/6/Copper_and_the_Ocean_Environment.pdf.

Evans, Monica. “Climate Fix? ‘Fertilizing’ Oceans with Iron Unlikely to Sequester More Carbon.” Mongabay Environmental News, 22 May 2020, news.mongabay.com/2020/03/climate-fix-fertilizing-oceans-with-iron-unlikely-to-sequester-more-carbon/.

Gao, K., Zhang, Y., & Häder, D. (2017, November 14). Individual and interactive effects of ocean acidification, global warming, and UV radiation on phytoplankton. Retrieved March 26, 2021, from https://link.springer.com/article/10.1007/s10811-017-1329-6

NOAA. “What Is Ocean Acidification?” National Ocean Service, 26 Feb. 2021, oceanservice.noaa.gov/facts/acidification.html.

Santos, Carmen B. de los, et al. “Interaction of Short-Term Copper Pollution and Ocean Acidification in Seagrass Ecosystems: Toxicity, Bioconcentration and Dietary Transfer.” Marine Pollution Bulletin, vol. 142, 2019, pp. 155–63. Crossref, doi:10.1016/j.marpolbul.2019.03.034.

Walsh, G. E. “Chapter 12: Toxic Effects of Pollutants on Plankton.” Principles of Ecotoxicology, 2006, pp. 257–274.