Stress response of bivalves: impact of copper on filtration rate and microbial communities of oysters (Crassostrea virginica)

Hannah Ryon Northwestern University Evanston, IL 60201

Advisor: Anne Giblin The Ecosystems Center Marine Biological Laboratory Woods Hole, MA 02543

Semester in Environmental Science 2016

Abstract Copper can have detrimental effects for organisms in aquatic environments, especially with increasing modern inputs from industrial waste, sewage, paint, and pressure treated lumber. Although there have been many studies on the lethal implications, there is not much known about the sub-lethal effects of copper on organisms. This study looks at the impact of copper exposure on filtration rate and microbial communities of eastern oysters. To test these two parameters, I setup tanks with varying concentrations of dissolved copper and a total of 20 oysters from Little Pond, in Falmouth, MA. I then measured the change in phytoplankton in the water over time to determine filtration rate. I also extracted and sequenced the DNA in the feces, pseudofeces, and on the shell. Finally, I determined the metal content of both the body and the gut of the exposed oysters. In this study, I found that the filtration rate of the oysters decreased with increasing concentrations of copper. However, the filtration rate rapidly recovers after being removed from tanks with copper. In the microbial component of this study, I determined that eastern oysters do not have a resident gut microbial community but they do have a resident shell community. This shell community is highly diverse, and it has denitrifying genera present. In the water and on the shell the microbial diversity decreases with exposure to copper. Finally, I determined that the oysters increase the amount of copper absorbed with increasing exposure. However, at around 600ppm the oysters become saturated with copper. Additionally, the gut contained higher concentrations of copper than the body of the oysters because the gut is used to expel excess copper. This data suggests that copper can have many sub-lethal effects on the eastern oyster at concentrations below the toxic concentration.

Introduction All organisms require trace amounts of copper in order to survive. Copper is a metal that can be naturally found in trace amounts in animals, soils, plants and water (Diseases and Contaminants). In aquatic systems, copper is a relatively common metal ion. However, when in excess it becomes one of the most toxic ions in aquatic systems (Solomon 2009). This is why it is so important to understand the interactions of organisms with copper in aquatic environments. Typically, natural background levels of copper enter water bodies through weathering and material blown in by the wind. However, in present day, copper concentrations in aquatic environments can be greatly increased due to inputs from air pollution, industrial waste, and sewage (Bryan 1971). In marine environments two other major sources of copper are boat bottom paint and pressure treated lumber (Schiff 2004 & Stook 2005). This increase in dissolved copper can have significant ramifications for the organisms in that environment. One way in which excess copper can be detrimental to organisms is by disrupting cellular processes. When copper is abundant in an organism it then displaces other ions by binding to metal-binding sites or other nonspecific sites in things like enzymes or DNA. This is highly disruptive to metabolic processes and can lead to the eventual death of the organism (Goldstein 1986). Additionally, excess copper in an organism can catalyze reactions, which produce oxyradicals. This is extremely dangerous to an organism because oxyradicals can cause their nervous system to shut down (Goldstein 1986). The gills of aquatic organisms can also be greatly damaged by excess copper in the body because the efficiency of respiratory mitochondria in the gills falls with increased copper concentration in oysters (Collins 2010). In this study I will be focusing on the impact of copper on eastern oysters (Crassotrea virginica). Oysters are suspension feeders, meaning they filter phytoplankton and other nutrient particles out of the water. The suspended particles that they siphon in are eventually separated into feces and pseudofeces or incorporated into the organism. The pseudofeces is the material that the oyster determines is not nutritious and it is rejected prior to digestion. The feces is excreted on the opposite end of the oyster after it is digested and the necessary material is extracted. These oysters pay a large role in maintaining ecosystems, as their consumption patterns allow nitrogen to cycle and they help to prevent eutrophication (Songsangjinda 1998). It has also been suggested that the microbes that they harbor assist in denitrification (Kellogg 2014). Another reason for the importance of the oysters in Cape Cod is because they are a large component of the shellfish industry, which boosts the economy and benefits tourism. As copper amounts increase in the environment, it is vital that we understand the implications of this on the Cape Cod oyster population (Diseases and Contaminants 2015). Many studies have shown the lethal effects of copper on organisms, however, little is known about the sub-lethal impact of copper on organisms. Previous studies have shown that reproduction can be severely hindered by exposure to increased copper concentrations (Hornberger 2000). This shows that organisms can have sub- lethal changes due to copper, and this experiment will aim to determine sub-lethal effects in a short-term setting. I will be using filtration rate as a test of these sub lethal effects on the organism, by showing the impact of copper on phytoplankton consumption. Additionally, I plan to examine the impact of copper on the residential microbial communities within the organism. Little is known about the species in these communities, or if the community is present at all, but any change in its composition could be an indicator of stress on the oyster. Additionally, it is important to understand the impact of copper on these communities, as they are involved in some vital processes in the environment such as denitrification. Finally, I will examine how the metal is incorporated into the organism and which parts of the body are most contaminated. This will determine if metal is actually absorbed by the organism, or if it just passes through its system. These tests can also point to the mechanism of storing or expelling the copper from the system.

Methods

Lab Setup This experiment was conducted entirely in a lab setting. I set up 5 different tanks with four oysters of similar size in each, as shown in Figure 1. The oysters were collected from the Little Pond oyster farm in Falmouth, MA. Each tank is filled with seawater filtered through a 20micron filter and a 1micron filter, in order to remove any phytoplankton present in the inflowing seawater. The water was also bubbled with air throughout the course of the entire experiment, to ensure uniform distribution of copper and phytoplankton. In each tank there was a different concentration of copper dissolved in the water: 0ppm, 0.05ppm, 0.1ppm, 0.2ppm, and 0.3ppm. The copper was added using a Cu (II) 1000ppm stock solution. The oysters were allowed to sit in the filtered seawater without any phytoplankton inputs or copper additions for two days. This was done in order to flush their systems of any prior food or other contaminants from their previous environments. I also predict that this will eliminate any microbiomes present due to anything they consumed prior to being moved to the lab. During test periods, the tanks were cleaned each morning and the water was replaced. Copper and algae was again added to the tanks after cleaning. The algae was obtained from the Marine Resources Center at the Marine Biological Laboratory of Woods Hole, and it was RotiGrow Plus Omega Algal Blend.

Filtration Rate The first objective of this experiment was to determine the impact of copper on the filtration rate of the organisms. In order to determine filtration rate in each tank, I measured the change in chlorophyll a concentration over time. As the algae is filtered out of the water column, the amount of chlorophyll a in the water will decrease. I measured chlorophyll a in the water by taking a subsample of the water from each tank and using a Turner Designs 10A Flourometer. I was then able to determine filtration rate by utilizing the following equation:

푉1−푉2 Filtration Rate = 푡 Where V is the concentration of chlorophyll a measured in ug/L, and t is time in hours.

Filtration rate was measured over 4 different trials of 1 day experiments, with fresh filtered sea water, copper, and food supply each day. There were typically approximately 2 days between each trial. During the second and third trials the oysters were placed on platforms for feces and pseudofeces collection, as shown in Figure 2. On the second day of test one of the oysters in the 0.2ppm tank died. The organisms were then starved without copper for two days and the filtration rate was measured to the organisms’ ability to recover.

Microbial Communities In order to determine the diversity of microbial communities present before and after copper exposure, I focused on microbes in the feces, pseudofeces, and on the shell of the oysters. During the third day of filtration tests, in the 0ppm and 0.3ppm tanks, I collected a subsample of feces and pseudofeces from the collection tin of three of the oysters. Only three oysters in each of the tanks were sampled due to limitations on costs. Additionally, I scraped the surface of the shells of these three oysters and collected any material present. After collecting the feces, pseudofeces, and shell matter from three oysters in both 0ppm and 0.3ppm, I then completed DNA extractions for each of these. DNA extractions were done using the Mo Bio PowerSoil DNA Isolation Kit. I then submitted the DNA to Emma White, in the Marine Biological Laboratory genetic sequencing lab, for the DNA to be sequenced and uploaded to Visualization and Analysis of Microbial Populatic (VAMPS).

Metal Absorption After running the filtration rate tests and sampling the microbial community, I euthanized the oysters by freezing them after two days without copper exposure or food and dissected all four oysters in each tank. I was able to separate the stomach from the remaining body parts, in order to determine the metal absorbed by these two different components. The extracted material was then dried at 50°C for 24 hours and both wet and dry weight were recorded. The samples were then digested using nitric acid and a 60°C water bath. Additionally, they were diluted to 100mL and filtered using Whatman 110mm Qualitative Filter Paper 1. The samples were then run on a Perkin Elmer Atomic Absorption Spectrophotemeter.

Results In initial trials it was immediately clear that there were visible differences in the feces and pseudofeces composition between the control and the highest concentration of copper, as shown in Figure 3. At 0ppm the oysters almost exclusively produced feces and minimal pseudofeces was excreted. In contrast, at 0.3ppm there was mostly pseudofeces produced. Additionally at 0ppm the excrement was dense and darkly colored. However, at 0.3ppm the excrement was light and much lighter in color. Filtration rate was then monitored during four different time periods of treatment over the course of two weeks. After the first day of exposure the change in filtration capabilities were immediately apparent. As shown in Figure 4, the filtration rate decreased as the concentration of copper in the tank increased. For the following two time periods the same trend was visible but the 0.3ppm oysters were slightly more effective at filtration than those in 0.2ppm (Figure 5 &6). On the third day of testing one oyster in the 0.2ppm tank died. On the final day of exposure the oysters again showed the pattern of decreased filtration with increasing copper, as shown in Figure 7. The organisms were then left for two days without food or copper in the water and on the third day I tested the filtration rate after recovery. As shown in Figure 8, the trends seen with copper entirely disappear. The oysters at all levels of copper exposure increase significantly from their filtration rates with copper present. However, the 0.3ppm oysters still had a lower filtration rate than all other organisms. On each of these graphs I plotted a trendline across the tops of the columns in order to compare the slopes (Table 1). This showed a slight variation across the different trials, with the most negative slope on the second day of exposure. However, all of the exposure trials had negative slopes. The recovery trial had a slope of almost zero. In the second part of this study I looked at the feces, pseudofeces, and shell microbial composition of oyster in the 0ppm tank and 0.3 ppm tank. As shown in Figure 9, the shell has the highest diversity, ranked by the Shannon Weaver Diversity Index. On these graphs the error bars represent range instead of error. The feces and pseudofeces have similar diversity values in their microbial communities. However, in feces, pseudofeces, and on the shell the microbial community is less diverse in 0.3ppm copper than without copper. I then compared the similarity of the composition of these microbial communities in the feces, pseudofeces, and shell to understand how they related to each other. As shown in Figure 10, the microbial composition of the pseodofeces and feces are highly similar, but the shell microbial community is entirely dissimilar from both feces and pseudofeces. However, in each category the 0ppm copper organisms are slightly dissimilar from the 0.3ppm copper organisms. I also analyzed the composition of the microbial communities in the feces, pseudofeces, and on the shell individually. In these analyses I looked at the communities on the genus level, and I only included microbes that made up at least 1% of the total microbial community. In order to determine the impact of copper on the microbial communities, I charted the composition of the feces with and without exposure of copper (Figure 11). In this comparison, I found that pseudoalteromonas is the dominant microbe in the feces regardless of copper exposure. It also shows that vibrio is not present in the copper exposed organisms’ feces. Additionally, with the addition of copper, amphritea becomes the dominant genus of microbe in the feces. I then made the same comparison for the pseudofeces of these organisms, as shown in Figure 12. The pseudofeces microbial composition had the same trends as the feces. The dominant microbe was also pseudoalteromonas. Additionally, with copper the vibrio died off and amphritea became the most abundant relative the microbial community. The final microbial analysis that I completed was examining the microbes found on the shell of the organisms (Figure 13). The most obvious trend in the shell microbial communities was the extreme diversity. There were minimal changes in the microbe composition with and without copper that were visible because the composition was highly diverse. Pseudoalteromonas was also present on the shell but it was not the dominant microbe. The dominant microbe on the shell was rhodobacteraceae. After two days without copper I then dissected the organisms and measured their weights and tested the concentration of copper in the oysters’ guts and bodies. In order to understand the impact of copper on the organisms’ body mass I compared the average shell length to the average dry weight of the oysters in each tank by first graphing the average dry weight (Figure 14) and then creating a weight: shell length ratio for each tank (Figure 15). The results of this showed 0ppm oyster to have the highest weight: shell length ratio. The 0.05ppm oysters had the lowest ratio and it increases at each increasing level of copper. When studying copper absorption, I found that as the copper concentration increases in the tank the amount of copper absorbed by the oyster increases, as shown in Figure 16. However, at a tank concentration of 0.1ppm, the copper absorbed levels off and does not increase despite increases in the copper concentration in the tank. I then compared the copper absorbed by the gut to the copper absorbed by the body of the oyster in Figure 17. While these two separate components had fairly similar values, the gut typically had higher concentrations of copper than the body. In both of these figures the dead oyster was excluded from the analysis, as it skewed the shape of the graph.

Discussion In the qualitative observations from this experiment I noticed that there was significantly more feces than pseudofeces in the tank without copper but the opposite was observed at the highest concentration of copper. I predict that this is because the copper confuses the organism’s system in sorting the particles that will benefit the oyster. This is shown in that the oysters in the 0.3ppm tank began excreting the algae in their pseudofeces, meaning it was rejected as inadequate for digestion. This could potentially lead to the starvation of the oysters if trials were continued. In measuring filtration rate I found a clear pattern in that the oysters become less effective at filtering when exposed to higher concentrations of copper. This shows that exposure to copper decreases the oysters’ ability to feed, as it rapidly degrades their health. However, in the second and third day of exposure the 0.3ppm tanks had higher filtration rates than those in 0.2ppm. I predict this is because the oysters in the 0.3ppm tank were propped up on platforms for feces collection. This potentially caused the organisms to be more effective at filtration than they would be when set on the bottom of the tank. Additionally, the 0.2ppm oysters may have been less effective at filtration because of the one oyster, which died on the third day of exposure. The recovery trials show that the filtration rates of the organisms were able to mostly recover from the stress of copper exposure. However, the 0.3ppm recovered less than the other copper levels did. This suggests that at levels higher then 0.3ppm copper the oysters may not have been able to recover. An interesting result in the recovery was that the tank where 0.2ppm copper had previously been added had the highest filtration rate. This is surprising because one of these oysters had already died, meaning that there were only three oysters in the tank, yet it still had the highest filtration rate. I suspect this is because this tank had a higher initial concentration of phytoplankton and therefor was able to filter more, despite having fewer oysters. The differences in the extremity of the slopes of the column graphs could simply be a result of the varied conditions across the different tanks. For example, temperature was not controlled to ensure the same conditions across tanks. Additionally, the amount of phytoplankton supplied to each tank often varied slightly, which may have caused differences in the amount they could filter. However, for the four days of exposure the slope was negative which proves that filtration rate consistently decreases with exposure to copper. Also because the slope was so close to 0 for the recovery trial this again points to the ability of the oysters to recover from the stress of copper. In the microbial component of this study, I determined that the shell was largely more diverse than the feces and pseudofeces. The shell contained a highly diverse collection of microbes and even eukaryotes. This diversity shows that the shell is a popular home to microbes of many genera. The decrease in the diversity of all of the communities with copper shows that there are a significant number of microbial genera that are incapable of surviving copper exposure, which could impact the role of the oyster in its environment. The similarity in microbes between the shells different oysters shows that despite the extreme diversity of the shell community, there are particular genera that are resident to oyster shells. However, it is important to note that these oysters were all collected in the same area and kept under similar conditions, which may contribute to the similarity in the microbes. Therefore, in future studies it would be interesting to determine if these communities differ across locations. The shell community was entirely dissimilar from the feces and pseudofeces, confirming that the shell microbes were not in the water column. The highly similar microbial composition of the feces and pseudofeces indicates that there is no resident gut microbial community in eastern oysters. I was able to prove this using the assumption that a resident gut community would cause a significant variation between the composition of the feces and the pseudofeces, with the microbes missing from the feces being the resident gut microbes. However, because the communities in the feces and pseudofeces were highly similar this confirms that the eastern oysters do not have a microbial gut community. This is also shown in the similarity of the specific genera in the feces and pseudofeces and the identical response to copper. This lack of a resident gut microbial community means that the changes that I observed in microbial communities of pseudofeces and feces are simply the impact of copper on the microbes in the surrounding water and the food source. This is logical, as the dominant microbe was pseudoalteromonas, which is commonly found in association with marine alga. However, the changes in the microbial communities with exposure to copper are still interesting, as this indicates the response of the oysters’ environment to the stress of copper. This study showed that vibrio are copper-sensitive, while amphritea are copper-resistant. The copper response of vibrio opens a field for future studies, as several species within vibrio are pathogens, and copper may be a potential means of eliminating these species when necessary (Heidelberg 2000). In contrast to the feces and pseudofeces, the microbial community of the shell can be assumed to be a resident community of the oysters. The shell of the oysters had an extremely diverse community of microbes and most of these microbes made up a very small amount of the total community. This allowed for minimal changes in the composition with copper because there were so many diverse genera. The dominant microbe genus found on the shell was rhodobacteraceae, which is an interesting finding, as this genus is known to have denitrifying characteristics (Heylen 2006). Therefore, this resident genus may contribute to oysters’ role in nitrogen management. In analyzing the weight: shell ratio it was logical that the oysters at 0ppm had the highest ratio. This is because I expected the exposure to copper to decrease the mass of the organisms because it degrades the system. However, I was surprised to find that the weight: shell length ratio increased from 0.05ppm to 0.3ppm. I predicted that 0.3ppm would have the lowest ratio because it was exposed to the most copper but this was not the case. The results from the metal absorption tests show that there is certain point at which the organisms become fully saturated with copper. The copper saturation point of the oysters appears to fall mostly around 600ug of copper per gram of oyster. Another potential cause for the leveling of the copper absorbed may have been due to the lesser filtration rates in the tanks with higher copper concentrations. This is because in these organisms less water was flowing through the oyster and therefore it may have been exposed to less copper. In the 0.2ppm copper tank there was a single outlier oyster in the copper in the gut and body. The reason for this organism’s abnormally high copper values is because it died during the final trial of exposure. This means that the organism was unable to flush its system of any copper not actually absorbed during the last two days of starvation, meaning that a significant amount of the copper measured inside of the organism was likely filtering through the oyster but not necessarily absorbed by it. For this reason, this oyster was excluded from the analysis. While the gut and body absorbed somewhat similar amounts of copper, the gut maintained higher concentrations than the body. I predict this is because one of the main mechanisms for excess copper expulsion in oysters is by transporting it to the gut and excreting it as feces (Rodney 2007). Additionally, bivalves often absorb metals when they are inhaling water through the gut for food consumption. However, the data suggests that the body and gut are similar in copper absorption, which suggests that although the main area of absorption is the gut, the copper is distributed across the entire organism. In conclusion, copper can have several sub-lethal implications for the filtration rate and microbial communities of eastern oysters. However, this study has shown that there is still a lot of research needed to understand these organisms’ ability to recover and the significance of these microbes in oysters’ role in the nitrogen cycle.

Acknowledgements

This project would not have been possible without the guidance of my two advisors, Anne Giblin and Jessica Mark Welch. I am also incredibly grateful for the patience and advice provided by the amazing TAs of SES 2016, especially Rich McHorney. I would also like to thank Emma White, for sequencing and processing the genetic data, despite all of the complications.

Literature Cited

Barrera-Escorcia, G., et al. 2010. Filtration rate, assimilation and assimilation efficiency in Crassostrea virginica (Gmelin) fed with Tetraselmis suecica under cadmium exposure. J Environ Sci Health A Tox Hazard Subst Environ Eng. 45: 14-22. Bryan, G.W. 1971. The effects of heavy metals (other than mercury) on marine and estuarine organisms. Proc. Roy. Soc. Lond. B. 177: 389-410. Collins S., et al. 2010. The effects of copper and copper blocking agents on gill mitochondrial O(2) Utilization of crassostrea virginica. In Vivo (Brooklyn). 32(1): 14- 19. Diseases and Contaminants. (2015). Centers for Disease Control and Prevention. Goldstein S, and Czapski G. 1986. The role and mechanism of metal ions and their complexes in enhancing damage in biological systems or in protecting these systems from the toxicity of O2-. J Free Radic Biol Med; 2(1):3-11.

Heidelberg, J. et al. 2000. DNA sequence of both chromosomes of the cholera pathogen vibrio cholerae. Nature 406: 477-483.

Held, P. 2010. Monitoring of Algal Growth Using Their Intrinsic Properties. BioTek.

Heylen, K., et al. 2006. Cultivation of denitrifying bacteria: optimization of isolation conditions and diversity study. Appl. Environ. Microbiol. 72(4): 2637-2643.

Hornberger et al. 2000. Linkage of bioaccumulation and biological effects to changes in pollutant loads to San Francisco Bay. Environmental Science and Technology 34: 2401-2409.

Kellogg, L., et al. 2014. Use of oysters to mitigate eutrophication in coastal waters. Elsevier 151: 156-168.

King GM, et al. 2012. Analysis of Stomach and Gut Microbiomes of the Eastern Oyster (Crassostrea virginica) from Coastal Louisiana, USA. PLoS ONE 7(12): e51475. doi:10.1371/journal.pone.0051475

Principle of Atomic Absorption/Emission Spectroscopy. Semester in Environmental Science Faculty. Marine Biological Laboratory.

Rodney, E., et al. 2007. Bioaccumulation and tissue distribution of arsenic, cadmium, copper, and zinc in Crassostra virginica grown at two different depths in Jamaica Bay, New York. In Vivo. 29(1): 16-27.

Schiff, K., et al. 2004. Copper emissions from antifouling paint on recreational vessels. Elsevier 48(3-4): 371-377.

Solomon, F. 2009. “Impacts of Copper on Aquatic Ecosystems and Human Health.” Environment and Communities.

Songsangjinda, P., et al. 1998. The Role of Suspended Oyster Culture on Nitrogen Cycle in Hiroshima Bay. Journal of Oceanography 56: 223-231.

Stook, K., et al. 2005. Relative leaching and aquatic toxicity of pressure-treated wood products using batch leaching tests. Environ. Sci. Technol. 39 (1): 155-163.

Sunila, I. 1981. Toxicity of copper and cadmium to Mytilus edulis L. (Bivalvia) in brackish water. Ann. Zool. Fennici 18: 213-223.

Tables

Table 1. The slopes of the column graph trendlines of filtration rate against concentration of copper.

Figures

Figure 1. The average length of the shells of the oysters in each tank. Figure 2. The general lab setup with tins separating the feces and pseudofeces that comes out on either side of the oyster. Figure 3. This image shows the visible differences in feces and pseudofeces composition with and without copper. Figure 4. Filtration rate at different levels of copper exposure during the first day of exposure. Figure 5. Filtration rate at different levels of copper exposure during the second day of exposure. Figure 6. Filtration rate at different levels of copper exposure during the third day of exposure. Figure 7. Filtration rate at different levels of copper exposure during the fourth day of exposure. Figure 8. Filtration rate of the oysters previously exposed to varying levels of copper after two days without copper exposure. Figure 9. The Shannon Weaver Diversity value with and without copper in the feces, pseudofeces, and on the shell. Error bars represent range, not error. Figure 10. Chart comparing feces, pseudofeces, and shell microbial composition with and without copper. Red indicates dissimilarity and blue indicates similarity. Figure 11. Microbial composition of oyster feces with and without copper. Different colors show different genera. Figure 12. Microbial composition of oyster pseudofeces with and without copper. Different colors show different genera. Figure 13. Microbial composition of oyster shell with and without copper. Different colors show different genera. Figure 14. The dry weight of the oysters after copper treatment. Figure 15. The ratio of average dry weight to shell length for the oysters after treatment Figure 16. Copper concentration absorbed by the oysters at different levels of exposure. Figure 17. The copper concentration absorbed by the gut and body of the oyster at different levels of exposure.

Tables

Table 1. The slopes of the column graph trendlines of filtration rate against concentration of copper.

Treatment Number Slope of Trendline

1 -0.1077 2 -0.3343

3 -0.1053 4 -0.177

Recovery 0.0244

Figures

Length of Shells in Each Tank 8

7

6 5 4 3

2 Length Length of shell(cm) 1 0 0ppm 0.05ppm 0.1ppm 0.2ppm 0.3ppm Copper Concentration

Figure 1. The average length of the shells of the oysters in each tank.

Figure 2. The general lab setup with tins separating the feces and pseudofeces that comes out on either side of the oyster.

Figure 3. This image shows the visible differences in feces and pseudofeces composition with and without copper.

Exposure 1

2.5

y = -0.1077x + 2.0903 1) 2 - R² = 0.8199

1 hr 1 1.5 -

1

Filtration Rate Filtration 0.5

(ug Chl L Chl (ug

0 0ppm 0.05ppm 0.1ppm 0.2ppm 0.3ppm Copper Concentration

Figure 4. Filtration rate at different levels of copper exposure during the first day of exposure.

Exposure 2

1) 1) 4 -

3.5 1 hr 1 - 3 y = -0.3343x + 3.8067 R² = 0.8071 2.5

2

Filtration Rate Filtration (ug Chl a L a Chl (ug 1.5 1 0.5 0 0ppm 0.05ppm 0.1ppm 0.2ppm 0.3ppm Copper Concentration

Figure 5. Filtration rate at different levels of copper exposure during the second day of exposure.

Exposure 3

1) 1) 2 -

1.8 1 hr 1

- 1.6 y = -0.1053x + 1.6787 1.4 R² = 0.5466 1.2

1 Filtration Rate Rate Filtration

(ug Chl a L a Chl (ug 0.8 0.6 0.4 0.2 0 0ppm 0.05ppm 0.1ppm 0.2ppm 0.3ppm Copper Concentration

Figure 6. Filtration rate at different levels of copper exposure during the third day of exposure.

Exposure 4

1) 1) 2.5 -

1 hr 1 2 y = -0.177x + 2.3199 - R² = 0.8768 1.5

Filtration Rate Rate Filtration 1 (ug Chl a L a Chl (ug

0.5

0 0ppm 0.05ppm 0.1ppm 0.2ppm 0.3ppm Copper Concentration

Figure 7. Filtration rate at different levels of copper exposure during the fourth day of exposure.

Recovery Trial 3 - y = 0.0244x + 2.2564

2.5 R² = 0.0292

1 hr 1 - 2

1.5

1) 1

0.5

0 0ppm 0.05ppm 0.1ppm 0.2ppm 0.3ppm

Filtration Rate (ug Chl a L a Chl (ug Rate Filtration Copper Concentration

Figure 8. Filtration rate of the oysters previously exposed to varying levels of copper after two days without copper exposure.

Shannon Weaver Diversity Index 2.5

2

1.5

0ppm Index

Weaver Diversity Diversity Weaver 1 0.3ppm -

0.5 Shannon 0 Feces Pseudofeces Shell

Figure 9. The Shannon Weaver Diversity value with and without copper in the feces, pseudofeces, and on the shell. Error bars represent range, not error.

Figure 10. Chart comparing feces, pseudofeces, and shell microbial composition with and without copper. Red indicates dissimilarity and blue indicates similarity.

Figure 11. Microbial composition of oyster feces with and without copper. Different colors show different genera.

Figure 12. Microbial composition of oyster pseudofeces with and without copper. Different colors show different genera.

Figure 13. Microbial composition of oyster shell with and without copper. Different colors show different genera.

Dry Weight of Oyster After Treatment 0.8 0.7 0.6 0.5 0.4

Weight Weight (g) 0.3 0.2 0.1 0 0ppm 0.05ppm 0.1ppm 0.2ppm 0.3ppm Copper Concentration

Figure 14. The dry weight of the oysters after copper treatment.

Weight: Shell Length Ratio 0.09 0.08

0.07 0.06 0.05 0.04 0.03

Weight: Weight: Length Shell 0.02 0.01 0 0ppm 0.05ppm 0.1ppm 0.2ppm 0.3ppm

Figure 15. The ratio of average dry weight to shell length for the oysters after treatment

Average Copper Absorbed

800

700

600 500 400 300

200 (ug Cu/g oyster) Cu/g (ug 100 Oyster Copper Absorbed Absorbed Copper Oyster 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Tank Copper Concentration (ppm)

Figure 16. Copper concentration absorbed by the oysters at different levels of exposure.

Copper Absorbed by Gut and Body

900 800

700 600 500 400 Gut 300 Body

(ug Cu/g oyster) Cu/g (ug 200 100

Osyter Copper Concentration Concentration Copper Osyter 0 0 0.1 0.2 0.3 0.4 Tank Copper (ppm)

Figure 17. The copper concentration absorbed by the gut and body of the oyster at different levels of exposure.