The Effects of Caffeine and Triclocarban on Gammarus pseudolimnaeus Jim Fietzer Department of Biological Sciences, University of Wisconsin – Whitewater Dr. Elisabeth Harrahy - Mentor Introduction The use of pharmaceuticals and personal care products (PPCPs) has become increasingly prevalent in the world in recent years. Over-the-counter pharmaceuticals such as aspirin and ibuprofen, prescription pharmaceuticals such as birth control pills, hypertension medication and anti- depressants, antimicrobials used in hand soaps and other personal care products, and the caffeine found in our coffee and soda are a regular part of daily life. Many of these compounds are only partially absorbed by the body through ingestion, and as a result, are routed through wastewater treatment plants (WWTPs). 1 Some of these PPCPs flow through the WWTPs as effluent, or are partitioned to sludge, which is ultimately released back into the environment as agricultural fertilizer. 2 The high volume use of these PPCPs has led to their accumulation in the aquatic environment, and measurable concentrations have been detected in surface waters, algae and fish. 1, 3, 4 Environmental levels of triclocarban and caffeine have been found to be as high as 12 2 and 6 5 µg/L, respectively. The objective of this study was to determine the acute and chronic toxicity of the antimicrobial triclocarban (TCC), and of the stimulant caffeine, to Gammarus pseudolimnaeus, a freshwater amphipod. Methods G. pseudolimnaeus kept in an environmental chamber set at 17°C, with a 16-h light/8-h dark cycle Amphipods fed a diet of mixed detritus Test solutions prepared through serial dilution using standardized water and test chemical Individual amphipods placed in 10 replicate beakers and exposed to five to seven different concentrations of the test chemical plus a control and carrier control (acetone; TCC tests only) Solutions renewed daily Temperature, dissolved oxygen, pH and conductivity measured daily Duration of exposure: 96 hours (acute) 14 days (chronic) Endpoints: survival (acute), survival and growth (as length; chronic) Results Figure 3. In the definitive acute test, triclocarban had a significant impact on survivorship at a concentration of only 22 µg/L (1-way ANOVA and Tukey’s multiple comparison test, p<0.05). Results to date of a chronic triclocarban test indicate effects on survival at a concentration of 10 µg/L and on growth at only 4 µg/L. Test Organism Gammarus pseudolimnaeus Macroinvertebrate found in freshwater streams Crustacean commonly known as scud or sideswimmer Serves as important detritivore, and food for many fish species Regularly used in aquatic toxicity tests G. pseudolimnaeus used in these studies were -collected from Bluff Creek outside Whitewater, WI -had an average length of 6.33mm. Image source: Conclusions Triclocarban was found to be lethal at concentrations currently being detected in surface waters Chronic test results to date indicate triclocarban is lethal at even lower concentrations under longer term exposure, and may have significant sublethal effects As use of PPCPs increases, we may find increased concentrations of these contaminants in the environment, with the potential for significant effects on populations of aquatic organisms Acknowledgments Funding was provided by the Merck/American Association for the Advancement of Science Undergraduate Science Research Program and by the University of Wisconsin – Whitewater College of Letters and Sciences A special thank you to Dean Mary Pinkerton for support of this program Analysis of triclocarban and caffeine will be conducted by the Wisconsin State Laboratory of Hygiene Lucas Kuehn assisted in all laboratory and field activities LITERATURE CITED 1 Thomas, P.M. and G.D. Foster Environmental Toxicology and Chemistry 24: Heidler et al Environmental Science and Technology 40: Coogan et al Chemosphere 67: Woodling et al Comparative Biochemistry and Physiology, Part C 144: Kolpin et al Environmental Science and Technology 36: Benefits/Future Studies Results will be useful in determining if additional treatment technologies are needed in the wastewater treatment process Results will be useful in calculating water quality criteria by regulatory agencies Future studies will assess: toxicity of additional PPCPs mixture effects of select PPCPs Results Figure 1. In the range-finding acute test, caffeine had a significant impact on survivorship at a concentration of 10 mg/L (1-way ANOVA and Tukey’s multiple comparison test, p<0.05). Figure 2. In the definitive acute test, caffeine had a significant impact on survivorship at a concentration of 360 mg/L (1-way ANOVA and Tukey’s multiple comparison test, p<0.05). aaaaa, b b c aa aaa a b aa b cc