1. Heather Thomas Diane O’Connell Rachelle Gleason Rebecca Byerly Philip Church Mentor: Amber Burgett Beth Biro

2. A Brief History The side effects of caffeine are not new to the scientific or economic world. Caffeine’s economic value stems to the 16 th century where Spanish conquestadors began exporting coffee beans back from South America for trade. Also in the 16 th century, the first coffee houses began to appear in South America. (They would not arrive in Europe until the 17 th century and ultimately in North America until nearly the 19 th century.) The popularity of the effects of coffee/caffeine is also thought to have been a key contributor as to why Britian sought and obtained control over islands like Jamaica, Barbados and Trinidad.

3. Where does it come from? Caffeine is a natural substance found in some plants but is also toxic to these plants – coffee, coco and Kola nuts – guarana (a popular additive in many energy drinks) – tea leaves.

4. Caffeine as a drug? Caffeine belongs to the group of chemicals called alkaloids and is the #1 drug used in society today. It is classified as a stimulant and is used medically as a diuretic or to increase cardiac function Some forms of caffeine also function as smooth muscle relaxers/vasodilators and cardiac stimulants. These forms are usually found in teas. The most popular uses today are generally related to the “pick-me-up” that caffeine gives.

5. Caffeine varies between beverages. Not included are the caffeine amounts in food products such as energy bars, candy bars etc. The Human LD50 level is roughly 150-200 mg per kg of body weight. This means that a 140 lb person would need to consume 9545 - 12727 mg of caffeine or approx. 63 cups of starbucks coffee in one sitting for caffeine to become lethal. Caffeine content based on data from www.energyfiend.com

6. Previous studies on caffeine using tadpoles No effect on oxygen consumption of bull frog tadpoles Brinley 1931. No effect on activity in American Toad tadpoles Smith and Burgett 2005 – Contrary to earlier study… Caffeine resulted in an increase in activity and a 100% survivorship of Leopard Frog tadpoles Fraker and Smith 2004 – If activity increases, will this make tadpoles more visible to predators or will this make them more evasive?

7. Caffeine in our water? Humans cannot fully break down caffeine and as a result excrete it. Recent tests in both Seattle and Boston have revealed small levels of caffeine in the water (140 to 1600 ng) Mickelson 2003 – “Just before 9am, for example, caffeine levels in the sewer system are nearly eight times as high as usual” Mickelson 2003 – Could have a high correlation to human morning caffeine consumption Aging and cracked sewer lines, excess food/beverages decomposing in landfills, inproper disposal all lead to water infiltrating our water systems.

8. Results of Mickelson’s Study Table 6 Target BNA Analytes (including Atrazine) and Detection Limits (  g/L) BNA Compound MDL RDL Acenaphthene 0.010 - 0.050 Acenaphthylene 0.010 - 0.050 Anthracene 0.010 - 0.050 Atrazine (Triazine Pesticide) 0.050 0.100 Benzo(a)anthracene 0.025 0.050 Benzo(a)pyrene 0.010 0.025 Benzo(b)fluoranthene 0.010 0.025 Benzo(g,h,i)perylene 0.100 0.250 Benzo(k)fluoranthene 0.010 0.025 Benzyl Butyl Phthalate 0.010 0.025 Bis(2-Ethylhexyl)Phthalate 0.010 0.025 Caffeine 0.010 - 0.025 Carbazole 0.025 0.050 2-Chloronaphthalene 0.010 0.050 Chrysene 0.025 0.050 Dibenzo(a,h)anthracene 0.100 0.250 Dibenzofuran 0.010 0.025 1,2-Dichlorobenzene 0.050 0.250 1,3-Dichlorobenzene 0.050 0.250 1,4-Dichlorobenzene 0.050 0.250 2,4-Dichlorophenol 0.500 1.000 Diethyl Phthalate 0.010 0.025 Di-N-Butyl Phthalate 0.010 0.025 Fluoranthene 0.010 0.025 Fluorene 0.010 0.025 Hexachlorobenzene 0.025 0.050 Indeno(1,2,3-Cd)Pyrene 0.100 0.250 2-Methylnaphthalene 0.100 0.500 Naphthalene 0.025 0.050 Pentachlorophenol 0.500 1.000 Phenanthrene 0.010 0.025 Phenol 0.500 1.000 Pyrene 0.010 0.025 2,4,6-Trichlorophenol 0.500 1.000

9. Sewer Problems

10. Run off and Infiltration

11. Hypothesis Caffeine increases the activity of tadpoles (Hyla versicolor ) in the presence of predators (Ambystoma maculatum ).

12. Experimental System Organism - Tadpoles (Hyla versicolor ) Caffeine - AWAKE brand (Walgreens version of 200mg per caplet) 6  g/L concentration which is the highest concentration that is not lethal (Smith and Burgett 2004) Predator – Salamanders (Ambystoma maculatum)

13. Predator

14. Methods Amber took Hyla versicolor tadpoles that have been hatched approximately 1 week. Tadpoles placed in 18 L control water and 18L at  g/L concentration of caffeine in water for 19 hours. (Thanks Amber)

15. Methods Took 4 plastic shoe box containers and put 2cm x 2 cm grids with sharpies on bottom.

16. Methods 1 No caffeine no predator 2. No caffeine predator 3. Caffeine no predator 4. Caffeine predator

17. Methods Made predator traps with bridal tool and duct taped tool to bottom and sides of box at 1 end for all 4 boxes.

18. Methods Filled tubs to about half with water. Took Ambystoma maculatum Salamanders out of outdoor ponds and placed 2 in each appropriate box.

19. Methods Transfer from holding bins to testing bins with turkey baster. Use clickers to keep track of how often each tadpole crossed a line to indicate activity rates in 10 minutes.

20. The effects caffeine has on activity levels without predators Critical T-Value at df=7 with p=.05 is 2.36 T-Value for caffeine effects w/o predators = 0.765

21. The effects of predators on activity levels with no caffeine Critical T-Value at df=7 with p=.05 is 2.36 T-Value for predator effects w/o caffeine = 2.114

22. Effects of Caffeine on Activity Levels in the presence of predators Activity level across Grid Critical T-Value at df=7 with p=.05 is 2.36 T-Value for caffeine effects w/predators = 3.954

23. Effects of Predators on Activity levels in the presence of Caffeine Critical T-Value at df=7 with p=.05 is 2.36 T-Value for predator effects w/caffeine = 1.820

24. Data Comparison Based on data accumulated, caffeine can be shown to alter the predator effect on tadpole activity

25. Conclusion Critical t-value = 2.36 Effects of caffeine alone: reject hypothesis (t-value: 0.765) Effects of predation alone: reject hypothesis (t-value: 2.114) Effects of caffeine in the presence of predators: accept our hypothesis (t-value: 3.954) Effects of predators in the presence of caffeine: reject hypothesis (t-value: 1.820)

26. Future Questions Should the FDA require companies to include an additive that would allow the body to break caffeine down more readily? What other variables should the EPA study when looking at the effect of chemicals in water quality test?

27. Resources Brinley, F.J., (1931). EFFECT OF CAFFEINE ON THE OXYGEN CONSUMPTION OF FISH AND TADPOLES. Journal of Pharmacology And Experimental Therapeutics. 42(1): 59-63. Cornelis, M.C., El-Sohemy, A., Kabagambe, E.K. and Campos, H. (2006) Coffee, CYP1A2 genotype, and Risk of Myocardial Infarction. JAMA. 295: 1135-41. Fraker, S.L., Smith G.R. Direct and interactive effects of ecologically relevant concentrations of organic wastewater contaminants on Rana pipiens tadpoles. Environmental Toxicology. 2004 Jun;19(3):250-6. Hardy, J. and J. Word. 1986. Contamination of the water surface of Puget Sound. Puget Sound Notes. 10:3-6. Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B. and Buxton, H.T. (2002). Phar- maceuticals, hormones, and other organic wastewater conaminants in U.S. streams1999–2000: a national reconnaissance. Environ. Sci. Technol. 36, 1202–11. Smith, G.R., Burgett, A.A. Effects of Three Organic Wastewater Contaminants on American Toad, Bufo americanus, Tadpoles EcotoxicologyEcotoxicology. 2005 May;14(4): 477-482.