1. Effects of the Kingston Ash Spill on Amphibians and Reptiles

2. December 22 2008 March 20 2009 April 2009 August 2009 www.wild-facts.com www.nationalgeographic.com www.oal-ash-spill.com media.knoxnews.com

3. Conceptual Exposure Model mayo.personcounty.net thegreatsmokeymountains.org

4. Photo credit: Dr. J.D. Wilson American Toad Anaxyrus americanus Family: Bufonidae Family: Hylidae (True Toads) (Treefrogs) Common species Common species Different Families Different Families Different Life Histories Different Life Histories

5. Kingston Fossil Plant Amphibians Sites = 5 n = 76 (2009) n = 121 (2010) n = 156 (2011) Reference Site: Timberlake

7. Two-way ANOVA within species across years and locations Combined reference and non-impacted sites within years (Wilcoxon-Mann-Whitney, p=0.629) Post hoc tests: Tukey-Kramer Significant differences found in 11 of 26 trace elements: BariumCalciumIronMagnesiumSodiumPotassiumSeleniumStrontiumCopper ZincManganese

8. One of the most toxic substances known to developing amphibians (Herfenist et al. 1989) Maternally transferred from parent to egg – retained in tissue as they undergo metamophosis (Hopkins et al. 2006; Snodgrass 2005) Teratogenic effects (Hopkins et al. 2000; Rowe et al 1996, 1998) D.J. Fort/The Stover Group Venesky et al. 2010

9. Location/Year Selenium Concentration in Whole Body (mg/kg) Selenium - Amphibians Location/Year Selenium Concentration in Whole Body (mg/kg) Location/Year Selenium Concentration in Whole Body (mg/L) Background North Embayment West Embayment 1.0 2.0 3.0 4.0 Location/Year Selenium Concentration in Whole Body (mg/kg) Spring Peeper Spring Peeper: 2-way ANOVA, Location (p=0.001), Year*Location (p=0.0317) Upland Chorus Frog: 2-way ANOVA, Location (p=0.006) Hopkins, W.A., Mendonça, M.T., C.L., and Congdon, J.D. 1998. Elevated trace element concentrations in southern toads, Bufo terrestris, exposed to coal combustion wastes. Archives of Environmental Contamination & Toxicology 35:325-329. Selenium Concentration in Whole Body (mg/kg) 1.0 1.5 2.0 2.5 Upland Chorus Frog Background North Embayment West Embayment Location/Year

10. Maternally transferred from parent to egg (Hopkins et al. 2006) No previous laboratory studies specifically examining impacts of strontium in amphibians Replaces calcium resulting in soft and brittle bones (Kabata- Pendias and Pendias, 1999, Szostek et al. 2009) www.tnfish.org

11. Location/Year Strontium Concentration in Whole Body (mg/kg) Strontium - Amphibians American Toads: 2-way ANOVA, Year*Location (p=0.012) Location/Year Strontium Concentration in Whole Body (mg/kg) Location/Year Strontium Concentration in Whole Body (mg/kg) Background North Embayment West Embayment 25 50 75 100 125 Location/Year Strontium Concentration in Whole Body (mg/kg) Spring Peeper Background North Embayment West Embayment Location/Year Strontium Concentration in Whole Body (mg/kg) 50 100 150 200 250 American Toad Spring Peeper: 2-way ANOVA, Year (p=0.048), Location (p<0.0001) Hopkins, W.A., Mendonça, M.T., C.L., and congdon, J.D. 1998. Elevated trace element concentrations in southern toads, Bufo terrestris, exposed to coal combustion wastes. Archives of Environmental Contamination & Toxicology 35:325-329.

12. Copper radicals lead to oxidative stress in the body – can damage proteins, lipids, DNA, etc. Chronic exposure may negatively affect growth of larvae (Lande and Guttman 1973) Impacts larval behavior, heart rate, and neuromuscular coordination (Kaplan and Yogh 1961) Loss of equilibrium and death (Rao and Madhayastha 1987) virginiaherpetologicalsociety.com

13. Location/Year Copper Concentration in Whole Body (mg/kg) Location/Year Copper Concentration in Whole Body (mg/kg) Location/Year Copper - Amphibians Upland Chorus Frog: 2-way ANOVA, Year (p=0.003), Interaction (p=0.003) Location/Year Selenium Concentration in Whole Body (mg/L) Background North Embayment West Embayment 10 20 30 40 Location/Year Copper Concentration in Whole Body (mg/kg) Spring Peeper Copper Concentration in Whole Body (mg/kg) Upland Chorus Frog Background North Embayment West Embayment Location/Year 10 20 30 40 Spring Peeper: 2-way ANOVA, Year (p=0.002), Location (p<0.0001) Hopkins, W.A., Mendonça, M.T., C.L., and Congdon, J.D. 1998. Elevated trace element concentrations in southern toads, Bufo terrestris, exposed to coal combustion wastes. Archives of Environmental Contamination & Toxicology 35:325-329. Hall, R.J., and B.M. Mulhern. 1984. Are anuran amphibians heavy metal accuculators? In: R.A. Seigel et al, (eds), Vertebrate ecology and systematics-A tribute to Henry S. Fitch. University of Kansas Museum of Natural History, Lawrence, Kansas. pgs 123-133

14. HerpnetTurtlesite.info a-z-animals.com

15. Feed in sediment on river bottom Feed in sediment on river bottom Eat any type of aquatic plant, animal or invertebrate(living or dead) Eat any type of aquatic plant, animal or invertebrate(living or dead) www.marshall.edu/herp/images/http://www.on.ec.gc.ca/wildlife/homepage/images/http://www.ctherpconsultant.com/images/ Bury themselves in sandy river bottom Seldom leave river Have permeable shell Eat insects, crayfish, fish Consumed by humans Utilize any aquatic environment Eat wide variety of plants, animals, invertebrates, frogs, salamanders, water snakes, turtles; actively hunt prey and are proficient scavengers Consumed by humans Musk /Mud Turtles Sternotherus sp./ Kinosternon sp. Eastern Spiny softshell Apalone spinifera spinifera Common Snapping Turtle Chelydra serpentina

16. Turtles Trap locations = 347 Total turtles captured n = 587 (2009) n = 3,806 (2010) Kingston Fossil Plant

17. 2010 Collections Target Species Snapping Turtles (58) Musk/Mud Turtles (350) Eastern Spiny Softshell Turtles (89) Non-Target Species Sliders Painted River Cooters Map

18. Kruskal-Wallis, Site differences Selenium Concentration in Blood (mg/kg wet weight) Selenium in Turtle Blood Selenium Concentration in Blood (mg/kg wet weight) Common Musk / Mud Turtle Blood Selenium Concentration in Blood (mg/kg wet weight) Eastern Spiny Softshell Turtle Blood Selenium Concentration in Blood (mg/kg wet weight) Snapping Turtle Blood Snapping Turtle (p=0.214); Common Musk / Mud Turtle (p<0.001); Softshell Turtle (p=0.102)

19. Clinch River Emory River Selenium in Turtle Blood 2-way ANOVA, Location (p=0.0002); Species (p<0.0001); Interaction (p=0.0192) Selenium Concentration in Turtle Blood (mg/kg wet weight) Upstream TN River Downstream TN River

20. Selenium in Turtle Blood

21. virginiaherpetologicalsociety.com www.pc.gc.ca/nature/

22. Adam Johnson Travis Walls Mirtha Martin Neil Carriker Wes James Jacqueline Iannuzzi Tom Bock Jason Brown David Greenberg Dr. William Hopkins U.S. Fish and Wildlife Service Tennessee Wildlife Resources Agency

23. bullmarketfrogs.com Questions?