1. Fish Bioaccumulation Studies Associated with the Kingston Fly Ash Release Marshall Adams - Oak Ridge National Lab Tyler Baker - TVA Allison Fortner - Arcadis Terry Matthews and Mary McCracken - ORNL

2. Presentation Outline - Research objectives - Study Design - Results and major findings - causal relationships - Relevance to overall fly ash project - integration with other studies

3. Objectives of Bioaccumulation Studies Determine if fly ash exposure is causing short and/or long- term health effects on representative (sentinel) fish populations (tomorrows presentation) Evaluate potential causal relationships between exposure to fly ash-associated metals and bioaccumulation in representative fish species Assess spatial and temporal patterns of metal bioaccumulation in sentinel fish species

4. Study Design Fish Species Sampled Spring - bluegill, largemouth bass, redear sunfish, and while crappie Fall - bluegill, largemouth bass, and channel catfish Sample Frequency spring and fall each year since spring 2009 Metals Analyzed - ICP suite of 25 metals including selenium, arsenic, etc. - metals measured in fillets (muscle) from all fish, liver and ovaries (spring), whole body (shad) Special Studies - whole body composite gizzard shad - reconstruction analysis on bluegill and LMB - trophic analysis on redear using stable isotopes Sample Sites - 6-7 sample sites along downstream gradient of Emory and Clinch Rivers - includes 2 instream reference sites

5. Bioaccumulation 25 metals + Hg Fly Ash Spill Exposure of Fish to Metals Whole fish MuscleLiverOvary Focus on sentinel species - sunfish - bass - catfish - crappie Analysis of Fish Health Assess Effects and Causality (different trophic levels and home ranges ) Physio- logical Repro- ductive Histo- path Bio- energetic

6. CRM 1.5 CRM 7 ERM 8 (Ref) CRM 8 ERM 0.9 ERM 3 Little Emory (Ref) CRM 25.0 (ref) Ash spill

7. Sentinel species for bioaccumulation and fish health studies Bluegill - mid trophic level/omnivore - restricted home range Largemouth bass - upper trophic level/predator - intermediate home range Channel catfish - bottom feeder/omnivore - large home range Gizzard Shad Redear Sunfish Types of Studies Bioaccumulation, fish health, and reconstruction analysis Bioaccumulation and fish health Bioaccumulation, fish health, and reconstruction analysis Bioaccumulation, fish health, & trophic analysis (stable isotopes) Bioaccumulation and role in food chain transfer of metals - exclusive molluscavore - high site fidelity - feeds on detritus & periphyton - large home range

8. Spatial Pattern Ash spill Ash spill Refs Downstream

9. Ash spill Spatial Pattern References below detection Refs

10. Reference Sites EPA tier 1 action level for assessing toxicity of selenium on fish and birds Ash spill Temporal Pattern Selenium in Bluegill Muscle EPA tier 1 action level for assessing toxicity of selenium on fish and birds References

11. Selenium in Ovarian Tissue EPA tier 2 action level for assessing toxicity of selenium on fish and birds References

12. Ash Spill Temporal Pattern

14. Selenium in whole body gizzard shad References Temporal Pattern

15. References

16. Selenium in Largemouth Bass (muscle) Selenium in whole body Gizzard Shad Range of Se in shad

17. Composite Bioaccumulation Index (Fillets—integrates 4 species and 4 seasons) Arsenic Selenium Composite Bioaccumulation Index DOE Legacy Sample Site Positive control Ash spill References

18. Composite Bioaccumulation Index (Liver– integrates 3 species and 2 seasons) Arsenic Selenium Composite Bioaccumulation Index DOE Legacy Positive control Sample Site Ash spill References

19. Relationship between selenium in fillet and whole body of bluegill based on reconstruction analysis

20. Relevance and Application

21. Ash Periphyton Adult mayflies emergence Piscivorous birds Metals, detritus, periphyton Tree swallow GB heronOsprey Mayfly nymph Sunfish LM bass Shad Racoon Mussel Integrated Aquatic Food Chain Studies Frogs Se +4 Se -2 To periphyton

22. Establishing causal relationships among environmental variables Possible direct cause & effect relationship Possible spatial correlations (indirect causality) Bird bioaccumulation Bird Health Fish bioaccumulation Fish Health Macroinvert bioaccumulation Metals in other biota - turtles - amphibians - raccoons Metals in water and sediment

23. Summary and Conclusions - Food chain studies provide a weight-of-evidence approach for evaluating possible causal relationships between exposure to fly ash metals and effects on ecological resources - For all fish species studied, there is spatial gradient in bioaccumulation of selenium and arsenic with highest levels downstream of the ash spill - In most cases, levels of selenium in fish collected below the ash spill are not high enough to trigger the EPA tiered monitoring scheme for assessing toxicity of selenium in fish - Spatial and temporal patterns in bioaccumulation suggest there are causal relationships between exposure to fly ash-associated metals and levels of metals in fish