1. Assessment of Fish Population Health in the Vicinity of the Kingston Fly Ash Release Marshall Adams - Oak Ridge National Lab Allison Fortner – Arcadis Swee Teh - Univ. California- Davis Mark Greeley - ORNL

2. Presentation Outline - Research objectives - Study Design - Results and major findings - individual responses - integrated (holistic) responses - causal relationships - Relevance and application

3. Objectives of Fish Health Studies Determine if fly ash exposure is causing short and/or long- term health effects on representative (sentinel) fish populations Evaluate potential causal relationships between bioaccumulation of metals in fish tissue and various indicators of fish health

4. Study Design Sample Frequency spring and fall each year since spring 2009 Health Indicators Measured - condition indices - bioenergetic indices - histopathology - organ dysfunction - carbohydrate-protein metabolism - electrolyte homeostasis - feeding and nutrition Fish Species Sampled Spring - bluegill, largemouth bass, redear sunfish, and while crappie Fall - bluegill, largemouth bass, and channel catfish 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 Sample Sites

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. Ecological Relevance Early warning, sensitivity, specificity Physiological Histopathological Reproductive Spring - Clutch size - Gonad condition - Egg quality - Oocyte atresia Indicators of: - organ dysfunction - electrolyte homeostasis - carbohydrate metabolism - protein metabolism - hematology - liver - gill - spleen - ovary (sp) Bioenergetic - condition factor - liver-somatic index - visceral-somatic index - lipid index - feeding & nutrition Measures of Fish Health Red = short-term and sensitive indicators Blue = longer term and more ecologically-relevant indicators Spring and Fall

9. Individual and Integrated Health Responses

10. Ash spill Bluegill Ref (Indicator of liver damage)

11. Ash spill Bluegill Ref

12. Ash spill Bluegill Ref (enzyme indicating liver damage) (indicator of gill damage)

13. Normal gill showing secondary lamellaeMucus cell hyperplasia in gill Mild mucus cell hyperplasia Severe mucus cell hyperplasia Example of Gill Histopathology

14. Bluegill Spring 2009 Bluegill Spring 2010 Condition factor Liver-somatic index Visceral-somatic index Feeding index Hematocrit Glucose Total protein Globulin Blood urea nitrogen Creatinine Liver enzyme Potassium Calcium Liver histopath Ovary histopath Condition factor Liver-somatic index Visceral-somatic index Feeding index Hematocrit Glucose Total protein Globulin Blood urea nitrogen Creatinine Liver enzyme Sodium Calcium Liver histopath Ovary histopath Variables used In analysis Integrated health responses 23 14 18 8 16 10 22 19

15. Largemouth Bass Ash spill Individual Responses Ref

16. Largemouth Bass Ash spill Ref (indicator of gill damage)

17. Largemouth bass Ash spill Ref (indicator of protein metabolism) (general stress indicator)

18. Largemouth bass Spring 2009 Largemouth bass Spring 2010 Condition factor Liver-somatic index Visceral-somatic index Feeding index Hematocrit Glucose Total protein Globulin Blood urea nitrogen Creatinine Liver enzyme Sodium Calcium Liver histopath Ovary histopath Condition factor Liver-somatic index Visceral-somatic index Feeding index Hematocrit Glucose Total protein Globulin Blood urea nitrogen Creatinine Liver enzyme Sodium Calcium Liver histopath Ovary histopath Variables use In analysis Integrated health responses (conical discriminant analysis ) 10 18 20 16 32 25 1429

19. Bluegill-fall 2009 Bluegill-fall 2010 Integrated health responses

20. Largemouth bass Fall 2009 Largemouth bass Fall 2010 CRM 8 Integrated health responses

21. Causal relationships between metal exposure and fish health

22. Selenium (mg/gmDW ) Bluegill Ref

23. Selenium (mg/gmDW ) (enzyme indicative of liver damage) Bluegill Ref

24. Selenium (mg/kgDW) Selenium in Fillets Largemouth Bass Ref

25. Largemouth Bass Selenium in Fillets Condition Factor Ref

26. Relevance and application of fish health studies

28. 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 water and sediment Metals in other biota - turtles - amphibians - raccoons

29. Summary - As demonstrated by individual bioindicators and the discriminant analysis procedure, the health of fish is generally poorer downstream of the fly ash spill than at reference areas - Food chain studies in combination with the use of multiple bioindicators provide a weight-of-evidence approach for evaluating possible causal relationships between exposure to fly ash metals and biological responses - Even though the health of fish immediately below the ash spill appears to be compromised to some degree, effects appears to be localized to a small area and to resident species - No significant health effects (i.e., damage or injury) have been observed in fish suggesting that: 1) metal exposure is not of sufficient magnitude or duration to elicit changes in biological responses, and/or 2) the rate of biological repair exceeds the rate of damage - From 2009 -2010, improvement in health status (recovery) seen in bluegill and bass at 2 of the 3 sites downstream of the ash spill