1. Physiological Overview of Rainbow Trout Grown In Reclaimed Mine-water Melody Danley, MS Division of Forestry, WVU, Morgantown, WV Patricia Mazik, PhD US Geological Survey, WV Coop Fish & Wildlife Research Unit, WVU, Morgantown, WV

2. Abundant mine water in WV: Approx. 868 million liters (or 232 million gallons) discharged daily Aquaculture identified as a potential opportunity for business diversity Introduction: Mine Water and Aquaculture in Appalachia

3. Experimental Question Can reclaimed mine water be used to grow… 1. Healthy fish? 2. Fish safe for human consumption?

4. Study Objectives To grow … 1. Healthy 2. Safe-to-eat … food fish using reclaimed mine water

5. Part 1. Fish Health  Monitored 4 general homeostasic parameters Balance and maintenance of an environment inside a body, different from the external surroundings

6. Part 2. Fillet Contaminants

7. EPA Food Safety Regulations in Mine Waters 1. Mercury 2. Cadmium 3. Selenium Rank based on: Occurrence in fish Potential to accumulate Toxicity to humans 3 priority pollutants considered as significant fish contaminants

8. Part 2. Fillet Contaminants  Fillets analyzed for EPA’s top 3 priority pollutants:  Mercury  Cadmium  Selenium

9. Materials and Methods Control fish grown at the USDA-ARS Aquaculture Center in Leetown, WV

10. Materials and Methods Treatment fish grown at reclaimed mine-water site in WV

11. Raceway set-up at reclaimed mine- water site in WV Materials and Methods Top View Flow 132 123 Fish Strain

12. Materials and Methods Sampling Procedure 1. Fish sampled monthly: Nov 2002 to May 2003 2. 3 strains of rainbow trout 3. 5 fish removed per raceway section/tank

13. Materials and Methods Sampling Procedure 4. Blood collected from caudal vein 5. Carcass wrapped in foil, bagged, and packed in ice 6. Samples frozen at -20 o C until analyzed

14. Materials and Methods Sample Analyses Blood: used standard medical-grade blood analyzers Carcass: filleted and analyzed by the National Research Center for Coal and Energy using EPA guidelines

15. Part 1. Fish Health Results All results are preliminary No replication

16. Blood measurements Whole blood: hematocrit Plasma: glucose lactate chloride Part 1. Fish Health Results

17. Part 1. Fish Health Results Hematocrit (%) Proportion of total blood volume = red blood cells (RBC) Indicates aerobic/respiratory status O 2 transport RBC content 100 % 40 %

18. Part 1. Fish Health Results Hematocrit (%) Sample Range TreatmentMeanLowHigh Control37.226.850.5 Mine35.416.064.5 Normal Hematocrit = 25 to 40 % No fish strain differences

19. Part 1. Fish Health Results Hematocrit Treatment n # Below Normal # Above Normal Control 90011 Mine180939 Normal Hematocrit = 25 to 40 %

20. Part 1. Fish Health Results Plasma Glucose Plasma = portion of blood with cells removed Primary energy source Indicator of aerobic metabolism

21. Part 1. Fish Health Results Plasma Glucose (mg/dL) Sample Range TreatmentMeanLowHigh Control92.044.4214.4 Mine77.036.3152.3 mg/dL Normal Glucose = 50 to 150 mg/dL No fish strain differences

22. Part 1. Fish Health Results Plasma Glucose Treatmentn # Below Normal # Above Normal Control9026 Mine18021 Normal Glucose = 50 to 150 mg/dL

23. Part 1. Fish Health Results Plasma Lactate A byproduct of metabolism when O 2 not available Indicator of anaerobic metabolism i.e. erratic and burst swimming

24. Part 1. Fish Health Results Plasma Lactate Sample Range TreatmentMeanLowHigh Control5.31.820.8 Mine9.62.034.4 mg/dL Normal Lactate = 2 to 25 mg/dL No fish strain differences

25. Part 1. Fish Health Results Plasma Lactate Treatment n # Below Normal # Above Normal Control 9020 Mine180012 Normal Lactate = 2 to 25 mg/dL

26. Involved in osmosis, fluid and cell membranes electrical charges Indicator of water and ion balance Fish Health Results Plasma Chloride Cl - Water Blood Gills H2OH2O H2OH2O H2OH2O

27. Fish Health Results Plasma Chloride Treatment

28. Fish Health Results Plasma Chloride Low chloride expected in control treatment due to high CO 2 in water Graph from Cameron and Iwama 1987 (mEq/L)

29. Fish Health Results Plasma Chloride Treatment n # Below Normal # Above Normal Control 90320 Mine18070 Normal Chloride = 105 to 140 mEq/L

30. Part 1. Fish Health Results Summary Some mine responses out of normal range: hematocrit, plasma lactate and chloride

31. Part 1. Fish Health Results Summary Some mine responses out of normal range: hematocrit, plasma lactate and chloride No fish strain differences

32. Part 1. Fish Health Results Summary Some mine responses out of normal range: hematocrit, plasma lactate and chloride No fish strain differences Most fish within normal ranges

33. Part 2. Fillet Contaminant Results

34. Fillet Measurements Metals: Mercury Cadmium Minerals: Selenium

35. Hg Contaminant Guidelines are Variable Depends on what government agency is involved FDA EPA State government

36. Hg Contaminant Guidelines are Variable Food and Drug Administration (FDA) Regulates state-to-state sales of food fish Uses an all-or-nothing approach: Hg < 1 ppm = unlimited Hg > 1 ppm = no consumption

37. Hg Contaminant Guidelines are Variable Environmental Protection Agency (EPA) No regulatory ability over food fish Develops risk-based guidelines and advisories Makes recommendations

38. Hg Contaminant Guidelines are Variable State Governments Regulate food fish sales within the state WV adopted the EPA risk-based method

39. Part 2. Fillet Contaminant Results for Mercury WV risk-based guidelines for Hg (adults): < 0.05 mg/Kg (ppm): Unlimited 0.05 – 0.22 ppm: 1 meal per week 0.22 – 0.95 ppm: 1 meal per month 0.95 – 1.89 ppm: 6 meals per year > 1.89 ppm: Do not eat

40. Part 2. Fillet Contaminant Results for Mercury (ppm) TreatmentMean Fish High% UC Control0.0290.10087 Mine0.0290.06095 If Hg < 0.05 ppm: Unlimited If Hg of 0.05 – 0.22 ppm: 1 meal per week % UC = percent of fish with unlimited consumption

41. Part 2. Fillet Contaminant Results for Cadmium No EPA or FDA cadmium limits established for food fish Max Cd

42. Part 2. Fillet Contaminant Results for Cadmium (ppm) Treatment% Fish BDL% SV Control100 Mine100 BDL = below detection limit of assay (4.7 ppm) EPA suggested screening value is 6 ppm (children) % SV = percent of fish below the screening value

43. Part 2. Fillet Contaminant Results Selenium No EPA or FDA Se limits established or any muscle-based foods Max Se

44. Part 2. Fillet Contaminant Results for Selenium (ppm) Treatment Fish High % Fish BDL% SV Control7.668100 Mine11.853100 BDL = Below detection limit of assay (8.47 ppm) EPA suggested screening value = 30 ppm (children) % SV = percent of fish below the screening value

45. Part 2. Summary of Fillet Contaminant Results Mercury: Concentrations were low Unlimited (to once weekly consumption Cadmium and selenium: All below EPA suggested screening limits

46. Summary and Conclusions Study objective was to grow healthy, safe-to-eat fish using reclaimed mine water

47. Summary and Conclusions Healthy Fish? Preliminary results: most physiological responses indicated healthy fish (> 80 % of samples) However, some hematocrit, plasma lactate and chloride concentrations were outside of the normal ranges (< 20 % of samples)

48. Summary and Conclusions Fillets safe to eat? Preliminary results: most mercury levels were low, consumption unlimited (95%) to once weekly (5%) Cadmium and selenium concentrations in fillets were below the EPA suggested screening values

49. Summary and Conclusions No replication so cannot determine if study objectives were met, but… Preliminary results were promising: aquaculture with reclaimed mine water may be a viable option for mining companies and other businesses

50. Study Conclusions The next phase of research should: Determine the source of unexpected physiological responses

51. Study Conclusions The next phase of research should: Determine the source of unexpected physiological responses Measure other potential contaminant concentrations in fillets

52. Acknowledgements USDA-CSREES for project funding The mining company for use of their reclaimed water treatment site Jeff Silverstein and the USDA National Center for Cool and Cold Water Aquaculture (Leetown, WV) for care and maintenance of control fish Kim Bjorgo, Sitima Jittinandana, Rodney Kiser, Chris Nelson, and Ken Stewart for helping with sample collection and analyses National Research Center for Coal and Energy Analytical Lab (Morgantown, WV) for fillet analyses USGS-WV Cooperative Fish and Wildlife Research Unit at WVU, the Division of Forestry and WVU for additional research support and facility use