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

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

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

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

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

Part 2. Fillet Contaminants

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

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

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

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

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

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

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

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

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

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

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 %

Part 1. Fish Health Results Hematocrit (%) Sample Range TreatmentMeanLowHigh Control Mine Normal Hematocrit = 25 to 40 % No fish strain differences

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

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

Part 1. Fish Health Results Plasma Glucose (mg/dL) Sample Range TreatmentMeanLowHigh Control Mine mg/dL Normal Glucose = 50 to 150 mg/dL No fish strain differences

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

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

Part 1. Fish Health Results Plasma Lactate Sample Range TreatmentMeanLowHigh Control Mine mg/dL Normal Lactate = 2 to 25 mg/dL No fish strain differences

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

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

Fish Health Results Plasma Chloride Treatment

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)

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

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

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

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

Part 2. Fillet Contaminant Results

Fillet Measurements Metals: Mercury Cadmium Minerals: Selenium

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

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

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

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

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

Part 2. Fillet Contaminant Results for Mercury (ppm) TreatmentMean Fish High% UC Control Mine 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

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

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

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

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

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

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

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)

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

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

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

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

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