2. Selenium Naturally occurring metalloid Associated with: Coal seams Phosphate deposits Other mineral formations

3. Increased mobilization due to: Irrigation Sewage Fly ash/coal combustion Mining

4. 4 Oxidation states Selenite (SeO 3 -2 ) Selenate (SeO 4 -2 ) Selenides (Se -2 ) Elemental selenium (Se) Insoluble Poorly assimilated by aquatic plants

5. Immobilized Selenate is oxidized to Selenite Selenite binds to: Clay Organic particles Forms precipitate with iron Precipitate settles out Sediment can be buried Remobilized by: Plant root absorption Consumption by inverts and fish

6. Benefits: Protects DNA, lipids, and proteins If fish are deficient: Loss coordination Reduced growth Reduced immune response

7. Impairs gas exchange Swollen lamellae Constricts blood vessels Binds to hemoglobin Unable to carry oxygen Induce cataracts Edema Seleno-proteins

8. Similar to sulfides Cells cannot discriminate Selenium substituted for sulfur Results in dysfunctional amino acids/proteins Accumulates in gonads, kidney, and liver

9. Seleno-proteins stored in ovarian tissues Proteins transferred to eggs Building blocks for tissue synthesis Metabolic fuel External feeding Critical period High mortality

10. Faulty proteins used for development Results in teratogenesis: Craniofacial deformities Spinal deformities Missing fins Edema Inability to feed leading to death

11. Kyphosis Lordosis Scoliosis

12. 1) Determine the effects of selenium on the aquatic ecosystem of the Upper Mud River Watershed, WV 2) Determine the effects of selenium on the survival of bluegill larvae

13. Lower Guyandotte River Watershed Lincoln and Boone Counties, WV 124 ha reservoir 4.5-6 m depth Inhabitants: Bluegill Largemouth bass Crappie Channel catfish

14. Paint Creek Watershed Fayette County, WV 81.75 ha reservoir 12.2 m depth Inhabitants: Bluegill Largemouth bass Channel catfish White crappie

16. October 2008-November 2010 Mud River (main stem and tributaries) 20 total sites Mined (13) Unmined (7) Reference Site 2 sites

17. Water quality

18. Plum Orchard Lake (Reference Site)

19. YSI: DO, pH, temperature, conductivity Total and dissolved Fe, total Se, TDS, sulfates, alkalinity, hardness, TSS and TOC Picture credit: Melissa ONeal

20. July 2009 Stantec, Inc. Stream (13) Reservoir (5) Transects Top 5 cm Picture credit: Stantec, Inc., Lexington, KY

23. July, August, September 2010 Mud River Reservoir (2) Reference Site (1) 10-20 min/ tow Surface and mid depth 95% ethanol

24. November 2009 and 2010 Mud River (11) Reference Site (2) 2009: 4 kick samples 2010: 6-8 kick samples Sorted into functional feeding group (FFG) Photo credit: Melissa ONeal

27. June 2009 and 2010 Spawning Mud River Reservoir Whole Body (n=118) Gonad (n=31) Reference Site Whole Body (n=86) Gonad (n=10) Egg selenium calculated Photo credit: John Howell

28. June 2009 and 2010 2009: Entire redd collected 2010: Only shells, rocks, and branches Individual redds collected Transported to WVU wet lab

30. Nest split into tanks A Tanks: 28 day early life stage test for chronic toxicity B Tanks: 9 day swim-up success Tanks contained water from original location 3-days after hatching fed fine fish food and brine shrimp twice daily

31. Multiple full water changes done daily Ammonia (daily) Nitrite, alkalinity, hardness (weekly) Temperature, DO, and pH (subset daily)

33. VariableMinedUnminedReference Temperature ( o C) 14.19 ± 0.42 a 13.60 ± 0.57 a 17.83 ± 1.45 b Conductivity (µs/cm) 1059.93 ± 33.91 a 152.23 ± 7.16 b 38.38 ± 0.66 c pH 7.77 ± 0.02 a 7.26 ± 0.05 b 7.05 ± 0.09 b DO (mg/L) 8.91 ± 0.32 a 8.23 ± 0.20 a 9.63 ± 1.88 a Hardness (mg/L CaCO 3 ) 640.81 ± 31.91 a 60.14 ± 5.49 b 13.35 ± 0.302 c Alkalinity (mg/L CaCO 3 ) 159.45 ± 5.54 a 37.09 ± 2.05 b 16.87 ± 1.37 c

34. VariableMinedUnminedReference TDS (mg/L) 1416.57 ± 217.61 a 139.16 ± 16.52 b 16.87 ± 1.37 c Sulfate (mg/L) 462.24 ± 17.11 a 39.87 ± 8.64 b 12.14 ± 0.64 c Dissolved Iron (mg/L) 0.068 ± 0.007 a 0.131 ± 0.009 b 0.208 ± 0.023 c Total Iron (mg/L) 0.606 ± 0.189 a 0.566 ± 0.093 ab 0.336 ± 0.037 b Total Selenium (µg/L) 6.52 ± 0.289 a 0.262 ± 0.015 b 0.194 ± 0.021 b TOC (mg/L) 3.46 ± 0.36 a 3.22 ± 0.308 a 3.15 ± 0.079 a

35. a a Site Type Unmined Mined Selenium (µg/g DW) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 a a

36. a a a a a

37. a a b

38. Site FiltererGathererScraperShredderComposite Unmined 2.69 ± 0.4 a 3.90 ± 1.0 a 3.7 ± 0.52 a 1.9 ± 0.72 a 3.4 ± 0.42 a Mined 11.7 ± 1.4 b 12.44 ± 1.8 b 8.1 ± 0.93 b 11.5 ± 2.45 b 11.1 ± 0.87 b Reference N/A2.67 ± 0.04 a N/A0.88 ± 0.12 a 2.1 ± 0.50 a

39. * Mud ReservoirReference 0 5 10 15 20 25 30 35 Site Selenium (µg/g DW) a b

40. * Mud ReservoirReference 0 5 10 15 20 25 30 Site Selenium (µg/g DW) a b

41. VariableReference Mud River Reservoir Temperature ( o C) 21.41 ± 0.12*22.69 ± 0.12 Dissolved O 2 (mg/L) 7.87 ± 0.097.99 ± 0.05 pH 7.73 ± 0.03*8.36 ± 0.008 Ammonia (mg/L) 0.766 ± 0.059*0.308 ± 0.041 Nitrite (mg/L) 0.01 ± 0.001*0.017 ± 0.002 Hardness (mg/L CaCO 3 ) 20 ± 0.0*250 ± 0.0 Alkalinity (mg/L CaCO 3 )16.35 ± 1.13*80 ± 0.0

43. The mine boundary is a good delineation for site types Sites in the mine and downstream were of high (> 5 µg/L) or moderate hazard (3-5 µg/L) based on the hazard profile by Lemly (1995) Levels as low as 2 µg/L can be bioaccumulated in the food chain and cause reproductive failure

44. All sediment samples considered no to low hazard ( 2 µg/g DW) Reservoir transects All samples also considered low to no hazard at < 2 µg/g DW Selenium seems to remain in the water column

45. Mud River Reservoir (mined and unmined) sites differed from the reference site Not sorted to species (zooplankton, etc.)

46. Mined areas greater than dietary threshold for fish (3 µg/g) and birds (7 µg/g) Did not see a significant difference between the reference site and unmined sites

47. No hazard category for whole body/gonad selenium concentrations Egg selenium (68.76 ± 1.95 µg/g ) High hazard (>20 µg/g)

48. Reference Mud Reservoir Toxic Effect Threshold: 5 ug/g is considered the threshold for the health and reproductive success of freshwater fish (Lemly 1995)

49. Did not observe deformities in larval fish Low survival possibly due to temperature and ammonia levels

50. No observations of deformaties Mined areas had higher selenium levels in the following categories: Water selenium concentrations Benthic macroinvertebrates Whole body/gonad/egg concentrations