1. U.S. Department of the Interior U.S. Geological Survey Bioavailability of Metals from Mining and Effects on Aquatic Organisms Christopher J. Schmitt* U.S. Geological Survey Columbia Environmental Research Center Columbia, MO 65201 cjschmitt@usgs.gov

2. *Acknowledgements FWS: D. Heckathorne, D. Martin, S. Dudding, T. Nash, C. Charbonneau, G. Allen, V. Tabor, J. Dwyer EPA: M. Doolan Assistance (2001) from J. Dalgarn (BIA, Miami, OK); D. Sappington (Quapaw Tribe of OK); and A. Donahue and J. Arms (CERC) CERC colleagues and co- authors S. Finger, M. Wildhaber, W. Brumbaugh, T. May, J. Whyte, D.Tillitt Spring River, Cherokee Co., KS Spring River, Ottawa Co., OK

3. Pb-Zn Mining Areas in MO, OK, & KS MO KS OK From Ferderer, D.A., 1996. U.S. Geological Survey, Open-File Report 96-549 IA IL Old Lead Belt (Pb/Zn; Inactive) Tri-States District (Zn/Pb; Inactive) New Lead Belt (Pb/Zn; Active) Prospecting Area

4. Bioavailability Not all the metals in sediment are available for uptake by aquatic organisms – two parts Chemical component Metal association (binding) with or to particles Assessment = sequential / partial extraction Particle size component Aquatic ecosystems are particle-driven; different size particles are utilized by organisms to differing degrees Metals not distributed uniformly among particle sizes; difficult to assess and often ignored Measure uptake / accumulation by organisms

5. Sequential Extraction (ca. 1981) 1-N HCl (AVS-SEM) Modified from Tessier et al. (1979, Anal Chem 51:884) Sulfide Minerals Pore Water Schmitt, C.J., S.E. Finger, T.W. May, and M.S. Kaiser, 1987. Bioavailability of lead and cadmium from mine tailings to the pocketbook mussel (Lampsilis ventricosa). Pp. 115-142 in R.J. Neves, ed. Proceedings of the Workshop on Die-offs of Freshwater Mussels in the United States. U.S. Fish and Wildlife Service and Upper Mississippi River Conservation Committee. Rock Island, IL.

6. Big R. Pb Fractions, 1981; also Zn Schmitt, C.J., S.E. Finger, T.W. May, and M.S. Kaiser, 1987. Bioavailability of lead and cadmium from mine tailings to the pocketbook mussel (Lampsilis ventricosa). Pp. 115-142 in R.J. Neves, ed. Proceedings of the Workshop on Die-offs of Freshwater Mussels in the United States. U.S. Fish and Wildlife Service and Upper Mississippi River Conservation Committee. Rock Island, IL.

7. Big R. Cd Fractions, 1981 Schmitt, C.J., S.E. Finger, T.W. May, and M.S. Kaiser, 1987. Bioavailability of lead and cadmium from mine tailings to the pocketbook mussel (Lampsilis ventricosa). Pp. 115-142 in R.J. Neves, ed. Proceedings of the Workshop on Die-offs of Freshwater Mussels in the United States. U.S. Fish and Wildlife Service and Upper Mississippi River Conservation Committee. Rock Island, IL.

8. Big R. Crayfish vs. Sediment Pb, 1981 Total Sediment Pb (ug/g dw) Crayfish Pb (ug/g dw) Schmitt, C.J., and S.E. Finger, 1982. The transport, fate, and effects of trace metals in the Big and Black River watersheds, Southeastern Missouri. U.S. Fish and Wildlife Service, Columbia National Fisheries Research Laboratory, Columbia, MO. Project Completion Report to the St. Louis District, U.S. Army Corps of Engineers, St. Louis, MO. 167 p. Orconectes luteus

9. Uptake of Pb and Cd by Lampsilis Tissue Pb, ug/g dw Tissue Cd, ug/g dw Indigenous Mussels Schmitt, C.J., S.E. Finger, T.W. May, and M.S. Kaiser, 1987. Bioavailability of lead and cadmium from mine tailings to the pocketbook mussel (Lampsilis ventricosa). Pp. 115-142 in R.J. Neves, ed. Proceedings of the Workshop on Die-offs of Freshwater Mussels in the United States. U.S. Fish and Wildlife Service and Upper Mississippi River Conservation Committee. Rock Island, IL. Pb Cd

10. Carcass Pb vs. Sediment Pb, 1989 Log Total Sediment Pb Log Carcass Pb Schmitt, C.J., M.L. Wildhaber, J.B. Hunn, T. Nash, M.N. Tieger, and B.L. Steadman, 1992. Lead in Missouri streams: Monitoring pollution from mining with an assay for δ-aminolevulinic acid dehydratase (ALA-D) in fish blood. U.S. Fish and Wildlife Service, Columbia, Missouri Field Office. 43 p. Hypentelium nigricans Moxostoma duquesnei Center Creek Big River

11. Blood Pb vs. Sediment Pb, 1989 Log Total Sediment Pb Log Blood Pb Hypentelium nigricans Moxostoma duquesnei Center Creek Big River Schmitt, C.J., M.L. Wildhaber, J.B. Hunn, T. Nash, M.N. Tieger, and B.L. Steadman, 1992. Lead in Missouri streams: Monitoring pollution from mining with an assay for δ-aminolevulinic acid dehydratase (ALA-D) in fish blood. U.S. Fish and Wildlife Service, Columbia, Missouri Field Office. 43 p.

12. Effects Span many levels of biological organization Biochemical / physiological to community Contaminant penetrates to target sites (tissue) Exposure pathway [Sediment-(Invertebrate?)-Fish] Documentation = laboratory + field studies Lab studies with field-collected sediments, spiked sediments, pore waters, waterborne metals Isolate effects of contaminants from other factors Field studies -- long-term cumulative effects Other factors difficult to tease out – Neosho madtom Resilience

13. Heme Biosynthesis and Pb HbFe Joselow, M. M. 1980. Blood zinc and lead poisoning. Pp. 171-181 in J. O. Nriagu, ed. Lead in the Environment.

14. -Aminolevulinic Acid Dehydratase (ALA-D) Catalyzes condensation of porphobilinogen (PBG), a porphyrin / heme precursor, from ALA Present in most cells; greatest in blood & other heme- rich tissues (liver, spleen, kidney, etc.) Activity inhibited by Pb; affected at Pb-B 0.5 mg/L (50 µg/dL) in fish Requires Zn for activation; activity inversely correlated w/ Zn-B in previous fish studies; reactivated by Zn (?) Activity can be measured; assay has a long history of use as Pb biomarker in humans, birds, and fish

15. ALA-D Activity vs. Blood Pb in Big R. Suckers, 1981 C. J. Schmitt et al., 1984. Can. J. Fish. Aquat. Sci. 41:1030- 1040. Blood Pb (ug/g ww) ALA-D/Hb (nmol/mg/h)

16. ALA-D Activity vs. Blood-Pb in Big R. Longear Sunfish, 1980 F. J. Dwyer et al., 1988. J Fish Biol 33:307-313. Blood Pb (ug/g ww) ALA-D/Hb (nmol/mg/h) Lepomis megalotis

17. ALA-D/Hb vs. Sediment Pb, 1989 Log ALA-D/Hb (nmol/mg/h Log Total Sediment Pb (ug/g dw) Schmitt, C.J., M.L. Wildhaber, J.B. Hunn, T. Nash, M.N. Tieger, and B.L. Steadman, 1992. Lead in Missouri streams: Monitoring pollution from mining with an assay for δ-aminolevulinic acid dehydratase (ALA-D) in fish blood. U.S. Fish and Wildlife Service, Columbia, Missouri Field Office. 43 p. Center Creek Big River

18. MO AR OK KS Picher Field (mines & tailings; Tribal lands) 3 Gauge Elm Ck. Neosho R. Miami 6 3 mi. 5 Grand L. Tar Ck. Spring R. 4 Gauge 2 1 Picher Zincville TSMD-OK 2001

19. TSMD Willow Creek Tar Creek Center Creek

20. BR / Desloge, MO Pb >1200 µg/g dw LB / Macon, MO BR LB CERC OzCf

21. 112 Fish / 6 Spp. / 10 Sites SRNRREFBR Sp1243(R)56 (TC)7(C)8(O)10(L)9Σ Carp4434356(b,l)231 ChC5154233(b)1235 FhC224 LmB25412121 SpB33129 WhC324312

22. Field Procedures 1 2 3 4

23. Laboratory Methods (Individual Fish) Age (scales only; cf not aged) Hb by azide-methemoglobin (HemoCue®) Blood metals (Fe, Pb, Cd, Zn) Freeeze-dried and digested in tube (clean!) Analyzed by ICPMS Liver (carp only) and carcass metals (Pb, Cd, Zn; not LB) by ICPMS (dw liver only) ALA-D by microtiter plate method (modified from Granick 1972 and Hodson 1976; described by Whyte et al. 2002)

24. PBG Standard Curve (X 3) Blank (X 3) ALA (X 3)) ALA-D Assay, 96-Well Microtiter Plate (12 samples + 9 standards) Color results from Ehrlichs reagent binding with PBG Plate is scanned with an automated plate reader Nmol PBG/µL blood/h Nmol PBG/mg Hb/h

25. BR LB Site 6 (NR-TC) Site 6 (NR-TC) Pb-B (ug/g dw) NAWQA carp range (0.05-2.20 μg/g dw, n = 182) 5.4 μ/g (n = 1) Pb-B vs. Pb-L in Carp, 2001* Pb-L (ug/g dw) y = x Site 3 (NR-Ref) n = 30, r = 0.88, p < 0.01 *2002 for LB

26. Pb*, Cd*, and Zn* in Carp, 2001** W. G. Brumbaugh et al. 2005, Arch Environ Contam Toxicol (in press). * Blood, liver and carcass; all ug/g dw ** 2002 for LB R 2 = 0.78 – 0.88 (L), 0.49 – 0.72 (C) R 2 = 0.69 (L), 0.61 (C) NS

27. SR & NR Discharge, Jun-Nov 2001 Collections originally scheduled for July 01 Postponed until October due to unusually high flows High flow may have induced carp to move Site 3 (SR) Site 4 (NR)

28. ALA-D/Hb vs. Pb-B, 2001* Pb-B (ug/g ww) All Micropterus ALA-D/Hb = 0.258 – 0.219 Pb-B, n = 29, r 2 = 0.68, p <0.01 BR ALA-D/Hb (nmol/mg/h) All catfish (mostly ChC) ALA-D/Hb = -1.206 – 0.237 Pb-B + 0.868 Zn-B, n = 35, R 2 =0.65 C. J. Schmitt et al., 2005. Environ Toxicol Chem 24, in press. *2002 for LB

29. Fe-B (or Hb) vs. Pb-B, 2001* BR Carp Pb-B (ug/g ww) LB CERC Fe-B (ug/g ww) C. J. Schmitt et al., 2005. Environ Toxicol Chem 24, in press. *2002 for LB

30. Summary (Bioavailability) Contamination by metals evident in TSMD fish Pb concentrations greater than background, but lower than in other historic mining areas (BR, etc.) Metals bioavailability issue resolved (blood, liver metals; correlations) Penetration to active sites / tissues Redundancy / QA vs. efficiency Variable results for carp suggest fish movement, possibly induced by flooding

31. ALA-D activity differed among sites (some taxa) Relationship vs Pb-B well documented in catfish and bass, less so in carp Differences among taxa apparent Sensitivity of ictalurids re. Neosho madtom? Bioavailability, unique biochemical response No detectable effects on Hb or Fe-B Exposure indicator* Higher level effects also evident (fish, invertebrates; dns) Future studies: Aquatic effects of Cd, Zn, and ? Summary (Effects)