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The Big River (BGR) drains much of the Old Lead Belt mining district (OLB) in southeastern Missouri, USA, which was historically among the largest producers of lead-zinc (Pb-Zn) ore in the world. We sampled benthic fish and crayfish in riffle habitats at eight sites in the BGR and conducted 56-day in situ exposures to the woodland crayfish (Orconectes hylas) and golden crayfish (Orconectes luteus) in cages at four sites affected to differing degrees by mining. Densities of fish and crayfish, physical habitat and water quality, and the survival and growth of caged crayfish were examined at sites with no known upstream mining activities (i.e., reference sites) and at sites downstream of mining areas (i.e., mining and downstream sites). Lead, zinc, and cadmium were analyzed in surface and pore water, sediment, detritus, fish, crayfish, and other benthic macro-invertebrates. Metals concentrations in all materials analyzed were greater at mining and downstream sites than at reference sites. Ten species of fish and four species of crayfish were collected. Fish and crayfish densities were significantly greater at reference than mining or downstream sites, and densities were greater at downstream than mining sites. Survival of caged crayfish was significantly lower at mining sites than reference sites; downstream sites were not tested. Chronic toxic-unit scores and sediment probable effects quotients indicated significant risk of toxicity to fish and crayfish, and metals concentrations in crayfish were sufficiently high to represent a risk to wildlife at mining and downstream sites. Collectively, the results provided direct evidence that metals associated with historical mining activities in the OLB continue to affect aquatic life in the BGR.

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