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ABSTRACT Objective Determine the 48-h acute toxicity of copper sulfate (CuSO4) to freshwater fish and snail species economically important to aquaculture in reconstituted waters ranging from “very soft” (low alkalinity/hardness/pH) to “very hard” (high alkalinity/hardness/pH). The 10 fish species studied were Channel Catfish Ictalurus punctatus, Golden Shiner Notemigonus crysoleucas, koi (a variant of Amur Carp Cyprinus rubrofuscus), Largemouth Bass Micropterus nigricans, Nile Tilapia Oreochromis niloticus, Rainbow Trout Oncorhynchus mykiss, Striped Bass Morone saxatilis, sunshine bass (female White Bass Morone chrysops × male Striped Bass Morone saxatilis), Walleye Sander vitreus, and White Bass Morone chrysops. Additional experiments examined CuSO4 toxicity in ghost rams-horn Biomphalaria havanensis and marsh rams-horn Planorbella trivolvis, which serve as intermediate hosts to parasites in many industry-relevant fish species, including those used in this study. Methods Acute toxicity of CuSO4 was determined during 48-h exposures to six nominal concentrations in each of the five reconstituted waters ranging from very soft to very hard. Bioassays were performed at water temperatures relevant to individual species (14–23.2°C). The lethal concentration to 50% of the population (LC50 values) and the No Observed Effect Concentration were determined for each species and reconstituted water combination. Results Using comparisons of the CuSO4 LC50 values to each species in the five reconstituted waters during separate bioassays, we determined that Rainbow Trout are most sensitive to CuSO4 compared with the least sensitive species, Channel Catfish, Nile Tilapia, and particularly Largemouth Bass. The No Observed Effect Concentration values would be considered the safe range for aquaculturists to use CuSO4 in a particular water type with a particular species. Snail species did not have similar trends or susceptibility to Cu (as CuSO4) as alkalinity increased. The CuSO4 toxicities were negatively correlated with temperatures. Conclusions These data benefit aquaculture producers when investigating treatments in various local waters or modeling by environmental scientists.