Contaminants of emerging concern (CECs; e.g. pharmaceuticals, flame retardants, pesticides, and industrial chemicals) are omnipresent throughout tributaries to the Great Lakes. Furthermore, CECs are often present at concentrations that are potentially hazardous to aquatic species. Since 2010, we characterized the presence of CECs at 309 sites within 47 Great Lakes tributaries and characterized responses of fathead minnows (Pimephales promelas) exposed to river water at a subset of 26 sites within 4 tributaries. Our work resulted in three independent lines of evidence related to the potential hazards of CEC exposure to fish. First, vulnerability (where ‘vulnerability’ refers to likelihood) of surface waters to CEC presence was predicted using select watershed characteristics. Second, hazard to fish (where ‘hazard’ means the potential for adverse biological responses) was predicted using screening values for a subset of CECs. Third, biological responses of fathead minnows exposed to river water in streamside exposures were measured. We assessed the congruence of these three lines of evidence for identifying sites with elevated hazards to CEC exposure. Predicted vulnerability and hazards agreed at 66% of all sites. Where the two indices did not agree, vulnerability often underestimated predicted hazard. When compared to measured biological responses from streamside exposures, predicted hazards agreed for 42% of samples. Furthermore, when predicted hazards for specific effect categories were compared to similar measured biomarkers, 26 and 46% of samples agreed for reproductive and physiological effect categories, respectively. Overall, vulnerability and hazard predictions tended to overestimate the measured biological responses, providing a protective estimate of the potential hazards of CEC exposure to fish. When used together, these three approaches can be useful for resource managers to prioritize management activities in efforts to minimize hazards of CEC exposure and can be used by researchers to prioritize studies focused on understanding the hazards of CEC exposure to fish.

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