This study evaluated potential effects of mercury (Hg) on benthic macroinvertebrates in the South River, Virginia, USA. The study used a multiple lines of evidence approach, including spatially and temporally matched sediment chemistry, biological, and toxicological information (Sediment Quality Triad), exposure and effect analysis in bulk and interstitial sediment and interstitial water, and critical body residue analysis. Ten‐day Chironomus dilutus and Hyalella azteca toxicity tests established site‐specific no‐effect concentrations (NOEC) at 18.9 μg/g total Hg (THg) and 102 ng/g methylmercury (MeHg). However, the benthic community at these locations was impaired, with lower mayfly and caddisfly composition. Few locations had concentrations of THg and MeHg that exceeded the NOEC in bulk or interstitial sediment. The THg concentrations in interstitial water were far below concentrations expected to reduce survival in benthic invertebrates, and only a low percentage of samples exceeded sublethal (growth) low‐effect concentrations (LOEC) for THg or MeHg. The THg concentrations in invertebrate tissue did not exceed the NOEC or LOEC in the South River, and MeHg concentrations exceeded the LOEC at all locations, including those with no evidence of benthic impairment, illustrating the uncertainty associated with this line of evidence. Finally, statistical modeling that evaluated diversity of sensitive invertebrate species as a function of Hg exposure, geomorphological parameters, and physicochemical variables indicated that physicochemical and geomorphological parameters were most predictive of benthic community; where Hg was indicated, we were unable to distinguish between models with or without interstitial water Hg. Overall, the lines of evidence indicate that Hg, while clearly toxic to invertebrates at sufficiently high exposure concentrations, is not negatively impacting invertebrate communities in the South River. This study illustrates the difficulty of assessing risks to invertebrates using traditional tools of risk assessment and identifies critical gaps in knowledge that complicate the management of Hg risk. Integr Environ Assess Manag 2019;00:000–000. © 2019 SETAC

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