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Abstract: Thiamethoxam is a widely used neonicotinoid insecticide that has been detected in surface water monitoring programs in North America and Europe. This has led to questions about its toxicity to nontarget insects, specifically those with an aquatic life stage. To address the uncertainty associated with possible impacts from environmental exposures, a chronic (35‐d) outdoor mesocosm study with a formulated product containing thiamethoxam was conducted. The specific focus of the study was the response of mayflies (Ephemeroptera), which have been reported to be particularly sensitive in laboratory studies. A range of concentrations (nominally 0.1, 0.3, 1.0, 3.0, and 10.0 µg/L thiamethoxam), plus untreated controls were tested, and the abundance and emergence of mayflies (Cloeon dipterum) were assessed weekly for 35 d. Mean measured time‐weighted average exposures were within 6% of nominal over the duration of the study, with the mean half‐life of thiamethoxam in each treatment ranging from 7 to 13 d. Statistically significant reductions in both larval abundance and adult emergence were observed at 10.0, 3.0, and 1.0 μg/L following 1, 2, and 3 wk of exposure, respectively. Exposure to 0.1 and 0.3 µg/L thiamethoxam had no statistically significant effect on larval mayfly abundance or adult emergence at any point in the study. These findings support a 35‐d no‐observed‐effect concentration (NOEC) of 0.3 µg thiamethoxam/L for mayflies (C. dipterum) under chronic conditions. Furthermore, because the 95th percentile of environmental concentrations has been reported to be 0.054 µg/L, these results indicate that populations of C. dipterum and similarly sensitive aquatic insects are unlikely to be significantly impacted by thiamethoxam exposure in natural systems represented by the conditions in our study. Environ Toxicol Chem 2018;37:1040–1050. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC. [ABSTRACT FROM AUTHOR]