Abstract   The present study evaluated the risk associated with the application and co-occurrence of 2 herbicides, atrazine and sulfentrazone, applied to a 32-ha corn and soybean rotational field. Field concentrations of the compounds were measured in soil, runoff water, and groundwater, with peak mean atrazine and sulfentrazone concentrations found in the soil (144 ng/g dry wt, and 318 ng/g dry wt, respectively). Individual and mixture laboratory bioassays were conducted to determine the effects of atrazine and sulfentrazone on the survival of Daphnia magna and Pimephales promelas, the germination of Lactuca sativa, and the growth of Pseudokirchneriella subcapita and Lemna minor. Pseudokirchneriella subcapita and L. minor were the most susceptible species tested, and the effects on growth of the herbicides in mixtures best fit an independent action model. Risk quotients and margin of safety of 10% (MOS10) values were used to estimate risk and were calculated using runoff water concentrations. The MOS10 values were more sensitive than risk quotients in estimating risk. The MOS10 value for sulfentrazone runoff water concentration effects on P. subcapita was 7.8, and for L. minor was 1.1, with MOS10 values < 1 indicating potential risk. Overall, the environmentally relevant concentrations fell below the effect concentrations; therefore, atrazine and sulfentrazone posed little to no risk to the nontarget species tested. Environ Toxicol Chem 2017;36:1301–1310. © 2016 SETAC