The occurrence of chemicals of emerging concern (CECs) and potential effects to aquatic biota in select tributaries of the St. Croix River in Minnesota and Wisconsin were measured. In 2011, treated wastewater effluent samples were collected from 22 sites in the St. Croix River Basin to determine total estrogenic activity. In 2012, wastewater effluent was collected at five select locations based on total estrogenicity and analyzed for CECs. In addition, surface water, bottom sediment, resident fish, and resident crayfish were collected upstream and downstream from effluent discharges. Water samples were analyzed for CECs (wastewater indicator compounds, steroid hormones, sterols, and pharmaceuticals). Bottom sediment samples were analyzed for a subset of CECs (wastewater indicator compounds, steroid hormones, and sterols). Fish and crayfish tissues were analyzed for select pharmaceuticals, antimicrobial compounds, plastic components, and alkylphenols. Multiple biological endpoints were assessed in resident and caged fish to assess biological effects of exposure to CECs.