Elevated Winter Stream Temperatures below Wastewater Treatment Plants Shift Reproductive Development of Female Johnny Darter Etheostoma nigrum : A Field and Histologic Approach

River water temperatures are increasing globally, particularly in urban systems. In winter, wastewater treatment plant (WWTP) effluent inputs are of particular concern because they increase water temperatures from near freezing to ~7-15 °C. Recent laboratory studies suggest that warm overwinter temperatures impact the reproductive timing of some fishes. To evaluate winter water temperature's influence in the wild, we sampled Johnny Darter from three urban South Platte River tributaries in Colorado upstream and downstream of WWTP effluent discharge sites. Fish were collected weekly during the spring spawning season of 2021 and reproductive development was determined from histological analysis of the gonads. Winter water temperatures were approximately 5-10 °C greater ~300 m downstream of the WWTP effluent compared to upstream sites, and approximately 3°C warmer at sampling sites ~5000 m downstream of the effluent discharge. Females collected downstream of WWTP effluent experienced accelerated reproductive development compared to upstream by 1-2 weeks. Water quality, including total estrogenicity, and spring water temperatures did not appear to explain varying reproductive development. It appears that small increases in winter water temperature influence the reproductive timing in . Further investigations into how shifts in reproductive timing influence other population dynamics are warranted.