In the Midwestern United States, waterways such as the Elkhorn River experience an annual spring pulse of runoff that carries sediments, nutrients, and organic compounds downstream. The objective of the present study was to elucidate relationships between contaminant load in Elkhorn River water and biological impacts on female fathead minnows throughout the entire spring agrichemical pulse. Fish were maintained in on-site outdoor microcosms at the Elkhorn River Research Station. The start of the spring pulse was determined using commercially available atrazine strips that detected atrazine when concentrations exceeded 3 ppb. Once the pulse began, 5 serial 7-d exposures were conducted. Concentrations of atrazine, its metabolites, and 5 other herbicides were quantified using gas chromatography-mass spectrometry. Agrichemicals peaked during the first and second weeks of the pulse, with a smaller peak occurring during week 4, but the peaks were not directly associated with runoff events (as estimated from river discharge). Elevated agrichemical concentrations were associated with biological impacts, but not solely responsible. In the present study, differences in the abiotic environment were found to play a significant role in the defeminization of exposed female fathead minnows.