The central research objective will be evaluated via two specific project objectives and associated methods: 1) Quantify changes in CEC complex mixture composition through space and time, and 2) Measure and relate the biological impacts of organisms to changing chemical exposure. During Year 1, bimonthly water samples will be gathered at four previously established sites. Chemical composition of the 15 most abundant compounds / metabolites from preliminary investigation will be quantified via liquid chromatography with tandem mass spectrometry (UIowa). Select subsamples will be quantified at the USGS National Water Quality Lab. Water samples will also be used for laboratory quantitative PCR-based fish gene expression tests (UW-M) and bioluminescent yeast (estrogenicity) nuclear receptor activation bioassays (USGS). During Year 2, instream caged fish experiments will be conducted to evaluate CEC exposure effects during reproduction (UW-M). Concurrent streamflow (USGS) and CEC chemical measurements will occur (UIowa). Water samples will be collected for laboratory batch tests (UIowa) to determine bulk chemical attenuation kinetics. During Year 3, data and statistical analysis will determine relationships between changing chemical composition exposure and quantified biological effects, providing predictive power to CEC attenuation, linking CEC presence with effects. Results interpretation and report writing will occur Year 3.

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