There is a great diversity of sources of chemical contaminants and stressors over large geographic areas. Chemical contaminant inputs and magnitude can potentially exhibit wide seasonal variation over large geographic areas. Together, these factors make linking exposure to monitored chemical contaminants and effects difficult. In practice, this linkage typically relies on relatively limited chemical occurrence data loosely coupled with individual effects, and population- or community-level assessments. Increased discriminatory power may be gained by approaching watershed level assessment in a more holistic manner, drawing from a number of disciplines that target endpoints spanning levels of the biological hierarchy. Using the Sacramento River as a case study, the present study aimed to 1) evaluate the performance of new analytical and biomarker tools in a real world setting and their potential for linking occurrence and effect; 2) characterize the effects of geographic and temporal variability through the integration of suborganismal, tissue, and individual level endpoints, as well as extensive chemical analyses; 3) identify knowledge gaps and research needs that limit the implementation of this holistic approach; and 4) provide an experimental design workflow for these types of assessments. Sites were selected to target inputs into the Sacramento River as it transitions from an agricultural to a mixed but primarily urban landscape. Chemical analyses were conducted on surface water samples at each site in both the spring and fall for pesticides, hormones, and active pharmaceutical ingredients (APIs). Active pharmaceutical ingredients were more often detected across sampling events in the fall; however, at the most downstream site the number of analytes detected and their concentrations were greater in the spring, which may be due to seasonal differences in rainfall. Changes in gene and protein expression targeting endocrine and reproductive effects were observed within each sampling event; however, they were inconsistent across seasons. Larval mortality at the most downstream site was seen in both seasons; however, behavioral changes were only observed in the spring. No clear linkages of specific analyte exposure to biological response were observed, nor were linkages across biological levels of organization. This failure may have resulted from limitations of the scope of molecular endpoints used, inconsistent timing of exposure, or discordance of analytical chemistry through grab sampling and longer term, integrative exposure. Together, results indicate a complicated view of the watershed.

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