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This Midwest Stream Quality Assessment (MSQA) simultaneously characterized watershed and stream-reach water-quality stressors along with instream biological conditions, to better understand regional stressor-effects relations. The MSQA design focused on effects from the widespread agriculture in the region and urban development because of their importance as ecological stressors of particular concern to Midwest region resource managers.A combined random stratified selection and a targeted selection based on land-use data were used to identify and select sites representing gradients in agricultural intensity across the region. During a 14-week period from May through August 2013, 100 sites were selected and sampled 12 times for contaminants, nutrients, and sediment. This 14-week water-quality \u201cindex\u201d period culminated with an ecological survey of habitat, periphyton, benthic macroinvertebrates, and fish at all sites. Sediment was collected during the ecological survey for analysis of sediment chemistry and toxicity testing. Of the 100 sites, 50 were selected for the MSQA random stratified group from 154 NRSA sites planned for the region, and the other 50 MSQA sites were selected as targeted sites to more evenly cover agricultural and urban stressor gradients in the study area. Of the 50 targeted sites, 12 were in urbanized watersheds and 21 represented \u201cgood\u201d biological conditions or \u201cleast disturbed\u201d conditions. The remaining 17 targeted sites were selected to improve coverage of the agricultural intensity gradient or because of historical data collection to provide temporal context for the study.This report provides a detailed description of the MSQA study components, including surveys of ecological conditions, routine water sampling, deployment of passive polar organic compound integrative samplers, and stream sediment sampling at all sites. 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