One of the biggest challenges when conducting a continental-scale assessment of streams is setting appropriate expectations for the assessed sites. The challenge occurs for 2 reasons: 1) tremendous natural environmental heterogeneity exists within a continental landscape and 2) reference sites vary in quality both across and within major regions of the continent. We describe the process used to set expectations for the multimetric index of biotic integrity (MIBI) and observed/expected (O/E) indices generated from predictive models used to assess stream condition for the US Wadeable Streams Assessment (WSA). The assessment was based on a reference-site approach, in which the least-disturbed sites in each region of the US were used to establish benchmarks for assessing the condition of macroinvertebrate assemblages at other sites. Reference sites were compiled by filtering WSA sample sites for disturbance using a series of abiotic variables. Additional reference sites were needed and were obtained from other state, university, and federal monitoring programs. This pool of potential reference sites was then assessed for uniformity in site quality and comparability of macroinvertebrate sample data. Ultimately, 1625 sites were used to set reference expectations for the WSA. Reference-site data were used to help define 9 large ecoregions that minimized the naturally occurring variation in macroinvertebrate assemblages associated with continental-wide differences in biogeography. These ecoregions were used as a basis for developing MIBI and O/E indices and for reporting results. A least-disturbed definition of reference condition was used nationally, but we suspect that the quality of the best extant sites in ecoregions, such as the Northern Plains and Temperate Plains, was lower than that of sites in other ecoregions. For the MIBI assessment, we used a simple modeling approach to adjust scores in ecoregions where gradients in reference-site quality could be demonstrated conclusively. The WSA provided an unparalleled opportunity to push the limits of our conceptual and technical understanding of how to best apply a reference-condition approach to a real-world need. Our hope is that we have learned enough from this exercise to improve the technical quality of the next round of national assessments.
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