Linking fecal indicator bacteria concentrations in large mixed-use watersheds back to diffuse human sources, such as septic systems, has met limited success. In this study, 64 rivers that drain 84% of Michigan's Lower Peninsula were sampled under baseflow conditions for Escherichia coli, Bacteroides thetaiotaomicron (a human source-tracking marker), landscape characteristics, and geochemical and hydrologic variables. E. coli and B. thetaiotaomicron were routinely detected in sampled rivers and an E. coli reference level was defined (1.4 log 10 most probable number·100 mL
−1). Using classification and regression tree analysis and demographic estimates of wastewater treatments per watershed, septic systems seem to be the primary driver of fecal bacteria levels. In particular, watersheds with more than 1,621 septic systems exhibited significantly higher concentrations of B. thetaiotaomicron. This information is vital for evaluating water quality and health implications, determining the impacts of septic systems on watersheds, and improving management decisions for locating, constructing, and maintaining on-site wastewater treatment systems.Escherichia coli | Bacteroides thetaiotaomicron | baseflow | reference conditions | septic system W ater quality degradation influenced by diffuse sources at large watershed scales has been difficult to describe. Human modifications of natural landscapes can permanently alter hydrologic cycles and affect water quality (1, 2). Deforestation (3) and increased impervious surface area (4) have been linked with decreased infiltration and thus increased surface runoff. Overland flows concentrate pollutants and rapidly transport them down gradient where they eventually enter surface water systems and affect water quality (5, 6). A number of models have been developed to calculate overland and surface water flows (7,8) and nutrient/chemical transport (9), but few studies have focused on microbial movement from land to water, particularly nontraditional fecal indicator bacteria that can be used to track human sources of pollution.Microbial contamination poses one of the greatest health risks to swimming areas, drinking water intakes, and fishing/shellfish harvesting zones where human exposures are highest (10-12). These highly visible areas often receive more attention than sources of contamination because identifying the origin of pollution in complex watersheds requires costly comprehensive investigation of environmental and hydrologic conditions across temporal and spatial scales (13). Grayson et al. (14) suggest using a "snapshot" approach that captures water quality characteristics at a single point in time across broad areas to provide information frequently missed during routine monitoring. Compared with long-term comprehensive investigations, the snapshot approach reduces the number of samples, cost, and personnel required to examine pollution sources.Escherichia coli concentrations are commonly used to describe the relative human health risk during water quality monitoring in li...