Characterization of sources and loadings of fecal pollutants using microbial source tracking assays in urban and rural areas of the Grand River Watershed, Southwestern Ontario

•Sources of fecal loading of a multiple-use watershed were evaluated over 10 months.•A Bacteroidales 16S rRNA gene assay was combined with land use and hydrological data.•Rainfall has a major role in host-specific Bacteroidales marker loading in rural areas.

Sources of fecal water pollution were assessed in the Grand River and two of its tributaries (Ontario, Canada) using total and host-specific (human and bovine) Bacteroidales genetic markers in conjunction with reference information, such as land use and weather. In-stream levels of the markers and culturable Escherichia coli were also monitored during multiple rain events to gain information on fecal loadings to catchment from diffuse sources. Elevated human-specific marker levels were accurately identified in river water impacted by a municipal wastewater treatment plant (WWTP) effluent and at a downstream site in the Grand River. In contrast, the bovine-specific marker showed high levels of cattle fecal pollution in two tributaries, both of which are characterized as intensely farmed areas. The bovine-specific Bacteroidales marker increased with rainfall in the agricultural tributaries, indicating enhanced loading of cattle-derived fecal pollutants to river from non-point sources following rain events. However, rain-triggered fecal loading was not substantiated in urban settings, indicating continuous inputs of human-originated fecal pollutants from point sources, such as WWTP effluent. This study demonstrated that the Bacteroidales source tracking assays, in combination with land use information and hydrological data, may provide additional insight into the spatial and temporal distribution of source-specific fecal contamination in streams impacted by varying land uses. Using the approach described in this study may help to characterize impacted water sources and to design targeted land use management plans in other watersheds in the future.