Microbial source tracking to identify fecal sources contaminating the Toronto Harbour and Don River watershed in wet and dry weather

Identification of sources of fecal pollution in urban areas is critical for protecting public health, the environment, and guiding remediation. We collected 670 water samples at 46 sites in 2018 to study fecal pollution in the Toronto Harbour and Don River watershed. Water samples were analyzed for E. coli, wastewater chemicals, and microbial source tracking DNA markers using a digital PCR technique. Microbial DNA markers were useful for interpreting the sources of elevated E. coli concentrations in the study. The HF183 marker for human sewage was the most frequently detected DNA marker, occurring in 100% of samples taken from the Don River and associated outfalls. It was also frequently detected in the Inner Harbour and outfalls along the Toronto harbour front. It was detected less frequently and at lower levels in the outer harbour. Detection of a human mitochondrial DNA marker and wastewater chemical markers such as caffeine provided additional evidence of widespread sewage contamination. The gull DNA marker was widely detected, but at lower frequencies and levels than human source DNA markers. A wet weather response of increased E. coli and human DNA marker concentrations occurred at most sites. However, human DNA markers were also widely detected on dry weather sampling days, indicating sewage cross-connections in stormwater and dry weather CSO systems. The cumulative impact of cross-connected stormwater outfalls is likely an under-recognized source of sewage contamination. E. coli and HF183 DNA marker levels observed can serve as benchmarks for evaluating future water quality improvements from wastewater infrastructure investments.