Evaluation of four human-associated fecal biomarkers in wastewater in Southern Ontario.

Human fecal biomarkers (HFBs) have a longstanding history in the field of microbial source tracking (MST) serving as indicators of human fecal contamination in drinking and recreational water. Further, HFBs have aided in recent efforts to monitor human pathogen transmission within communities. The dilution of wastewater from various sources throughout the sewershed cannot be controlled and human fecal biomarkers (HFBs) can be used to normalize target human pathogen concentrations so that fluctuations in fecal matter in wastewater can be accounted for. In the current study, we monitored the prevalence of four HFBs - including two viruses, Pepper mild mottle virus (PMMoV), cross-assembly phage (crAssphage), as well as two human-associated Bacteroides markers, HF183 and BacHuman - in wastewater samples from ten Southern Ontario wastewater treatment plants and evaluated their temporal and spatial variation in context of environmental factors that may impact the ability of HFB to normalize pathogen concentrations in wastewater. Environmental variables including precipitation, wastewater flow rate, temperature, and concentrated mass were also analyzed for their potential correlation with HFB variation in wastewater. The four HFBs were detected at high concentrations across all 10 sampling locations. The median concentrations across all sampling sites were: PMMoV 3.6 Log gene copies (GC)/mL; crAssphage 5.0 Log GC/mL; HF183 6.8 Log GC/mL and BacHuman 6.9 Log GC/mL. All HFBs were found to be similarly stratified across all 10 sites, and the bacterial markers were consistently found at higher concentration compared to the viral HFBs at all sites. The coefficient of variation (CV) for each HFB was used to characterize the variability of each biomarker at each sewershed. BacHuman and crAssphage were found to have lower CV than PMMoV and HF183, indicating that BacHuman and crAssphage may perform better in reflecting the variations in abundance of human feces in wastewater or MST applications.