Volatile Solids Outperform Pepper Mild Mottle Virus (PMMoV) as a Normalizing Parameter in Wastewater-Based Epidemiology: A 3-Year Multi-Site Comparative Study

Aims Wastewater-based epidemiology (WBE) requires reliable normalization to account for dilution and population variability across sampling locations. Although Pepper Mild Mottle Virus (PMMoV) is widely used as a viral fecal marker for normalization, its effectiveness varies considerably across sewersheds. This study aims to evaluate volatile solids (VS), a routine physicochemical wastewater parameter, as an alternative normalization marker over a multi-year, multi-site investigation. Methods and Results We analyzed 1,611 wastewater samples from five distinct locations in Eastern Ontario, including four wastewater treatment plants and one university campus, over three years. PMMoV and VS concentrations were measured and compared to assess their temporal stability and correlation with population size. VS exhibited markedly lower variability (monthly robust coefficients of variance 0.11-0.18) compared to PMMoV (0.30-0.52), whose concentrations fluctuated up to 100-fold within single sites and declined year-wise. VS concentrations remained stable with less than 6-fold variation. Linear regression showed that VS was strongly associated with population size (R² = 0.721, p = 0.044), whereas PMMoV showed no significant correlation (R² = 0.054, p = 0.706). Negative correlations between PMMoV and wastewater pH (p < 0.001) across multiple sites suggest that pH-dependent degradation affects PMMoV stability. Conclusions VS provided a more consistent normalization parameter than PMMoV in this multi-year, multi-site dataset, suggesting that routinely measured VS can serve as a practical normalization option for WBE in similar sewersheds. It requires simple, cost-effective measurement techniques and reflects fecal content independently of dietary fluctuations affecting molecular markers.