Identification of environmental and methodological factors driving variability of Pepper Mild Mottle Virus (PMMoV) across three wastewater treatment plants in the City of Toronto

PMMoV has been widely used to normalize the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, influenza, and respiratory syncytial virus (RSV) to account for variations in the fecal content of wastewater. PMMoV is also used as an internal RNA recovery control for wastewater-based epidemiology (WBE) tests. While potentially useful for the interpretation of WBE data, previous studies have suggested that PMMoV concentration can be affected by various physico-chemical characteristics of wastewater. There is also the possibility that laboratory methods, particularly the variability in centrifugation steps to remove supernatant from pellets can cause PMMoV variability. The goal of this study is to improve our understanding of the main drivers of PMMoV variability by assessing the relationship between PMMoV concentration, the physico-chemical characteristics of wastewater, and the methodological approach for concentrating wastewater samples. We analyzed 24-hour composite wastewater samples collected from the influent stream of three wastewater treatment plants (WWTPs) located in the City of Toronto, Ontario, Canada. Samples were collected 3 to 5 times per week starting from the beginning of March 2021 to mid-July 2023. The influent flow rate was used to partition the data into wet and dry weather conditions. Physico-chemical characteristics (e.g., total suspended solids (TSS), biological oxygen demand (BOD), alkalinity, electrical conductivity (EC), and ammonia (NH