From food to sewer: Dietary inputs support the temporal and population-wide stability of pepper mild mottle virus in wastewater

• PMMoV was consistently detected at high levels in wastewater across diverse Swedish WWTPs • Average PMMoV daily levels scale almost proportionally with catchment population size • Only minor temporal trends were detected over days, months and years • No associations found with population structure, wastewater temperature or weekday • High PMMoV levels in spices and processed foods like sauces, snacks and ready meals Pepper Mild Mottle Virus (PMMoV) is widely used in wastewater-based surveillance as a faecal indicator and population size marker. It is highly abundant in domestic wastewater and originates from the consumption of Capsicum -containing foods. Despite its widespread use, data on the variability of PMMoV concentrations in wastewater remain limited, and its primary dietary sources are not fully characterised. To evaluate the robustness of PMMoV as a human faecal marker, we analysed wastewater from multiple locations in Sweden over different timescales, ranging from daily to weekly sampling for up to nearly three years. We also investigated dietary sources by screening a broad range of foods. Wastewater consistently contained high PMMoV levels, averaging 10.34 log 10 genome copies per inhabitant per day. The average daily PMMoV load scaled almost proportionally with the number of inhabitants connected to each catchment area. No clear temporal trends were detected over either short or long time periods, and no substantial associations were found with potentially influencing factors such as demographic or socioeconomic characteristics of the catchment population, season, wastewater temperature or weekday. Of 60 foods tested, 45 contained PMMoV, with concentrations ranging from 5.70 to 12.21 log 10 genome copies per serving. The highest levels were found in spices, such as paprika powder, as well as in ready meals, spreads and snack products. These widely consumed foods likely contribute to the consistently high PMMoV levels observed in domestic wastewater. Taken together, our findings support the potential of PMMoV as a robust and broadly applicable human faecal marker.