Sewer transport conditions and their role in the decay of endogenous SARS-CoV-2 and pepper mild mottle virus from source to collection

This study presents a comprehensive analysis of the decay patterns of endogenous SARS-CoV-2 and Pepper mild mottle virus (PMMoV) within wastewaters spiked with stool from infected patients expressing COVID-19 symptoms, and hence explores the decay of endogenous SARS-CoV-2 and PMMoV targets in wastewaters from source to collection of the sample. Stool samples from infected patients were used as endogenous viral material to more accurately mirror real-world decay processes compared to more traditionally used lab-propagated spike-ins. As such, this study includes data on early decay stages of endogenous viral targets in wastewaters that are typically overlooked when performing decay studies on wastewaters harvested from wastewater treatment plants that contain already-degraded endogenous material. The two distinct sewer transport conditions of dynamic suspended sewer transport and bed and near-bed sewer transport were simulated in this study at temperatures of 4 °C, 12 °C and 20 °C to elucidate decay under these two dominant transport conditions within wastewater infrastructure. The dynamic suspended sewer transport was simulated over 35 h, representing typical flow conditions, whereas bed and near-bed transport extended to 60 days to reflect the prolonged settling of solids in sewer systems during reduced flow periods. In dynamic suspended sewer transport, no decay was observed for SARS-CoV-2, PMMoV, or total RNA over the 35-h period, and temperature ranging from 4 °C to 20 °C had no noticeable effect. Conversely, experiments simulating bed and near-bed transport conditions revealed significant decreases in SARS-CoV-2 and total RNA concentrations by day 2, and PMMoV concentrations by day 3. Only PMMoV exhibited a clear trend of increasing decay constant with higher temperatures, suggesting that while temperature influences decay dynamics, its impact may be less significant than previously assumed, particularly for endogenous RNA that is bound to dissolved organic matter in wastewater. First order decay models were inadequate for accurately fitting decay curves of SARS-CoV-2, PMMoV, and total RNA in bed and near-bed transport conditions. F-tests confirmed the superior fit of the two-phase decay model compared to first order decay models across temperatures of 4 °C–20 °C. Finally, and most importantly, total RNA normalization emerged as an appropriate approach for correcting the time decay of SARS-CoV-2 exposed to bed and near-bed transport conditions. These findings highlight the importance of considering decay from the point of entry in the sewers, sewer transport conditions, and normalization strategies when assessing and modelling the impact of viral decay rates in wastewater systems. This study also emphasizes the need for ongoing research into the diverse and multifaceted factors that influence these decay rates, which is crucial for accurate public health monitoring and response strategies.