Persistence of Endogenous SARS-CoV-2 and Pepper Mild Mottle Virus RNA in Wastewater-Settled Solids

[Image: see text] Limited information is available on the decay rate of endogenous SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA in wastewater and primary settled solids, potentially limiting an understanding of how transit or holding times within wastewater infrastructure might impact RNA measurements and their relationship to community COVID-19 infections. In this study, primary settled solids samples were collected from two wastewater treatment plants in the San Francisco Bay Area. Samples were thoroughly mixed, aliquoted into subsamples, and stored at 4, 22, and 37 °C for 10 days. The concentrations of SARS-CoV-2 (N1 and N2 targets) and PMMoV RNA were measured using an RT-ddPCR. Limited decay (<1 log(10) reduction) was observed in the detection of viral RNA targets at all temperature conditions, suggesting that SARS-CoV-2 and PMMoV RNA can be highly persistent in solids. First-order decay rate constants ranged from 0.011 to 0.098 day(–1) for SARS-CoV-2 RNA and from 0.010 to 0.091 day(–1) for PMMoV RNA depending on the temperature conditions. A slower decay was observed for SARS-CoV-2 RNA in primary settled solids compared to previously reported decay in wastewater influent. Further research is needed to understand if solid content and wastewater characteristics might influence the persistence of viral RNA targets.