Environmental and microbial factors shaping SARS-CoV-2 RNA decay in wastewater: insights from batch tests and a lab-scale sewer pipeline simulator

Wastewater-based surveillance (WBS) is a valuable tool for providing early warnings of disease outbreaks, but viral concentrations in sewers can be underestimated due to analytical limitations, wastewater characteristics, and transport times. We examined how pH, temperature, microbial concentrations, suspended solids (SS), and travel distance affect viral RNA loss, using human coronavirus NL63 (HCoV-NL63) as a surrogate. Batch tests under different pH values (2, 5, 7, 8), temperatures (20°C, 30°C), and SS concentrations (74, 133, 216 mg L⁻¹) showed that higher microbial concentrations markedly increased decay rates. In raw wastewater at 30°C, the first-order viral decay rate constant ( k ) reached k  = 2.21 d⁻¹, while filtration and/or microbial suppression reduced k to 1.12–0.47 d⁻¹. A lab-scale sewer pipeline simulator confirmed that decay rates increased with transport distance, with wastewater decaying faster than dechlorinated tap water at 25°C ( k  = 0.52 vs 0.28 d⁻¹). Across all tests, microbial activity was the dominant factor driving viral RNA loss. These findings can be applied to adjust SARS-CoV-2 RNA levels in wastewater for environmental conditions in different regions, improving the accuracy of WBS data and supporting timely public health responses.