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) can provide early warning of outbreaks, but wastewater RNA signals may be underestimated due to analytical limitations and in-sewer attenuation driven by matrix conditions and conveyance. Using human coronavirus NL63 (HCoV-NL63) as a BSL-2 surrogate to characterize coronavirus RNA decay (distinct from SARS-CoV-2), we quantified loss kinetics as a function of pH (2, 5, 7, 8), temperature (20, 30 °C), microbial abundance, suspended solids (SS; 74–216 mg L− 1), and transport distance. Batch tests showed that higher microbial concentrations markedly increased decay rates: in raw wastewater at 30 °C, the first-order decay constant k reached 2.21 d− 1, whereas filtration and/or microbial suppression reduced k to 1.12–0.47 d− 1. A lab-scale sewer pipeline simulator further showed faster decay with increasing transport distance, and faster decay in wastewater than in dechlorinated tap water at 25 °C (k = 0.52 vs. 0.28 d− 1). Across the conditions evaluated, microbially mediated processes were the dominant drivers of viral RNA loss. These decay kinetics provide a basis to interpret—and, where appropriate, adjust—SARS-CoV-2 wastewater RNA measurements across diverse environmental and conveyance conditions.