Particle association and size fraction of molecular viral fecal pollution indicators in wastewater

Fecal indicator bacteria currently used for water quality monitoring inadequately represent viral fate in water systems, motivating the development of viral fecal pollution indicators. Molecular viral fecal pollution indicators such as crAssphage and pepper mild mottle virus (PMMoV) have emerged as leading viral fecal pollution indicator candidates due to ease and speed of measurement and target specificity. Elucidating the fate of molecular viral fecal indicators in water systems is necessary to facilitate their development, broader adoption, and ultimately their association with infectious risk. A significant mechanism controlling the behavior of viral indicators in environmental waters is association with particles, as this would dictate removal via settling and transport characteristics. In this study, we investigated the particle associations of six molecular fecal pollution targets (crAssphage, PMMoV, adenovirus, human polyomavirus, norovirus, HF183/BacR287) in wastewater using a cascade filtration approach. Four different filters were employed representing large settleable particles (180 mu m), larger (20 mu m) and smaller suspended particles (0.45 mu m), and non-settleable particles (0.03 mu m). All molecular targets were detected on all particle size fractions; however, all targets had their highest concentrations on the 0.45 mu m (percent contribution ranging from 40% to 80.5%) and 20 mu m (percent contribution ranging from 3.9% to 39.4%) filters. The association of viral fecal pollution targets with suspended particles suggests that particle association will dictate transport in environmental waters and that sample concentration approaches based upon particle collection will be effective for these targets.