Variability of RNase P, PMMoV and CrAssphage concentration in wastewater across distinct urban sewershed scales, Kentucky, USA

Understanding the human fecal concentration in a pooled wastewater sample is important for interpreting results of wastewater disease surveillance studies at different scales. Fecal indicator positive controls can be used for quantifying this concentration. Wastewater monitoring studies must also be designed to best incorporate knowledge of defecation frequency; while many studies rely on 24-hour composite samples, it is known that 60-70% of child-bearing age individuals do not have a regular 24-hour defecation cycle (Heaton et al., 1992). The aim of this work is to report on the variability of three commonly used fecal indicator targets (RNase P, PMMoV and CrAssphage) in 24-hour composite wastewater samples across 28 urban sewersheds of varying scales collected in four counties of Kentucky from August to December 2020. Results from the untransformed wastewater data indicate concentration ranges across five orders of magnitude: fecal indicator density of human RNase P ranged from 5.1 x 101 to 1.15 x 106 copies/ml; PMMoV ranged from 7.23 x 103 to 3.53 x 107 copies/ml; and CrAssphage ranged from 9.69 x 103 to 1.85 x 108 copies/ml. Despite stationary sample collection sites during COVID-19 lockdown, regional and temporal variability for fecal indicators was seen, with no clear relationship between fecal indicator concentrations and sewershed size or population income level. When used for public health, wastewater disease surveillance requires better regional and temporal baseline data on positive controls representing human contribution to a pooled wastewater sample.