SARS-CoV-2 RNA removal by wastewater treatment plants with and without disinfection

Introduction: SARS-CoV-2 RNA is excreted in the feces of infected persons, and thereby enters wastewater streams. In communities with developed sewage infrastructure, wastewater travels to wastewater treatment plants (WWTPs), where it is then processed to remove major chemical and biological pollutants. The treated effluent is then often released into nearby surface waters or reused (e.g. for irrigation). SARS-CoV-2 RNA is regularly quantified in the raw sewage influents of many WWTPs as a means of monitoring and predicting local infection rates; however, it has only rarely been quantified in treated WWTPs effluents. Therefore, there is limited data available regarding the removal and/or persistence of SARS-CoV-2 RNA during sewage treatment processes. The purpose of this research is to address this knowledge gap by comparing SARS-CoV-2 concentrations between raw sewage and treated WWTPs effluents. Sample locations: We collected samples from the raw sewage influents and final effluents of seven WWTPs in the Greater Chicago area: the Calumet, Terrence J. O'Brien (O'Brien), Stickney, Egan, Hanover Park, Kirie, and Lemont Water Reclamation Plants. The daily processing volumes and populations served by these WWTPs are shown in Table 1. Primary treatment at these WWTPs consists of mechanical filtration and aerated grit tanks followed by settling tanks at all seven WWTPs except Kirie, which does not include primary settling tanks, and Lemont, which does not include aerated grit tanks. All seven WWTPs utilize secondary treatment in the form of aerated tanks that facilitate the growth of aerobic heterotrophic bacteria, followed by additional settling tanks. In addition, the O'Brien and Calumet WWTPs utilize tertiary disinfection treatments in the form of UV exposure and chlorination/dechlorination, respectively. These disinfection methods are employed seasonally because the surface waters that receive outfall from the Calumet and O'Brien WWTPs are classified as recreational during warm seasons, making the discharges subject to additional regulatory limits for fecal indicator bacteria. At the O'Brien WWTP, disinfection was inactive between November 24, 2020 and February 20, 2021; at the Calumet WWTP, disinfection was inactive between November 24, 2020 and February 22, 2021. Disinfection was active at both the Calumet and O'Brien WWTPs during the rest of the sampling period, making it possible to compare the effect of treatment with and without disinfection at the same WWTPs. Sample processing: Samples were collected as 24-hour composites using autosamplers, with influent and effluent samples being collected simultaneously. At Calumet, O'Brien, and Stickney WWTPs, samples were collected once weekly from October 28, 2020 until December 9, 2020, and twice weekly from December 16, 2020 until March 2, 2021. At the Egan, Hanover Park, Kirie, and Lemont WWTPs, samples were collected twice weekly from December 16, 2020 until March 23, 2021. 25 mL of each sample was concentrated by filtration through mixed cellulose ester filters, with MgCl2 added to facilitate viral adsorption to the filter membrane. RNA extraction from the filter membranes was performed using the QIAamp Viral RNA MiniPrep kit. In order to quantify SARS-CoV-2 RNA concentrations, we amplified the N1 and N2 regions of the SARS-CoV-2 nucleocapsid gene using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). We then compared SARS-CoV-2 RNA concentrations between influent and effluent samples. Additionally, to evaluate the impact of disinfection at Calumet and O'Brien WWTPs, we compared the effluent:influent proportions of SARS-CoV-2 RNA concentrations between the periods when disinfection was active versus inactive. Results: Median influent and effluent concentrations of SARS-CoV-2 RNA at each WWTP, the mean proportion of effluent:influent concentrations, and P-values from statistical comparisons between influent and effluent are shown in Table 2. For all WWTPs except Stickney, P-values are generated from Wilcoxon signed-rank tests comparing influent and effluent concentrations. Because Stickney had two influent streams which joined at the WWTP to produce a single effluent, a proportion of effluent:influent concentrations could not be calculated, and a Friedman test comparing the three sites was used rather than a Wilcoxon test. Effluent concentrations were significantly lower than influent concentrations at all seven WWTPs, with mean proportions of effluent:influent concentrations at each WWTP ranging from 6.35 x 10-3 to 3.39 x 10-1 for N1 and 1.61 x 10-2 to 8.42 x 10-2 for N2 (Figure 1). In order to assess the effect of disinfection at the O'Brien and Calumet WWTPs, proportions of effluent:influent concentrations were between samples collected on the same days, and the set of proportions from samples collected while disinfection was active was compared to the set collected while disinfection was inactive using Wilcoxon rank-sum tests. These comparisons did not show and significant differences related to disinfection status (Figure 2). Conclusions: For all seven WWTPs studied here, primary and secondary treatment significantly reduced SARS-CoV-2 RNA concentrations, but did not consistently eliminate SARS-CoV-2 RNA altogether. At the two WWTPs where disinfection was used, there was no detectable impact of either UV treatment or chlorination/dechlorination on the amount of SARS-CoV-2 RNA removed. The data presented here provide insight into the effect of wastewater treatment, both with and without disinfection, on SARS-CoV-2 RNA concentrations.