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Primary and secondary treatment of municipal wastewater contributes to virus removal upstream of advanced purification to produce water for potable reuse. In this study, virus occurrence by cultivable and molecular methods was measured over a 24-month period in raw wastewater influents and secondary effluents from two municipal wastewater treatment plants that together provide the recycled water source for an advanced water purification facility. Using a rank-paired, covariance-based statistical approach, virus log removal values were determined for four wastewater treatment processes that operate in parallel at the two facilities (two activated sludge processes, trickling filter process, and trickling filter/solids contactor process). The trickling filter process exhibited the lowest observed removal of cultivable enteric virus with a median removal of 1.0 log10 (or 90% removal) and a 5th percentile log removal of 0.73 (or 82%), compared to the greatest removal observed for one of the activated sludge processes (median log removal of 2.4 or 99.6% and 5th percentile of 2.1 or 99.2%). Median log removal observed for male-specific (MS) and somatic (SOM) coliphage was 1.8 (98.6% removal) and 0.5 (70%), respectively, for trickling filter and 2.9 (99.9%) and 2.0 (99%) for activated sludge. Thus, coliphage removal was fairly similar to removal observed for cultivable enteric virus. The cultivable enteric virus 5th percentile removal (0.7) from the trickling filter treatment process was proposed to the state regulator for credit towards state requirements for virus removal related to groundwater augmentation with purified recycled water. Receiving pathogen removal credits for secondary wastewater treatment would allow for an improved margin (safety factor) of credits beyond the minimum required; and in this case may also increase the number of viable future groundwater recharge sites closer to drinking water production wells by reducing the underground travel time otherwise required to obtain sufficient credits.
The objectives of the present study were to measure virus occurrence and removal during conventional wastewater treatment using cultivable and molecular methods, along with other microbial indicators, and to select and execute an appropriate statistical approach to use the virus data to conservatively calculate the total removal of virus (log removal value) toward potential regulatory credit for potable reuse treatment. This was done by monitoring microbial targets over 24 sampling events in raw wastewater influents and secondary effluents from one wastewater collection agency that operates two municipal wastewater treatment plants. One plant currently serves as the source of recycled water for an advanced water purification facility, with the other plant to begin contributing supply in the near future [6]. While the original objective was to complete the 24 events in a one-year period (covering all seasons), sampling was extended over a two-year period due to the SARS-CoV-2 pandemic. Two different statistical approaches for data analysis were compared as recommended by an independent expert committee [10, 11], including a Monte Carlo simulation-based approach and a rank-paired, covariance-based approach. Microbial concentration data were analyzed using each distinct approach to generate probability distributions and calculate virus log removal values. During the sampling period, routine WWTP operational performance data collected by the facilities was analyzed to establish the operational envelope representative of normal performance and conditions.
ORVs were derived for each secondary treatment process supplying influent to OCWD AWPF, i.e., P1 TF, AS1, AS2 and P2 TF/SC for key plant operational parameters (namely, mean cell residence time (MCRT) for AS1, AS2, and TF/SC, and total biological oxygen demand [BOD-T] for TF). An exceedance of a designated ORV represents a deviation from normal treatment performance. In other words, an exceedance would indicate that the WWTP is operating outside the normal operating envelope, and virus log credit would not be granted for that period. This deviation could be related to an unexpected event but also due to planned activities such as operational maintenance of treatment systems or flow adjustments by the OC San operators. For detailed information on ORVs and values calculated for this study, (see S7 Text and S3 Table).
Raw influent samples and secondary effluent samples were analyzed for enteric viruses, male-specific (MS) coliphage, somatic (SOM) coliphage, and total and fecal coliform. To analyze samples for enteric viruses, samples were split and analyzed using culture-based and molecular detection methods as described in EPA Method 1615 [15]. For MS and SOM coliphage enumeration, two-liter grab samples were collected in sterile polypropylene Nalgene bottles and processed using EPA Method 1602 with double agar layer modification [16]. For total and fecal coliform analysis, 100 mL grab samples were collected in sterile IDEXX bottles and enumerated according to standard methods 9222B and 9222D, respectively [17]. Details on methods used for samples analysis, including minor modifications to EPA 1615 method for molecular detection of viruses is provided in Polanco et al. [6].
Secondary effluent concentrates were extracted from each ultrafilter using a high-volume elution canister consisting of pressurized 0.075% Tween 20 phosphate buffer solution. Ultrafilter eluents were filtered through sterile 0.22 μm pore filter and divided into a series of subsamples for culture-based enteric virus analysis and for DNA extraction (molecular detection) [18]. Detailed information on the ultrafiltration procedure can be found in the S2 Text.
A total of 24 sampling events were completed over the course of the study. Samples were collected from each of the six sampling locations and spanned between one to three days constituting one sampling event. On average, two sampling events were scheduled per month and spanned a total of 24 months due to pandemic-related postponement of sampling. Duplicate samples were also collected for all microbial targets on a staggered schedule at each site. With exception to the P2 raw influent duplicate sample, which was collected once, a total of two duplicate samples were collected for all sites during the study. Grab samples for coliphage evaluation were collected once a month from all six locations contemporaneously with enteric virus samples, such that a total of 12 coliphage sampling events were reported over the study. All samples were shipped and processed immediately to comply with sample hold time criteria and quality assurance.
No attempt was made to time collection of the effluent versus influent samples according to the average system hydraulic residence time, i.e. Lagrangian sampling, since this is generally difficult and unreliable and because the anticipated data analysis method to determine log removal did not require it. Both of the data analysis approaches utilized for this study calculate removal of virus based on a statistical evaluation of the observed influent and effluent distribution of concentrations. Considering the use of a statistical analysis that utilizes the full sampling distribution may be considered superior to same day influent-effluent pairing [19].
To determine virus recovery efficiency, matrix spike recovery (MSR) samples were collected contemporaneously with a subset of the enteric virus and coliphage samples. A total of 9 raw influent MSR samples were collected per raw influent sampling location over the 24 total samples. For each secondary effluent sampling location, five MSR samples were collected, which given four secondary effluent sampling locations resulted in 20 total secondary effluent MSR samples over the course of the study. Male-specific (MS) coliphage (ATCC 15597-B1), somatic (SOM) coliphage (ATCC 13706-B1) and poliovirus (ATCC VR1562) of known concentration were spiked into raw influent samples directly and into the ultrafiltration unit prior to elution to determine and evaluate method recovery. For ddPCR recoveries, armored RNA targets for enterovirus and norovirus GII were spiked into raw influent samples and into the ultrafiltration unit at known concentrations. Additional information on how MSR samples were processed can be found in the (see S3 and S4 Texts).
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