Modeled and measured SARS-CoV-2 virus in septic tank systems for wastewater surveillance.

SARS-CoV-2 wastewater surveillance (WWS) at wastewater treatment plants (WWTPs) can reveal sewered community COVID-19 prevalence. For unsewered areas using septic tank systems (STSs) or holding tanks, how to conduct WWS remains unexplored. Here, two large STSs serving Zuma Beach (Malibu, CA) were studied. Supernatant and sludge SARS-CoV-2 concentrations from the directly-sampled STSs parameterized a dynamic solid-liquid separation, mass balance-based model for estimating the infection rate of users. Pumped septage before hauling and upon WWTP disposal was also sampled and assessed. Most (96%) STS sludge samples contained SARS-CoV-2 N1 and N2 genes, with concentrations exceeding the supernatant and increasing with depth while correlating with total suspended solids (TSS). The trucked septage contained N1 and N2 genes which decayed (coefficients: 0.09-0.29 h-1) but remained detectable. Over approximately 5 months starting in December 2020, modeled COVID-19 prevalence estimations among users ranged from 8 to 18%, mirroring a larger metropolitan area for the first 2 months. The approaches herein can inform public health intervention and augment conventional WWS in that: (1) user infection rates for communal holding tanks are estimable and (2) pumped and hauled septage can be assayed to infer where disease is spreading in unsewered areas.

Municipal separate storm sewer system (MS4) dry weather flows and potential flow sources as assessed by conventional and advanced bacterial analyses.

Municipal separate storm sewer systems (MS4s) function in urbanized areas to convey flows during both wet weather (i.e., stormwater) and dry weather (i.e., urban runoff as well as subsurface sources of flow) to receiving waters. While urban stormwater is known to contain microbial and chemical pollutants, MS4 dry weather flows, or non-stormwater discharges (NSWDs), are much less studied, although they are also known to contain pollutants, especially when these flows include raw sewage. In addition, some natural NSWDs (e.g., from groundwater infiltrating MS4 pipes) are critical for aquatic habitat protection. Thus, it is important to distinguish NSWD sources to prevent non-natural flows while retaining natural waters (i.e., groundwater). Here, MS4 dry weather flows were assessed by analyzing water samples from MS4 outfalls across multiple watersheds and water provider service areas in south Orange County, CA; potential NSWD sources including sewage, recycled water, potable water, and groundwater were sampled and analyzed for their likely contributions to overall NSWDs. Geochemical and microbiological water quality indicators, as well as bacterial communities, differed across NSWDs, yet water quality within most locations did not vary significantly diurnally or by sampling date. Meanwhile, NSWD source waters had distinctly different bacterial taxa abundances and specific bacterial genera. Shared geochemical and microbial characteristics of certain sources and outfall flows suggested the contributions of sources to outfall flows. The average proportions by sources contributing to MS4 outfalls were further estimated by SourceTracker and FEAST, respectively. The results of this study highlight the use of multiple tools when assessing chemical and microbiological water quality to predict sources of NSWDs contributing to urban MS4 flows during dry weather. This information can be used to support management actions to reduce unnatural and high risk sources of dry weather drainage while preserving natural sources important to environmental health in downstream receiving waters.

Assessing multiple fecal sources to surf zone waters of two recreational beaches by bacterial community analysis.

Fecal sources to recreational surf zone waters should be identified to protect public health. While watershed origins of human and other fecal sources are often discoverable by quantitative polymerase chain reaction (qPCR) of fecal markers using spatially stratified samples, similarly assessing wastewater treatment plant (WWTP) outfall and other offshore contributions to surf zones is challenged by individual marker fate and transport. Here, bacterial communities were assessed for relatedness between all hypothesized fecal sources and surf zone waters for two urban California recreational beaches, by sequencing genes encoding 16S rRNA and analyzing data using SourceTracker and FEAST. Ambient marine bacterial communities dominated the surf zone, while fecal (human, dog, or gull) or wastewater (sewage or treated WWTP effluent) bacterial communities were present at low proportions and those from recycled water were absent. Based on the relative abundances of bacterial genera specifically associated with human feces, the abundances of HF183 in bacterial community sequences, and FEAST and SourceTracker results when benchmarked to HF183, the major sources of HF183 to surf zone waters were human feces and treated WWTP effluent. While surf zone sequence proportions from human sources (feces, sewage and treated WWTP effluent) appeared uncorrelated to previously obtained qPCR HF183 results, the proportions of human fecal and potential human pathogen sequences in surf zone waters were elevated when there were more swimmers (i.e. during weekday afternoons, holidays and busy weekends, and race events), thus confirming previously-published qPCR-based conclusions that bather shedding contributed low levels of human fecal contamination. Here, bacterial community sequencing also showed evidence that treated WWTP effluent from an offshore outfall was entering the surf zone, thereby resolving a prior uncertainty. Thus, bacterial community sequencing not only confirms qPCR HF183-based human marker detections, but further allows for confirming fecal sources for which individual marker quantification results can be equivocal.

Sources of Low Level Human Fecal Markers in Recreational Waters of Two Santa Barbara, CA Beaches: Roles of WWTP Outfalls and Swimmers.

Worldwide, fecal indicator bacteria (FIB) evidence coastal water contamination for which sources are unknown. Here, for two FIB-impacted Santa Barbara recreational beaches, hypothesized fecal sources were investigated over three dry seasons (summers) using nearly 2000 field samples of water (ocean, creek, groundwater), sand, sediments, effluent and fecal sources. In years 1 and 2, gull and dog feces were identified as the probable main FIB sources to surf zone waters, yet HF183 human fecal markers were consistently detected. Determining HF183 sources was therefore prioritized, via year 3 sub-studies. In lower watersheds, human and dog wastes were mobilized by small storms into creeks, but no storm drain outfalls or creeks discharged into surf zones. Beach area bathrooms, sewers, and a septic system were not sources: dye tracing discounted hydraulic connections, and shallow groundwater was uncontaminated. Sediments from coastal creeks and downstream scour ponds, nearshore marine sediments, and sands from inter- and supratidal zones contained neither HF183 nor pathogens. Two nearby wastewater treatment plant (WWTP) outfalls discharged HF183 into plumes that were either deep or distant with uncertain onshore transport. Regardless, local sources were evidenced, as surf zone HF183 detection rates mostly exceeded those offshore and nearshore (around boat anchorages). The presence of swimmers was associated with surf zone HF183, as swimmer counts (on weekdays, holidays, weekends, and during races) significantly correlated (p<0.05, n = 196) to HF183 detections. Besides comprehensively assessing all possible fecal sources, this study provides new explanations of chronic low-level human markers in recreational beach surf zones, suggesting likely lowest achievable HF183 thresholds.

Bather Shedding as a Source of Human Fecal Markers to a Recreational Beach.

Microbial source tracking (MST) can identify and locate surf zone fecal indicator bacteria (FIB) sources. However, DNA-based fecal marker results may raise new questions, since FIB and DNA marker sources can differ. Here, during 2 years of summertime (dry season) MST for a Goleta, California recreational beach, surf zone FIB were mainly from gulls, yet low level human-associated DNA-based fecal marker (HF183) was detected in 25 and 14% of surf zone water samples, respectively. Watershed sources were hypothesized because dry weather creek waters had elevated FIB, and runoff-generating rain events mobilized human (and dog) fecal markers and Salmonella spp. into creeks, with human marker HF183 detected in 40 and 50% of creek water samples, dog markers detected in 70 and 50% of samples, and Salmonella spp. in 40 and 33.3% of samples, respectively over 2 years. However, the dry weather estuary outlet was bermed in the first study year; simultaneously, creek fecal markers and pathogens were lower or similar to surf zone results. Although the berm breached in the second year, surf zone fecal markers stayed low. Watershed sediments, intertidal beach sands, and nearshore sediments were devoid of HF183 and dog-associated DNA markers. Based on dye tests and groundwater sampling, beach sanitary sewers were not leaking; groundwater was also devoid of HF183. Offshore sources appeared unlikely, since FIB and fecal markers decreased along a spatial gradient from the surf zone toward nearshore and offshore ocean waters. Further, like other regional beaches, surf zone HF183 corresponded significantly to bather counts, especially in the afternoons when there were more swimmers. However, morning detections of surf zone HF183 when there were few swimmers raised the possibility that the wastewater treatment plant (WWTP) offshore outfall discharged HF183 overnight which transported to the surf zone. These findings support that there may be lowest achievable limits of surf zone HF183 owing to several chronic and permanent, perhaps diurnal, low concentration sources.

Assessing sediment recontamination from metals in stormwater.

Recontamination of sediments by stormwater is a major concern when evaluating the potential effectiveness of sediment remediation. Stormwater and sediment sampling were conducted in a mixed-use watershed at Paleta Creek in San Diego, CA to evaluate methods for assessing sediment recontamination by metals. Size-segregated stormwater contaminant loads with simultaneous receiving water and sediment measurements were used to identify dominant sources and contaminants with respect to their impact on sediment recontamination. Most of the stormwater contaminant loads of Cd, Cu, Pb, and Zn were associated with residential and highway sources from the upstream portions of the watershed and As, Ni and Hg were more significantly influenced by the downstream area of the watershed. Cd was strongly associated with large particles (>63 µm) and observed to settle in near shore areas with some attenuation due to mixing and dilution. Cu, in contrast, was associated more with the filtered fraction (<0.45 µm) and clay fraction (0.45-5 µm), resulting in less near shore sediment recontamination. Depositing sediment and other metals, particularly Cu and Hg, exhibited greater accumulation in settling traps than could be attributed to stormwater loads indicating the importance of other sources or resuspension of bay sediments on surficial sediment concentrations. Pb, Zn, Ni, and As showed influences of both stormwater and other sources. The study showed that measurement of size-segregated stormwater contaminant mass and concentrations combined with simultaneous measurements of deposition in sediment traps could differentiate between recontamination by stormwater and that of other sources.

Solar Inactivation of Enterococci and Escherichia coli in Natural Waters: Effects of Water Absorbance and Depth.

The decay of sewage-sourced Escherichia coli and enterococci was measured at multiple depths in a freshwater marsh, a brackish water lagoon, and a marine site, all located in California. The marine site had very clear water, while the waters from the marsh and lagoon contained colored dissolved organic matter that not only blocked light but also produced reactive oxygen species. First order decay rate constants of both enterococci and E. coli were between 1 and 2 d(-1) under low light conditions and as high as 6 d(-1) under high light conditions. First order decay rate constants were well correlated to the daily average UVB light intensity corrected for light screening incorporating water absorbance and depth, suggesting endogenous photoinactivation is a major pathway for bacterial decay. Additional laboratory experiments demonstrated the presence of colored dissolved organic matter in marsh water enhanced photoinactivation of a laboratory strain of Enterococcus faecalis, but depressed photoinactivation of sewage-sourced enterococci and E. coli after correcting for UVB light screening, suggesting that although the exogenous indirect photoinactivation mechanism may be active against Ent. faecalis, it is not for the sewage-source organisms. A simple linear regression model based on UVB light intensity appears to be a useful tool for predicting inactivation rate constants in natural waters of any depth and absorbance.

Wastewater compounds in urban shallow groundwater wells correspond to exfiltration probabilities of nearby sewers.

Wastewater compounds are frequently detected in urban shallow groundwater. Sources include sewage or reclaimed wastewater, but origins are often unknown. In a prior study, wastewater compounds were quantified in waters sampled from shallow groundwater wells in a small coastal California city. Here, we resampled those wells and expanded sample analyses to include sewage- or reclaimed water-specific indicators, i.e. pharmaceutical and personal care product chemicals or disinfection byproducts. Also, we developed a geographic information system (GIS)-based model of sanitary sewer exfiltration probability--combining a published pipe failure model accounting for sewer pipe size, age, materials of construction, with interpolated depths to groundwater--to determine if sewer system attributes relate to wastewater compounds in urban shallow groundwater. Across the wells, groundwater samples contained varying wastewater compounds, including acesulfame, sucralose, bisphenol A, 4-tert-octylphenol, estrone and perfluorobutanesulfonic acid (PFBS). Fecal indicator bacterial concentrations and toxicological bioactivities were less than known benchmarks. However, the reclaimed water in this study was positive for all bioactivity tested. Excluding one well intruded by seawater, the similarity of groundwater to sewage, based on multiple indicators, increased with increasing sanitary sewer exfiltration probability (modeled from infrastructure within ca. 300 m of each well). In the absence of direct exfiltration or defect measurements, sewer exfiltration probabilities modeled from the collection system's physical data can indicate potential locations where urban shallow groundwater is contaminated by sewage.

Microbial source tracking in a coastal California watershed reveals canines as controllable sources of fecal contamination.

Elevated levels of fecal indicator bacteria (FIB), including Escherichia coli and enterococci, trigger coastal beach advisories and signal public health risks. Solving FIB pollution in suburban coastal watersheds is challenging, as there are many potential sources. The Arroyo Burro watershed in Santa Barbara, CA is an example, with its popular, but chronically FIB-contaminated beach. To address, a microbial source tracking study was performed. Surface waters were sampled over 2 years, FIB were quantified, and DNA was analyzed for host-associated fecal markers. Surf zone FIB were only elevated when the coastal lagoon was discharging. Among the fecal sources into the lagoon, including upstream human sources and coastal birds, canines were the most important. Canine sources included input via upstream creek water, which decreased after creek-side residences were educated about proper pet waste disposal, and direct inputs to the lagoon and surf zone, where dog waste could have been tidally exchanged with the lagoon. Based on this study, canine waste can be an influential, yet controllable, fecal source to suburban coastal beaches.

Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods.

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study.

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.

Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources.

Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR(®) Green qPCR method, and two TaqMan(®) qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.

Characterization of fecal concentrations in human and other animal sources by physical, culture-based, and quantitative real-time PCR methods.

The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers.

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources.

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation.