Wastewater-based surveillance as a tool for public health action: SARS-CoV-2 and beyond.

Wastewater-based surveillance (WBS) has undergone dramatic advancement in the context of the coronavirus disease 2019 (COVID-19) pandemic. The power and potential of this platform technology were rapidly realized when it became evident that not only did WBS-measured SARS-CoV-2 RNA correlate strongly with COVID-19 clinical disease within monitored populations but also, in fact, it functioned as a leading indicator. Teams from across the globe rapidly innovated novel approaches by which wastewater could be collected from diverse sewersheds ranging from wastewater treatment plants (enabling community-level surveillance) to more granular locations including individual neighborhoods and high-risk buildings such as long-term care facilities (LTCF). Efficient processes enabled SARS-CoV-2 RNA extraction and concentration from the highly dilute wastewater matrix. Molecular and genomic tools to identify, quantify, and characterize SARS-CoV-2 and its various variants were adapted from clinical programs and applied to these mixed environmental systems. Novel data-sharing tools allowed this information to be mobilized and made immediately available to public health and government decision-makers and even the public, enabling evidence-informed decision-making based on local disease dynamics. WBS has since been recognized as a tool of transformative potential, providing near-real-time cost-effective, objective, comprehensive, and inclusive data on the changing prevalence of measured analytes across space and time in populations. However, as a consequence of rapid innovation from hundreds of teams simultaneously, tremendous heterogeneity currently exists in the SARS-CoV-2 WBS literature. This manuscript provides a state-of-the-art review of WBS as established with SARS-CoV-2 and details the current work underway expanding its scope to other infectious disease targets.

A Bayesian framework for modeling COVID-19 case numbers through longitudinal monitoring of SARS-CoV-2 RNA in wastewater.

Wastewater-based surveillance has become an important tool for research groups and public health agencies investigating and monitoring the COVID-19 pandemic and other public health emergencies including other pathogens and drug abuse. While there is an emerging body of evidence exploring the possibility of predicting COVID-19 infections from wastewater signals, there remain significant challenges for statistical modeling. Longitudinal observations of viral copies in municipal wastewater can be influenced by noisy datasets and missing values with irregular and sparse samplings. We propose an integrative Bayesian framework to predict daily positive cases from weekly wastewater observations with missing values via functional data analysis techniques. In a unified procedure, the proposed analysis models severe acute respiratory syndrome coronavirus-2 RNA wastewater signals as a realization of a smooth process with error and combines the smooth process with COVID-19 cases to evaluate the prediction of positive cases. We demonstrate that the proposed framework can achieve these objectives with high predictive accuracies through simulated and observed real data.

Surveillance for SARS-CoV-2 and its variants in wastewater of tertiary care hospitals correlates with increasing case burden and outbreaks.

Wastewater-based SARS-CoV-2 surveillance enables unbiased and comprehensive monitoring of defined sewersheds. We performed real-time monitoring of hospital wastewater that differentiated Delta and Omicron variants within total SARS-CoV-2-RNA, enabling correlation to COVID-19 cases from three tertiary-care facilities with >2100 inpatient beds in Calgary, Canada. RNA was extracted from hospital wastewater between August/2021 and January/2022, and SARS-CoV-2 quantified using RT-qPCR. Assays targeting R203M and R203K/G204R established the proportional abundance of Delta and Omicron, respectively. Total and variant-specific SARS-CoV-2 in wastewater was compared to data for variant specific COVID-19 hospitalizations, hospital-acquired infections, and outbreaks. Ninety-six percent (188/196) of wastewater samples were SARS-CoV-2 positive. Total SARS-CoV-2 RNA levels in wastewater increased in tandem with total prevalent cases (Delta plus Omicron). Variant-specific assessments showed this increase to be mainly driven by Omicron. Hospital-acquired cases of COVID-19 were associated with large spikes in wastewater SARS-CoV-2 and levels were significantly increased during outbreaks relative to nonoutbreak periods for total SARS-CoV2, Delta and Omicron. SARS-CoV-2 in hospital wastewater was significantly higher during the Omicron-wave irrespective of outbreaks. Wastewater-based monitoring of SARS-CoV-2 and its variants represents a novel tool for passive COVID-19 infection surveillance, case identification, containment, and potentially to mitigate viral spread in hospitals.

Amino Acids as Potential Precursors to Odorous Compounds in Tap Water during Spring Runoff Events.

The onset of spring runoff in northern climates and tap water odor events are difficult to predict because common water quality parameters cannot fully explain the intermittent odor events that occurred over past decades. Studies have shown that small polar water-soluble compounds, such as amino acids (AAs), leach first from ice/snowmelt. AAs are known to produce odorous compounds, such as aldehydes and chloroaldimines, upon chlorination. Therefore, we proposed that AAs may serve as markers for small and soluble organics that contribute to the odor of chlorinated tap water. Here, we studied the occurrence of AAs in source water collected at two water treatment plants and the odor profiles of tap water at >300 homes during the 2021 and 2022 spring runoff events. AA concentrations were at baseline levels (<100 ng/L) during the 2021 runoff but much higher (up to 5500 ng/L) in 2022 and associated with an escalation in odor complaints. AA concentrations peaked at the onset of the 2022 spring runoff and corresponded with the strongest reported odor intensities in tap water. We obtained high resolution MS and MS/MS spectra of chloroaldimines and confirmed the formation of chloroaldimines under chlorination of the six AAs detected in source water. The results indicate that AAs signal the onset of spring runoff and represent small polar water-soluble compounds that may contribute to tap water odor problems.

A critical review of risk matrices used in water safety planning: improving risk matrix construction.

Risk matrices are used in water safety planning to prioritize improvements to drinking water systems. While water safety plans (WSPs) are promoted globally, no study has evaluated whether risk matrices are comprehensively constructed to accurately assess risk. We used risk matrix criteria adapted from previous risk matrix research to evaluate risk matrices found in twelve templates across global jurisdictions. WSP templates were found using the WSPortal website and definitions of likelihood and impact were extracted from each template to assist in the evaluation of WSP risk matrices. Application of the criteria developed from a detailed mathematical analysis by revealed that 11 of 12 risk matrices evaluated contravene at least one of the risk matrix criteria. Furthermore, definitions of likelihood and impact varied widely across different jurisdictions, due in part to the system specific nature of the WSP methodology. To improve risk matrix construction, we recommend: setting clearer risk level boundary criteria, aligning specific impact category definitions with water system objectives, and selecting specific impact categories as opposed to defining impact in several ways. Finally, we recommend risk matrix construction be reviewed as part of the WSP process to ensure accurate identification of key risks in a water system.

An efficient method to enhance recovery and detection of SARS-CoV-2 RNA in wastewater.

Wastewater surveillance (WS) of SARS-CoV-2 currently requires multiple steps and suffers low recoveries and poor sensitivity. Here, we report an improved analytical method with high sensitivity and recovery to quantify SARS-CoV-2 RNA in wastewater. To improve the recovery, we concentrated SARS-CoV-2 viral particles and RNA from both the solid and aqueous phases of wastewater using an electronegative membrane (EM). The captured viral particles and RNA on the EM were incubated in our newly developed viral inactivation and RNA preservation (VIP) buffer. Subsequently, the RNA was concentrated on magnetic beads and inhibitors removed by washing. Without eluting, the RNA on the magnetic beads was directly detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Analysis of SARS-CoV-2 pseudovirus (SARS-CoV-2 RNA in a noninfectious viral coat) spiked to wastewater samples showed an improved recovery of 80%. Analysis of 120 wastewater samples collected twice weekly between May 2021 and February 2022 from two wastewater treatment plants showed 100% positive detection, which agreed with the results independently obtained by a provincial public health laboratory. The concentrations of SARS-CoV-2 RNA in these wastewater samples ranged from 2.4×102 to 2.9×106 copies per 100 mL of wastewater. Our method's capability of detecting trace and diverse concentrations of SARS-CoV-2 in complex wastewater samples is attributed to the enhanced recovery of SARS-CoV-2 RNA and efficient removal of PCR inhibitors. The improved method for the recovery and detection of viral RNA in wastewater is important for wastewater surveillance, complementing clinical diagnostic tests for public health protection.

Number of COVID-19 cases required in a population to detect SARS-CoV-2 RNA in wastewater in the province of Alberta, Canada: Sensitivity assessment.

With a unique and large size of testing results of 1,842 samples collected from 12 wastewater treatment plants (WWTP) for 14 months through from low to high prevalence of COVID-19, the sensitivity of RT-qPCR detection of SARS-CoV-2 RNA in wastewater that correspond to the communities was computed by using Probit analysis. This study determined the number of new COVID-19 cases per 100,000 population required to detect SARS-CoV-2 RNA in wastewater at defined probabilities and provided an evidence-based framework of wastewater-based epidemiology surveillance (WBE). Input data were positive and negative test results of SARS-CoV-2 RNA in wastewater samples and the corresponding new COVID-19 case rates per 100,000 population served by each WWTP. The analyses determined that RT-qPCR-based SARS-CoV-2 RNA detection threshold at 50%, 80% and 99% probability required a median of 8 (range: 4-19), 18 (9-43), and 38 (17-97) of new COVID-19 cases /100,000, respectively. Namely, the positive detection rate at 50%, 80% and 99% probability were 0.01%, 0.02%, and 0.04% averagely for new cases in the population. This study improves understanding of the performance of WBE SARS-CoV-2 RNA detection using the large datasets and prolonged study period. Estimated COVID-19 burden at a community level that would result in a positive detection of SARS-CoV-2 in wastewater is critical to support WBE application as a supplementary warning/monitoring system for COVID-19 prevention and control.

Tracking Emergence and Spread of SARS-CoV-2 Omicron Variant in Large and Small Communities by Wastewater Monitoring in Alberta, Canada.

Wastewater monitoring of SARS-CoV-2 enables early detection and monitoring of the COVID-19 disease burden in communities and can track specific variants of concern. We determined proportions of the Omicron and Delta variants across 30 municipalities covering >75% of the province of Alberta (population 4.5 million), Canada, during November 2021-January 2022. Larger cities Calgary and Edmonton exhibited more rapid emergence of Omicron than did smaller and more remote municipalities. Notable exceptions were Banff, a small international resort town, and Fort McMurray, a medium-sized northern community that has many workers who fly in and out regularly. The integrated wastewater signal revealed that the Omicron variant represented close to 100% of SARS-CoV-2 burden by late December, before the peak in newly diagnosed clinical cases throughout Alberta in mid-January. These findings demonstrate that wastewater monitoring offers early and reliable population-level results for establishing the extent and spread of SARS-CoV-2 variants.

The devil is in the details: emerging insights on the relevance of wastewater surveillance for SARS-CoV-2 to public health.

The severe health consequences and global spread of the COVID-19 pandemic have necessitated the rapid development of surveillance programs to inform public health responses. Efforts to support surveillance capacity have included an unprecedented global research response into the use of genetic signals of SARS-CoV-2 in wastewater following the initial demonstration of the virus' detectability in wastewater in early 2020. The confirmation of fecal shedding of SARS-CoV-2 from asymptomatic, infected and recovering individuals further supports the potential for wastewater analysis to augment public health conventional surveillance techniques based on clinical testing of symptomatic individuals. We have reviewed possible capabilities projected for wastewater surveillance to support pandemic management, including independent, objective and cost-effective data generation that complements and addresses attendant limitations of clinical surveillance, early detection (i.e., prior to clinical reporting) of infection, estimation of disease prevalence, tracking of trends as possible indicators of success or failure of public health measures (mask mandates, lockdowns, vaccination, etc.), informing and engaging the public about pandemic trends, an application within sewer networks to identify infection hotspots, monitoring for presence or changes in infections from institutions (e.g., long-term care facilities, prisons, educational institutions and vulnerable industrial plants) and tracking of appearance/progression of viral variants of concern.

Ethics Guidance for Environmental Scientists Engaged in Surveillance of Wastewater for SARS-CoV-2.

The COVID-19 pandemic has given rise to rapid and widespread international pursuit of wastewater surveillance for genetic signals of SARS-CoV-2, the virus causing the pandemic. Environmental scientists and engineers familiar with the techniques required for this endeavor have responded. Many of the environmental scientists engaged in these investigations have not necessarily had experience with the ethical obligations associated with generating and handling human health data. The Canadian Water Network facilitated adoption of these surveillance methods by creating a national coalition, which included a public health advisory group that recognized a need for ethics guidance for the wastewater approach to public health surveillance. This Policy Analysis addresses that need and is based on a review of relevant ethics literature tightly focused on ethics applicable to public health surveillance. That review revealed that classical health bioethics governing clinical practice and general public health ethics guidance did not adequately address key issues in wastewater surveillance. The 2017 World Health Organization guidelines, directly based on a systematic literature review, specifically addressed ethical issues in public health surveillance. The application of relevant ethical guidance to wastewater surveillance is analyzed and summarized for environmental scientists.

Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada.

Detection of SARS-CoV-2 RNA in wastewater is a promising tool for informing public health decisions during the COVID-19 pandemic. However, approaches for its analysis by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) are still far from standardized globally. To characterize inter- and intra-laboratory variability among results when using various methods deployed across Canada, aliquots from a real wastewater sample were spiked with surrogates of SARS-CoV-2 (gamma-radiation inactivated SARS-CoV-2 and human coronavirus strain 229E [HCoV-229E]) at low and high levels then provided "blind" to eight laboratories. Concentration estimates reported by individual laboratories were consistently within a 1.0-log10 range for aliquots of the same spiked condition. All laboratories distinguished between low- and high-spikes for both surrogates. As expected, greater variability was observed in the results amongst laboratories than within individual laboratories, but SARS-CoV-2 RNA concentration estimates for each spiked condition remained mostly within 1.0-log10 ranges. The no-spike wastewater aliquots provided yielded non-detects or trace levels (<20 gene copies/mL) of SARS-CoV-2 RNA. Detections appear linked to methods that included or focused on the solids fraction of the wastewater matrix and might represent in-situ SARS-CoV-2 to the wastewater sample. HCoV-229E RNA was not detected in the no-spike aliquots. Overall, all methods yielded comparable results at the conditions tested. Partitioning behavior of SARS-CoV-2 and spiked surrogates in wastewater should be considered to evaluate method effectiveness. A consistent method and laboratory to explore wastewater SARS-CoV-2 temporal trends for a given system, with appropriate quality control protocols and documented in adequate detail should succeed.

N-Nitrosamine formation from chloramination of two common ionic liquids.

Ionic liquids (ILs) are a class of solvents increasingly used as "green chemicals." Widespread applications of ILs have led to concerns about their accidental entry to the environment. ILs have been assessed for some environmental impacts; however, little has been done to characterize their potential impacts on drinking water if ILs accidentally enter surface water. IL cations are often aromatic or alkyl quaternary amines that resemble structures of previously confirmed N-nitrosamine (NA) precursors. Therefore, this study has evaluated two common ILs, 1-ethyl-3-methylimidazolium bromide (EMImBr) and 1-ethyl-1-methylpyrrolidinium bromide (EMPyrBr), for their NA formation potential. Each IL species was reacted with pre-formed monochloramine under various laboratory conditions. The reaction mixtures were extracted using liquid-liquid extraction and analyzed for NAs using high performance liquid chromatography tandem mass spectrometry. At low concentration of IL (250 µmol/L), the yields of NAs (NMEA or NPyr) increased with increasing doses of monochloramine from both IL species. The total NA yield was as high as 2.5 ±â€¯0.3 ng/mg from EMImBr, and as high as 8.6 ±â€¯0.8 ng/mg from EMPyrBr. At high concentration of IL (5 mmol/L), the NA yield reached a maximum at 2.5 mmol/L NH2Cl, and then decreased with subsequent increases in the reactant concentrations, demonstrating ILs' solvent effects. This study re-emphasizes the importance of preventing discharge of ILs to water bodies to prevent secondary impacts on drinking water.

Nontargeted identification of peptides and disinfection byproducts in water.

A broad range of organic compounds are known to exist in drinking water sources and serve as precursors of disinfection byproducts (DBPs). Epidemiological findings of an association of increased risk of bladder cancer with the consumption of chlorinated water has resulted in health concerns about DBPs. Peptides are thought to be an important category of DBP precursors in water. However, little is known about the actual presence of peptides and their DBPs in drinking water because of their high sample complexity and low concentrations. To address this challenge and identify peptides and non-chlorinated/chlorinated peptide DBPs from large sets of organic compounds in water, we developed a novel high throughput analysis strategy, which integrated multiple solid phase extraction (SPE), high performance liquid chromatography (HPLC) separation, and non-target identification using precursor ion exclusion (PIE) high resolution mass spectrometry (MS). After MS analysis, structures of candidate compounds, particularly peptides, were obtained by searching against the Human Metabolome Database (HMDB). Using this strategy, we successfully detected 625 peptides (out of 17,205 putative compounds) and 617 peptides (out of 13,297) respectively in source and finished water samples. The source and finished water samples had 501 peptides and amino acids in common. The remaining 116 peptides and amino acids were unique to the finished water. From a subset of 30 putative compounds for which standards were available, 25 were confirmed using HPLC-MS analysis. By analyzing the peptides identified in source and finished water, we successfully confirmed three disinfection reaction pathways that convert peptides into toxic DBPs.

Determination of 14 nitrosamines at nanogram per liter levels in drinking water.

N-Nitrosamines, probable human carcinogens, are a group of disinfection byproducts under consideration for drinking water regulation. Currently, no method can determine trace levels of alkyl and tobacco-specific nitrosamines (TSNAs) of varying physical and chemical properties in water by a single analysis. To tackle this difficulty, we developed a single solid-phase extraction (SPE) method with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination of 14 nitrosamines of health concern with widely differing properties. We made a cartridge composed of a vinyl/divinylbenzene polymer that efficiently concentrated the 14 nitrosamines in 100 mL of water (in contrast to 500 mL in other methods). This single SPE-HPLC-MS/MS technique provided calculated method detection limits of 0.01-2.7 ng/L and recoveries of 53-93% for the 14 nitrosamines. We have successfully demonstrated that this method can determine the presence or absence of the 14 nitrosamines in drinking water systems (eight were evaluated in Canada and the U.S.), with occurrence similar to that in other surveys. N-Nitrosodimethylamine (NDMA), N-nitrosodiphenylamine, and the TSNA 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were identified and quantified in authentic drinking water. Formation potential (FP) tests demonstrated that NDMA and TSNA precursors were present in (1) water samples in which tobacco was leached and (2) wastewater-impacted drinking water. Our results showed that prechlorination or ozonation destroyed most of the nitrosamine precursors in water. Our new single method determination of alkylnitrosamines and TSNAs significantly reduced the time and resource demands of analysis and will enable other studies to more efficiently study precursor sources, formation mechanisms, and removal techniques. It will be useful for human exposure and health risk assessments of nitrosamines in drinking water.

Drinking water microbial myths.

Accounts of drinking water-borne disease outbreaks have always captured the interest of the public, elected and health officials, and the media. During the twentieth century, the drinking water community and public health organizations have endeavored to craft regulations and guidelines on treatment and management practices that reduce risks from drinking water, specifically human pathogens. During this period there also evolved misunderstandings as to potential health risk associated with microorganisms that may be present in drinking waters. These misunderstanding or "myths" have led to confusion among the many stakeholders. The purpose of this article is to provide a scientific- and clinically-based discussion of these "myths" and recommendations for better ensuring the microbial safety of drinking water and valid public health decisions.

Evaluating Evidence for Association of Human Bladder Cancer with Drinking-Water Chlorination Disinfection By-Products.

Exposure to chlorination disinfection by-products (CxDBPs) is prevalent in populations using chlorination-based methods to disinfect public water supplies. Multifaceted research has been directed for decades to identify, characterize, and understand the toxicology of these compounds, control and minimize their formation, and conduct epidemiologic studies related to exposure. Urinary bladder cancer has been the health risk most consistently associated with CxDBPs in epidemiologic studies. An international workshop was held to (1) discuss the qualitative strengths and limitations that inform the association between bladder cancer and CxDBPs in the context of possible causation, (2) identify knowledge gaps for this topic in relation to chlorine/chloramine-based disinfection practice(s) in the United States, and (3) assess the evidence for informing risk management. Epidemiological evidence linking exposures to CxDBPs in drinking water to human bladder cancer risk provides insight into causality. However, because of imprecise, inaccurate, or incomplete estimation of CxDBPs levels in epidemiologic studies, translation from hazard identification directly to risk management and regulatory policy for CxDBPs can be challenging. Quantitative risk estimates derived from toxicological risk assessment for CxDBPs currently cannot be reconciled with those from epidemiologic studies, notwithstanding the complexities involved, making regulatory interpretation difficult. Evidence presented here has both strengths and limitations that require additional studies to resolve and improve the understanding of exposure response relationships. Replication of epidemiologic findings in independent populations with further elaboration of exposure assessment is needed to strengthen the knowledge base needed to better inform effective regulatory approaches.

Precursors of Halobenzoquinones and Their Removal During Drinking Water Treatment Processes.

Halobenzoquinones (HBQs) widely occur in drinking water treatment plant (DWTP) effluents; however, HBQ precursors and their removal by treatments remain unclear. Thus, we have investigated HBQ precursors in plant influents and their removal by each treatment before chlorination in nine DWTPs. The levels of HBQ precursors were determined using formation potential (FP) tests for 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,3,6-trichloro-1,4-benzoquinone (TCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), and 2,6-dibromo-1,4-benzoquinone (DBBQ). HBQ precursors were present in all plant influents. DCBQ precursors were the most abundant (DCBQ FP up to 205 ng/L). Coagulation removed dissolved organic carbon (DOC) (up to 56%) and HBQ precursors (up to 39% for DCBQ). The level of removal of DOC was significantly greater than the level of removal of HBQ FP, suggesting that organic matter removed by coagulation had a high proportion of non-HBQ-precursor material. Granular activated carbon (GAC) decreased the level of HBQ FPs by 10-20%, where DOC removal was only 0.2-4.7%, suggesting that the GAC was not in the adsorption mode and biodegradation of HBQ precursors may have been occurring. Ozonation destroyed/transformed HBQ FPs by 10-30%, whereas anthracite/sand filtration and UV irradiation appeared to have no impact. The results demonstrated that the combined treatments did not substantially reduce HBQ precursor levels in water.

Analytical and toxicity characterization of halo-hydroxyl-benzoquinones as stable halobenzoquinone disinfection byproducts in treated water.

Exposure to chlorination disinfection byproducts (DBPs) is potentially associated with an increased risk of bladder cancer. Four halobenzoquinones (HBQs) have been detected in treated drinking water and have shown potency in producing reactive oxygen species and inducing damage to cellular DNA and proteins. These HBQs are unstable in drinking water. The fate and behavior of these HBQs in drinking water distribution systems is unclear. Here we report the high-resolution mass spectrometry identification of the transformation products of HBQs as halo-hydroxyl-benzoquinones (OH-HBQs) in water under realistic conditions. To further examine the kinetics of transformation, we developed a solid-phase extraction with ultrahigh-performance liquid chromatography tandem mass spectrometry (SPE-UHPLC-MS/MS) method to determine both the HBQs and OH-HBQs. The method provides reproducible retention times (SD < 0.05 min), limits of detection (LODs) at subnanogram per liter levels, and recoveries of 68%-96%. Using this method, we confirmed that decrease of HBQs correlated with increase of OH-HBQs in both the laboratory experiments and several distribution systems, supporting that OH-HBQs were more stable forms of HBQ DBPs. To understand the toxicological relevance of the OH-HBQs, we studied the in vitro toxicity with CHO-K1 cells and determined the IC50 of HBQs and OH-HBQs ranging from 15.9 to 72.9 µM. While HBQs are 2-fold more toxic than OH-HBQs, both HBQs and OH-HBQs are substantially more toxic than the regulated DBPs.