The susceptibility of waterborne viruses to disinfection is known to vary between viruses and even between closely related strains, yet the extent of this variation is not known. Here, different ...enteroviruses (six strains of coxsackievirus B5, two strains of coxsackievirus B4 and one strain of coxackievirus B1) were isolated from wastewater and inactivated by UV254, sunlight, free chlorine (FC), chlorine dioxide (ClO2), and heat. Inactivation kinetics of these isolates were compared with those of laboratory enterovirus strains (CVB5 Faulkner and echovirus 11 Gregory) and MS2 bacteriophage. FC exhibited the greatest (10-fold) variability in inactivation kinetics between different strains, whereas inactivation by UV254 differed only subtly. The variability in inactivation kinetics was greater between serotypes than it was among the seven strains of the CVB5 serotype. MS2 was a conservative surrogate of enterovirus inactivation by UV254, sunlight, or heat but frequently underestimated the disinfection requirements for FC and ClO2. Similarly, laboratory strains did not always reflect the inactivation behavior of the environmental isolates. Overall, there was considerable variability in inactivation kinetics among and within enteroviruses serotypes, as well as between laboratory and environmental isolates. We therefore recommend that future disinfection studies include a variety of serotypes and environmental isolates.
Wastewater treatment ponds (lagoons) are one of the most common types of technologies used for wastewater management worldwide, especially in small cities and towns. They are particularly well-suited ...for systems where the effluent is reused for irrigation. However, the efficiency of virus removal in wastewater treatment pond systems is not very well understood. The main objective of this paper is to critically review the major findings related to virus removal in wastewater treatment pond systems and to statistically analyze results reported in the literature from field studies on virus removal in these systems. A comprehensive analysis of virus removal reported in the literature from 71 different wastewater treatment pond systems reveals only a weak to moderate correlation of virus removal with theoretical hydraulic retention time. On average, one log10 reduction of viruses was achieved for every 14.5–20.9 days of retention, but the 95th percentile value of the data analyzed was 54 days. The mechanisms responsible for virus removal in wastewater treatment ponds were also reviewed. One recent finding is that sedimentation may not be a significant virus removal mechanism in some wastewater ponds. Recent research has also revealed that direct and indirect sunlight-mediated mechanisms are not only dependent on pond water chemistry and optics, but also on the characteristics of the virus and its genome. MS2 coliphage is considered to be the best surrogate for studying sunlight disinfection in ponds. The interaction of viruses with particles, with other microorganisms, and with macroinvertebrates in wastewater treatment ponds has not been extensively studied. It is also unclear whether virus internalization by higher trophic-level organisms has a protective or a detrimental effect on virus viability and transport in pond systems. Similarly, the impact of virus-particle associations on sunlight disinfection in ponds is not well understood. Future research should focus on the interactions of viruses with particles and with other organisms, as well as the development of a model for virus removal in pond systems that can be used for design purposes, and to inform future editions of the WHO Guidelines for Wastewater Use in Agriculture.
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•Previously published virus removal data from 71 different systems were analyzed.•Weak to moderate correlation between virus removal and hydraulic retention time.•14.5 days of retention for each log10 reduction of viruses (geometric mean).•54 days of retention for each log10 reduction of viruses (95th percentile).
•Wastewater PLOD values were determined for six SARS-CoV-2 assays.•The US CDC N1 RT-dPCR and RT-qPCR assays were the most sensitive assays.•The US CDC N2 and E_Sarbeco assays were the least sensitive ...assays.•At seeding levels > 2.32 × 104 GC/50 mL, consistent amplification was observed.•At seeding levels < 2.32 × 103 GC/50 mL, inconsistent amplification was observed.
Effective wastewater surveillance of SARS-CoV-2 RNA requires the rigorous characterization of the limit of detection resulting from the entire sampling process - the process limit of detection (PLOD). Yet to date, no studies have gone beyond quantifying the assay limit of detection (ALOD) for RT-qPCR or RT-dPCR assays. While the ALOD is the lowest number of gene copies (GC) associated with a 95% probability of detection in a single PCR reaction, the PLOD represents the sensitivity of the method after considering the efficiency of all processing steps (e.g., sample handling, concentration, nucleic acid extraction, and PCR assays) to determine the number of GC in the wastewater sample matrix with a specific probability of detection. The primary objective of this study was to estimate the PLOD resulting from the combination of primary concentration and extraction with six SARS-CoV-2 assays: five RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab (CCDC N and CCDC ORF1ab), and E_Sarbeco RT-qPCR, and one RT-dPCR assay (US CDC N1 RT-dPCR) using two models (exponential survival and cumulative Gaussian). An adsorption extraction (AE) concentration method (i.e., virus adsorption on membrane and the RNA extraction from the membrane) was used to concentrate gamma-irradiated SARS-CoV-2 seeded into 36 wastewater samples. Overall, the US CDC N1 RT-dPCR and RT-qPCR assays had the lowest ALODs (< 10 GC/reaction) and PLODs (<3,954 GC/50 mL; 95% probability of detection) regardless of the seeding level and model used. Nevertheless, consistent amplification and detection rates decreased when seeding levels were < 2.32 × 103 GC/50 mL even for US CDC N1 RT-qPCR and RT-dPCR assays. Consequently, when SARS-CoV-2 RNA concentrations are expected to be low, it may be necessary to improve the positive detection rates of wastewater surveillance by analyzing additional field and RT-PCR replicates. To the best of our knowledge, this is the first study to assess the SARS-CoV-2 PLOD for wastewater and provides important insights on the analytical limitations for trace detection of SARS-CoV-2 RNA in wastewater.
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•Indicator organisms do not tell the whole story for the safety of water and sanitation systems.•Considering only fecal indicator groups may provide a falsely reduced sense of risk.•Consideration of ...pathogens matters for meeting SDG6.
Water and wastewater utilities, water and sanitation hygiene (WASH) practitioners, and regulating bodies, particularly in developing nations, rely heavily on indicator microorganisms, as opposed to pathogens, for much of their regulatory decisions. This commentary illustrates the importance of considering pathogens and not relying only on indicator organisms when making decisions regarding water and sanitation, especially with respect to meeting the current targets of the Sustainable Development Goal (SDG) 6. We use quantitative microbial risk assessment (QMRA) to present three common scenarios that WASH and public health practitioners encounter to illustrate our point. These include 1) chlorination of surface water for drinking, 2) land application of latrine waste as a fertilizer, and 3) recreation/domestic use of surface waters impacted by wastewater discharge. We show that the calculated probabilities of risk of infection are statistically significantly higher when using treatment/survival information for pathogens versus using indicator species data. Thus, demonstrating that relying solely on indicators for sanitation decision making is inadequate if we truly want to achieve the SDG6 targets of safely managed water and sanitation services.
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Human enteric pathogens are a major global concern, as they are responsible for thousands of preventable deaths every year. New pathogens in wastewater are constantly emerging. For example, ...SARS-CoV-2 has been recently detected in domestic sewage and primary sludge. Knowledge about the reduction of viruses in wastewater treatment and their partitioning between the treated liquid effluent versus the sludge or biosolids is still very scarce, especially in countries with emerging economies and tropical climates. Upflow anaerobic sludge blanket (UASB) reactors are among the top three most commonly used technologies for the treatment of sewage in Latin America and the Caribbean, and their use has become increasingly common in many other low- and middle-income countries. High-rate algal ponds (HRAP) are regarded as a sustainable technology for the post-treatment of UASB effluent. This study evaluated the overall reduction and the liquid-solid partitioning of somatic coliphages, F-specific coliphages, and E. coli in a pilot-scale system comprised of a UASB reactor followed by HRAPs treating real wastewater. Average log removal for somatic and F-specific coliphages were 0.40 and 0.56 for the UASB reactor, and 1.15 and 1.70 for HRAPs, respectively. The overall removal of both phages in the system was 2.06-log. Removal of E. coli was consistently higher. The number of viruses leaving the system in the UASB solids and algal biomass was less than 10% of the number leaving in the clarified liquid effluent. The number of E. coli leaving the system in solids residuals was estimated to be approximately one order of magnitude higher than the number of E. coli leaving in the liquid effluent. Results from this study demonstrate the suitability of UASB-HRAP systems to reduce viral and bacterial indicators from domestic sewage and the importance of adequately treating sludge for pathogen reduction before they are used as biosolids.
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•Virus and bacteria removal and solids partitioning evaluated in UASB-HRAP system.•90% of surviving E. coli left the system in the UASB sludge.•>90% of surviving coliphages left the system in the liquid effluent.•Coliphages did not show any affinity for associating with sludge or biomass.
Heat treatment, or thermal disinfection, is one of the simplest disinfection methods, and is widely used in the water, sanitation, and food sectors, especially in low resource settings. Pathogen ...reductions achieved during heat treatment are influenced by a combination of temperature and exposure time. The objective of this paper was to construct updated time-temperature pathogen inactivation curves to define “safety zones” for the reduction of four pathogen groups (bacteria, viruses, protozoan (oo)cysts, and helminth eggs) during heat treatment in a variety of matrices. A systematic review and meta-analysis were conducted to determine the times needed to achieve specified levels of pathogen reduction at different temperatures. Web of Science was searched using a Boolean string to target studies of heat treatment and pasteurization systems that exposed pathogens in water, wastewater, biosolids, soil, or food matrices to temperatures between 20 °C and 95 °C. Data were extracted from tables or figures and regression was used to assess the relationship between time and temperature. Our findings indicate that the temperatures and times needed to achieve a 1-log10 reduction of all pathogen groups are likely higher and longer, respectively, than previously reported. The type of microorganism and the matrix significantly impact T90 values reported at different temperatures. At high temperatures, the time-temperature curves are controlled by thermally stable viruses such as hepatitis A virus. Data gaps include the lack of data on protozoa, and the lack of data on all pathogen groups at low temperatures, for long exposure times, and with high log10 reductions. The findings from this study can be used by engineers, food safety specialists for the planning and design of engineered water, sanitation, and food pasteurization and treatment systems.
Sewer overflows and exfiltration can potentially contaminate water bodies with pathogens from wastewater. Microbial source tracking (MST) methods such as the detection of the HF183 gene target of
...Bacteroides
have been proposed to monitor human fecal pollution inputs to surface waters; however, the persistence of HF183 and other MST markers in water flushed from soils after contamination events is not well understood. In this study, the persistence and decay of two culture-based fecal indicators,
Escherichia coli
and enterococci, and two molecular MST markers, HF183 and pepper mild mottle virus (PMMoV), were evaluated in riverbank soils spiked with untreated sewage, which were left idle for 1, 14, 28, 60, and 121 days under dark conditions and then flushed with synthetic rainwater. All four microbial indicators were still detected in flush water 4 months after the soil was contaminated. PMMoV persisted much longer and had a slower decay rate than the other microbial indicators, and
E. coli
degraded most rapidly. In consecutive flushing experiments with fresh (1 day) sewage-spiked soils, HF183,
E. coli
, and enterococci were all detected after 20 consecutive flushes with rainwater, but PMMoV was not detected after the fifth flush. Our findings indicate that water (e.g., stormwater interflow) flushing through riverbank soils that have previously been contaminated by sewer overflows or sewer exfiltration can potentially be a source of microbial pollution to surface waters, even for several months after the contamination occurs. Results from this study also demonstrate the benefits of using multiple human-associated fecal indicators to distinguish pollution from different microbial groups in water bodies.
Graphical abstract
Fluorescence spectroscopy has been increasingly used to detect sewage and other anthropogenic contaminants in surface waters. Despite progress in successfully detecting bacterial and sewage inputs to ...rivers over diverse spatial scales, the use of fluorescence-based in-situ sensors to track contaminant inputs during storm events and to discern bacterial contamination from background natural organic matter (NOM) fluorescence have received less attention. A portable, submersible fluorometer equipped with tryptophan (TRP)-like and humic-like fluorescence sensors was used to track inputs of untreated wastewater added to natural creek water in a laboratory sewage spill simulation. Significant, positive correlations were observed between TRP fluorescence, the TRP:humic ratio, percent wastewater, and Escherichia coli concentrations, indicating that both the TRP sensor and the TRP:humic ratio tracked wastewater inputs against the background creek water DOM fluorescence. The portable fluorometer was subsequently deployed in an urban creek during a storm in 2018. The peak in TRP fluorescence was found to increase with the rising limb of the hydrograph and followed similar temporal dynamics to that of caffeine and fecal indicator bacteria, which are chemical and biological markers of potential fecal pollution. Results from this study demonstrate that tracking of TRP fluorescence intensity and TRP:humic ratios, with turbidity correction of sensor outputs, may be an appropriate warning tool for rapid monitoring of sewage or other bacterial inputs to aquatic environments.
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•In-situ fluorescence monitoring of pollutant inputs in lab and field was evaluated.•TRP fluorescence and TRP:humic correlated with % wastewater added to creek water.•TRP fluorescence and TRP:humic correlated with E. coli in experimental simulations.•In-situ fluorescence sensor deployed during a storm tracked contaminant inputs.•TRP:humic ratio tracked flux of E. coli and caffeine in urban creek during storm.
The use of partially treated and untreated wastewater for irrigation is beneficial in agriculture but may be associated with human health risks. Reports from different locations globally have linked ...microbial outbreaks with agricultural reuse of wastewater. This article reviews the epidemiological evidence and health risks associated with this practice, aiming toward evidence-based conclusions. Exposure pathways that were addressed in this review included those relevant to agricultural workers and their families, consumers of crops, and residents close to areas irrigated with wastewater (partially treated or untreated). A meta-analysis gave an overall odds ratio of 1.65 (95% CI: 1.31, 2.06) for diarrheal disease and 5.49 (95% CI: 2.49, 12.10) for helminth infections for exposed agricultural workers and family members. The risks were higher among children and immunocompromised individuals than in immunocompetent adults. Predominantly skin and intestinal infections were prevalent among individuals infected mainly via occupational exposure and ingestion. Food-borne outbreaks as a result of crops (fruits and vegetables) irrigated with partially or untreated wastewater have been widely reported. Contamination of crops with enteric viruses, fecal coliforms, and bacterial pathogens, parasites including soil-transmitted helminthes (STHs), as well as occurrence of antibiotic residues and antibiotic resistance genes (ARGs) have also been evidenced. The antibiotic residues and ARGs may get internalized in crops along with pathogens and may select for antibiotic resistance, exert ecotoxicity, and lead to bioaccumulation in aquatic organisms with high risk quotient (RQ). Appropriate mitigation lies in adhering to existing guidelines such as the World Health Organization wastewater reuse guidelines and to Sanitation Safety Plans (SSPs). Additionally, improvement in hygiene practices will also provide measures against adverse health impacts.
•Viruses tend to adsorb to solids in wastewater due to their isoelectric point.•Low removal and reduction of human viruses compared to fecal indicators.•Human viruses may have a higher affinity for ...solids than coliphages.•Suitable indicators are needed for the liquid-solid partitioning of human viruses.
Investigating waterborne viruses is of great importance to minimizing risks to public health. Viruses tend to adsorb to sludge particles from wastewater processes by electrostatic and hydrophobic interactions between virus, aquatic matrix, and particle surface. Sludge is often re-used in agriculture; therefore, its evaluation is also of great interest to public health. In the present study, a pilot scale system treating real domestic wastewater from a large city in Brazil was used to evaluate the removal, the overall reduction, and liquid-solid partitioning of human adenovirus (HAdV), the novel coronavirus (SARS-CoV-2) and fecal indicators (F-specific coliphages and E. coli). The system consists of a high-rate algal pond (HRAP) post-treating the effluent of an upflow anaerobic sludge blanket (UASB) reactor. Samples were collected from the influent and effluent of each unit, as well as from the sludge of the UASB and from the microalgae biomass in the HRAP. Pathogens and indicators were quantified by quantitative polymerase chain reaction (qPCR) (for HAdV), qPCR with reverse transcription (RTqPCR) (for SARS-CoV-2), the double agar plaque assay (for coliphages), and the most probable number (MPN) method (for E. coli). The removal and overall reduction of HAdV and SARS-CoV-2 was greater than 1-log10. Almost 60% of remaining SARS-CoV-2 RNA and more than 70% of remaining HAdV DNA left the system in the sludge, demonstrating that both viruses may have affinity for solids. Coliphages showed a much lower affinity to solids, with only 3.7% leaving the system in the sludge. The system performed well in terms of the removal of organic matter and ammoniacal nitrogen, however tertiary treatment would be necessary to provide further pathogen reduction, if the effluent is to be reused in agriculture. To our knowledge, this is the first study that evaluated the reduction and partitioning of SARS-CoV-2 and HAdV through the complete cycle of a wastewater treatment system consisting of a UASB reactor followed by HRAPs.
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