Site-specific risk-based threshold (RBT) concentrations for sewage-associated markers in estuarine swimming waters.

This study establishes site-specific risk-based threshold (RBT) concentrations for sewage-associated markers, including Bacteroides HF183 (HF183), Lachnospiraceae Lachno3 (Lachno3), cross-assembly phage (CrAssphage), and pepper mild mottle virus (PMMoV), utilizing quantitative microbial risk assessment (QMRA) for recreational estuarine waters (EW). The QMRA model calculates a RBT concentration corresponding to a selected target illness risk for ingestion of EW contaminated with untreated sewage. RBT concentrations were estimated considering site-specific decay rates and concentrations of markers and reference pathogen (human norovirus; HNoV), aiding in the identification of high-risk days during the swimming season. Results indicated varying RBT concentrations for fresh (Day 0) and aged (Days 1 to 10) sewage contamination scenarios over 10 days. HF183 exhibited the highest RBT concentration (26,600 gene copis (GC)/100 mL) initially but decreased rapidly with aging (2570 to 3120 GC/100 mL on Day 10) depending on the decay rates, while Lachno3 and CrAssphage remained relatively stable. PMMoV, despite lower initial RBT (3920 GC/100 mL), exhibited increased RBT (4700 to 6440 GC/100 mL) with aging due to its slower decay rate compared to HNoV. Sensitivity analysis revealed HNoV concentrations as the most influential parameter. Comparison of marker concentrations in estuarine locations with RBT concentrations showed instances of marker exceedance, suggesting days of potential higher risks. The observed discrepancies between bacterial and viral marker concentrations in EW highlight the need for optimized sample concentration method and simultaneous measurement of multiple markers for enhanced risk predictions. Future research will explore the utility of multiple markers in risk management. Overall, this study contributes to better understanding human health risks in recreational waters, aiding regulators, and water quality managers in effective decision-making for risk prioritization and mitigation strategies.

Assessing the nucleic acid decay of human wastewater markers and enteric viruses in estuarine waters in Sydney, Australia.

This research investigated the in-situ decay rates of four human wastewater-associated markers (Bacteroides HF183 (HF183), Lachnospiraceae Lachno3 (Lachno3), cross-assembling phage (crAssphage), pepper mild mottle virus (PMMoV) and three enteric viruses (human adenovirus 40/41 (HAdV 40/41), enterovirus (EV) and human norovirus GII (HNoV GII) in two estuarine water environments (Davidson Park (DP) and Hen and Chicken Bay (HCB) in temperate Sydney, NSW, Australia, employing qPCR and RT-qPCR assays. The study also aimed to compare decay rates observed in mesocosms with previously published laboratory microcosms, providing insights into the persistence of markers and viruses in estuarine environments. Results indicated varying decay rates between DP and HCB mesocosms, with HF183 exhibiting relatively faster decay rates compared to other markers and enteric viruses in sunlight and dark mesocosms. In DP mesocosms, HF183 decayed the fastest, contrasting with PMMoV, which exhibited the slowest. Sunlight induced higher decay rates for all markers and viruses in DP mesocosms. In HCB sunlight mesocosms, HF183 nucleic acid decayed most rapidly compared to other markers and enteric viruses. In dark mesocosms, crAssphage showed the fastest decay, while PMMoV decayed at the slowest rate in both sunlight and dark mesocosms. Comparisons with laboratory microcosms revealed faster decay of markers and enteric viruses in laboratory microcosms than the mesocosms, except for crAssphage and HAdV 40/41 in dark, and PMMoV in sunlight mesocosms. The study concludes that decay rates of markers and enteric viruses vary between estuarine mesocosms, emphasizing the impact of sunlight exposure, which was potentially influenced by the elevated turbidity at HCB estuarine waters. The generated decay rates contribute valuable insights for establishing site-specific risk-based thresholds of human wastewater-associated markers.

Comparing the decay of human wastewater-associated markers and enteric viruses in laboratory microcosms simulating estuarine waters in a temperate climatic zone using qPCR/RT-qPCR assays.

This study investigated the decay rates of wastewater-associated markers and enteric viruses in laboratory microcosms mimicking estuarine water environments in temperate Sydney, NSW, Australia using qPCR and RT-qPCR assays. The results demonstrated the reduction in concentrations of Bacteroides HF183, Lachnospiraceae Lachno3, cross-assembly phage (crAssphage), pepper mild mottle virus (PMMoV), human adenovirus (HAdV 40/41), and enterovirus (EV) over a span of 42 days under spring/summer temperatures, presence/absence of microbiota, and different light conditions. The study found that HF183, Lachno3, crAssphage, PMMoV, HAdV 40/41, and EV exhibited varying decay rates depending on the experimental conditions. The average T90 values ranged from a few days to several months, indicating the rapid decay or prolonged persistence of these markers and enteric viruses in the estuarine environment. Furthermore, the study examined the effects of indigenous microbiota and spring/summer temperatures on wastewater-associated markers and enteric viruses decay rates. It was found that the presence of microbiota and temperature significantly influenced the decay rates of HF183 and PMMoV. Additionally, the study compared the effects of artificial sunlight and spring/summer temperatures on marker decay rates. Bacterial markers decayed faster than viral markers, although among viral markers crAssphage decay rates were relatively faster when compared to PMMoV. The exposure to artificial sunlight significantly accelerated the decay rates of bacterial markers, viral markers, and enteric viruses. Temperature also had an impact on the decay rates of Lachno3, crAssphage, and HAdV 40/41. In conclusion, this study provides valuable insights into the decay rates of wastewater-associated markers and enteric viruses under different experimental conditions that mimicked temperate environmental conditions. The findings contribute to our understanding of the fate and persistence of these markers in the environment which is crucial for assessing and managing risks from contamination by untreated human wastewater.

Tracking contaminants of concern in wet-weather sanitary sewer overflows.

Four representative sites in the greater city of Sydney, Australia, were selected for a study of the wet-weather overflow of sanitary (separate to stormwater) sewerage systems. Water samples were collected by autosamplers from up to eight wet weather overflow events over 16 months and from companion receiving water sites. The objective was to identify the risks posed by sewage contaminants to aquatic biota in the receiving waters, to aid in prioritising management actions. Twelve organic contaminants were identified in influents across the four sites under rainfall ingress diluted conditions, with measurements showing that the highest concentrations were restricted to the anti-inflammatory acetaminophen and the diabetes medication metformin. Lesser contaminants included theobromine, ibuprofen, sucralose, and three benzotriazoles (mainly 1-H benzotriazole). An assessment of the toxicity of the identified organic chemicals indicated that none appeared to pose concerns for ecosystem health before wet-weather ingress dilution, and this was even less likely after dilution in the receiving waters. Metal concentrations were low; however, ammonia concentrations in the influent did pose a risk to ecosystem health, although receiving water dilution diminished this risk at four of the five receiving water locations studied.

Reduction of human fecal markers and enteric viruses in Sydney estuarine waters receiving wet weather overflows.

The current microbial source tracking (MST) study tracked the reduction of the culturable fecal indicator bacteria enterococci, four human fecal markers (Bacteroides HF183, Lachnospiraceae Lachno3, cross-assembly phage (CrAssphage) and pepper mild mottle virus (PMMoV)) along with four enteric viruses - human adenovirus 40/41 (HAdV 40/41), enterovirus (EV), human norovirus GI (HNoV GI) and GII (HNoV GII) post wet weather overflows (WWOs) at two estuarine water sites from two depths under separate six-day sampling campaigns over seven and 12 days in Sydney, NSW, Australia. Neither HNoV GI nor GII was detected, while 13.9 % (10/72) of estuarine water samples had detections of EV. Quantifiable concentrations (0.64 to 2.00 log10 gene copies (GC)/100 mL) for HAdV 40/41 were returned from 65.2 % (47/72) of samples collected across the two sites and two depths with 30 quantifications recorded in the surface layer samples. In contrast the presence of HF183, Lachno3, CrAssphage, and PMMoV markers was observed in all 36 (100 %) estuarine water samples collected from the surface layer from both sites. Detection frequencies of these markers were slightly lower at 1 m above the bottom surface. The concentrations of the human fecal markers were compared to established gastrointestinal (GI) risk benchmarks. The concentrations of HF183, Lachno3 and CrAssphage marker only exceeded the GI risk benchmark until day 3, while concentrations of PMMoV marker were indicative of exceedance of the GI risk benchmark on day 7 post WWOs that was much longer than indicated by culturable enterococci concentrations that were within this GI risk benchmark by day 2 and day 4 for the two sites, respectively.

Microbial source tracking of untreated human wastewater and animal scats in urbanized estuarine waters.

The study assessed the performance characteristics of host sensitivity, host specificity and concentration for seven human wastewater- and six animal scat-associated marker genes by analysing human wastewater and animal scat samples from urban catchments of the mega-coastal city of Sydney, Australia. Absolute host sensitivity was exhibited across three criteria used to assess seven human wastewater-associated marker genes of cross-assembly phage (CrAssphage), human adenovirus (HAdV), Bacteroides HF183 (HF183), human polyomavirus (HPyV), Lachnospiraceae (Lachno3), Methnobrevibacter smithii nifH (nifH) and pepper mild mottle virus (PMMoV). In contrast, only the horse scat-associated marker gene Bacteroides HoF597 (HoF597) exhibited absolute host sensitivity. The absolute host specificity value of 1.0 was returned for the wastewater-associated marker genes of HAdV, HPyV, nifH and PMMoV for each of the three applied host specificity calculation criteria, while values of >0.9 were returned for CrAssphage and Lachno3. Ruminants and cow scat-associated marker genes of BacR and CowM2, respectively exhibited the absolute host specificity value of 1.0. Concentrations of Lachno3 were greater in most human wastewater samples followed by CrAssphage, HF183, nifH, HPyV, PMMoV and HAdV. Human wastewater marker genes were detected in several scat samples from cats and dogs, and this suggests concordant sampling of animal scat-associated marker genes and at least two human wastewater-associated marker genes will be required to assist in interpretation of fecal sources in environmental waters. A greater prevalence, together with several samples with greater concentrations of human wastewater-associated marker genes PMMoV and CrAssphage warrant consideration by water quality managers for the detection of diluted human fecal pollution in estuarine waters.

Distribution of human fecal marker genes and their association with pathogenic viruses in untreated wastewater determined using quantitative PCR.

Quantitative microbial risk assessment (QMRA) of human health risks using human fecal marker genes (HFMGs) is an useful water quality management tool. To inform accurate QMRA analysis, generation of probability distribution functions for HFMGs, and reference pathogenic viruses can be improved by input of correlation and ratios based upon measurement of HFMGs and gene copies (GC) of pathogenic viruses in untreated wastewater. The concentrations of four HFMGs (Bacteroides HF183, Lachnospiraceae Lachno3, CrAssphage and pepper mild mottle virus (PMMoV)), and GC of three reference pathogenic viruses human adenovirus 40/41 (HAdV 40/41), human norovirus GI + GII HNoV GI + GII and enterovirus (EV) were measured in untreated wastewater samples collected over a period of 12 months from two wastewater treatment plants in Sydney, Australia using quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR). Over the course of the study, the GC of potential pathogenic viruses were 3-5 orders of magnitude lower than HFMGs in untreated wastewater. The GC of pathogenic viruses were highly variable over the course of the study, which contrasted with the concentrations of HFMGs that were quite stable with little variation observed within and between WWTPs. Among the HFMGs, HF183, CrAssphage and PMMoV correlated well with pathogenic virus GC, whereas weak or negative correlations were observed between Lachno3 and pathogenic virus GC. While the two assessed WWTPs had dissimilar population service sizes, the ratios between log10 transformed pathogenic virus GC and HFMGs demonstrated similar central tendency and variability for the same combinations between WWTP A and WWTP B with no difference between the WWTPs. This suggests the widespread presence of these HFMGs in both populations serviced by these two WWTPs. The observed correlation and ratios of HFMGs and GC of reference pathogenic viruses can contribute to improved QMRA of human health risks in environmental waters subject to fresh sewer overflows.

Interlaboratory accuracy and precision among results of three sewage-associated marker genes in urban environmental estuarine waters and freshwater streams.

The application of quantitative polymerase chain reaction (qPCR) based microbial source tracking (MST) marker genes are increasingly being used to identify contaminating sources and inform management decisions. In this study, we assessed interlaboratory agreement on duplicate environmental water samples collected from estuarine and freshwater locations, by comparing results of qPCR based testing for Bacteroides HF183, crAssphage CPQ_056, and pepper mild mottle virus (PMMoV). The overall agreements (co-detection and non-co-detection) between CSIRO Land and Water (CLW) laboratory and Sydney Water (SW) laboratory for the HF183, crAssphage CPQ_056 and PMMoV marker genes for duplicate water samples were 74, 75 and 74%, respectively. Cohene's kappa (k) revealed fair to moderate agreements and acceptable relative percent difference (RPD) values of <15% for duplicate samples. The pooled mean abundances of HF183, CPQ_056, and PMMoV in measurable samples at the CLW laboratory were 5.19 ± 0.93, 5.12 ± 0.82, and 4.42 ± 0.65 log10 copies/L, respectively. However, the pooled mean abundances were significantly lower at the SW laboratory, HF183 (4.58 ± 0.84 log10 copies/L), crAssphage CPQ_056 (4.20 ± 0.63 log10 copies/L), and PMMoV (3.89 ± 0.41 log10 copies/L). At individual sample level, most of the paired samples had <1 log10 difference. Significant positive Spearman rank correlations were obtained between two laboratories for the HF183 (Rs = 0.65; p < 0.05), CPQ_056 (Rs = 0.79; p < 0.05), and PMMoV (Rs = 0.54; p < 0.05) marker genes. Several factors such as standards, qPCR platforms, PCR inhibitors, nucleic acid extraction efficiency and low levels of targets in some samples may have contributed to the observed discrepancies. Results presented in this study highlight the importance of standardized protocol, laboratory equipment (such as digital PCR), sample processing strategies and appropriate quality controls that may need implementation to further improve accuracy and precision of results between laboratories.

Prevalence and abundance of traditional and host-associated fecal indicators in urban estuarine sediments: Potential implications for estuarine water quality monitoring.

This study aimed to determine the prevalence and abundance of sewage and animal fecal contamination of sediment at seven estuarine locations in Sydney, NSW, Australia. Sediment samples were tested for the occurrence of microbial targets including molecular marker genes of enterococci (ENT), Bacteroides HF183 (HF183), Methanobrevibacter smithii (nifH), human adenovirus (HAdV) and emerging sewage-associated marker genes crAssphage (CPQ_056) and Lachnospiraceae (Lachno3) and animal feces-associated marker genes, including avian feces-associated Helicobacter spp. (GFD), canine-feces associated Bacteroides (DogBact), cattle-feces associated (cowM2) and horse feces-associated Bacteroides (HoF597). Results from this study showed that urban estuarine sediment can act as a reservoir of fecal indicator bacteria (FIB) and several microbial source tracking (MST) marker genes, including previously unreported Lachno3. The sewage-associated marker gene CPQ_056 was most prevalent, in 63.8% of sediment samples, while the avian associated marker gene GFD had the highest mean abundance. The GFD marker gene was highly abundant and widely detected in sediment samples from all seven locations compared to the other animal feces-associated marker genes. In all, 31 (44.9%) sediment samples were positive for at least two sewage-associated marker genes. However, the non-quantifiable detection of the HAdV marker gene did not always align with the detection of two or more sewage-associated marker genes. In addition, the most frequent wet weather overflow exposure occurred at locations that did not have a consistent pattern of detection of the sewage-associated marker genes, suggesting sediments may not be a suitable measure of recent sewage contamination. To assist water quality and public health managers better understand past microbial contamination of estuarine sediment, further studies seem justified to explore the role of decay of MST marker genes in sediment. Further work is also needed on the role of resuspension of MST marker genes from sediment during storm events to the water column as a source of contamination for both the GFD and sewage-associated marker genes.

Sewage-associated marker genes illustrate the impact of wet weather overflows and dry weather leakage in urban estuarine waters of Sydney, Australia.

This study investigates the impact of wet weather overflows (WWOs) at three estuarine locations in Sydney, NSW, Australia. WWOs can occur when infiltration of stormwater leads to an excess volume of flow within the sewerage system, resulting in the release of diluted sewage into the environment. Sewage contamination poses a risk to human health due to the presence of pathogens. The magnitude of sewage contamination was monitored using established and novel sewage-associated marker genes, Bacteroides HF183, pepper mild mottle virus (PMMoV), crAssphage CPQ_056, Lachnospiraceae (Lachno3) marker genes along with culturable fecal indicator bacteria (FIB) Escherichia coli (E. coli) and enterococci. Water samples were collected at two water depths (0.5 m below the water surface and 1 m above the bottom surface) during one dry weather and two storm events. Analysis of sewage-associated marker genes showed greater (i.e., 3-5 orders of magnitude) concentrations in water samples collected during the storm events compared to dry weather event. Water samples were also analysed for four animal feces-associated marker genes targeting avian (GFD), dog (BacCan-UCD), cow (cowM2) and horse (HoF597) species to determine the extent of animal fecal contamination. Among the four marker genes, cowM2 and HoF597 could not be detected, while GFD marker gene was consistently present and BacCan-UCD was occasionally detected. Overall results suggested that after rainfall, untreated sewage from WWOs was present at sampling locations. In addition, microbial source tracking (MST) monitoring was able to distinguish the presence of a leaking sewer impacting on the recreational area during dry weather condition. This study demonstrated the capability of the MST monitoring approach to understand sources (sewage or animal) of fecal contamination. This capability will greatly enhance management decisions assisting in the prioritisation of remediation efforts of the sewerage system to improve estuarine bathing water quality and diminish human health risk.

Enhanced insights from human and animal host-associated molecular marker genes in a freshwater lake receiving wet weather overflows.

This study investigated the magnitude of wet weather overflow (WWO)-driven sewage pollution in an urban lake (Lake Parramatta) located in Sydney, New South Wales, Australia. Water samples were collected during a dry weather period and after two storm events, and tested for a range of novel and established sewage- [Bacteroides HF183, crAssphage CPQ_056 and pepper mild mottle virus (PMMoV)] and animal feces-associated (Bacteroides BacCan-UCD, cowM2 and Helicobacter spp. associated GFD) microbial source tracking marker genes along with the enumeration of culturable fecal indicator bacteria (FIB), namely Escherichia coli (E. coli) and Enterococcus spp. The magnitude of general and source-specific fecal pollution was low in water samples collected during dry weather compared to storm events. The levels of HF183, crAssphage and PMMoV in water samples collected during storm events were as high as 6.39, 6.33 and 5.27 log10 GC/L of water, respectively. Moderate to strong positive correlations were observed among the quantitative occurrence of sewage-associated marker genes. The concentrations of HF183 and PMMoV in most storm water samples exceeded the risk benchmark threshold values established in the literature for primary contact recreators. None of the samples tested was positive for the cowM2 (cow) marker gene, while BacCan-UCD (dog) and GFD (avian) animal-associated markers were sporadically detected in water samples collected from both dry weather and storm events. Based on the results, the ongoing advice that swimming should be avoided for several days after storm events appears appropriate. Further research to determine the decay rates of sewage-associated marker genes in relation to each other and enteric viruses would help refine current advice. Microbial source tracking approaches employed in this study provided insights into sources of contamination over currently used FIB.

A duplex PCR assay for the simultaneous quantification of Bacteroides HF183 and crAssphage CPQ_056 marker genes in untreated sewage and stormwater.

The HF183 marker gene, derived from the 16S rRNA gene of Bacteroides dorei, has been widely used to identify sewage pollution in environmental waters. CrAssphages are recently discovered DNA bacteriophages that are highly abundant in untreated sewage and have shown promises for tracking sewage contamination in environmental waters. In this paper, we report the development of a duplex quantitative PCR (qPCR) assay for simultaneous quantification of HF183 and crAssphage CPQ_056 marker genes in untreated sewage and sewage impacted stormwater. Same primer and probe sequences were used in the duplex qPCR assay as used in published simplex qPCR assays. The performance characteristics of the duplex qPCR assay were similar to its simplex counterparts. We validated the performance of the duplex assay in a collaborative laboratory study with the aim to evaluate reproducibility, sensitivity and concordance for field study. The concordance values between the simplex vs. duplex qPCR assays for HF183 and crAssphage CPQ_056 marker genes ranged from 96.7 to 100% and the mean concentrations of HF183 and CPQ_056 in environmental water samples were remarkably similar or in some cases slightly greater for the duplex qPCR assay suggesting the reliability of this assay for monitoring HF183 and CPQ_056 simultaneously. The newly developed duplex qPCR assay will be a valuable addition to the MST toolbox for sewage pollution monitoring and would allow rapid and comparative sample analysis.

Novel crAssphage marker genes ascertain sewage pollution in a recreational lake receiving urban stormwater runoff.

Considerable efforts have been made in recent years in developing novel marker genes for fecal pollution tracking in environmental waters. CrAssphage are recently discovered DNA bacteriophage that are highly abundant in human feces and untreated sewage. In this study, we evaluated the host-sensitivity and -specificity of the newly designed crAssphage qPCR assays (Stachler et al., 2017) CPQ_056 and CPQ_064 (i.e., marker genes) in fecal samples collected from various human and several animal host groups in Australia. We also investigated the utility of these marker genes to detect sewage pollution in an urban recreational lake (i.e., Lake Parramatta) in Sydney, NSW. The mean concentrations of CPQ_056 and CPQ_064 marker genes in untreated sewage were 9.43 ±â€¯0.14 log10 GC/L and 8.91 ±â€¯0.17 log10 GC/L, respectively, 2 to 3 orders of magnitude higher than other sewage-associated viruses used in microbial source tracking studies. Among 177 animal fecal samples tested from 11 species, the host-specificity values for CPQ_056 and CPQ_064 marker genes were 0.95 and 0.93, respectively. Limited cross-reactivity was observed with cat fecal and cattle wastewater samples. Abundance of crAssphage markers were monitored in an urban lake that receives stormwater runoff. The concentrations of both markers were higher (CPQ_056 ranging from 3.40 to 6.04 log10 GC/L and CPQ_064 ranging from 2.90 to 5.47 log10 GC/L) in 20 of 20 (for CPQ_056) and 18 of 20 (for CPQ_064) samples collected after storm events with gauged sewer overflows compared to dry weather event (10 of 10 samples were qPCR negative for the CPQ_056 and 8 of 10 were negative for the CPQ_064 marker genes) suggesting sewage pollution was transported by urban stormwater runoff to Lake Parramatta. The results of the study may provide context for management of sewage pollution from gauged overflow points of the sewerage system in the catchment.