Decay of Cryptosporidium parvfum DNA in cowpats in subtropical environments determined using qPCR.

Cryptosporidium oocysts pose a significant threat to public health due to its ability to contaminate environmental waters, leading to outbreaks of waterborne diseases and emphasizing the crucial need for effective water treatment and monitoring systems. This study aimed to investigate the decay of Cryptosporidium oocyst DNA in cow fecal matter under different environmental conditions prevalent in sub-tropical Southeast Queensland (SEQ) during summer and winter seasons. The effects of ambient sunlight and shaded conditions on the decay rates of C. parvum DNA in cow fecal samples were evaluated. The results showed that measurable levels of C. parvum DNA were observed for up to 60 days during the summer experiments, with a slower decay rate on the surface (k = -0.029) and sub-surface (k = -0.043) of the cowpat under shaded conditions than those on the surface (k = -0.064) and sub-surface (k = -0.079) under sunlight conditions. The decay rates of C. parvum DNA on the surface and sub-surface of the cowpat under shaded conditions were significantly slower (p = 0.004; p = 0.004) than those on the surface and sub-surface under sunlight conditions during summer experiments. During the winter treatments, measurable levels of C. parvum DNA were observed for up to 90 days, and the decay rates were slower on the surface (k = -0.036) and sub-surface (k = -0.034) of the cowpat under shaded conditions than those under sunlight conditions (k = -0.067 for surface and k = -0.057 for sub-surface). The decay rates of C. parvum DNA on the surface and sub-surface of the cowpat under shaded conditions were significantly slower than those on the surface (p = 0.009) and sub-surface (p = 0.041) under sunlight conditions during winter experiments. Moreover, the decay rate in the summer sunlight surface treatment (k = -0.064) was significantly faster from those in the winter shaded surface (k = -0.036; p = 0.018) and sub-surface (k = -0.034; p = 0.011) treatments. Similar results were also observed for summer sunlight sub-surface (k = -0.079), which was significantly faster than winter shaded surface (k = -0.036; p = 0.0008) and sub-surface (k = -0.034; p = 0.0005) treatments. Overall, these findings are important to enhance our understanding on the degradation of C. parvum DNA in cow fecal matter in SEQ, particularly in relation to seasonal variations and environmental conditions.

Household Food Insecurity Negatively Impacts Diet Diversity in the Vietnamese Mekong Delta: A Cross-Sectional Study.

Healthy, diverse diets are vital for life. In low/middle-income countries, however, the focus is more on food quantity rather than diet quality. This study assessed household diet diversity (HDD) in the Vietnamese Mekong Delta and its associations with household food insecurity (HFI) and household food availability (HFA) controlling for socioeconomic factors. Primary food-preparers in 552 randomly selected households in two rural provinces were interviewed about socioeconomic factors, HDD, HFI, and HFA. More than 80% of households predominantly consumed energy-dense foods, whereas less than 20% consumed nutrient-dense foods. Lower HDD was associated with HFI, lower HFA, for the Khmer ethnic minority, and low livelihood capitals (landlessness, low expenditure, debt) and low utensil scores. The study highlighted the need to provide improved food and nutrition policies that increase availability and access to diverse and healthy foods as well as reduce poverty and increase incomes for at-risk rural and ethnic minority groups.

Household water and food insecurity negatively impacts self-reported physical and mental health in the Vietnamese Mekong Delta.

INTRODUCTION: Household food insecurity and inadequate water, sanitation, and hygiene (WASH) contribute to ill health. However, the interactions between household food insecurity, WASH and health have been rarely assessed concurrently. This study investigated compounded impacts of household food insecurity and WASH on self-reported physical and mental health of adults in the Vietnamese Mekong Delta. MATERIALS AND METHODS: This cross-sectional survey interviewed 552 households in one northern and one southern province of the Vietnamese Mekong Delta. The survey incorporated previously validated tools such as the Short Form 12-item Health Survey, Household Food Insecurity Assessment Scale, and the Access and Behavioural Outcome Indicators for Water, Sanitation, and Hygiene. Physical and mental health were quantified using the physical health composite score (PCS) and mental health composite score (MCS), respectively. These measures were the dependent variables of interest for this study. RESULTS: Statistical analysis revealed that household food insecurity and using <50 litres of water per person per day (pppd) were independently associated with lower PCS (p<0.05), after adjusting for socio-economic confounders. Household food insecurity and lack of food availability, using <50 litres of water pppd, and the use of untreated drinking water were associated with lower MCS (p<0.05), with water usage being an effect modifier of the relationship between household food insecurity and MCS. The results indicate that being food insecure and having limited potable quality water had a compounding effect on MCS, compared to being individually either food insecure or having limited water. CONCLUSION: This study is one of only a few that have established a link between potable water availability, food insecurity and poorer physical and mental health. The results also indicate a need to validate national data with fine-scale investigations in less populous regions to evaluate national initiatives with local populations that may be at higher risk. Adopting joint dual-action policies for interventions that simultaneously address water and food insecurity should result in larger improvements in health, particularly mental health, compared to targeting either food or water insecurity in isolation.

Virological Characterization of Roof-Harvested Rainwater of Densely Urbanized Low-Income Region.

Roof-harvested rainwater (RHRW) is considered relatively clean water, even though the possible presence of pathogens in the water may pose human health risks. In this study, we investigated the occurrence of enteric viruses in the first flush (10 mm) of RHRW from a densely populated and low-income urbanized region of Rio de Janeiro. One hundred samples (5 L) were collected from 10 rainfall events between April 2015 and March 2017. RNA and DNA viruses were concentrated using the skimmed milk flocculation method and analyzed using the TaqMan® quantitative RT-qPCR and qPCR. Human adenoviruses, noroviruses, rotaviruses A, and avian parvoviruses were detected in 54%, 31%, 12%, and 12% of the positive samples. JC polyomavirus, also targeted, was not detected. Virus concentrations ranged from 1.09 × 101 to 2.58 × 103 genome copies/Liter (GC/L). Partial nucleotide sequence confirmed the presence of HAdV type 41, norovirus genotype GII.4, and avian parvovirus 1. The results suggest that the first flush diversion devices may not adequately remove enteric virus from the rainwater. Additional treatment of RHRW is required to mitigate potential health risks from potable use of captured water.

Comparative decay of culturable faecal indicator bacteria, microbial source tracking marker genes, and enteric pathogens in laboratory microcosms that mimic a sub-tropical environment.

Enteric pathogens can be present in drinking water catchments due to several point and non-point sources of faecal contamination. Pathogen and contaminant signatures will decay due to environmental stresses, such as temperature, Ultra Violet (UV) radiation, salinity, and predation. In this study, we determined the decay of the culturable faecal indicator bacterium (FIB) Escherichia coli (E. coli), two sewage-associated marker genes (Bacteroides HF183 and crAssphage CPQ_056), and enteric pathogens (Campylobacter spp., human adenovirus 40/41, and Cryptosporidium parvum) in two freshwater laboratory microcosms using culture-based, quantitative PCR (qPCR) and vital dye (determine the fraction of viable Cryptosporidium oocysts) assays. Freshwater samples from the Lake Wappa and Lake Wivenhoe (Australia) were seeded with untreated sewage and C. parvum oocysts, and their declining concentrations were measured over a 28-day period. Moreover, 16S rRNA amplicon sequencing was also undertaken to determine the change/shift in sewage-associated bacterial communities using SourceTracker. Overall, culturable E. coli and the HF183 marker gene decayed significantly (p < 0.05) faster than did the qPCR measured enteric pathogens suggesting that the absence of culturable FIB or qPCR HF183 in water samples may not indicate the absence of pathogens. The decay of crAssphage was similar to that of HAdV 40/41 and other pathogens tested, suggesting crAssphage may be a better surrogate for enteric viruses in sub-tropical catchment waters. The decay rates were greater at 25 °C compared to 15 °C, suggesting that FIB and pathogens persist longer in the winter season compared to summer. Overall decay rates of the tested microorganisms in this microcosm study suggest that sub-tropical conditions, especially temperature, have a negative impact on the persistence of tested microorganisms. Sewage-associated bacterial communities also showed similar patterns. Based on the results, which showed differences in simulated summer and winter temperatures for pathogen decay, corresponding management options and treatment need to be adjusted accordingly to minimize human health risks effectively.

A review on microbial contaminants in stormwater runoff and outfalls: Potential health risks and mitigation strategies.

Demands on global water supplies are increasing in response to the need to provide more food, water, and energy for a rapidly growing population. These water stressors are exacerbated by climate change, as well as the growth and urbanisation of industry and commerce. Consequently, urban water authorities around the globe are exploring alternative water sources to meet ever-increasing demands. These alternative sources are primarily treated sewage, stormwater, and groundwater. Stormwater including roof-harvested rainwater has been considered as an alternative water source for both potable and non-potable uses. One of the most significant issues concerning alternative water reuse is the public health risk associated with chemical and microbial contaminants. Several studies to date have quantified fecal indicators and pathogens in stormwater. Microbial source tracking (MST) approaches have also been used to determine the sources of fecal contamination in stormwater and receiving waters. This review paper summarizes occurrence and concentrations of fecal indicators, pathogens, and MST marker genes in urban stormwater. A section of the review highlights the removal of fecal indicators and pathogens through water sensitive urban design (WSUD) or Best Management Practices (BMPs). We also discuss approaches for assessing and mitigating health risks associated with stormwater, including a summary of existing quantitative microbial risk assessment (QMRA) models for potable and non-potable reuse of stormwater. Finally, the most critical research gaps are identified for formulating risk management strategies.

Quantification of hookworm ova from wastewater matrices using quantitative PCR.

A quantitative PCR (qPCR) assay was used to quantify Ancylostoma caninum ova in wastewater and sludge samples. We estimated the average gene copy numbers for a single ovum using a mixed population of ova. The average gene copy numbers derived from the mixed population were used to estimate numbers of hookworm ova in A. caninum seeded and unseeded wastewater and sludge samples. The newly developed qPCR assay estimated an average of 3.7×103 gene copies per ovum, which was then validated by seeding known numbers of hookworm ova into treated wastewater. The qPCR estimated an average of (1.1±0.1), (8.6±2.9) and (67.3±10.4) ova for treated wastewater that was seeded with (1±0), (10±2) and (100±21) ova, respectively. The further application of the qPCR assay for the quantification of A. caninum ova was determined by seeding a known numbers of ova into the wastewater matrices. The qPCR results indicated that 50%, 90% and 67% of treated wastewater (1L), raw wastewater (1L) and sludge (~4g) samples had variable numbers of A. caninum gene copies. After conversion of the qPCR estimated gene copy numbers to ova for treated wastewater, raw wastewater, and sludge samples, had an average of 0.02, 1.24 and 67 ova, respectively. The result of this study indicated that qPCR can be used for the quantification of hookworm ova from wastewater and sludge samples; however, caution is advised in interpreting qPCR generated data for health risk assessment.

Optimization of sampling strategy to determine pathogen removal efficacy of activated sludge treatment plant.

Large-scale wastewater schemes rely on multi-barrier approach for the production of safe and sustainable recycled water. In multi-barrier wastewater reclamation systems, conventional activated sludge process (ASP) often constitutes a major initial treatment step. The main aim of this research was to determine most appropriate sampling approach to establish pathogen removal efficacy of ASP. The results suggest that ASP is capable of reducing human adenovirus (HAdV) and polyomavirus (HPyV) by up to 3 log10. The virus removal data suggests that HAdV removal is comparable to somatic bacteriophage belonging to Microviridae family. Due to the high removal of Escherichia coli (>3 log10) and very poor correlation with the enteric virus, it is not recommended that E. coli be used as a surrogate for enteric virus removal. The results also demonstrated no statistically significant differences (t test, P > 0.05) in calculated log removal values (LRVs) for HAdV, HPyV, and Microviridae from samples collected on hydraulic retention time (HRT) or simultaneous paired samples collected for influent and effluent. This indicates that a more practical approach of simultaneous sampling for influent and effluent could be used to determine pathogen removal efficiency of ASP. The results also suggest that a minimum of 10, preferably 20 samples, are required to fully capture variability in the removal of virus. In order to cover for the potential seasonal prevalence of viruses such as norovirus and rotavirus, sampling should be spread across all seasons.

Comparative prevalence of Escherichia coli carrying virulence genes and class 1 and 2 integrons in sub-tropical and cool temperate freshwater.

Aquatic environments are now recognized secondary habitat of potentially pathogenic Escherichia coli. In this study, PCR-based analyses were used to determine the phylogenetic composition and frequency of occurrence of eight clinically significant virulence genes (VGs) in E. coli isolates from sub-tropical Brisbane and cool temperate Tasmania freshwater in Australia. In Brisbane, non-commensal E. coli isolates belonging to the B2 and D phylogenetic group were dominant (72%). A significantly higher number (P < 0.05) of E. coli carrying VGs were detected in the sub-tropical freshwaters compared to the cool temperate water. Furthermore, diarrheagenic pathotype (EHEC) was also observed in the sub-tropical freshwater. The genes east1 and eaeA were significantly more common (P < 0.00001) than other VGs. The eaeA gene which codes for intimin protein along with toxin genes east1, stx 1 , stx 2 , and LT1 were mostly detected in phylogenetic groups B2 and D. The ANOVA results also suggested a statistically significant difference (P < 0.016) between the VGs carried by phylogenetic groups B2 and D. Class 1 integrase (intl1) and class 2 integrase (intl2) genes were detected in 38 (24.83%) and 23 (15.03%) of E. coli isolates, respectively. The Gretna site (Tasmania) with known fecal input from bovine and ovine sources had the highest number of E. coli carrying intl1 (29%) and intl2 (13%) genes. In addition, class 2 integron was more commonly detected in the phylogenetic group B2. The results of this study highlight the need to better understand sources and reasons for the high prevalence of E. coli carrying clinically significant VGs in a sub-tropical environment and its public health implications.

Human health risks for Legionella and Mycobacterium avium complex (MAC) from potable and non-potable uses of roof-harvested rainwater.

A quantitative microbial risk assessment (QMRA) of opportunistic pathogens Legionella pneumophila (LP) and Mycobacterium avium complex (MAC) was undertaken for various uses of roof-harvested rainwater (RHRW) reported in Queensland, Australia to identify appropriate usages and guide risk management practices. Risks from inhalation of aerosols due to showering, swimming in pools topped up with RHRW, use of a garden hose, car washing, and toilet flushing with RHRW were considered for LP while both ingestion (drinking, produce consumption, and accidental ingestion from various activities) and inhalation risks were considered for MAC. The drinking water route of exposure presented the greatest risks due to cervical lymphadenitis and disseminated infection health endpoints for children and immune-compromised populations, respectively. It is therefore not recommended that these populations consume untreated rainwater. LP risks were up to 6 orders of magnitude higher than MAC risks for the inhalation route of exposure for all scenarios. Both inhalation and ingestion QMRA simulations support that while drinking, showering, and garden hosing with RHRW may present the highest risks, car washing and clothes washing could constitute appropriate uses of RHRW for all populations, and toilet flushing and consumption of lettuce irrigation with RHRW would be appropriate for non- immune-compromised populations.

Seasonal Assessment of Opportunistic Premise Plumbing Pathogens in Roof-Harvested Rainwater Tanks.

A seasonal study on the occurrence of six opportunistic premise plumbing pathogens (OPPPs) in 24 roof-harvested rainwater (RHRW) tanks repeatedly sampled over six monthly sampling events (n = 144) from August 2015 to March 2016 was conducted using quantitative qPCR. Fecal indicator bacteria (FIB) Escherichia coli (E. coli) and Enterococcus spp. were enumerated using culture-based methods. All tank water samples over the six events were positive for at least one OPPP (Legionella spp., Legionella pneumophila, Mycobacterium avium, Mycobacterium intracellulare, Pseudmonas aeruginosa, or Acanthamoeba spp.) during the entire course of the study. FIB were positively but weakly correlated with P. aeruginosa (E. coli vs P. aeruginosa τ = 0.090, p = 0.027; Enterococcus spp. vs P. aeruginosa τ = 0.126, p = 0.002), but not the other OPPPs. FIBs were more prevalent during the wet season than the dry season, and L. pneumophila was only observed during the wet season. However, concentrations of Legionella spp., M. intracellulare, Acanthamoeba spp., and M. avium peaked during the dry season. Correlations were assessed between FIB and OPPPs with meteorological variables, and it was determined that P. aeruginosa was the only OPPP positively associated with an increased antecedent dry period, suggesting stagnation time may play a role for the occurrence of this OPPP in tank water. Infection risks may exceed commonly cited benchmarks for uses reported in the rainwater usage survey such as pool top-up, and warrant further exploration through quantitative microbial risk assessment (QMRA).

Amplicon-based profiling of bacteria in raw and secondary treated wastewater from treatment plants across Australia.

In this study, we investigated the use of Illumina high-throughput sequencing of 16S ribosomal RNA (rRNA) amplicons to explore microbial diversity and community structure in raw and secondary treated wastewater (WW) samples from four municipal wastewater treatment plants (WWTPs A-D) across Australia. Sequence reads were analyzed to determine the abundance and diversity of bacterial communities in raw and secondary treated WW samples across the four WWTPs. In addition, sequence reads were also characterized to phenotypic features and to estimate the abundance of potential pathogenic bacterial genera and antibiotic-resistant genes in total bacterial communities. The mean coverage, Shannon diversity index, observed richness (S obs), and abundance-based coverage estimate (ACE) of richness for raw and secondary treated WW samples did not differ significantly (P > 0.05) among the four WWTPs examined. Generally, raw and secondary treated WW samples were dominated by members of the genera Pseudomonas, Arcobacter, and Bacteroides. Evaluation of source contributions to secondary treated WW, done using SourceTracker, revealed that 8.80-61.4% of the bacterial communities in secondary treated WW samples were attributed to raw WW. Twenty-five bacterial genera were classified as containing potential bacterial pathogens. The abundance of potentially pathogenic genera in raw WW samples was higher than that found in secondary treated WW samples. Among the pathogenic genera identified, Pseudomonas and Arcobacter had the greatest percentage of the sequence reads. The abundances of antibiotic resistance genes were generally low (<0.5%), except for genes encoding ABC transporters, which accounted for approximately 3% of inferred genes. These findings provided a comprehensive profile of bacterial communities, including potential bacterial pathogens and antibiotic-resistant genes, in raw and secondary treated WW samples from four WWTPs across Australia and demonstrated that Illumina high-throughput sequencing can be an alternative approach for monitoring WW quality in order to protect environmental and human health.

Attachment and Detachment Behavior of Human Adenovirus and Surrogates in Fine Granular Limestone Aquifer Material.

The transport of human adenovirus, nanoparticles, and PRD1 and MS2 bacteriophages was tested in fine granular limestone aquifer material taken from a borehole at a managed aquifer recharge site in Adelaide, South Australia. Comparison of transport and removal of virus surrogates with the pathogenic virus is necessary to understand the differences between the virus and surrogate. Because experiments using pathogenic viruses cannot be done in the field, laboratory tests using flow-through soil columns were used. Results show that PRD1 is the most appropriate surrogate for adenovirus in an aquifer dominated by calcite material but not under high ionic strength or high pH conditions. It was also found that straining due to size and the charge of the colloid were not dominant removal mechanisms in this system. Implications of this study indicate that a certain surrogate may not represent a specific pathogen solely based on similar size, morphology, and/or surface charge. Moreover, if a particular surrogate is representative of a pathogen in one aquifer system, it may not be the most appropriate surrogate in another porous media system. This was apparent in the inferior performance of MS2 as a surrogate, which is commonly used in virus transport studies.

Assessment of Genetic Markers for Tracking the Sources of Human Wastewater Associated Escherichia coli in Environmental Waters.

In this study, we have evaluated the performance characteristics (host-specificity and -sensitivity) of four human wastewater-associated Escherichia coli (E. coli) genetic markers (H8, H12, H14, and H24) in 10 target (human) and nontarget (cat, cattle, deer, dog, emu, goat, horse, kangaroo, and possum) host groups in Southeast Queensland, Australia. The overall host-sensitivity values of the tested markers in human wastewater samples were 1.0 (all human wastewater samples contained the E. coli genetic markers). The overall host-specificity values of these markers to differentiate between human and animal host groups were 0.94, 0.85, 0.72, and 0.57 for H8, H12, H24, and H14, respectively. Based on the higher host-specificity values, H8 and H12 markers were chosen for a validation environmental study. The prevalence of the H8 and H12 markers was determined among human wastewater E. coli isolates collected from a wastewater treatment plant (WWTP). Among the 97 isolates tested, 44 (45%) and 14 (14%) were positive for the H8 and H12 markers, respectively. A total of 307 E. coli isolates were tested from environmental water samples collected in Brisbane, of which 7% and 20% were also positive for the H8 and H12 markers, respectively. Based on our results, we recommend that these markers could be useful when it is important to identify the source(s) of E. coli (whether they originated from human wastewater or not) in environmental waters.

Evaluating two infiltration gallery designs for managed aquifer recharge using secondary treated wastewater.

As managed aquifer recharge (MAR) becomes increasingly considered for augmenting water-sensitive urban areas, fundamental knowledge of the achievable scale, longevity and maintenance requirements of different options will become paramount. This paper reports on a 39 month pilot scale MAR scheme that infiltrated secondary treated wastewater through unsaturated sand into a limestone and sand aquifer. Two types of infiltration gallery were constructed to compare their hydraulic performance, one using crushed, graded gravel, the other using an engineered leach drain system (Atlantis Leach System(®)). Both galleries received 25 kL of nutrient-rich, secondary treated wastewater per day. The Atlantis gallery successfully infiltrated 17 ML of treated wastewater over three years. The slotted distribution pipe in the gravel gallery became clogged with plant roots after operating for one year. The infiltration capacity of the gravel gallery could not be restored despite high pressure cleaning, thus it was replaced with an Atlantis system. Reduction in the infiltration capacity of the Atlantis system was only observed when inflow was increased by about 3 fold for two months. The performance of the Atlantis system suggests it is superior to the gravel gallery, requiring less maintenance within at least the time frame of this study. The results from a bromide tracer test revealed a minimum transport time of 3.7 days for the recharged water to reach the water table below 9 m of sand and limestone. This set a limit on the time available for attenuation by natural treatment within the unsaturated zone before it recharged groundwater.

Urban stormwater harvesting and reuse: a probe into the chemical, toxicology and microbiological contaminants in water quality.

Stormwater is one of the last major untapped urban water resources that can be exploited as an alternative water source in Australia. The information in the current Australian Guidelines for Water Recycling relating to stormwater harvesting and reuse only emphasises on a limited number of stormwater quality parameters. In order to supply stormwater as a source for higher value end-uses, a more comprehensive assessment on the potential public health risks has to be undertaken. Owing to the stochastic variations in rainfall, catchment hydrology and also the types of non-point pollution sources that can provide contaminants relating to different anthropogenic activities and catchment land uses, the characterisation of public health risks in stormwater is complex, tedious and not always possible through the conventional detection and analytical methods. In this study, a holistic approach was undertaken to assess the potential public health risks in urban stormwater samples from a medium-density residential catchment. A combined chemical-toxicological assessment was used to characterise the potential health risks arising from chemical contaminants, while a combination of standard culture methods and quantitative polymerase chain reaction (qPCR) methods was used for detection and quantification of faecal indicator bacteria (FIB) and pathogens in urban stormwater. Results showed that the concentration of chemical contaminants and associated toxicity were relatively low when benchmarked against other alternative water sources such as recycled wastewater. However, the concentrations of heavy metals particularly cadmium and lead have exceeded the Australian guideline values, indicating potential public health risks. Also, high numbers of FIB were detected in urban stormwater samples obtained from wet weather events. In addition, qPCR detection of human-related pathogens suggested there are frequent sewage ingressions into the urban stormwater runoff during wet weather events. Further water quality monitoring study will be conducted at different contrasting urban catchments in order to undertake a more comprehensive public health risk assessment for urban stormwater.

Occurrence of virulence genes associated with Diarrheagenic pathotypes in Escherichia coli isolates from surface water.

Escherichia coli isolates (n = 300) collected from six sites in subtropical Brisbane, Australia, prior to and after storm events were tested for the presence of 11 virulence genes (VGs) specific to diarrheagenic pathotypes. The presence of eaeA, stx(1), stx(2), and ehxA genes specific for the enterohemorrhagic E. coli (EHEC) pathotype was detected in 56%, 6%, 10%, and 13% of isolates, respectively. The VGs astA (69%) and aggR (29%), carried by enteroaggregative (EAEC) pathotypes, were frequently detected in E. coli isolates. The enteropathogenic E. coli (EPEC) gene bfp was detected in 24% of isolates. In addition, enteroinvasive E. coli (EIEC) VG ipaH was also detected in 14% of isolates. During dry periods, isolates belonging to the EAEC pathotype were most commonly detected (23%), followed by EHEC (11%) and EPEC (11%). Conversely, a more uniform prevalence of pathotypes, EPEC (14%), EAEC (12%), EIEC (10%), EHEC (7%), and ETEC (7%), was observed after the storm events. The results of this study highlight the widespread occurrence of potentially diarrheagenic pathotypes in the urban aquatic ecosystems. While the presence of VGs in E. coli isolates alone is insufficient to determine pathogenicity, the presence of diarrheagenic E. coli pathotypes in high frequency after the storm events could lead to increased health risks if untreated storm water were to be used for nonpotable purposes and recreational activities.

Sensitive detection of human adenovirus from small volume of primary wastewater samples by quantitative PCR.

An accurate quantitative detection of enteric viruses from the primary wastewater requires, sample concentration followed by extraction of nucleic acid with high purity. A highly efficient and sensitive method was developed for the concentration and quantitative detection of human adenovirus (HAdv) from wastewater samples. The two-step method which combines concentration of virus from 10 mL sample with centrifugal filters followed by extraction and purification of DNA with commercially available nucleic acid extraction kit resulted in high purity DNA for downstream quantitative PCR (qPCR). The results obtained on analytical sensitivities of five commercial nucleic acid extraction kits show that they differ in their ability for DNA yield and purity. Nevertheless, despite variable analytical sensitivities extracted nucleic acid was found to be relatively PCR inhibition free. The genomic copy numbers of HAdv detected from the same concentrated wastewater sample were significantly higher (P<0.01) when Qiagen Blood and Tissue kit (1.54×10(6) L(-1)) was used as compared to Mo-Bio PowerSoil kit (5.30×10(5) L(-1)) which suggests that the nucleic acid extraction kit can influence the sensitivity of qPCR assays. The method developed in this study is simple, rapid, sensitive, and can be applicable for the qPCR detection of adenovirus and other DNA virus in wastewater.

Water quality assessment using the AREc32 reporter gene assay indicative of the oxidative stress response pathway.

The reporter gene assay AREc32 is based on the induction of the Nrf2 mediated oxidative stress response pathway in the human breast cancer cell line MCF7, where eight copies of the antioxidant response element (ARE) are linked to a reporter gene encoding for luciferase. The Nrf2-ARE pathway is responsive to many chemicals that cause oxidative stress, among them a large number of pesticides and skin irritants. We adopted and validated the AREc32 bioassay for water quality testing. tert-Butylhydroquinone served as the positive control, phenol as the negative control and other reactive chemicals were assessed for their specificity. An environmentally relevant reference chemical, benzo(a)pyrene was the most potent inducer of all tested chemicals. The concentration causing an induction ratio (IR) of 1.5 (EC(IR1.5)) was chosen as the effect benchmark value. The assay was applied to 21 water samples ranging from sewage to drinking water, including secondary treatment and various tertiary treatment options (ozonation, biologically activated carbon filtration, membrane filtration, reverse osmosis, advanced oxidation, chlorination, chloramination). The samples were enriched by solid phase extraction. In most samples the oxidative stress response was far more sensitive than cytotoxicity. The primary and secondary treated effluent exceeded the effect threshold IR 1.5 at a relative enrichment factor (REF) of 1, i.e., the native samples were active. All tertiary treated samples were less potent and their EC(IR1.5) lay between REF 1 and 10. The Nrf2 pathway was induced at a REF of approximately 10 for surface waters and drinking water, and above this enrichment cytotoxicity took over in most samples and quenched the induction. The blank (ultrapure water run through the sample enrichment process) was cytotoxic at an REF of 100, which is the limit of concentrations range that can be evaluated. Treatment typically decreased both the cytotoxicity and oxidative stress response apart from drinking water treatment where chlorination caused an increase in oxidative stress response, presumably due to the formation of disinfection by-products. This study demonstrates the relevance and applicability of the oxidative stress response pathway for water quality monitoring.

Managed aquifer recharge of treated wastewater: water quality changes resulting from infiltration through the vadose zone.

Secondary treated wastewater was infiltrated through a 9 m-thick calcareous vadose zone during a 39 month managed aquifer recharge (MAR) field trial to determine potential improvements in the recycled water quality. The water quality improvements of the recycled water were based on changes in the chemistry and microbiology of (i) the recycled water prior to infiltration relative to (ii) groundwater immediately down-gradient from the infiltration gallery. Changes in the average concentrations of several constituents in the recycled water were identified with reductions of 30% for phosphorous, 66% for fluoride, 62% for iron and 51% for total organic carbon when the secondary treated wastewater was infiltrated at an applied rate of 17.5 L per minute with a residence time of approximately four days in the vadose zone and less than two days in the aquifer. Reductions were also noted for oxazepam and temazepam among the pharmaceuticals tested and for a range of microbial pathogens, but reductions were harder to quantify as their magnitudes varied over time. Total nitrogen and carbamazepine persisted in groundwater down-gradient from the infiltration galleries. Infiltration does potentially offer a range of water quality improvements over direct injection to the water table without passage through the unsaturated zone; however, additional treatment options for the non-potable water may still need to be considered, depending on the receiving environment or the end use of the recovered water.