Screening-level quantitative microbial risk assessments (QMRA) undertaken to assess health risks associated with the reticulation of recycled water have identified distribution pipe biofilms and their ability to accumulate enteric and opportunistic pathogens as potential sources of public health concern. A simplistic model to assess human health risks associated with enteric virions (extracellular viruses) present in recycled water for domestic use was therefore developed. Recycled water biofilms formed on glass and stainless steel coupons in a laboratory-scale distribution system were challenged with model enteric virions (B40-8, MS-2 and FX174 bacteriophages). Approximately 1% of bacteriophages present in the adjacent bulk water was incorporated into 3 month-old biofilms and a persistent sub-population (0.01%) of the model enteric virions remained infectious in biofilms throughout an experimental period of 30 days. Significant potential for virions to accumulate within biofilms was therefore demonstrated, and indeed biofilm uptake could mask treatment failures assessed through analysis of the bulk water (infectious virions would not be detected after 1.5 km even if 10.L-1 virions present in treated effluent). Subsequent sloughing of biofilm into the bulk water phase could therefore release infective virions within mobilized aggregates of biofilm and infect susceptible consumers. During normal operating conditions (one virion in 100 L of water) sufficient virions may accumulate within distribution pipe biofilms that a 50% sloughing event could present a Pi (annual) of 1.9 × 10-4 for the consumption of 1 mL of water, exceeding the US-EPA benchmark of 10-4.
A pilot-scale plant was employed to validate the performance of a proposed full-scale advanced water treatment plant (AWTP) in Sydney, Australia. The primary aim of this study was to develop a chemical monitoring program that can demonstrate proper plant operation resulting in the removal of priority chemical constituents in the product water. The feed water quality to the pilot plant was tertiary-treated effluent from a wastewater treatment plant. The unit processes of the AWTP were comprised of an integrated membrane system (ultrafiltration, reverse osmosis) followed by final chlorination generating a water quality that does not present a source of human or environmental health concern. The chemical monitoring program was undertaken over 6 weeks during pilot plant operation and involved the quantitative analysis of pharmaceuticals and personal care products, steroidal hormones, industrial chemicals, pesticides, N-nitrosamines and halomethanes. The first phase consisted of baseline monitoring of target compounds to quantify influent concentrations in feed waters to the plant. This was followed by a period of validation monitoring utilising indicator chemicals and surrogate measures suitable to assess proper process performance at various stages of the AWTP. This effort was supported by challenge testing experiments to further validate removal of a series of indicator chemicals by reverse osmosis. This pilot-scale study demonstrated a simplified analytical approach that can be employed to assure proper operation of advanced water treatment processes and the absence of trace organic chemicals.
The persistence of two model enteric virions (Bacteroides fragilis phage B40-8 and coliphage MS-2) within pipe biofilms was investigated in situ in an urban distribution system. Biofilms were allowed to develop on uPVC and stainless steel (SS) coupons in a modified Robbins' device for 70 d within a 150 mm uPVC reticulation main. Coupons were then placed in annular reactors and slug dosed with B40-8 and MS-2 phages (108 pfu/mL). Pipe water velocity, pH and free chlorine were recorded during the experimental period. Biofilms on uPVC were generally more abundant (based on total bacterial counts, HPCs, total protein and total carbohydrate). Both B40-8 and MS-2 were incorporated into biofilms formed on uPVC and SS coupons (>104 and >103 pfu/μg protein respectively) and persisted for >30 d and 6 d respectively, reflecting biofilm biomass on the two pipe surfaces. Virion loss/inactivation from biofilm followed an initial rapid phase, followed by a very slow phase representing approximately 0.01% of the original virion population. Virions, therefore, have the potential to accumulate within distribution biofilm and problems could arise when clusters of biofilm-associated enteric virions become detached from the substrata by hydrodynamic forces or sudden changes in disinfection regime.
The occurrence of Aeromonas spp. within biofilms formed on stainless steel (SS), unplasticized polyvinyl chloride (uPVC) and glass (GL) substrata was investigated in modified Robbins Devices (MRD) in potable (MRD-p) and recycled (MRD-r) water systems, a Biofilm Reactor™ (BR) and a laboratory-scale pipe loop (PL) receiving simulated recycled wastewater. No aeromonads were isolated from the MRD-p whereas 3–10% of SS and uPVC coupons (mean 3.85 CFU cm−2 and 12.8 CFU cm−2, respectively) were aeromonad-positive in the MRD-r. Aeromonads were isolated from six SS coupons (67%) (mean 63.4 CFU cm−2) and nine uPVC coupons (100%) (mean 6.50×102 CFU cm−2) in the BR™ fed with recycled water and from all coupons (100%) in the simulated recycled water system (PL). Mean numbers of aeromonads on GL and SS coupons were 5.83×102 CFU cm−2 and 8.73×102 CFU cm−2, respectively. No isolate was of known human health significance (i.e. Aeromonas caviae, A. hydrophila or A. veronii), though they were confirmed as Aeromonas spp. by PCR and fluorescence in situ hybridization (FISH). Challenging the PL biofilms with a slug dose of A. hydrophila (ATCC 14715) showed that biofilm in the PL accumulated in the order of 103–104A. hydrophila cm−2, the number of which decreased over time, though could not be explained in terms of conventional 1st order decay kinetics. A sub-population of FISH-positive A. hydrophila became established within the biofilm, thereby demonstrating their ability to incorporate and persist in biofilms formed within distribution pipe systems. A similar observation was not made for culturable aeromonads, though the exact human health significance of this remains unknown. These findings, however, further question the adequacy of culture-based techniques and their often anomalous discrepancy with direct techniques for the enumeration of bacterial pathogens in environmental samples.
A simplistic quantitative microbial risk assessment (QMRA) based on the maximum risk curve (r = 1) was developed for Legionella within a water distribution system. Both biofilms and a thermophilic isolate of acanthamoebae were shown to increase the resistance of Legionella to conventional thermal disinfection by between one and two logs respectively. The level of risk presented to consumers was shown to exceed the USEPA 10(-4) benchmark in many cases tested. This was caused, in part, by the sensitivity of the risk model but also through a lack of reliable dose-response data for Legionella. Not withstanding this, the current study provided comparative information on the efficacy of conventional disinfection against Legionella. Combined chlorine was shown to reduce the risk of infection by as much as 1-log when compared to free chlorine, although thermal disinfection provided the most effective means of risk reduction. Biofilm detachment and the interaction of Legionella with acanthamoebae were two important ecological factors that significantly increased the risk of legionellosis, and thus should be further considered in the refinement of QMRA models.
Through their many sorption sites, microbial biofilms can accumulate both organic and inorganic particulate and colloidal material from bulk water environments. An application of such first principles to the ecology of “biocolloidal” enteric virions would suggest that they too may be concentrated by biofilms in a similar way. Though previous studies have isolated human gastrointestinal (enteric) virions from microbial biofilms, the exact human health significance of this has been neither fully investigated nor completely understood. Through an assessment of the location, accumulation and persistence of model enteric virions (φX174, MS2 and B40-8 bacteriophages as well as 20 nm fluorescent latex microspheres) within biofilms, the aim of the current study was to investigate whether the interaction of enteric virions with distribution pipe biofilms could provide a secondary source of public health concern to consumers. Model enteric virions were found to be incorporated into biofilms at concentrations representing 1% of those present in the adjacent bulk water environment. A sub-population (0.01%) of these persisted throughout an experimental period of 30 days, inferring their potential to accumulate over time. Furthermore, model enteric virions were partitioned into bacterial microcolonies, environments where biofilm bacteria can persist and re-grow, even in the presence of “acceptable” levels of disinfection. A risk model for enteric virion accumulation and release from distribution pipe biofilms suggested that associated risks may exceed USEPA benchmark values. These findings could have wide-reaching implications in water treatment and distribution strategies, and necessitate a re-appraisal of current water guideline values.
Reliance on coliform monitoring of ground waters is slowly changing as is reflected in the proposed US EPA Ground Water Rule. In line with this we have investigated the use of an expanded range of faecal indicators and potential surrogate analytes within the Gwelup and Jandakot borefields in Perth, Western Australia. The aims of the study included comparing contamination in bores and surface waters in vulnerable locations, quantifying aquifer removal of microorganisms, trialing novel biochemical pollution indicators such as faecal sterols, assessing Escherichia coli as a measure of groundwater contamination and generating data for risk assessments. Sampling was undertaken of nine production bores, nine monitoring bores and four surface waters for 32 parameters comprising seven microbial indicators, 12 physico-chemical parameters and 13 biomarkers (including 8 faecal sterols and caffeine) at sampling stations potentially impacted by urban development. Concentrations of microbial indicators and biomarkers followed the pattern: basins >> monitoring bores >> production bores. Only one production bore sample contained bacterial indicators (0.1 enterococci.100 mL-1 on 1 occasion). Of the faecal biomarkers, coprostanol was generally at background levels. Cholesterol appeared to be a more sensitive measure of infiltration, but was also effectively removed. E. coli appeared to be a less sensitive indicator than enterococci. None of the physico-chemical parameters were useful surrogates. Overall apparent faecal microbial removal by aquifer filtration averaged >4-5 logs (not accounting for viruses). To maximise warning time and assay sensitivity it is suggested that enterococci be considered as the key bacterial indicator rather than E. coli and that different combinations of indicators and biomarkers be used to identify aquifer locations at risk, the presence of significant faecal material, and the likely presence of pathogens.
Distribution pipe biofilms can provide sites for the concentration of a wide range of microbial pathogens, thereby acting as a potential source of continual microbial exposure and furthermore can affect the aesthetic quality of water. In a joint project between Stockholm Water, the MISTRA "Sustainable Urban Water" program, the Swedish Institute for Infectious Disease Control and the Royal Technical University, Stockholm, the aim of the current study was to investigate biofilms formed in an urban water distribution system, and quantify the impact of such biofilms on potential pathogen accumulation and persistence within the Greater Stockholm Area, Sweden. When used for primary disinfection, ultra-violet (UV) treatment had no measurable influence on biofilm formation within the distribution system when compared to conventional chlorination. Biofilms produced within a model pilot-plant were found to be representative to those that had formed within the larger municipal water distribution system, demonstrating the applicability of the novel pilot-plant for future studies. Polystyrene microspheres (1.0 microm) and Salmonella bacteriophages demonstrated their ability to accumulate and persist within the model pilot-plant system, where the means of primary disinfection (UV-treatment, chlorination) had no influence on such phenomena. With the exception of aeromonads, potential pathogens and faecal indicators could not be detected within biofilms from the Stockholm water distribution system. Results from this investigation may provide information for water treatment and distribution management strategies, and fill key data gaps that presently hinder the refinement of microbial risk models.
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