Evaluating potential applications of faecal sterols in distinguishing sources of faecal contamination from mixed faecal samples

Faecal samples from humans, herbivores, carnivores and birds as well as samples from septic tanks and effluents from a sewage treatment plant (STP) were extracted and analyzed by gas chromatography-mass spectrometry for faecal sterols including coprostanol, epicoprostanol, cholestanol, cholesterol, stigmasterol, campesterol, 24-ethylcoprostanol and beta-sitosterol. Coprostanol was observed in the highest concentrations from the human derived samples, but it was also present in substantial quantities in a range of herbivores. There was no unique marker of human faecal contamination. Multivariate analyses revealed that the faecal sterol profiles were significantly different between the four groups of animals (Wilks' lambda=0.007, P<0.002), and coprostanol and 24-ethylcoprostanol were the major discriminant factors. However, when faecal samples were mixed, the confounding of faecal sterol levels prevented accurate identification of contributing species. Conversely, faecal sterol ratios were highly efficient at identifying which mixtures contained human contribution, but could not appropriately determine percentage contributions of sources.     

Comparisons of water quality parameters from diverse catchments during dry periods and following rain events

In this study, 12 catchments sites located along the north coast of New South Wales in Australia were grouped into the four categories of septic, cattle, sewage treatment plant (STP) and forested sites via cluster analysis based on their land use patterns. Water samples from all these sites were collected between October 2004 and June 2006 at a regular monthly interval and within 48 h of rain events. The samples were analyzed for bacterial counts including faecal coliform and total coliform; faecal sterols including coprostanol, epicoprostanol, cholesterol, cholestanol, 24-ethylcoprostanol, campesterol, stigmasterol and beta-sitosterol; and the elements including Na, Rb, Sr, Ag, Cd, Sn, Cs, Ba, Hg, Tl, Pb, Bi, U, Mg, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, K, As, Se, P and Mo. Over the course of the sampling period, the STP site had the highest average coprostanol level of 1693+/-567 ng/L which was significantly higher (p<0.05) than the septic sites (190+/-71 ng/L), the cattle sites (163+/-94 ng/L) and forested sites (14+/-4 ng/L). As expected, the forested sites had significantly lower average level of faecal coliforms (373+/-87 cfu/100 mL) compared with the STP (1395+/-574 cfu/100 mL), septic (1243+/-494 cfu/100 mL) and cattle sites (535+/-112 cfu/100 mL). The concentrations of coprostanol were not correlated with the numbers of faecal coliform bacteria when the entire data set was evaluated. The forested sites generally had the lowest average levels of elemental compositions, with significantly lower levels noted for Na, U, Mg, V, Cu, Sr, K, As, P and Mo, whereas Fe was the only element notably higher in the forested sites. Temporal and rain events analyses of the data set revealed that elevated levels of both coprostanol and faecal coliforms were not exclusive to rain events. The average coprostanol levels in rain event samples at each site were not significantly different compared with the corresponding dry event samples. Conversely, faecal coliform numbers increased by 2-4 times in rain events samples from septic, cattle and forested sites, but did not alter in the STP site. Multivariate analyses identified coprostanol and Sr as major contributing factors for the discrimination of septic, cattle, STP and forested sites for both rain and dry events samples. It was clear that each land use type of catchment could be characterized by biochemical, bacteriological and elemental parameters.     

Chemometric approach to validating faecal sterols as source tracer for faecal contamination in water

Faecal sterols detection is a promising method for identifying sources of faecal pollution. In this study, faecal contamination in water samples from point source (sewage treatment plants, chicken farms, quail farms and horse stables) was extracted using the solid phase extraction (SPE) technique. Faecal sterols (coprostanol, cholesterol, stigmasterol, β-sitosterol and stigmastanol) were selected as parameters to differentiate the source of faecal pollution. The results indicated that coprostanol, cholesterol and β-sitosterol were the most significant parameters that can be used as source tracers for faecal contamination. Chemometric techniques, such as cluster analysis, principal component analysis and discriminant analysis were applied to the data set on faecal contamination in water from various pollution sources in order to validate the faecal sterols’ profiles. Cluster analysis generated three clusters: coprostanol was in cluster 1, cholesterol and β-sitosterol formed cluster 2, while cluster 3 contained stigmasterol and stigmastanol. Discriminant analysis suggested that coprostanol, cholesterol and β-sitosterol were the most significant parameters to discriminate between the faecal pollution source. The use of chemometric techniques provides useful and promising indicators in tracing the source of faecal contamination.     

Development of microbial and chemical MST tools to identify the origin of the faecal pollution in bathing and shellfish harvesting waters in France

The microbiological quality of coastal or river waters can be affected by faecal pollution from human or animal sources. An efficient MST (Microbial Source Tracking) toolbox consisting of several host-specific markers would therefore be valuable for identifying the origin of the faecal pollution in the environment and thus for effective resource management and remediation. In this multidisciplinary study, after having tested some MST markers on faecal samples, we compared a selection of 17 parameters corresponding to chemical (steroid ratios, caffeine, and synthetic compounds), bacterial (host-specific Bacteroidales, Lactobacillus amylovorus and Bifidobacterium adolescentis) and viral (genotypes I-IV of F-specific bacteriophages, FRNAPH) markers on environmental water samples (n = 33; wastewater, runoff and river waters) with variable Escherichia coli concentrations. Eleven microbial and chemical parameters were finally chosen for our MST toolbox, based on their specificity for particular pollution sources represented by our samples and their detection in river waters impacted by human or animal pollution; these were: the human-specific chemical compounds caffeine, TCEP (tri(2-chloroethyl)phosphate) and benzophenone; the ratios of sitostanol/coprostanol and coprostanol/(coprostanol+24-ethylcopstanol); real-time PCR (Polymerase Chain Reaction) human-specific (HF183 and B. adolescentis), pig-specific (Pig-2-Bac and L. amylovorus) and ruminant-specific (Rum-2-Bac) markers; and human FRNAPH genogroup II.     

Concentrations of coprostanol that correspond to existing bacterial indicator guideline limits

Coprostanol is a faecal sterol that has been proposed as an alternative measure of faecal pollution. While the technique has been used successfully to trace sewage-derived organic matter in a range of environments, it has not been embraced for use as a water quality indicator. This is mostly because of a lack of epidemiological evidence relating coprostanol abundance to any health risk. However, there is a valuable reason why the concentration of coprostanol should be related as quantitatively as possible to the abundance of bacterial indicators currently used to measure faecal pollution. The measurement of coprostanol (and concurrently other faecal sterols) offers many diagnostic and quantitative advantages over traditional techniques for detecting human sewage pollution versus faecal contamination from animal sources. Knowing the amount of coprostanol expected given a certain amount of human sewage pollution would provide a measure against which water managers could quantitatively assess faecal pollution as a whole and relate that assessment to variables with which they are more familiar. This study determines the relationships between coprostanol concentrations and indicator bacterial counts and synthesises the results from several environments to propose coprostanol concentrations broadly equivalent to existing bacterial standards. Our data suggest that 60 and 400 ng L⁻¹ of coprostanol correspond to currently defined primary and secondary contact limits for bacteria measured as thermotolerant coliforms (commonly referred to as faecal coliforms) or enterococci.     

Sedimentary coprostanol and hydrocarbon distribution adjacent to a sewage outfall

The concentrations of coprostanol and hydrocarbons were measured in the effluent from the Chesapeake-Elizabeth sewage treatment plant (STP) and surface sediments from the area surrounding the effluent discharge site. Most of the coprostanol (> 84%) and hydrocarbons (> 91%) were associated with particulates in the effluent. The area was found to be dynamic where changes in the percentage and distribution of fine grained sediments occur over periods of months and the movement of these sediments is an important determinant of the distribution of sewage derived contaminants. The Chesapeake-Elizabeth STP was found to be responsible for only 7% of the hydrocarbon contamination of the sediments in the study area. The sediment hydrocarbon distribution also indicates that the Bay Bridge tunnel may be a unique source of hydrocarbons to the lower Chesapeake Bay. This study shows the usefulness of coprostanol in providing a better understanding of the fate and importance of sewage derived contaminants in areas around sewage outfalls.     

Persistence of microbial and chemical pig manure markers as compared to faecal indicator bacteria survival in freshwater and seawater microcosms

Natural seawater and freshwater microcosms inoculated with pig manure were set up to determine the persistence of pig faecal microbial and chemical markers in these two types of surface water. The concentrations of Lactobacillus amylovorus, the Bacteroidales Pig-2-Bac 16S rRNA genetic marker, five stanols and the evolution of two ratios of stanols, R₁ (coprostanol to the sum of coprostanol and 24-ethylcoprostanol) and R₂ (sitostanol to coprostanol) were analyzed during two months along with the concentration of Faecal Indicator Bacteria (FIB). Pig manure was inoculated to unfiltered water microcosms incubated aerobically at 18 °C in the dark. The faecal contamination load represented by the concentrations of culturable Escherichia coli and/or enterococci remained for two months in the freshwater and seawater microcosms water column. These concentrations followed a biphasic decay pattern with a 97% reduction of the initial amount during a first rapid phase (<6 days) and a remaining proportion undergoing a slower or null second decline. The L. amylovorus marker and five stanols persisted as long as the indicators in both treatments. The Pig-2-Bac marker persisted 20 and 27 days in seawater and freshwater, respectively. The ratios R₁ and R₂ were in the range specific to pig manure until day 6 in both types of water. These results indicate that Pig-2-Bac, L. amylovorus and stanol ratios might be used in combination to complement FIB testing to determine the pig source of fecal pollution. However, stanol ratios are to be used when the time point of the discharge is known.     

Occurrence and densities of fungi from northern Greek coastal bathing waters and their relation with faecal pollution indicators

The objective of this study was to determine the frequency and densities of yeasts and filamentous fungi in coastal water samples as well as their correlation with the indicator bacteria of faecal pollution. The prevalence of fungi was investigated in parallel with the standard pollution indicator microorganisms in 197 marine water samples from six northern Greek prefectures during the bathing season May–October 1999. Filamentous fungi were isolated from all the examined samples and yeasts from 29 (14.7%) of them; among the positive samples, their mean counts were 90.9 and 38.4 cfu/100 ml, respectively. A total of 23 genera of filamentous fungi and four genera of yeasts were identified. Prevailing genera of filamentous fungi were Penicillium, Aspergillus and Alternaria spp., whereas Candida spp. was the most frequently isolated yeast. Counts of yeasts were significantly (p<0.01) correlated with those of total and faecal coliforms, whereas no correlation was found between filamentous fungi and the indicator bacteria of faecal pollution. Significantly higher counts of total and faecal coliforms (p<0.05), and enterococci (p<0.001), were found during the months with the higher water temperatures and bather numbers. In the six prefectures, significant differences were observed in the counts of filamentous fungi and yeasts as well as in the counts of all the faecal pollution indicators. The results of this study indicate that coastal water can be a path for contamination of swimmers with yeasts and filamentous fungi and that the pollution indicator microorganisms cannot always predict their presence.     

The application of a recently isolated strain of Bacteroides (GB-124) to identify human sources of faecal pollution in a temperate river catchment

Recent work has suggested that bacteriophages infecting Bacteroides are a potential tool for faecal source tracking, but that different host strains may be needed for different geographic areas. This study used a recently identified strain of Bacteroides (GB-124) to detect human sources of faecal pollution in a river catchment in southeast England (UK). A total of 306 river water, municipal wastewater and animal samples were obtained over a 16-month period. Bacteriophages capable of infecting GB-124 were present in all municipal wastewaters but were not detected in faecal samples from animals, and were detected at significantly lower levels (P< 0.001) in river waters directly downstream of a dairy farm. This last observation was despite the presence of high levels of faecal indicator bacteria at this site. The study suggests that GB-124 appears to be specific to human faeces. As such it may represent an effective and low-cost method of faecal source identification.     

Integrated analysis of water quality parameters for cost-effective faecal pollution management in river catchments

In many parts of the world, microbial contamination of surface waters used for drinking, recreation, and shellfishery remains a pervasive risk to human health, especially in Less Economically Developed Countries (LEDC). However, the capacity to provide effective management strategies to break the waterborne route to human infection is often thwarted by our inability to identify the source of microbial contamination. Microbial Source Tracking (MST) has potential to improve water quality management in complex river catchments that are either routinely, or intermittently contaminated by faecal material from one or more sources, by attributing faecal loads to their human or non-human sources, and thereby supporting more rational approaches to microbial risk assessment. The River Ouse catchment in southeast England (U.K.) was used as a model with which to investigate the integration and application of a novel and simple MST approach to monitor microbial water quality over one calendar year, thereby encompassing a range of meteorological conditions. A key objective of the work was to develop simple low-cost protocols that could be easily replicated. Bacteriophages (viruses) capable of infecting a human specific strain of Bacteroides GB-124, and their correlation with presumptive Escherichia coli, were used to distinguish sources of faecal pollution. The results reported here suggest that in this river catchment the principal source of faecal pollution in most instances was non-human in origin. During storm events, presumptive E. coli and presumptive intestinal enterococci levels were 1.1-1.2 logs higher than during dry weather conditions, and levels of the faecal indicator organisms (FIOs) were closely associated with increased turbidity levels (presumptive E. coli and turbidity, r = 0.43). Spatio-temporal variation in microbial water quality parameters was accounted for by three principal components (67.6%). Cluster Analysis, reduced the fourteen monitoring sites to six representative ‘sentinel’ sites. The correlation coefficient between presumptive E. coli and phages of Bacteroides GB-124 was very small (r = 0.05) whilst that between turbidity and suspended solids was high (r = 0.62). Variations in climate, animal and anthropogenic interferences were all, either directly or indirectly, related to faecal contamination. The findings show the importance of meteorological conditions, such as storm events, on microbial water quality, and suggest that any future increases in the frequency of storm events (associated with climate change) are likely to result in a greater incidence of FIO/pathogen loads. This low-cost approach could help to predict spatio-temporal ‘hotspots’ of elevated waterborne disease risk. The work also represents an important step towards integrating novel MST tools into river catchment modelling.     

Using faecal sterols from humans and animals to distinguish faecal pollution in receiving waters

The sterol content of faeces from humans and 14 species of animals common to rural or urban environments were examined. The major human faecal sterol was the 5β-stanol, coprostanol which constituted ≈ 60% of the total sterols found in human faeces. The sterol profiles of herbivores were dominated by C₂₉ sterols and 5β-stanols were generally in equal or greater abundance than 5-stanols. The principal faecal biomarker of herbivores was 24-ethylcoprostanol. The sterol content of bird faeces was extremely variable and largely dependent on the animals diet. Both 5β and 5 stanols were in very low abundance in birds and dogs faeces from this study presumably due to the absence or low activity of the necessary anaerobic biota required to reduce Δ⁵- or Δ^5,22-sterols to stanols. Cats and pigs were the only animals that had similar faecal sterol profiles to humans. However, the concentration of the principal human biomarker coprostanol was some 10 times more abundant on a dry weight basis in the faeces of humans than in those of cats and pigs. The source specificity of faecal sterol biomarkers is a combination of sterol intake, metabolic production of sterols and the biota resident within the animal's digestive tract. The “sterol fingerprints” of the faeces of humans and animals are sufficiently distinctive to be of diagnostic value in determining whether faecal pollution in water samples are of human or animal origin.     

Sourcing faecal pollution: a combination of library-dependent and library-independent methods to identify human faecal pollution in non-sewered catchments

Library-dependent (LD) (biochemical fingerprinting of Escherichia coli and enterococci) and library-independent (LI) (PCR detection of human-specific biomarkers) methods were used to detect human faecal pollution in three non-sewered catchments. In all, 550 E. coli isolates and 700 enterococci isolates were biochemically fingerprinted from 18 water samples and compared with metabolic fingerprint libraries of 4508 E. coli and 4833 enterococci isolates. E. coli fingerprints identified human unique biochemical phenotypes (BPTs) in nine out of 18 water samples; similarly, enterococci fingerprints identified human faecal pollution in 10 water samples. Seven samples were tested by PCR for the detection of biomarkers. Human-specific HF134 Bacteroides and enterococci surface protein (esp) biomarkers were detected in five samples. Four samples were also positive for HF183 Bacteroides biomarker. The combination of biomarkers detected human faecal pollution in six out of seven water samples. Of the seven samples analysed for both the indicators/markers, at least one indicator/marker was detected in every sample. Four of the seven PCR-positive samples were also positive for one of the human-specific E. coli or enterococci BPTs. The results indicated human faecal pollution in the studied sub-catchments after storm events. LD and LI methods used in this study complimented each other and provided additional information regarding the polluting sources when one method failed to detect human faecal pollution. Therefore, it is recommended that a combination of methods should be used to identify the source(s) of faecal pollution where possible.     

Risk factors contributing to microbiological contamination of shallow groundwater in Kampala,Uganda

A study of water quality variation in shallow protected springs in Kampala was undertaken over a 12-month period to assess the causes of microbiological contamination. The microbiological quality of water was assessed using thermotolerant coliforms and faecal streptococci. Sanitary inspections and hazard assessments were undertaken to identify faecal sources (hazards), contaminant pathways and contributory factors. Data were collected on rainfall and population as additional factors potentially exerting an influence on microbiological quality. Initial analysis of the data showed a significant relationship between median level of contamination and rainfall, in particular to short-term rainfall events. Total sanitary risk score showed a significant relationship with median level of contamination, but population density may be a confounding factor. The raw microbiological data were transformed into five water quality targets: <1 and < or =10 cfu 100ml(-1) for faecal streptococci; and <1, < or =10 and < or=50 cfu 100 ml(-1) for thermotolerant coliforms. The presence of individual risk factors as well as variables for rainfall and population density were analysed with respect to failure to meet these water quality targets using contingency tables. Logistic regression models were developed for each of the five water quality targets. The analysis strongly suggested that there is rapid recharge of the springs after rainfall and this leads to microbiological contamination. On-site sanitation was less important than other sources of faecal matter, which was consistent with a low sanitation coverage in the study area. The study suggested that improving sanitary completion and local environmental hygiene was more important than controlling on-site sanitation in improving the quality of these springs.     

Comparison of the efficacy of an existing versus a locally developed metabolic fingerprint database to identify non-point sources of faecal contamination in a coastal lake

A comparison of the efficacy of an existing large metabolic fingerprint database of enterococci and Escherichia coli with a locally developed database was undertaken to identify the sources of faecal contamination in a coastal lake, in southeast Qld., Australia. The local database comprised of 776 enterococci and 780 E. coli isolates from six host groups. In all, 189 enterococci and 245 E. coli biochemical phenotypes (BPTs) were found, of which 118 and 137 BPTs were unique (UQ) to host groups. The existing database comprised of 295 enterococci UQ-BPTs and 273 E. coli UQ-BPTs from 10 host groups. The representativeness and the stability of the existing database were assessed by comparing with isolates that were external to the database. In all, 197 enterococci BPTs and 179 E. coli BPTs were found in water samples. The existing database was able to identify 62.4% of enterococci BPTs and 64.8% of E. coli BPTs as human and animal sources. The results indicated that a representative database developed from a catchment can be used to predict the sources of faecal contamination in another catchment with similar landuse features within the same geographical area. However, the representativeness and the stability of the database should be evaluated prior to its application in such investigation.     

Sustainable microbial water quality monitoring programme design using phage-lysis and multivariate techniques

Contamination of surface waters is a pervasive threat to human health, hence, the need to better understand the sources and spatio-temporal variations of contaminants within river catchments. River catchment managers are required to sustainably monitor and manage the quality of surface waters. Catchment managers therefore need cost-effective low-cost long-term sustainable water quality monitoring and management designs to proactively protect public health and aquatic ecosystems. Multivariate and phage-lysis techniques were used to investigate spatio-temporal variations of water quality, main polluting chemophysical and microbial parameters, faecal micro-organisms sources, and to establish ‘sentry’ sampling sites in the Ouse River catchment, southeast England, UK. 350 river water samples were analysed for fourteen chemophysical and microbial water quality parameters in conjunction with the novel human-specific phages of Bacteroides GB-124 (Bacteroides GB-124). Annual, autumn, spring, summer, and winter principal components (PCs) explained approximately 54%, 75%, 62%, 48%, and 60%, respectively, of the total variance present in the datasets. Significant loadings of Escherichia coli, intestinal enterococci, turbidity, and human-specific Bacteroides GB-124 were observed in all datasets. Cluster analysis successfully grouped sampling sites into five clusters. Importantly, multivariate and phage-lysis techniques were useful in determining the sources and spatial extent of water contamination in the catchment. Though human faecal contamination was significant during dry periods, the main source of contamination was non-human. Bacteroides GB-124 could potentially be used for catchment routine microbial water quality monitoring. For a cost-effective low-cost long-term sustainable water quality monitoring design, E. coli or intestinal enterococci, turbidity, and Bacteroides GB-124 should be monitored all-year round in this river catchment.     

Faecal contamination of source-separated human urine based on the content of faecal sterols

Transmissible pathogens in source-separated human urine, intended for reuse in agriculture, mainly originate from faeces that cross-contaminate the urine. The health risks associated with the enteric pathogens will largely be dependent on their initial concentration and their inactivation during storage in the urine. Faecal sterols have proven stable in urine and can, rather than indicator bacteria, be used to quantify the faecal cross-contamination. In this study, urine collection tanks were sampled and ratios between various faecal sterols were used to determine if the urine was contaminated by faeces. Twenty-two percent of samples from the upper part of the tanks and 37% of samples from the bottom sludge were found to be contaminated. Coprostanol concentrations in the contaminated urine samples corresponded to a mean faecal contamination of 9.1+/-5.6 mg l(-1) urine. E. coli was absent in a majority of the samples. Faecal streptococci and clostridia were enumerated but not found to correlate with coprostanol concentrations in contaminated samples.     

Validation of host-specific Bacteriodales 16S rRNA genes as markers to determine the origin of faecal pollution in Atlantic Rim countries of the European Union

The recent implementation of the Revised Bathing Water Directive in the European Union has highlighted the need for development of effective methods to differentiate between sources of faecal contamination. It had previously been shown that amplification of 16S rRNA genes of host-specific Bacteriodales species using the HF183F and CF128F primers could be used as markers for human and bovine faecal contamination in the United States. This paper determined the sensitivity and specificity of these markers in four Atlantic Rim countries (France, Ireland, Portugal and the United Kingdom) to evaluate their usefulness in determining the origin of faecal contamination. It was shown that the HF183F marker displayed high sensitivity (80-100%) and specificity (91-100%), and is reliable as an indication of human faecal contamination. The CF128F marker displayed 100% sensitivity in all four countries. However, strong regional variations in specificity (41-96%) were observed, highlighting the need for local validation before this marker is employed in source tracking of faecal contamination.     

Differential persistence of F-specific RNA phage subgroups hinders their use as single tracers for faecal source tracking in surface water

The four subgroups of F-specific RNA bacteriophages (I-IV) have been proposed as potential tracers for faecal source tracking. Groups II and III predominate in human sources while groups I and IV are most abundant in animal sources. The four subgroups of naturally occurring F-specific RNA bacteriophages were identified in different samples by plaque hybridization with genotype-specific probes and the persistence of each subgroup was evaluated. The proportions of the F-specific RNA bacteriophage subgroups were measured in wastewaters, after inactivation in surface waters or after wastewater treatment and in mixtures of wastewater of human and animal origin. Our results indicate that phage groups differ in their persistence in the environment and to different disinfecting treatments. The greater survival of subgroups I and II in treated samples hinders the interpretation of results obtained with F-specific RNA bacteriophages. The phages of subgroups III and IV were the least resistant to all treatments. These results should be considered when using genotypes of F-specific RNA as sole tracers for faecal source tracking.     

Degradation of faecal sterols in urine for assessment of faecal cross-contamination in source-separated human urine and urine storage tank sediment

The degradation of the faecal sterol coprostanol and its precursor cholesterol, were determined in source-separated human urine and urine tank sediment. The aim was to assess their potential as biomarkers of faecal cross-contamination in source-separated waste systems since traditional microbial indicators have been shown not to be reliable in this context. Investigations were performed at 4 and 20°C in collected human urine and urine tank sediment to which human faeces and synthetic cholesterol and coprostanol were added. The individual degradation experiments were performed over periods of up to 118 days and represented a wide concentration range both for the urine and the urine tank sediment. Coprostanol showed no significant degradation in either the urine or urine tank sediment when faeces or synthetic coprostanol were added. Neither the normalised variance for the individual slopes, nor the combined regression analysis showed any significant difference from the common slope of zero. For cholesterol no significant degradation was found in the urine tank sediment, but based on pooled data, a moderate decrease occurred in the urine. This was most evident for endogenous cholesterol in the urine at low concentrations where 38 and 55% of the original amounts were lost at 4 and 20°C, respectively. The daily rate of loss of cholesterol based on the regression analysis was 0.73 μg l⁻¹ per day. It is concluded that amounts of coprostanol in urine collection tanks can be used as an indicator of faecal cross-contamination cumulatively occurring over the collection period. Together with die-off data of indicator organisms and pathogens, establishment of a baseline risk assessment would now be possible in relation to storage, handling and reuse of the urine.     

Relationship between F-specific RNA phage genogroups, faecal pollution indicators and human adenoviruses in river water

Recent studies have shown the increasing interest of F-specific RNA phage genotyping to identify major sources of faecal contamination in waters. This study, conducted in a river located in an urbanized watershed with recognized anthropogenic influences, was aimed at evaluating the relevance of direct phage genotyping by real-time RT-PCR. One hundred percent of positive results were obtained with a 5 mL aliquot of river water (n = 31). Phage distribution was modified after cultivation, since the ratio of the two most abundant genogroups (II and I) reached 1.51 log₁₀ by direct RT-PCR-based method versus 0.30 log₁₀ after cultivation (n = 8). For the first time, positive correlations between the concentrations of genogroup II, bacterial indicators and human adenoviruses were observed, which may indicate a human faecal pollution. No correlation between genogroups II and I has been revealed. The concentration of genogroup I was only correlated with water turbidity, suggesting an animal pollution coming from upstream after rainfall events. Among the microbiological parameters studied, only genogroup II/genogroup I ratio shows variations occurring in the major sources of faecal pollution.