Spatial and hydrologic variation of Bacteroidales, adenovirus and enterovirus in a semi-arid, wastewater effluent-impacted watershed.

Bacteroidales and viruses were contemporaneously measured during dry and wet weather conditions at a watershed-scale in a semi-arid watershed impacted by a mixture of agricultural runoff, municipal wastewater effluent and municipal runoff. The results highlight the presence of municipal wastewater effluent as a confounding factor for microbial source tracking (MST) studies, and thus data were segregated into groups based on whether they were impacted by wastewater effluent. In semi-arid environments such as the Calleguas Creek watershed, located in southern California, the relative contribution of municipal wastewater effluent is dependent on hydrology as storm events lead to conditions where agricultural and municipal stormwater dominate receiving waters (rather than municipal wastewater, which is the case during dry weather). As such, the approach to data segregation was dependent on hydrology/storm conditions. Storm events led to significant increases in ruminant- and dog-associated Bacteroidales concentrations, indicating that overland transport connects strong non-human fecal sources with surface waters. Because the dataset had a large number of non-detect samples, data handling included the Kaplan-Meir estimator and data were presented graphically in a manner that reflects the potential effect of detection limits. In surface water samples with virus detections, Escherichia coli concentrations were often below (in compliance with) the recreational water quality criteria. In fact, sites downstream of direct inputs of municipal wastewater effluent exhibited the lowest concentrations of E. coli, but the highest concentrations of human-associated Bacteroidales and highest detection rates of human viruses. The toolkit, comprised of the four Bacteroidales assays and human virus assays used, can be successfully applied to inform watershed managers seeking to comply with recreational water quality criteria. However, care should be taken when analyzing data to account for the effect of non-detect samples, sources with differing microbial viability, and diverging hydrologic conditions.

Discharge-based QMRA for estimation of public health risks from exposure to stormwater-borne pathogens in recreational waters in the United States.

This study is the first to report a quantitative microbial risk assessment (QMRA) on pathogens detected in stormwater discharges-of-concern, rather than relying on pathogen measurements in receiving waters. The pathogen concentrations include seven "Reference Pathogens" identified by the U.S. EPA: Cryptosporidium, Giardia, Salmonella, Norovirus, Rotavirus, Enterovirus, and Adenovirus. Data were collected from 12 sites representative of seven discharge types (including residential, commercial/industrial runoff, agricultural runoff, combined sewer overflows, and forested land), mainly during wet weather conditions during which times human health risks can be substantially elevated. The risks calculated herein therefore generally apply to short-term conditions (during and just after rainfall events) and so the results can be used by water managers to potentially inform the public, even for waters that comply with current criteria (based as they are on a 30-day mean risk). Using an example waterbody and mixed source, pathogen concentrations were used in QMRA models to generate risk profiles for primary and secondary water contact (or inhalation) by adults and children. A number of critical assumptions and considerations around the QMRA analysis are highlighted, particularly the harmonization of the pathogen concentrations measured in discharges during this project with those measured (using different methods) during the published dose-response clinical trials. Norovirus was the most dominant predicted health risk, though further research on its dose-response for illness (cf. infection) is needed. Even if the example mixed-source concentrations of pathogens had been reduced 30 times (by inactivation and mixing), the predicted swimming-associated illness rates - largely driven by Norovirus infections - can still be appreciable. Rotavirus generally induced the second-highest incidence of risk among the tested pathogens while risks for the other Reference Pathogens (Giardia, Cryptosporidium, Adenovirus, Enterovirus and Salmonella) were considerably lower. Secondary contact or inhalation resulted in considerable reductions in risk compared to primary contact. Measurements of Norovirus and careful incorporation of its concentrations into risk models (harmonization) should be a critical consideration for future QMRA efforts. The discharge-based QMRA approach presented herein is particularly relevant to cases where pathogens cannot be reliably detected in receiving waters with detection limits relevant to human health effects.

Quo vadis source tracking? Towards a strategic framework for environmental monitoring of fecal pollution.

Advances in microbial source tracking (MST) have largely been driven by the need to comply with water quality standards based on traditional indicator bacteria. Recently, a number of culture-independent, and library-independent methods based on polymerase chain reaction (PCR) have been gaining popularity among source trackers. However, only a limited number of these methods have been successfully used in field applications, primarily due to the fact that many of them are still being developed. In this critical outlook, we examine different viewpoints associated with the practical use of MST to identify critical research gaps, propose a priority-based timeline to address them, and outline emerging technologies that will likely impact the future of source tracking. We propose that it is necessary to consider each of these aspects in order to advance towards a unifying framework in source identification, so that fecal pollution monitoring can be reliably used for comprehensive environmental microbial monitoring, to develop risk assessment models, and to implement and validate adequate management practices.

16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal Bacteroidales: a Bayesian approach.

We report the design and validation of new TaqMan((R)) assays for microbial source tracking based on the amplification of fecal 16S rRNA marker sequences from uncultured cells of the order Bacteroidales. The assays were developed for the detection and enumeration of non-point source input of fecal pollution to watersheds. The quantitative "universal"Bacteroidales assay BacUni-UCD detected all tested stool samples from human volunteers (18 out of 18), cat (7 out of 7), dog (8 out of 8), seagull (10/10), cow (8/8), horse (8/8), and wastewater effluent (14/14). The human assay BacHum-UCD discriminated fully between human and cow stool samples but did not detect all stool samples from human volunteers (12/18). In addition, there was 12.5% detection of dog stool (1/8), but no cross-reactivity with cat, horse, or seagull fecal samples. In contrast, all wastewater samples were positive for the BacHum-UCD marker, supporting its designation as 100% sensitive for mixed-human source identification. The cow-specific assay BacCow-UCD fully discriminated between cow and human stool samples. There was 38% detection of horse stool (3/8), but no cross-specificity with any of the other animal stool samples tested. The dog assay BacCan-UCD discriminated fully between dog and cow stool or seagull guano samples and detected 62.5% stool samples from dogs (5/8). There was some cross-reactivity with 22.2% detection of human stool (4/18), 14.3% detection of cat stool (1/7), and 28.6% detection of wastewater samples (4/14). After validation using stool samples, single-blind tests were used to further demonstrate the efficacy of the developed markers; all assays were sensitive, reproducible, and accurate in the quantification of mixed fecal sources present in aqueous samples. Finally, the new assays were compared with previously published sequences, which showed the new methodologies to be more specific and sensitive. Using Bayes' Theorem, we calculated the conditional probability that the four assays would correctly identify general and host-specific fecal pollution in a specific watershed in California for which 73 water samples had been analyzed. Such an approach allows for a direct comparison of the efficacy of different MST methods, including those based on library-dependent methodologies. For the universal marker BacUni-UCD, the probability that fecal pollution is present when the marker is detected was 1.00; the probability that host-specific pollution is present was 0.98, 0.84, and 0.89 for the human assay HF160F, the cow assay BacCow-UCD, and the dog assay BacCan-UCD, respectively. The application of these markers should provide meaningful information to assist with efforts to identify and control sources of fecal pollution to impaired watersheds.

Seasonal and spatial patterns of metals at a restored copper mine site. I. Stream copper and zinc.

Seasonal and spatial variations in metal concentrations and pH were found in a stream at a restored copper mine site located near a massive sulfide deposit in the Foothill copper-zinc belt of the Sierra Nevada, California. At the mouth of the stream, copper concentrations increased and pH decreased with increased streamflow after the onset of winter rain and, unexpectedly, reached extreme values 1 or 2 months after peaks in the seasonal hydrographs. In contrast, aqueous zinc and sulfate concentrations were highest during low-flow periods. Spatial variation was assessed in 400 m of reach encompassing an acidic, metal-laden seep. At this seep, pH remained low (2-3) throughout the year, and copper concentrations were highest. In contrast, the zinc concentrations increased with downstream distance. These spatial patterns were caused by immobilization of copper by hydrous ferric oxides in benthic sediments, coupled with increasing downstream supply of zinc from groundwater seepage.

Seasonal and spatial patterns of metals at a restored copper mine site II. Copper in riparian soils and Bromus carinatus shoots.

Soil and plants were sampled throughout winter and spring near a perennial stream traversing a restored mine site in a winter-rainy climate. Within 1m of an acidic reach of the stream, soil had pH 3-5 and 50-100 microg/g "bioavailable" copper (extractable with 0.01 M CaCl2). Soil 2-3 m from the stream had pH 5-8 and lower (less than 3 microg/g) bioavailable copper. "Oxide-bound" copper (extractable with 2N HCl) was 50-100 microg/g at most locations. Copper concentrations in the shoots of field-collected Bromus carinatus declined from 20 microg/g in winter to 2 microg/g in spring at all sampling sites. A similar temporal pattern was found in plants grown under controlled conditions. Thus B. carinatus has a developmental program for control of shoot copper concentration, causing a seasonally-varying pattern of copper phytoaccumulation over a large range of copper availability in the soil.