SARS-CoV-2 RNA in Wastewater Settled Solids Is Associated with COVID-19 Cases in a Large Urban Sewershed.

Wastewater-based epidemiology may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the preanalytical and analytical approaches and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlated positively and significantly with COVID-19 clinically confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARS-CoV-2 in influent.

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA.

Preserving biodiversity is a global challenge requiring data on species' distribution and abundance over large geographic and temporal scales. However, traditional methods to survey mobile species' distribution and abundance in marine environments are often inefficient, environmentally destructive, or resource-intensive. Metabarcoding of environmental DNA (eDNA) offers a new means to assess biodiversity and on much larger scales, but adoption of this approach for surveying whole animal communities in large, dynamic aquatic systems has been slowed by significant unknowns surrounding error rates of detection and relevant spatial resolution of eDNA surveys. Here, we report the results of a 2.5 km eDNA transect surveying the vertebrate fauna present along a gradation of diverse marine habitats associated with a kelp forest ecosystem. Using PCR primers that target the mitochondrial 12S rRNA gene of marine fishes and mammals, we generated eDNA sequence data and compared it to simultaneous visual dive surveys. We find spatial concordance between individual species' eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 m. eDNA reliably detected vertebrates with low false-negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.

Absolute Quantification of Enterococcal 23S rRNA Gene Using Digital PCR.

We evaluated the ability of chip-based digital PCR (dPCR) to quantify enterococci, the fecal indicator recommended by the United States Environmental Protection Agency (USEPA) for water-quality monitoring. dPCR uses Poisson statistics to estimate the number of DNA fragments in a sample with a specific sequence. Underestimation may occur when a gene is redundantly encoded in the genome and multiple copies of that gene are on one DNA fragment. When genomic DNA (gDNA) was extracted using two commercial DNA extraction kits, we confirmed that dPCR could discern individual copies of the redundant 23s rRNA gene in the enterococcal genome. dPCR quantification was accurate when compared to the nominal concentration inferred from fluorometer measurements (linear regression slope = 0.98, intercept = 0.03, R(2) = 0.99, and p value <0.0001). dPCR quantification was also consistent with quantitative PCR (qPCR) measurements as well as cell counts for BioBall reference standard and 24 environmental water samples. qPCR and dPCR quantification of enterococci in the 24 environmental samples were significantly correlated (linear regression slope =1.08, R(2) of 0.96, and p value <0.0001); the group mean of the qPCR measurements was 0.19 log units higher than that of the dPCR measurements. At environmentally relevant concentrations, dPCR quantification was more precise (i.e., had narrower 95% confidence intervals than qPCR quantification). We observed that humic acid caused a similar level of inhibition in both dPCR and qPCR, but calcium inhibited dPCR to a lesser degree than qPCR. Inhibition of dPCR was partially relieved when the number of thermal cycles was increased. Based on these results, we conclude that dPCR is a viable option for enumerating enterococci in ambient water.

Quantification of Environmental DNA (eDNA) Shedding and Decay Rates for Three Marine Fish.

Analysis of environmental DNA (eDNA) to identify macroorganisms and biodiversity has the potential to significantly augment spatial and temporal biological monitoring in aquatic ecosystems. Current monitoring methods relying on the physical identification of organisms can be time consuming, expensive, and invasive. Measuring eDNA shed from organisms provides detailed information on the presence and abundance of communities of organisms. However, little is known about eDNA shedding and decay in aquatic environments. In the present study, we designed novel Taqman qPCR assays for three ecologically and economically important marine fish-Engraulis mordax (Northern Anchovy), Sardinops sagax (Pacific Sardine), and Scomber japonicas (Pacific Chub Mackerel). We subsequently measured fish eDNA shedding and decay rates in seawater mesocosms. eDNA shedding rates ranged from 165 to 3368 pg of DNA per hour per gram of biomass. First-order decay rate constants ranged from 0.055 to 0.101 per hour. We also examined the size fractionation of eDNA and concluded eDNA is both intra- and extracellular. Finally, we derived a simple mass-balance model to estimate fish abundance from eDNA concentration. The mesocosm-derived shedding and decay rates inform the interpretation of eDNA concentrations measured in environmental samples and future use of eDNA as a monitoring tool.

Temporal stability of the microbial community in sewage-polluted seawater exposed to natural sunlight cycles and marine microbiota.

Billions of gallons of untreated wastewater enter the coastal ocean each year. Once sewage microorganisms are in the marine environment, they are exposed to environmental stressors, such as sunlight and predation. Previous research has investigated the fate of individual sewage microorganisms in seawater but not the entire sewage microbial community. The present study used next-generation sequencing (NGS) to examine how the microbial community in sewage-impacted seawater changes over 48 h when exposed to natural sunlight cycles and marine microbiota. We compared the results from microcosms composed of unfiltered seawater (containing naturally occurring marine microbiota) and filtered seawater (containing no marine microbiota) to investigate the effect of marine microbiota. We also compared the results from microcosms that were exposed to natural sunlight cycles with those from microcosms kept in the dark to investigate the effect of sunlight. The microbial community composition and the relative abundance of operational taxonomic units (OTUs) changed over 48 h in all microcosms. Exposure to sunlight had a significant effect on both community composition and OTU abundance. The effect of marine microbiota, however, was minimal. The proportion of sewage-derived microorganisms present in the microcosms decreased rapidly within 48 h, and the decrease was the most pronounced in the presence of both sunlight and marine microbiota, where the proportion decreased from 85% to 3% of the total microbial community. The results from this study demonstrate the strong effect that sunlight has on microbial community composition, as measured by NGS, and the importance of considering temporal effects in future applications of NGS to identify microbial pollution sources.

Diversity and transport of microorganisms in intertidal sands of the California coast.

Forced by tides and waves, large volumes of seawater are flushed through the beach daily. Organic material and nutrients in seawater are remineralized and cycled as they pass through the beach. Microorganisms are responsible for most of the biogeochemical cycling in the beach; however, few studies have characterized their diversity in intertidal sands, and little work has characterized the extent to which microbes are transported between different compartments of the beach. The present study uses next-generation massively parallel sequencing to characterize the microbial community present at 49 beaches along the coast of California. In addition, we characterize the transport of microorganisms within intertidal sands using laboratory column experiments. We identified extensive diversity in the beach sands. Nearly 1,000 unique taxa were identified in sands from 10 or more unique beaches, suggesting the existence of a group of "cosmopolitan" sand microorganisms. A biogeographical analysis identified a taxon-distance relationship among the beaches. In addition, sands with similar grain size, organic carbon content, exposed to a similar wave climate, and having the same degree of anthropogenic influence tended to have similar microbial communities. Column experiments identified microbes readily mobilized by seawater infiltrating through unsaturated intertidal sands. The ease with which microbes were mobilized suggests that intertidal sands may represent a reservoir of bacteria that seed the beach aquifer where they may partake in biogeochemical cycling.

Occurrence and persistence of bacterial pathogens and indicator organisms in beach sand along the California coast.

This report documents the presence of fecal indicators and bacterial pathogens in sand at 53 California marine beaches using both culture-dependent and -independent (PCR and quantitative PCR [QPCR]) methods. Fecal indicator bacteria were widespread in California beach sand, with Escherichia coli and enterococci detected at 68% and 94% of the beaches surveyed, respectively. Somatic coliphages and a Bacteroidales human-specific fecal marker were detected at 43% and 13% of the beaches, respectively. Dry sand samples from almost 30% of the beaches contained at least one of the following pathogens: Salmonella spp., Campylobacter spp., Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA), which were detected at 15%, 13%, 14%, and 3% of tested beaches, respectively. Fecal indicators and pathogens were poorly correlated to one another and to land cover. Sands were dry at the time of collection, and those with relatively high moisture tended to have higher concentrations or a more frequent occurrence of both indicators and pathogens. Using culture-dependent assays, fecal indicators decayed faster than pathogens in microcosm experiments using unaltered beach sand seeded with sewage and assessed by culture-dependent assays. The following order of persistence was observed (listed from most to least persistent): Campylobacter > Salmonella > somatic coliphages > enterococci > E. coli > F(+) phages. In contrast, pathogens decayed faster than fecal indicators in culture-independent assays: enterococci > Bacteroidales human-specific marker > Salmonella > Campylobacter. Microcosm experiments demonstrated that both indicators and pathogens were mobilized by wetting with seawater. Decay rates measured by QPCR were lower than those measured with culture-dependent methods. Enterococcal persistence and possible growth were observed for wetted microcosms relative to unwetted controls.

Mobilization and transport of naturally occurring enterococci in beach sands subject to transient infiltration of seawater.

This study explores the transport of enterococci (ENT) from naturally contaminated beach sands to the groundwater table via infiltrating seawater using field, laboratory, and modeling experiments. ENT were readily mobilized and transported through the unsaturated zone during infiltration events in both the field and laboratory column experiments. Detachment mechanisms were investigated using a modified version of HYDRUS-1D. Three models for detachment kinetics were tested. Detachment kinetics that are first order with respect to the rate of change in the water content and attached surface bacterial concentrations were found to provide a best fit between predicted and observed data. From these experimental and model results we conclude that detachment mechanisms associated with the rapid increases in pore water content such as air-water interface scouring and thin film expansion are likely drivers of ENT mobilization in the investigated system. These findings suggest that through-beach transport of ENT may be an important pathway through which ENT from beach sands are transported to beach groundwater where they may be discharged to coastal waters via submarine groundwater discharge.

A Raman spectral reference library of potential anthropogenic and biological ocean polymers.

Microplastics have been extensively documented in marine ecosystems and food webs with devastating impacts. To solve this global crisis, identifying the polymer composition is key for resolving the material origin, geographic source, and ecosystem life cycle of ocean plastics. Visually based techniques, importantly, are not diagnostic. Raman spectroscopy is an increasingly preferred identification method for its accuracy and reduced likelihood of misinterpretation, though it can be inaccessible due to cost of paywalled spectral libraries and availability of relevant polymer spectra for comparison. Here, we provide an open-access reference library of high-quality, broad-spectrum Raman spectra of major polymer categories germane to marine environments. The library includes high-quality spectra from: (a) pristine anthropogenic polymers newly sourced from manufacturers (n = 40), (b) weathered anthropogenic polymers collected from used consumer, beachcast, agricultural, and fishery sources (n = 22), and (c) biological polymers representing diverse marine taxa, trophic levels, and tissues (n = 17). We hope this reference library can help this rapidly expanding scientific community and facilitate progress in the global plastic pollution crisis.

Quantitative PCR assays to detect whales, rockfish, and common murre environmental DNA in marine water samples of the Northeastern Pacific.

Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.

Application of molecular source tracking and mass balance approach to identify potential sources of fecal indicator bacteria in a tropical river.

Microbial source tracking and a mass balance approach were used to identify sources of fecal indicator bacteria (FIB) in the Hanalei River, Kaua'i, Hawai'i. Historically, concentrations enterococci and Clostridium perfringens were significantly higher during storm flows compared to non-storm flows in the Hanalei River, and correlated to total suspended solids in the river. During targeted dry weather studies, the Hanalei River bed sediments and streambank soils were documented to harbor E. coli, enterococci, and the human- and pig-specific fecal markers in Bacteroidales, suggesting that sediments and soils may be potential sources of these microorganisms to the Hanalei river. The human-specific marker in Bacteroidales was four times as likely to be detected in sediment and soil samples as in water samples. Furthermore, the occurrence of host-specific source tracking markers is indicative that a portion of FIB present in the Hanalei River are of fecal origin. A mass balance approach was used to explore causes of observed FIB loadings and losses along different reaches of the river. Resuspension or deposition of FIB-laden river sediments cannot account for changes in E. coli and enterococci concentrations along the river during dry weather. Additionally, losses due to bacterial inactivation were insignificant. Groundwater and ditches draining agricultural and urban lands were shown to provide sufficient FIB fluxes to account for the observed loads along some river reaches. The presence of the human-specific Bacteroidales marker in the river water, sediments and adjacent soils, as well as the presence of the human enterovirus marker in the water, suggests that there is widespread human fecal contamination in the Hanalei River that is likely a result of nearby wastewater disposal systems.

Robotic environmental DNA bio-surveillance of freshwater health.

Autonomous water sampling technologies may help to overcome the human resource challenges of monitoring biological threats to rivers over long time periods and across large geographic areas. The Monterey Bay Aquarium Research Institute has pioneered a robotic Environmental Sample Processor (ESP) that overcomes some of the constraints associated with traditional sampling since it can automate water sample filtration and preservation of the captured material. The ESP was originally developed for marine environment applications. Here we evaluated whether the ESP can provide reliable, timely information on environmental (e)DNA detections of human and fish pathogens and introduced fishes at U.S. Geological Survey streamgage sites in freshwater rivers. We compared eDNA collected via ESP at high frequency (e.g., every 3 h) with manual eDNA collections collected at lower frequency (e.g., weekly). We found that water samples filtered and preserved by ESPs successfully detected the DNA of human pathogens, fish pathogens and introduced fishes. Both ESP and manually collected samples provided similar information about target DNA presence. We suggest that the greatest current benefit of the ESP is the cost savings of high frequency, bio-surveillance at remote or hard to access sites. The full potential of robotic technologies like the ESP will be realized when they can more easily execute in situ analyses of water samples and rapidly transmit results to decision-makers.

Growth of enterococci in unaltered, unseeded beach sands subjected to tidal wetting.

Enterococci are indicator bacteria used to assess the risk of acquiring enteric disease from swimming in marine waters. Previous work identified beach sands as reservoirs of enterococci which can be transported from the sand to the sea, where they may instigate beach advisories. The present study establishes that naturally occurring enterococci can replicate in beach sands under environmentally relevant conditions. In unseeded, nonsterile microcosm experiments, it was shown that intermittent wetting of sands by seawater, like that which would occur at the high tide line, stimulates the transient replication of enterococci at rates of 0.20 to 0.63 per day (equivalent to doubling times of 1.1 to 3.5 days). Replication was not observed in control microcosms that were not subjected to wetting. Enterococci were enumerated using both culture-dependent (membrane filtration and mEI media) and culture-independent (quantitative PCR [QPCR], 23S rRNA gene based) techniques, which allowed tracking of both culturable and total enterococcus populations. Inhibition of QPCR and DNA extraction efficiencies were accounted for in the interpretation of the QPCR results. The results provide evidence that enterococci may not be an appropriate indicator of enteric disease risk at recreational beaches subject to nonpoint sources of pollution.

Impacts of a changing earth on microbial dynamics and human health risks in the continuum between beach water and sand.

Although infectious disease risk from recreational exposure to waterborne pathogens has been an active area of research for decades, beach sand is a relatively unexplored habitat for the persistence of pathogens and fecal indicator bacteria (FIB). Beach sand, biofilms, and water all present unique advantages and challenges to pathogen introduction, growth, and persistence. These dynamics are further complicated by continuous exchange between sand and water habitats. Models of FIB and pathogen fate and transport at beaches can help predict the risk of infectious disease from beach use, but knowledge gaps with respect to decay and growth rates of pathogens in beach habitats impede robust modeling. Climatic variability adds further complexity to predictive modeling because extreme weather events, warming water, and sea level change may increase human exposure to waterborne pathogens and alter relationships between FIB and pathogens. In addition, population growth and urbanization will exacerbate contamination events and increase the potential for human exposure. The cumulative effects of anthropogenic changes will alter microbial population dynamics in beach habitats and the assumptions and relationships used in quantitative microbial risk assessment (QMRA) and process-based models. Here, we review our current understanding of microbial populations and transport dynamics across the sand-water continuum at beaches, how these dynamics can be modeled, and how global change factors (e.g., climate and land use) should be integrated into more accurate beachscape-based models.

Using environmental DNA to census marine fishes in a large mesocosm.

The ocean is a soup of its resident species' genetic material, cast off in the forms of metabolic waste, shed skin cells, or damaged tissue. Sampling this environmental DNA (eDNA) is a potentially powerful means of assessing whole biological communities, a significant advance over the manual methods of environmental sampling that have historically dominated marine ecology and related fields. Here, we estimate the vertebrate fauna in a 4.5-million-liter mesocosm aquarium tank at the Monterey Bay Aquarium of known species composition by sequencing the eDNA from its constituent seawater. We find that it is generally possible to detect mitochondrial DNA of bony fishes sufficient to identify organisms to taxonomic family- or genus-level using a 106 bp fragment of the 12S ribosomal gene. Within bony fishes, we observe a low false-negative detection rate, although we did not detect the cartilaginous fishes or sea turtles present with this fragment. We find that the rank abundance of recovered eDNA sequences correlates with the abundance of corresponding species' biomass in the mesocosm, but the data in hand do not allow us to develop a quantitative relationship between biomass and eDNA abundance. Finally, we find a low false-positive rate for detection of exogenous eDNA, and we were able to diagnose non-native species' tissue in the food used to maintain the mesocosm, underscoring the sensitivity of eDNA as a technique for community-level ecological surveys. We conclude that eDNA has substantial potential to become a core tool for environmental monitoring, but that a variety of challenges remain before reliable quantitative assessments of ecological communities in the field become possible.

Characterization of fecal concentrations in human and other animal sources by physical, culture-based, and quantitative real-time PCR methods.

The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers.

Bacterial pathogens in Hawaiian coastal streams--associations with fecal indicators, land cover, and water quality.

This work aimed to understand the distribution of five bacterial pathogens in O'ahu coastal streams and relate their presence to microbial indicator concentrations, land cover of the surrounding watersheds, and physical-chemical measures of stream water quality. Twenty-two streams were sampled four times (in December and March, before sunrise and at high noon) to capture seasonal and time of day variation. Salmonella, Campylobacter, Staphylococcus aureus, Vibrio vulnificus, and V. parahaemolyticus were widespread -12 of 22 O'ahu streams had all five pathogens. All stream waters also had detectable concentrations of four fecal indicators and total vibrio with log mean ± standard deviation densities of 2.2 ± 0.8 enterococci, 2.7 ± 0.7 Escherichia coli, 1.1 ± 0.7 Clostridium perfringens, 1.2 ± 0.8 F(+) coliphages, and 3.6 ± 0.7 total vibrio per 100 ml. Bivariate associations between pathogens and indicators showed enterococci positively associated with the greatest number of bacterial pathogens. Higher concentrations of enterococci and higher incidence of Campylobacter were found in stream waters collected before sunrise, suggesting these organisms are sensitive to sunlight. Multivariate regression models of microbes as a function of land cover and physical-chemical water quality showed positive associations between Salmonella and agricultural and forested land covers, and between S. aureus and urban and agricultural land covers; these results suggested that sources specific to those land covers may contribute these pathogens to streams. Further, significant associations between some microbial targets and physical-chemical stream water quality (i.e., temperature, nutrients, turbidity) suggested that organism persistence may be affected by stream characteristics. Results implicate streams as a source of pathogens to coastal waters. Future work is recommended to determine infectious risks of recreational waterborne illness related to O'ahu stream exposures and to mitigate these risks through control of land-based runoff sources.

Persistence of nucleic acid markers of health-relevant organisms in seawater microcosms: implications for their use in assessing risk in recreational waters.

In the last decade, the use of culture-independent methods for detecting indicator organisms and pathogens in recreational waters has increased and has led to heightened interest in their use for routine water quality monitoring. However, a thorough understanding of the persistence of genetic markers in environmental waters is lacking. In the present study, we evaluate the persistence of enterococci, enterovirus, and human-specific Bacteroidales in seawater microcosms. Two microcosms consisted of seawater seeded with human sewage. Two additional seawater microcosms were seeded with naked Enterococcus faecium DNA and poliovirus RNA. One of each replicate microcosm was exposed to natural sunlight; the other was kept in complete darkness. In the sewage microcosms, concentrations of enterococci and enterovirus were measured using standard culture-dependent methods as well as QPCR and RT-QPCR respectively. Concentrations of human-specific Bacteroidales were determined with QPCR. In the naked-genome microcosms, enterococci and enterovirus markers were enumerated using QPCR and RT-QPCR, respectively. In the sewage microcosm exposed to sunlight, concentrations of culturable enterococci fell below the detection limit within 5 days, but the QPCR signal persisted until the end of the experiment (day 28). Culturable enterococci did not persist as long as infectious enteroviruses. The ability to culture enteroviruses and enterococci was lost before detection of the genetic markers was lost, but the human-specific Bacteroidales QPCR signal persisted for a similar duration as infectious enteroviruses in the sewage microcosm exposed to sunlight. In the naked-genome microcosms, DNA and RNA from enterococci and enterovirus, respectively, persisted for over 10d and did not vary between the light and dark treatments. These results indicate differential persistence of genetic markers and culturable organisms of public health relevance in an environmental matrix and have important management implications.

Covariation and photoinactivation of traditional and novel indicator organisms and human viruses at a sewage-impacted marine beach.

Sunlight modulates concentrations of Escherichia coli and enterococci in marine waters. However, the mechanism of photoinactivation is poorly understood. Additionally, little is known about photoinactivation of other fecal indicators and human viruses in recreational waters. We sampled nearshore waters at Avalon Beach, California hourly for 72 h for reactive oxygen species (ROS), traditional indicator bacteria (E. coli and enterococci, and QPCR-based detection of enterococci), F+ (DNA and RNA) and somatic coliphages, the human-specific marker in Bacteroidales (HF marker), human enterovirus, and human adenovirus. E. coli and enterococci (regardless of measurement technique) covaried with each other and the coliphages suggesting similar sources and fates. The occurrence of the HF and enterovirus markers was correlated, but their occurrence was not positively correlated with the other indicators. Lower concentrations or occurrence of all microbes, excluding the HF and enterovirus markers, were observed during sunlit as opposed to dark hours, pointing to the importance of photoinactivation. Empirical-deterministic models for a subset of microbial indicators were created to determine field-relevant sunlight inactivation rates while accounting for time dependent sources and sinks. Photoinactivation rates of enterococci and E. coli, enterococci measured by QPCR, and somatic coliphage were estimated at 7, 6, 3, and 28 d(-1) I(-1), respectively, where I is UVB intensity in W/m(2). Average H(2)O(2) was 183 nM and the maximum singlet oxygen steady state concentration was 6.6 fM. Given the clarity of the water, direct genomic damage of bacteria and coliphage, as well as indirect endogenous damage of bacteria, were likely the most important inactivation mechanisms, but we cannot rule out a contribution by indirect mechanisms involving the H(2)O(2) and singlet oxygen produced exogenously.