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Urban stormwater is a major threat to ecological health, causing a range of adverse, mostly sublethal effects. In western North America, urban runoff is acutely lethal to adult coho salmon ( Oncorhynchus kisutch) that spawn each fall in freshwater creeks. Although the mortality syndrome is correlated to urbanization and attributed to road runoff contaminant(s), the causal agent(s) remain unknown. We applied high-resolution mass spectrometry to isolate a coho mortality chemical signature: a list of nontarget and identified features that co-occurred in waters lethal to coho spawners (road runoff from controlled exposures and urban receiving waters from two field observations of symptomatic coho). Hierarchical cluster analysis indicated that tire wear particle (TWP) leachates were most chemically similar to the waters with observed toxicity, relative to other vehicle-derived sources. Prominent road runoff contaminants in the signature included two groups of nitrogen-containing compounds derived from TWP, polyethylene glycols, octylphenol ethoxylates, and polypropylene glycols. A (methoxymethyl)melamine compound family, previously unreported in North America, was detected in road runoff and urban creeks at concentrations up to ∼9 and ∼0.3 μg/L, respectively. The results indicate TWPs are an under-appreciated contaminant source in urban watersheds and should be prioritized for fate and toxicity assessment.

Recent evidence suggests macroalgal blooms may play a role in the worldwide decline in seagrass, but the shape of the functional relationship between seagrass health and dominant bloom-forming macroalgae is poorly characterized. We tested whether the impact of varying abundances of two cosmopolitan bloom-forming macroalgal genera caused linear/quasi-linear or sudden threshold changes in measures of eelgrass, Zostera marina, meadow health. We conducted two caging experiments in a shallow Z. marina bed (∼1 m depth) in Bodega Harbor, California, USA where we maintained six densities within the range of natural abundances of macroalgae, Ulva (0-4.0 kg m -2 ) and Gracilariopsis (0-2.0 kg m -2 ), as well as uncaged controls over a 10-week period. Shoot density, blade growth, and epiphyte load were measured every two weeks and algal treatments reset. We did not find support for threshold transitions between algal abundance and measures of seagrass bed health using sigmoidal and broken-stick regression analyses for each data set; these models are commonly used to identify threshold patterns in ecological shifts. Instead, final measurements of shoot density and epiphyte load were best modelled as linear or slightly non-linear declines with increasing Ulva abundance. A negative linear relationship also existed between shoot density and Gracilariopsis abundance and a trend towards linear negative effects on epiphyte load. The similar shape of these functional relationships across different types of algae suggests the relationship may be generalizable. At algal abundances that are commonly observed, we found smooth and predictable negative impacts to Z. marina by decline in shoot density and potential impacts to food webs by loss of epiphytes rather than sudden threshold shifts or "ecological surprises". Our work contrasts with the growing body of literature suggesting highly non-linear shifts in response to human impact; thus, it is important to broaden understanding of shifts to more than just pattern but to the processes that drive different patterns of shifts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

One application of nanocopper is as a wood-preserving pesticide in pressure-treated lumber. Recent research has shown that pressure-treated lumber amended with micronized copper azole (MCA), which contains nanosized copper, releases copper under estuarine and marine conditions. The form of copper released (i.e., ionic, nanocopper [1-100 nm in size]) is not fully understood but will affect the bioavailability and toxicity of the metal. In the present study, multiple lines of evidence, including size fractionation, ion-selective electrode electrochemistry, comparative toxicity, and copper speciation were used to determine the form of copper released from lumber blocks and sawdust. The results of all lines of evidence supported the hypothesis that ionic copper was released from MCA lumber and sawdust, with little evidence that nanocopper was released. For example, copper concentrations in size fractionations of lumber block aqueous leachates including unfiltered, 0.1 μm, and 3 kDa were not significantly different, suggesting that the form of copper released was in the size range operationally defined as dissolved. These results correlated with the ion-selective electrode data which detects only ionic copper. In addition, comparative toxicity testing resulted in a narrow range of median lethal concentrations (221-257 μg/L) for MCA lumber blocks and CuSO 4 . We conclude that ionic copper was released from the nanocopper pressure-treated lumber under estuarine and marine conditions. Environ Toxicol Chem 2018;37:1969-1979. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America., (Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

Little is known about the release of metal engineered nanomaterials (ENMs) from consumer goods, including lumber treated with micronized copper. Micronized copper is a recent form of antifouling wood preservative containing nanosized copper particles for use in pressure-treated lumber. The present study investigated the concentrations released and the release rate of total copper over the course of 133 d under freshwater, estuarine, and marine salinity conditions (0, 1, 10, and 30‰) for several commercially available pressure-treated lumbers: micronized copper azole (MCA) at 0.96 and 2.4 kg/m 3 , alkaline copper quaternary (ACQ) at 0.30 and 9.6 kg/m 3 , and chromated copper arsenate (CCA) at 40 kg/m 3 . Lumber was tested as blocks and as sawdust. Overall, copper was released from all treated lumber samples. Under leaching conditions, total release ranged from 2 to 55% of the measured copper originally in the lumber, with release rate constants from the blocks of 0.03 to 2.71 (units per day). Generally, measured release and modeled equilibrium concentrations were significantly higher in the estuarine conditions compared with freshwater or marine salinities, whereas rate constants showed very limited differences between salinities. Furthermore, organic carbon was released during the leaching and demonstrated a significant relationship with released copper concentrations as a function of salinity. The results indicate that copper is released into estuarine/marine waters from multiple wood treatments including lumber amended with nanoparticle-sized copper. Environ Toxicol Chem 2018;37:1956-1968. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America., (Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

There are numerous reasons to conduct scientific research within protected areas, but research activities may also negatively impact organisms and habitats, and thus conflict with a protected area's conservation goals. We developed a quantitative ecological decision-support framework that estimates these potential impacts so managers can weigh costs and benefits of proposed research projects and make informed permitting decisions. The framework generates quantitative estimates of the ecological impacts of the project and the cumulative impacts of the proposed project and all other projects in the protected area, and then compares the estimated cumulative impacts of all projects with policy-based acceptable impact thresholds. We use a series of simplified equations (models) to assess the impacts of proposed research to: a) the population of any targeted species, b) the major ecological assemblages that make up the community, and c) the physical habitat that supports protected area biota. These models consider both targeted and incidental impacts to the ecosystem and include consideration of the vulnerability of targeted species, assemblages, and habitats, based on their recovery time and ecological role. We parameterized the models for a wide variety of potential research activities that regularly occur in the study area using a combination of literature review and expert judgment with a precautionary approach to uncertainty. We also conducted sensitivity analyses to examine the relationships between model input parameters and estimated impacts to understand the dominant drivers of the ecological impact estimates. Although the decision-support framework was designed for and adopted by the California Department of Fish and Wildlife for permitting scientific studies in the state-wide network of marine protected areas (MPAs), the framework can readily be adapted for terrestrial and freshwater protected areas., Competing Interests: The authors have declared that no competing interests exist.

The multivariate AMBI (M-AMBI) is an extension of the AZTI Marine Biotic Index (AMBI) that has been used extensively in Europe, but not in the United States. In a previous study, we adapted AMBI for use in US coastal waters (US AMBI), but saw biases in salinity and score distribution when compared to locally calibrated indices. In this study we modified M-AMBI for US waters and compared its performance to that of US AMBI. Index performance was evaluated in three ways: 1) concordance with local indices presently being used as management tools in three geographic regions of US coastal waters, 2) classification accuracy for sites defined a priori as good or bad and 3) insensitivity to natural environmental gradients. US M-AMBI was highly correlated with all three local indices and removed the compression in response seen in moderately disturbed sites with US AMBI. US M-AMBI and US AMBI did a similar job correctly classifying sites as good or bad in local validation datasets (83 to 100% accuracy vs. 84 to 95%, respectively). US M-AMBI also removed the salinity bias of US AMBI so that lower salinity sites were not more likely to be incorrectly classified as impaired. The US M-AMBI appears to be an acceptable index for comparing condition across broad-scales such as estuarine and coastal waters surveyed by the US EPA's National Coastal Condition Assessment, and may be applicable to areas of the US coast that do not have a locally derived benthic index.

Along southern California beaches, the concentrations of fecal indicator bacteria (FIB) used to quantify the potential presence of fecal contamination in coastal recreational waters have been previously documented to be higher during wet weather conditions (typically winter or spring) than those observed during summer dry weather conditions. FIB are used for management of recreational waters because measurement of the bacterial and viral pathogens that are the potential causes of illness in beachgoers exposed to stormwater can be expensive, time-consuming, and technically difficult. Here, we use droplet digital Polymerase Chain Reaction (digital PCR) and digital reverse transcriptase PCR (digital RT-PCR) assays for direct quantification of pathogenic viruses, pathogenic bacteria, and source-specific markers of fecal contamination in the stormwater discharges. We applied these assays across multiple storm events from two different watersheds that discharge to popular surfing beaches in San Diego, CA. Stormwater discharges had higher FIB concentrations as compared to proximal beaches, often by ten-fold or more during wet weather. Multiple lines of evidence indicated that the stormwater discharges contained human fecal contamination, despite the presence of separate storm sewer and sanitary sewer systems in both watersheds. Human fecal source markers (up to 100% of samples, 20-12440 HF183 copies per 100 ml) and human norovirus (up to 96% of samples, 25-495 NoV copies per 100 ml) were routinely detected in stormwater discharge samples. Potential bacterial pathogens were also detected and quantified: Campylobacter spp. (up to 100% of samples, 16-504 gene copies per 100 ml) and Salmonella (up to 25% of samples, 6-86 gene copies per 100 ml). Other viral human pathogens were also measured, but occurred at generally lower concentrations: adenovirus (detected in up to 22% of samples, 14-41 AdV copies per 100 ml); no enterovirus was detected in any stormwater discharge sample. Higher concentrations of avian source markers were noted in the stormwater discharge located immediately downstream of a large bird sanctuary along with increased Campylobacter concentrations and notably different Campylobacter species composition than the watershed that had no bird sanctuary. This study is one of the few to directly measure an array of important bacterial and viral pathogens in stormwater discharges to recreational beaches, and provides context for stormwater-based management of beaches during high risk wet-weather periods. Furthermore, the combination of culture-based and digital PCR-derived data is demonstrated to be valuable for assessing hydrographic relationships, considering delivery mechanisms, and providing foundational exposure information for risk assessment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Seabirds are often cited as sentinels of the marine environment, but are rarely used in traditional ocean and coastal contaminant monitoring. Four classes of persistent organic pollutants (POPs, n=68) and three trace elements (mercury, selenium, and arsenic) were measured in the eggs of California least terns (Sterna antillarum browni), caspian terns (Hydroprogne caspia), double-crested cormorants (Phalacrocorax auritus), and western gulls (Larus occidentalis) that nest in the Southern California Bight. Building on a periodic five year regional monitoring program, we measured contaminant exposure and assessed the utility of seabirds as regional contaminant biomonitors. We found that the eggs of larger, more piscivorous species generally had the highest concentrations of POPs and trace elements while California least terns had the lowest concentrations, except for mercury which was higher in least terns. As expected, DDT concentrations were elevated near the Palos Verdes Superfund site. However, we also detected a previously unknown latitudinal pattern in PBDE concentrations in least terns. POP congener profiles also confirmed differences in contamination in urban least tern colonies closest to urban centers. Though toxicants were at detectable levels across species and sites, concentrations were below those known to cause adverse effects in avian taxa and are steady or declining compared to previous studies in this region. Our results suggest that regional seabird monitoring can inform site-specific remediation and support management and protection of regionally-threatened wildlife and coastal systems. Integration of seabird contaminant data with traditional sediment, water, bivalve and fish monitoring is needed to further our understanding of exposure pathways and food web contaminant transfer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This work presents the results of an international interlaboratory comparison on ex situ passive sampling in sediments. The main objectives were to map the state of the science in passively sampling sediments, identify sources of variability, provide recommendations and practical guidance for standardized passive sampling, and advance the use of passive sampling in regulatory decision making by increasing confidence in the use of the technique. The study was performed by a consortium of 11 laboratories and included experiments with 14 passive sampling formats on 3 sediments for 25 target chemicals (PAHs and PCBs). The resulting overall interlaboratory variability was large (a factor of ∼10), but standardization of methods halved this variability. The remaining variability was primarily due to factors not related to passive sampling itself, i.e., sediment heterogeneity and analytical chemistry. Excluding the latter source of variability, by performing all analyses in one laboratory, showed that passive sampling results can have a high precision and a very low intermethod variability (

High-throughput cell assays that detect and integrate the response of multiple chemicals acting via a common mode of action have the potential to enhance current environmental monitoring practices. Establishing the linkage between in vitro and in vivo responses is key to demonstrating that in vitro cell assays can be predictive of ecologically relevant outcomes. The present study investigated the potency of 17β-estradiol (E2), estrone (E1), nonylphenol (NP), and treated wastewater effluent using the readily available GeneBLAzer ® estrogen receptor transactivation assay and 2 life stages of the inland silverside (Menidia beryllina). In vitro estrogenic potencies were ranked as follows: E2 > E1 >> NP. All 3 model estrogens induced vitellogenin and choriogenin expression in a dose-dependent manner in larvae and juveniles. However, apical effects were only found for E2 and E1 exposures of juveniles, which resulted in female-skewed sex ratios. Wastewater effluent samples exhibiting low in vitro estrogenicity (below the 10% effective concentration [EC10]), did not cause significant changes in M. beryllina. Significant induction of estrogen-responsive genes was observed at concentrations 6 to 26 times higher than in vitro responses. Gonadal feminization occurred at concentrations at least 19 to 26 times higher than the in vitro responses. These findings indicated that in vitro cell assays were more sensitive than the fish assays, making it possible to develop in vitro effect thresholds protective of aquatic organisms. Environ Toxicol Chem 2018;37:884-892. © 2017 SETAC., (© 2017 SETAC.)

Swimming and recreating in lakes, oceans, and rivers is common, yet the literature suggests children may be at greater risk of illness following such exposures. These effects might be due to differences in immunity or differing behavioral factors such as poorer hygiene, longer exposures to, and greater ingestion of potentially contaminated water and sand. We pooled data from 12 prospective cohorts (n=68,685) to examine exposures to potentially contaminated media such as beach water and sand among children compared with adults, and conducted a simulation using self-reported time spent in the water and volume of water swallowed per minute by age to estimate the total volume of water swallowed per swimming event by age category. Children aged 4-7 and 8-12 years had the highest exposures to water, sand, and algae compared with other age groups. Based on our simulation, we found that children (6-12 years) swallow a median of 36 ml (90th percentile=150 ml), whereas adults aged ≥35 years swallow 9 ml (90th percentile=64 ml) per swimming event, with male children swallowing a greater amount of water compared with females. These estimates may help to reduce uncertainty surrounding routes and durations of recreational exposures and can support the development of chemical and microbial risk assessments.

Some current-use pesticides are chiral and have nonsuperimposable mirror images called enantiomers that exhibit identical physical-chemical properties but can behave differently when in contact with other chiral molecules (e.g., regarding degradation and uptake). These differences can result in variations in enantiomer presence in the environment and potentially change the toxicity of pesticide residues. Several current-use chiral pesticides are applied in urban and agricultural areas, with increased potential to enter watersheds and adversely affect aquatic organisms. The present study describes a stereoselective analytical method for the current-use pesticides fipronil, cis-bifenthrin, cis-permethrin, cypermethrin, and cyfluthrin. We show use of the method by characterizing enantiomer fractions in environmental sample extracts (sediment and water), and laboratory-dosed fish and concrete extracts previously collected by California organizations. Enantiomer fractions for most environmental samples are the same as racemic standards (equal amounts of enantiomers, enantiomer fraction = 0.5) and therefore are not expected to differ in toxicity from racemic mixtures typically tested. In laboratory-derived samples, enantiomer fractions are more frequently nonracemic and favor the less toxic enantiomer; permethrin enantiomer fractions range from 0.094 to 0.391 in one type of concrete runoff and enantiomer fractions of bifenthrin in dosed fish range from 0.378 to 0.499. We use enantiomer fractions as a screening tool to understand environmental exposure and explore ways this uncommon measurement could be used to better understand toxicity and risk. Environ Toxicol Chem 2018;37:99-106. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America., (Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

Quantitative descriptions of chemical, physical, and biological characteristics of estuaries are critical for developing an ecological understanding of drivers of change. Historical trends and relationships between key species of dissolved inorganic nitrogen (ammonium, nitrate/nitrite, total) from the Delta region of the San Francisco Estuary were modeled with an estuarine adaptation of the Weighted Regressions on Time, Discharge, and Season (WRTDS). Analysis of flow-normalized data revealed trends that were different from those in the observed time-series. Flow-normalized data exhibited changes in magnitude and even reversal of trends relative to the observed data. Modelled trends demonstrated that nutrient concentrations were on average higher in the last twenty years relative to the earlier periods of observation, although concentrations have been slowly declining since the mid-1990s and early 2000s. We further describe mechanisms of change with two case studies that evaluated 1) downstream changes in nitrogen following upgrades at a wastewater treatment plant, and 2) interactions between biological invaders, chlorophyll, macro-nutrients (nitrogen and silica), and flow in Suisun Bay. WRTDS results for ammonium trends showed a distinct signal as a result of upstream wastewater treatment plant upgrades, with specific reductions observed in the winter months during low-flow conditions. Results for Suisun Bay showed that chlorophyll a production in early years was directly stimulated by flow, whereas the relationship with flow in later years was indirect and influenced by grazing pressure. Although these trends and potential causes of change have been described in the literature, results from WRTDS provided an approach to test alternative hypotheses of spatiotemporal drivers of nutrient dynamics in the Delta., Competing Interests: The authors declare no competing financial interest.

Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and management. However, there are currently no standardized approaches for field implementation and interpretation of qPCR data. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and a novel Bayesian weighted average approach to establish a human fecal contamination score (HFS) that can be used to prioritize sampling sites for remediation based on measured human waste levels. The HFS was then used to investigate 975 study design scenarios utilizing different combinations of sites with varying sampling intensities (daily to once per week) and number of qPCR replicates per sample (2-14 replicates). Findings demonstrate that site prioritization with HFS is feasible and that both sampling intensity and number of qPCR replicates influence reliability of HFS estimates. The novel data analysis strategy presented here provides a prescribed approach for the implementation and interpretation of human-associated HF183/BacR287 qPCR data with the goal of site prioritization based on human fecal pollution levels. In addition, information is provided for future users to customize study designs for optimal HFS performance., (Published by Elsevier Ltd.)

The coupling of disposable solid-phase microextraction (SPME) with performance reference compounds (PRCs) has been recently introduced to measure time-averaged freely dissolved concentrations (C free ) of hydrophobic organic contaminants in sediments under laboratory conditions. To explore the use of PRC-SPME for in situ sampling in seawater, disposable PDMS fibers (35-μm and 100-μm coating) preloaded with stable isotope labeled analogues as PRCs were deployed at six stations (each with three depths) in the open ocean of the Palos Verdes Shelf (CA, USA) Superfund site for 33d to measure C free of DDT and its degradates. The observed values of fractional equilibration (f eq ) of PRCs were mostly <0.85, suggesting nonequilibrium conditions at the end of deployment. The observed f eq s for the samplers varied with compound, sampling station and depth, validating the need for calibration to derive accurate C free . The C free values of DDE and DDD determined with PRC-SPME were in good agreement with those previously measured by in situ large-volume water sampling or polyethylene devices. The highest C free in seawater 5m off the ocean floor was 750pgL -1 for o,p'-DDE, 2170pgL -1 for p,p'-DDE, 24pgL -1 for o,p'-DDD, and 75pgL -1 for p,p'-DDD. Results of this study demonstrated the feasibility and advantages of using disposable PDMS fiber coupled with PRCs for in situ sampling., (Copyright © 2017 Elsevier B.V. All rights reserved.)

San Francisco Bay (SFB), USA, is highly enriched in nitrogen and phosphorus, but has been resistant to the classic symptoms of eutrophication associated with over-production of phytoplankton. Observations in recent years suggest that this resistance may be weakening, shown by: significant increases of chlorophyll- a ( chl-a ) and decreases of dissolved oxygen (DO), common occurrences of phytoplankton taxa that can form Harmful Algal Blooms (HAB), and algal toxins in water and mussels reaching levels of concern. As a result, managers now ask: what levels of chl-a in SFB constitute tipping points of phytoplankton biomass beyond which water quality will become degraded, requiring significant nutrient reductions to avoid impairments? We analyzed data for DO, phytoplankton species composition, chl-a , and algal toxins to derive quantitative relationships between three indicators (HAB abundance, toxin concentrations, DO) and chl-a . Quantile regressions relating HAB abundance and DO to chl-a were significant, indicating SFB is at increased risk of adverse HAB and low DO levels if chl-a continues to increase. Conditional probability analysis (CPA) showed chl-a of 13 mg m -3 as a "protective" threshold below which probabilities for exceeding alert levels for HAB abundance and toxins were reduced. This threshold was similar to chl-a of 13 - 16 mg m -3 that would meet a SFB-wide 80 % saturation Water Quality Criterion (WQC) for DO. Higher "at risk" chl-a thresholds from 25 - 40 mg m -3 corresponded to 0.5 probability of exceeding alert levels for HAB abundance, and for DO below a WQC of 5.0 mg L -1 designated for lower South Bay (LSB) and South Bay (SB). We submit these thresholds as a basis to assess eutrophication status of SFB and to inform nutrient management actions. This approach is transferrable to other estuaries to derive chl-a thresholds protective against eutrophication.

The impact of unmonitored contaminants, also known as contaminants of emerging concern (CECs), on freshwater streams remains largely uncharacterized. Water samples from 31 streams representing urban, agricultural and undeveloped (i.e., open space) land use in Southern California (USA) were analyzed for in vitro and in vivo bioactivity. The extent and magnitude of bioactivity screened using endocrine-responsive cell bioassays and a fish embryo screening assay were low. In contrast, a wider gradient of responses for the aryl hydrocarbon receptor (AhR) assay was observed, which was negatively correlated with a measure of benthic community structure. Both aromatic and non-aromatic CECs were tentatively identified in these samples, but polycyclic aromatic hydrocarbons (PAHs), known AhR agonists in urban environments, were not present at detectable levels. These results suggest that a combination of in vitro and in vivo show potential as screening techniques for biological condition in situ, but that more advanced, comprehensive analytical methods are needed to identify bioactive contaminants.

We modeled the risk of gastrointestinal (GI) illness associated with recreational exposures to marine water following storm events in San Diego County, California. We estimated GI illness risks via quantitative microbial risk assessment (QMRA) techniques by consolidating site specific pathogen monitoring data of stormwater, site specific dilution estimates, literature-based water ingestion data, and literature based pathogen dose-response and morbidity information. Our water quality results indicated that human sources of contamination contribute viral and bacterial pathogens to streams draining an urban watershed during wet weather that then enter the ocean and affect nearshore water quality. We evaluated a series of approaches to account for uncertainty in the norovirus dose-response model selection and compared our model results to those from a concurrently conducted epidemiological study that provided empirical estimates for illness risk following ocean exposure. The preferred norovirus dose-response approach yielded median risk estimates for water recreation-associated illness (15 GI illnesses per 1000 recreation events) that closely matched the reported epidemiological results (12 excess GI illnesses per 1000 wet weather recreation events). The results are consistent with norovirus, or other pathogens associated with norovirus, as an important cause of gastrointestinal illness among surfers in this setting. This study demonstrates the applicability of QMRA for recreational water risk estimation, even under wet weather conditions and describes a process that might be useful in developing site-specific water quality criteria in this and other locations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Host-associated genetic markers that allow for fecal source identification have been used extensively as a diagnostic tool to determine fecal sources within watersheds, but have not been used in routine monitoring to prioritize remediation actions among watersheds. Here, we present a regional assessment of human marker prevalence among drainages that discharge to the U.S. southern California coast. Approximately 50 samples were analyzed for the HF183 human marker from each of 22 southern California coastal drainages under summer dry weather conditions, and another 50 samples were targeted from each of 23 drainages during wet weather. The HF183 marker was ubiquitous, detected in all but two sites in dry weather and at all sites during wet weather. However, there was considerable difference in the extent of human fecal contamination among sites. Similar site ranking was produced regardless of whether the assessment was based on frequency of HF183 detection or site average HF183 concentration. However, site ranking differed greatly between dry and wet weather. Site ranking also differed greatly when based on enterococci, which do not distinguish between pollution sources, vs. HF183, which distinguishes higher risk human fecal sources from other sources, indicating the additional value of the human-associated marker as a routine monitoring tool., Competing Interests: The authors declare no conflict of interest.

In this study, the aging of culturable FIB and DNA representing genetic markers for Enterococcus spp. (ENT1A), general Bacteroides (GB3), and human-associated Bacteroides (HF183) in freshwater sediments was evaluated. Freshwater sediment was collected from four different sites within the upper and lower reach of the Topanga Creek Watershed and two additional comparator sites within the Santa Monica Bay, for a total of six sites. Untreated (ambient) and oven-dried (reduced microbiota) sediment was inoculated with 5% sewage and artificial freshwater. Microcosms were held for a 21-day period and sampled on day 0, 1, 3, 5, 7, 12, and 21. There were substantial differences in decay among the sediments tested, and decay rates were related to sediment characteristics. In the ambient sediments, smaller particle size and higher levels of organic matter and nutrients (nitrogen and phosphorus) were associated with increased persistence of the GB3 marker and culturable Escherichia coli (cEC) and enterococci (cENT). The HF183 marker exhibited decay rates of -0.50 to -0.96 day -1 , which was 2-5 times faster in certain ambient sediments than decay of culturable FIB and the ENT1A and GB3 markers. The ENT1A and GB3 markers decayed at rates of between -0.07 and -0.28 and -0.10 to -0.44 day -1 , and cEC and cENT decayed at rates of between -0.22 and -0.81 and -0.03 and -0.40 day -1 , respectively. In the oven-dried sediments, increased persistence of all indicators and potential for limited growth of culturable FIB and the GB3 and ENT1A markers was observed. A simplified two-box model using the HF183 marker and cENT decay rates generated from the microcosm experiments was applied to two reaches within the Topanga Canyon watershed in order to provide context for the variability in decay rates observed. The model predicted lower ambient concentrations of enterococci in sediment in the upper (90 MPN g -1 ) versus lower Topanga watershed (530 MPN g -1 ) and low ambient levels of the HF183 marker (below the LLOQ) in sediments in both lower and upper watersheds. It is important to consider the variability in the persistence of genetic markers and FIB when evaluating indicators of fecal contamination in sediments, even within one watershed., (Copyright © 2017. Published by Elsevier Ltd.)

Toxin producing cyanobacterial blooms have increased globally in recent decades in both frequency and intensity. Despite the recognition of this growing risk, the extent and magnitude of cyanobacterial blooms and cyanotoxin prevalence is poorly characterized in the heavily populated region of southern California. Recent assessments of lentic waterbodies (depressional wetlands, lakes, reservoirs and coastal lagoons) determined the prevalence of microcystins and, in some cases, additional cyanotoxins. Microcystins were present in all waterbody types surveyed although toxin concentrations were generally low across most habitats, as only a small number of sites exceeded California's recreational health thresholds for acute toxicity. Results from passive samplers (Solid Phase Adsorption Toxin Tracking (SPATT)) indicated microcystins were prevalent throughout lentic waterbodies and that traditional discrete samples underestimated the presence of microcystins. Multiple cyanotoxins were detected simultaneously in some systems, indicating multiple stressors, the risk of which is uncertain since health thresholds are based on exposures to single toxins. Anatoxin-a was detected for the first time from lakes in southern California. The persistence of detectable microcystins across years and seasons indicates a low-level, chronic risk through both direct and indirect exposure. The influence of toxic cyanobacterial blooms is a more complex stressor than presently recognized and should be included in water quality monitoring programs., Competing Interests: The authors declare no conflict of interest.

The pteropod Limacina helicina frequently experiences seasonal exposure to corrosive conditions (Ω ar   < 1) along the US West Coast and is recognized as one of the species most susceptible to ocean acidification (OA). Yet, little is known about their capacity to acclimatize to such conditions. We collected pteropods in the California Current Ecosystem (CCE) that differed in the severity of exposure to Ω ar conditions in the natural environment. Combining field observations, high-CO 2 perturbation experiment results, and retrospective ocean transport simulations, we investigated biological responses based on histories of magnitude and duration of exposure to Ω ar  < 1. Our results suggest that both exposure magnitude and duration affect pteropod responses in the natural environment. However, observed declines in calcification performance and survival probability under high CO 2 experimental conditions do not show acclimatization capacity or physiological tolerance related to history of exposure to corrosive conditions. Pteropods from the coastal CCE appear to be at or near the limit of their physiological capacity, and consequently, are already at extinction risk under projected acceleration of OA over the next 30 years. Our results demonstrate that Ω ar exposure history largely determines pteropod response to experimental conditions and is essential to the interpretation of biological observations and experimental results.

Rising global temperatures are causing increases in the frequency and severity of extreme climatic events, such as floods, droughts, and heat waves. We analyze changes in summer temperatures, the frequency, severity, and duration of heat waves, and heat-related mortality in India between 1960 and 2009 using data from the India Meteorological Department. Mean temperatures across India have risen by more than 0.5°C over this period, with statistically significant increases in heat waves. Using a novel probabilistic model, we further show that the increase in summer mean temperatures in India over this period corresponds to a 146% increase in the probability of heat-related mortality events of more than 100 people. In turn, our results suggest that future climate warming will lead to substantial increases in heat-related mortality, particularly in developing low-latitude countries, such as India, where heat waves will become more frequent and populations are especially vulnerable to these extreme temperatures. Our findings indicate that even moderate increases in mean temperatures may cause great increases in heat-related mortality and support the efforts of governments and international organizations to build up the resilience of these vulnerable regions to more severe heat waves.

The United States's Clean Water Act stipulates in section 303(d) that states must identify impaired water bodies for which total maximum daily loads (TMDLs) of pollution inputs into water bodies are developed. Decision-making procedures about how to list, or delist, water bodies as impaired, or not, per Clean Water Act 303(d) differ across states. In states such as California, whether or not a particular monitoring sample suggests that water quality is impaired can be regarded as a binary outcome variable, and California's current regulatory framework invokes a version of the exact binomial test to consolidate evidence across samples and assess whether the overall water body complies with the Clean Water Act. Here, we contrast the performance of California's exact binomial test with one potential alternative, the Sequential Probability Ratio Test (SPRT). The SPRT uses a sequential testing framework, testing samples as they become available and evaluating evidence as it emerges, rather than measuring all the samples and calculating a test statistic at the end of the data collection process. Through simulations and theoretical derivations, we demonstrate that the SPRT on average requires fewer samples to be measured to have comparable Type I and Type II error rates as the current fixed-sample binomial test. Policymakers might consider efficient alternatives such as SPRT to current procedure., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cyclooxygenase (COX) inhibitors are ubiquitous in aquatic systems and have been detected in fish tissues. The exposure of fish to these pharmaceuticals is concerning because COX inhibitors disrupt the synthesis of prostaglandins (PGs), which modulate a variety of essential biological functions, including reproduction. In this study, we investigated the effects of well-characterized mammalian COX inhibitors on female fathead minnow reproductive health. Fish (n = 8) were exposed for 96 h to water containing indomethacin (IN; 100 µg/l), ibuprofen (IB; 200 µg/l) or celecoxib (CX; 20 µg/l), and evaluated for effects on liver metabolome and ovarian gene expression. Metabolomic profiles of IN, IB and CX were not significantly different from control or one another. Exposure to IB and CX resulted in differential expression of comparable numbers of genes (IB = 433, CX = 545). In contrast, 2558 genes were differentially expressed in IN-treated fish. Functional analyses (canonical pathway and gene set enrichment) indicated extensive effects of IN on PG synthesis pathway, oocyte meiosis, and several other processes consistent with physiological roles of PGs. Transcriptomic data were congruent with PG data; IN-reduced plasma PG F2α concentration, whereas IB and CX did not. Five putative AOPs were developed linking the assumed molecular initiating event of COX inhibition, with PG reduction and the adverse outcome of reproductive failure via reduction of: (1) ovulation, (2) reproductive behaviors mediated by exogenous or endogenous PGs, and (3) oocyte maturation in fish. These pathways were developed using, in part, empirical data from the present study and other publicly available data., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Blooms of toxic cyanobacteria in freshwater ecosystems have received considerable attention in recent years, but their occurrence and potential importance at the land-sea interface has not been widely recognized. Here we present the results of a survey of discrete samples conducted in more than fifty brackish water sites along the coastline of southern California. Our objectives were to characterize cyanobacterial community composition and determine if specific groups of cyanotoxins (anatoxins, cylindrospermopsins, microcystins, nodularins, and saxitoxins) were present. We report the identification of numerous potentially harmful taxa and the co-occurrence of multiple toxins, previously undocumented, at several locations. Our findings reveal a potential health concern based on the range of organisms present and the widespread prevalence of recognized toxic compounds. Our results raise concerns for recreation, harvesting of finfish and shellfish, and wildlife and desalination operations, highlighting the need for assessments and implementation of monitoring programs. Such programs appear to be particularly necessary in regions susceptible to urban influence.

To catalog the diversity and abundance of halogenated organic compounds (HOCs) accumulating in high trophic marine species from the southwestern Atlantic Ocean, tissue from bottlenose dolphins (Tursiops truncatus) stranded or incidentally captured along the coast of Rio de Janeiro, Brazil, were analyzed by a nontargeted approach based on GC×GC/TOF-MS. A total of 158 individual HOCs from 32 different structural classes were detected in the blubber of 4 adult male T. truncatus. Nearly 90% of the detected compounds are not routinely monitored in the environment. DDT-related and mirex/dechlorane-related compounds were the most abundant classes of anthropogenic origin. Methoxy-brominated diphenyl ethers (MeO-BDEs) and chlorinated methyl- and dimethyl bipyrroles (MBPs and DMBPs) were the most abundant natural products. Reported for the first time in southwestern Atlantic cetaceans and in contrast to North American marine mammals, chlorinated MBPs and DMBPs were more abundant than their brominated and/or mixed halogenated counterparts. HOC profiles in coastal T. truncatus from Brazil and California revealed a distinct difference, with a higher abundance of MeO-BDEs, mirex/dechloranes and chlorinated bipyrroles in the Brazilian dolphins. Thirty-six percent of the detected HOCs had an unknown structure. These results suggest broad geographical differences in the patterns of bioaccumulative chemicals found in the marine environment and indicate the need to develop more complete catalogs of HOCs from various marine environments.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17β-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell "kits", can be adapted to screen for endocrine activity in water.

Nontargeted GC×GC-TOF/MS analysis of blubber from 8 common bottlenose dolphins (Tursiops truncatus) inhabiting the Southern California Bight was performed to identify novel, bioaccumulative DDT-related compounds and to determine their abundance relative to the commonly studied DDT-related compounds. We identified 45 bioaccumulative DDT-related compounds of which the majority (80%) is not typically monitored in environmental media. Identified compounds include transformation products, technical mixture impurities such as tris(chlorophenyl)methane (TCPM), the presumed TCPM metabolite tris(chlorophenyl)methanol (TCPMOH), and structurally related compounds with unknown sources, such as hexa- to octachlorinated diphenylethene. To investigate impurities in pesticide mixtures as possible sources of these compounds, we analyzed technical DDT, the primary source of historical contamination in the region, and technical Dicofol, a current use pesticide that contains DDT-related compounds. The technical mixtures contained only 33% of the compounds identified in the blubber, suggesting that transformation products contribute to the majority of the load of DDT-related contaminants in these sentinels of ocean health. Quantitative analysis revealed that TCPM was the second most abundant compound class detected in the blubber, following DDE, and TCPMOH loads were greater than DDT. QSPR estimates verified 4,4',4″-TCPM and 4,4'4,″-TCPMOH are persistent and bioaccumulative.

Understanding the fate of enteric viruses in water is vital for protection of water quality. However, the decay of enteric viruses is not well characterized, and its uncertainty has not been examined yet. In this study, the decay of coliphages, an indicator for enteric viruses, was investigated in situ under both sunlit and shaded conditions as well as in summer and winter. The decay rates of coliphages and their uncertainties were analyzed using a Bayesian approach. The results from the summer experiments revealed that the decay rates of somatic coliphages were significantly higher in sunlight (1.29 ± 0.06 day -1 ) than in shade (0.96 ± 0.04 day -1 ), but the decay rates of male-specific (F+) coliphages were not significantly different between sunlight (1.09 ± 0.09 day -1 ) and shaded treatments (1.11 ± 0.08 day -1 ). The decay rates of both F+ coliphages (0.25 ± 0.02 day -1 ) and somatic coliphages (0.12 ± 0.01 day -1 ) in winter were considerably lower than those in summer. Temperature and chlorophyll a (chla) concentration varied significantly (p < 0.001) between the two seasons, suggesting that these parameters might be important contributors to the seasonal variation of coliphage decay. Additionally, the Bayesian approach provided full distributions of decay rates and reduced the uncertainty, offering useful information for comparing decay rates under different conditions.

The State of California conducted an extensive and systematic survey of mercury (Hg) in fish from the California coast in 2009 and 2010. The California survey sampled 3483 fish representing 46 species at 68 locations, and demonstrated that methylHg in fish presents a widespread exposure risk to fish consumers. Most of the locations sampled (37 of 68) had a species with an average concentration above 0.3μg/gwet weight (ww), and 10 locations an average above 1.0μg/gww. The recent and robust dataset from California provided a basis for a broader examination of spatial and temporal patterns in fish Hg in coastal waters of Western North America. There is a striking lack of data in publicly accessible databases on Hg and other contaminants in coastal fish. An assessment of the raw data from these databases suggested the presence of relatively high concentrations along the California coast and in Puget Sound, and relatively low concentrations along the coasts of Alaska and Oregon, and the outer coast of Washington. The dataset suggests that Hg concentrations of public health concern can be observed at any location on the coast of Western North America where long-lived predator species are sampled. Output from a linear mixed-effects model resembled the spatial pattern observed for the raw data and suggested, based on the limited dataset, a lack of trend in fish Hg over the nearly 30-year period covered by the dataset. Expanded and continued monitoring, accompanied by rigorous data management procedures, would be of great value in characterizing methylHg exposure, and tracking changes in contamination of coastal fish in response to possible increases in atmospheric Hg emissions in Asia, climate change, and terrestrial Hg control efforts in coastal watersheds., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Although chemosynthetic ecosystems are known to support diverse assemblages of microorganisms, the ecological and environmental factors that structure microbial eukaryotes (heterotrophic protists and fungi) are poorly characterized. In this study, we examined the geographic, geochemical and ecological factors that influence microbial eukaryotic composition and distribution patterns within Hydrate Ridge, a methane seep ecosystem off the coast of Oregon using a combination of high-throughput 18S rRNA tag sequencing, terminal restriction fragment length polymorphism fingerprinting, and cloning and sequencing of full-length 18S rRNA genes. Microbial eukaryotic composition and diversity varied as a function of substrate (carbonate versus sediment), activity (low activity versus active seep sites), sulfide concentration, and region (North versus South Hydrate Ridge). Sulfide concentration was correlated with changes in microbial eukaryotic composition and richness. This work also revealed the influence of oxygen content in the overlying water column and water depth on microbial eukaryotic composition and diversity, and identified distinct patterns from those previously observed for bacteria, archaea and macrofauna in methane seep ecosystems. Characterizing the structure of microbial eukaryotic communities in response to environmental variability is a key step towards understanding if and how microbial eukaryotes influence seep ecosystem structure and function., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Objectives: To provide summary estimates of gastroenteritis risks and illness burden associated with recreational water exposure and determine whether children have higher risks and burden., Methods: We combined individual participant data from 13 prospective cohorts at marine and freshwater beaches throughout the United States (n = 84 411). We measured incident outcomes within 10 days of exposure: diarrhea, gastrointestinal illness, missed daily activity (work, school, vacation), and medical visits. We estimated the relationship between outcomes and 2 exposures: body immersion swimming and Enterococcus spp. fecal indicator bacteria levels in the water. We also estimated the population-attributable risk associated with these exposures., Results: Water exposure accounted for 21% of diarrhea episodes and 9% of missed daily activities but was unassociated with gastroenteritis leading to medical consultation. Children aged 0 to 4 and 5 to 10 years had the most water exposure, exhibited stronger associations between levels of water quality and illness, and accounted for the largest attributable illness burden., Conclusions: The higher gastroenteritis risk and associated burden in young children presents important new information to inform future recreational water quality guidelines designed to protect public health.

To examine the occurrence and fate of contaminants of emerging concern (CECs) and inform future monitoring of CECs in coastal urban waterways, water, sediment, and fish tissue samples were collected and analyzed for a broad suite of pharmaceuticals and personal care products (PPCPs), commercial and/or household chemicals, current use pesticides, and hormones in an effluent-dominated river and multiple embayments in southern California (USA). In the Santa Clara River, which receives treated wastewater from several facilities, aqueous phase CECs were detectable at stations nearest discharges from municipal wastewater treatment plants but were attenuated downstream. Sucralose and the chlorinated phosphate flame retardants tris(1-chloro-2-propyl) phosphate (TCPP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP), and tris(2-chloroethyl) phosphate (TCEP) were most abundant in water, with maximum concentrations of 35 μg/L, 3.3 μg/L, 1.4 μg/L, and 0.81 μg/L, respectively. Triclocarban, an antimicrobial agent in use for decades, was more prevalent in water than triclosan or nonylphenol. Maximum concentrations of bifenthrin, permethrin, polybrominated diphenyl ethers (PBDEs), and degradates of fipronil exceeded CEC-specific monitoring trigger levels recently established for freshwater and estuarine sediments by factors of 10 to 1000, respectively. Maximum fish tissue concentrations of PBDEs varied widely (370 ng/g and 7.0 ng/g for the Santa Clara River and coastal embayments, respectively), with most species exhibiting concentrations at the lower end of this range. These results suggest that continued monitoring of pyrethroids, PBDEs, and degradates of fipronil in sediment is warranted in these systems. In contrast, aqueous pharmaceutical concentrations in the Santa Clara River were not close to exceeding current monitoring trigger levels, suggesting a lower priority for targeted monitoring in this medium. Environ Toxicol Chem 2016;35:1986-1994. © 2016 SETAC., (© 2016 SETAC.)

The chemical-specific risk-based paradigm that informs monitoring and assessment of environmental contaminants does not apply well to the many thousands of new chemicals that are being introduced into ambient receiving waters. We propose a tiered framework that incorporates bioanalytical screening tools and diagnostic nontargeted chemical analysis to more effectively monitor for contaminants of emerging concern (CECs). The framework is based on a comprehensive battery of in vitro bioassays to first screen for a broad spectrum of CECs and nontargeted analytical methods to identify bioactive contaminants missed by the currently favored targeted analyses. Water quality managers in California have embraced this strategy with plans to further develop and test this framework in regional and statewide pilot studies on waterbodies that receive discharge from municipal wastewater treatment plants and stormwater runoff. In addition to directly informing decisions, the data obtained using this framework can be used to construct and validate models that better predict CEC occurrence and toxicity. The adaptive interplay among screening results, diagnostic assessment and predictive modeling will allow managers to make decisions based on the most current and relevant information, instead of extrapolating from parameters with questionable linkage to CEC impacts. Integr Environ Assess Manag 2016;12:540-547. © 2015 SETAC., (© 2015 SETAC.)

High concentrations of DDT and metabolites (ΣDDT) have been detected in sediment and the demersal flatfish hornyhead turbot (Pleuronichtys verticalis) collected from Palos Verdes (PV), California, USA, a site contaminated with over 100 metric tons of DDT throughout 1960s-70s. This study was conducted to assess the transfer of ΣDDT from PV-sediment into polychaetes (Neanthes arenaceodentata) and hornyhead turbot, and to investigate if the responses in turbots from two different laboratory exposures mimic those in turbots caught in PV (PV-turbot). Turbot fed PV-sediment-contaminated polychaete for 7 days had liver concentrations of ΣDDT similar to PV-turbot. After 28 days, ΣDDT also accumulated in livers of turbot gavaged with a ΣDDT mixture. In vitro cell bioassays indicated significant increases of 17β-estradiol equivalents (EEQ) in turbot bile extracts as compared to the control in the 7-day study. These responses corresponded to those measured in PV-fish. Glucocorticoid receptor (GR), anti-androgen receptor (anti-AR), estrogen receptor (ER) or aryl hydrocarbon receptor (AhR) activities were also observed in extracts of PV-sediment, and PV-sediment-exposed worm. Anti-AR, AhR and GR activities were significantly higher in PV-sediment than reference sediment (San Diego, SD). Higher transcripts of hepatic VTG, ERα and ERβ were found in PV-turbot than SD-turbot, but were unaltered in fish exposed to sediment-contaminated worms for the 7-day study. In contrast, liver extracts from the 28-day treatment of ΣDDT showed lower EEQ but similar hepatic VTG and ERβ transcripts relative to those of PV-turbot. These data indicated that trophic transfer of sediment-associated DDT in 7-day exposures corresponded to field measurements of DDT residues and in vitro ER bioactivities, but failed to mimic in vivo biological effects observed in field fish. In contrast, treatment with ΣDDT alone for 28 days mimicked in vivo biological effects of DDTs in PV fish, but did not correspond to liver concentrations or in vitro bioactivities., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The decay of sewage-sourced Escherichia coli and enterococci was measured at multiple depths in a freshwater marsh, a brackish water lagoon, and a marine site, all located in California. The marine site had very clear water, while the waters from the marsh and lagoon contained colored dissolved organic matter that not only blocked light but also produced reactive oxygen species. First order decay rate constants of both enterococci and E. coli were between 1 and 2 d(-1) under low light conditions and as high as 6 d(-1) under high light conditions. First order decay rate constants were well correlated to the daily average UVB light intensity corrected for light screening incorporating water absorbance and depth, suggesting endogenous photoinactivation is a major pathway for bacterial decay. Additional laboratory experiments demonstrated the presence of colored dissolved organic matter in marsh water enhanced photoinactivation of a laboratory strain of Enterococcus faecalis, but depressed photoinactivation of sewage-sourced enterococci and E. coli after correcting for UVB light screening, suggesting that although the exogenous indirect photoinactivation mechanism may be active against Ent. faecalis, it is not for the sewage-source organisms. A simple linear regression model based on UVB light intensity appears to be a useful tool for predicting inactivation rate constants in natural waters of any depth and absorbance.

Introduction: Advances in molecular methods provide new opportunities for directly measuring pathogens or host-associated markers of fecal pollution instead of relying on fecal indicator bacteria (FIB) alone for beach water quality monitoring. Adoption of new indicators depends on identifying relationships between either the presence or concentration of the indicators and illness among swimmers. Here we present results from three epidemiologic studies in which a broad range of bacterial and viral indicators of fecal contamination were measured simultaneously by either culture or molecular methods along with Enterococcus to assess whether they provide better health risk prediction than current microbial indicators of recreational water quality., Methods: We conducted prospective cohort studies at three California beaches -- Avalon Bay (Avalon), Doheny State Beach (Doheny), Surfrider State Beach (Malibu) -- during the summers of 2007, 2008 and 2009. The studies enrolled 10,785 swimmers across the beaches and recorded each swimmer's water exposure. Water and sand samples were collected several times per day at multiple locations at each beach and analyzed for up to 41 target indicators using 67 different methodologies. Interviewers contacted participants by phone 10-14 days later and recorded symptoms of gastrointestinal illness occurring after their beach visit. Regression models were used to evaluate the association between water quality indicators and gastrointestinal illness among swimmers at each beach., Results: F+ coliphage (measured using EPA Method 1602) exhibited a stronger association with GI illness than did EPA Method 1600 at the two beaches where it was measured, while a molecular method, F+ RNA Coliphage Genotype II, was the only indicator significantly associated with GI illness at Malibu. MRSA, a known pathogen, had the strongest association with GI illness of any microbe measured at Avalon. There were two methods targeting human-associated fecal anaerobic bacteria that were more strongly associated with GI illness than EPA Method 1600, but only at Avalon. No indicator combinations consistently had a higher odds ratio than EPA Method 1600, but one composite indicator, based on the number of pathogens detected at a beach, was significantly associated with gastrointestinal illness at both Avalon and Doheny when freshwater flow was high., Discussion: While EPA Method1600 performed adequately at two beaches based on its consistency of association with gastrointestinal illness and the precision of its estimated associations, F+ coliphage measured by EPA Method 1602 had a stronger association with GI illness under high risk conditions at the two beaches where it was measured. One indicator, F+ Coliphage Genotype II was the only indicator significantly associated with GI illness at Malibu. Several indicators, particularly those targeting human associated bacteria, exhibited relationships with GI illness that were equal to or greater than that of EPA Method 1600 at Avalon, which has a focused human fecal source. Our results suggest that site-specific conditions at each beach determine which indicator or indicators best predict GI illness., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta-delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta-delta tended to bias recoveries from apparent partially inhibitory samples on the high side which could help in avoiding potential underestimates of enterococci--an important consideration in a public health context. Control assay and delta-delta recovery results were largely consistent across the range of habitats sampled, and among laboratories. The methodological option that best balanced acceptable estimated target gene recoveries with method sensitivity and avoidance of underestimated enterococci densities was Method 1609 without extract dilution and using the delta-delta calculation method. The applicability of this method can be extended by the analysis of diluted extracts to sites where interference is indicated but, particularly in these instances, should be confirmed by augmenting the control assays with analyses for target gene recoveries from spiked target organisms., (Published by Elsevier B.V.)

Enterococci are fecal indicator bacteria used to monitor fecal pollution of recreational waters. When enterococci levels exceed health standards, fecal pollution is assumed as the cause. Enterococci growing on plants limit their usefulness as fecal indicator bacteria. Here we examined enterococcal growth on eelgrass in Mission Bay, CA where enterococci levels have exceeded water quality thresholds. A total of 69 eelgrass samples were collected from six sites, shaken to remove enterococci attached to plant surfaces and the eluant filtered onto culture media. Isolates were then identified to species using biochemical methods, and DNA typing by pulsed-field gel electrophoresis was done to assess clonality of strains. Enterococci concentrations among eelgrass ranged from 8 to 14 000 CFU g(-1) dry weight. The most predominant enterococcal species found were Enterococcus casseliflavus and E. hirae followed by E. faecalis. Cluster analysis indicated a high level of clonality among isolates across all species, with clonal isolates consistently associated with individual eelgrass samples. Finding high densities of E. casseliflavus, E. hirae and E. faecalis on eelgrass that included clonal strains indicates the capability of enterococcal growth on eelgrass. Amplification of enterococci on eelgrass presents challenges for regulatory agencies that interpret elevated levels of these bacteria as an indication of fecal pollution., (Published by Oxford University Press on behalf of FEMS 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Microorganisms are vital in mediating the earth's biogeochemical cycles; yet, despite our rapidly increasing ability to explore complex environmental microbial communities, the relationship between microbial community structure and ecosystem processes remains poorly understood. Here, we address a fundamental and unanswered question in microbial ecology: 'When do we need to understand microbial community structure to accurately predict function?' We present a statistical analysis investigating the value of environmental data and microbial community structure independently and in combination for explaining rates of carbon and nitrogen cycling processes within 82 global datasets. Environmental variables were the strongest predictors of process rates but left 44% of variation unexplained on average, suggesting the potential for microbial data to increase model accuracy. Although only 29% of our datasets were significantly improved by adding information on microbial community structure, we observed improvement in models of processes mediated by narrow phylogenetic guilds via functional gene data, and conversely, improvement in models of facultative microbial processes via community diversity metrics. Our results also suggest that microbial diversity can strengthen predictions of respiration rates beyond microbial biomass parameters, as 53% of models were improved by incorporating both sets of predictors compared to 35% by microbial biomass alone. Our analysis represents the first comprehensive analysis of research examining links between microbial community structure and ecosystem function. Taken together, our results indicate that a greater understanding of microbial communities informed by ecological principles may enhance our ability to predict ecosystem process rates relative to assessments based on environmental variables and microbial physiology.

Stormwater is a challenging source of coastal pollution to abate because stormwater also involves complex natural processes, and differentiating these processes from anthropogenic excesses is difficult. The goal of this study was to identify the natural concentrations of stormwater constituents along the 1377 km coastline of California, USA. Twenty-eight ocean reference sites, a priori defined by lack of human disturbance in its adjacent watershed, were collected following 78 site-events and measured for 57 constituents and toxicity. Results indicated a complete lack of toxicity and undetectable levels of anthropogenic constituents (i.e., pesticides). The range of concentrations in ocean receiving waters for naturally-occurring constituents (i.e., total suspended solids, nutrients, trace metals) typically ranged three orders of magnitude. Regional differences and storm characteristics did not explain much of the variations in concentration. The reference site information is now being used to establish targets for marine protected areas subject to runoff from developed watersheds., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Transcriptomic analysis can complement traditional ecotoxicology data by providing mechanistic insight, and by identifying sub-lethal organismal responses and contaminant classes underlying observed toxicity. Before transcriptomic information can be used in monitoring and risk assessment, it is necessary to determine its reproducibility and detect key steps impacting the reliable identification of differentially expressed genes. A custom 15K-probe microarray was used to conduct transcriptomics analyses across six laboratories with estuarine amphipods exposed to cyfluthrin-spiked or control sediments (10 days). Two sample types were generated, one consisted of total RNA extracts (Ex) from exposed and control samples (extracted by one laboratory) and the other consisted of exposed and control whole body amphipods (WB) from which each laboratory extracted RNA. Our findings indicate that gene expression microarray results are repeatable. Differentially expressed data had a higher degree of repeatability across all laboratories in samples with similar RNA quality (Ex) when compared to WB samples with more variable RNA quality. Despite such variability a subset of genes were consistently identified as differentially expressed across all laboratories and sample types. We found that the differences among the individual laboratory results can be attributed to several factors including RNA quality and technical expertise, but the overall results can be improved by following consistent protocols and with appropriate training., (Published by Elsevier Ltd.)

Most Enterococcus faecalis and E. faecium are harmless to humans; however, strains harboring virulence genes, including esp, gelE, cylA, asa1, and hyl, have been associated with human infections. E. faecalis and E. faecium are present in beach waters worldwide, yet little is known about their virulence potential. Here, multiplex PCR was used to compare the distribution of virulence genes among E. faecalis and E. faecium isolated from beaches in Southern California and Puerto Rico to isolates from potential sources including humans, animals, birds, and plants. All five virulence genes were found in E. faecalis and E. faecium from beach water, mostly among E. faecalis. gelE was the most common among isolates from all source types. There was a lower incidence of asa1, esp, cylA, and hyl genes among isolates from beach water, sewage, septage, urban runoff, sea wrack, and eelgrass as compared to human isolates, indicating that virulent strains of E. faecalis and E. faecium may not be widely disseminated at beaches. A higher frequency of asa1 and esp among E. faecalis from dogs and of asa1 among birds (mostly seagull) suggests that further studies on the distribution and virulence potential of strains carrying these genes may be warranted.

In coastal waters, pesticides and parasites are widespread stressors that may separately and interactively affect the physiology, behavior, and survival of resident organisms. We investigated the effects of the organophosphate pesticide chlorpyrifos and the trematode parasite Euhaplorchis californiensis on three important traits of California killifish (Fundulus parvipinnis): neurotransmitter activity, release of the stress hormone cortisol, and behavior. Killifish were collected from a population without E. californiensis, and then half of the fish were experimentally infected. Following a 30 day period for parasite maturation, infected and uninfected groups were exposed to four concentrations of chlorpyrifos (solvent control, 1-3ppb) prior to behavior trials to quantify activity, feeding behavior, and anti-predator responses. Water-borne cortisol release rates were measured non-invasively from each fish prior to infection, one-month post-infection, and following pesticide exposure. Killifish exposed to 3ppb chlorpyrifos exhibited a 74.6±6.8% and 60.5±8.3% reduction in brain and muscle acetylcholinesterase (AChE) activity relative to controls. The rate of cortisol release was suppressed by each chlorpyrifos level relative to controls. Killifish exposed to the medium (2ppb) and high (3ppb) pesticide concentrations exhibited reduced activity and a decrease in mean swimming speed following a simulated predator attack. Muscle AChE was positively related to swimming activity while brain AChE was positively related to foraging behavior. ​No effects of the parasite were observed, possibly because of low metacercariae densities achieved through controlled infections. We found that sublethal pesticide exposure has the potential to modify several organismal endpoints with consequences for reduced fitness, including neurological, endocrine, and behavioral responses in an ecologically abundant fish., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Real-time quantitative PCR (qPCR) is increasingly being used for ambient water monitoring, but development of digital polymerase chain reaction (digital PCR) has the potential to further advance the use of molecular techniques in such applications. Digital PCR refines qPCR by partitioning the sample into thousands to millions of miniature reactions that are examined individually for binary endpoint results, with DNA density calculated from the fraction of positives using Poisson statistics. This direct quantification removes the need for standard curves, eliminating the labor and materials associated with creating and running standards with each batch, and removing biases associated with standard variability and mismatching amplification efficiency between standards and samples. Confining reactions and binary endpoint measurements to small partitions also leads to other performance advantages, including reduced susceptibility to inhibition, increased repeatability and reproducibility, and increased capacity to measure multiple targets in one analysis. As such, digital PCR is well suited for ambient water monitoring applications and is particularly advantageous as molecular methods move toward autonomous field application.