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In April 2015, the Governor of California mandated a 25% statewide reduction in water consumption (relative to 2013 levels) by urban water suppliers. The more than 400 public water agencies affected by the regulation were also required to report monthly progress towards the conservation goal to the State Water Resources Control Board. This paper uses the reported data to assess how the water utilities have responded to this mandate and to estimate the electricity savings and greenhouse gas (GHG) emissions reductions associated with reduced operation of urban water infrastructure systems. The results show that California succeeded in saving 524 000 million gallons (MG) of water (a 24.5% decrease relative to the 2013 baseline) over the mandate period, which translates into 1830 GWh total electricity savings, and a GHG emissions reduction of 521 000 metric tonnes of carbon dioxide equivalents (MT CO2e). For comparison, the total electricity savings linked to water conservation are approximately 11% greater than the savings achieved by the investor-owned electricity utilities' efficiency programs for roughly the same time period, and the GHG savings represent the equivalent of taking about 111 000 cars off the road for a year. These indirect, large-scale electricity and GHG savings were achieved at costs that were competitive with existing programs that target electricity and GHG savings directly and independently. Finally, given the breadth of the results produced, we built a companion website, called 'H2Open' (https://cwee.shinyapps.io/greengov/), to this research effort that allows users to view and explore the data and results across scales, from individual water utilities to the statewide summary.

Using data from the water service area of the East Bay Municipal Utility District in Northern California, we develop and discuss a method for assessing, at a high resolution, the energy intensity of water treated and delivered to customers of a major metropolitan water district. This method extends previous efforts by integrating hourly data from supervisory control and data acquisition systems with calculations based on the actual structure of the engineered infrastructure to produce a detailed understanding of energy use in space and time within the territory of a large-scale urban water provider. We found significant variations in the energy intensity of delivered potable water resulting from seasonal and topographic effects. This method enhances our understanding of the energy inputs for potable water systems and can be applied to the entire delivery and postuse water life cycle. A nuanced understanding of water's energy intensity in an urban setting enables more intelligent, targeted efforts to jointly conserve water and energy resources that take seasonal, distance, and elevation effects into account.

Machine learning (ML) is revolutionizing groundwater quality research by enhancing predictive accuracy and management strategies for contamination. This comprehensive review explores the evolution of ML technologies and their integration into environmental science, assessing 230 papers to understand the advancements and challenges in groundwater quality research. It reveals that a substantial portion of the research neglects critical preprocessing steps, crucial for model accuracy, with 83 % of the studies overlooking this phase. Furthermore, while model optimization is more commonly addressed, being implemented in 65 % of the papers, there is a noticeable gap in model interpretability, with only 15 % of the research providing explanations for model outcomes. Comparative evaluation of ML algorithms and careful selection of evaluation metrics are deemed essential for determining model fitness and reliability. The review underscores the need for interdisciplinary collaboration, methodological rigor, and continuous innovation to advance ML in groundwater management. By addressing these challenges and implementing solutions, the full potential of ML can be harnessed to tackle complex environmental issues and ensure sustainable groundwater management. This comprehensive and critical review paper can serve as a guiding framework to establish minimum standards for developing ML in groundwater quality studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Environmental concerns about microplastics (MPs) have motivated research of their sources, occurrence, and fate in aquatic and soil ecosystems. To mitigate the environmental impact of MPs, biodegradable plastics are designed to naturally decompose, thus reducing the amount of environmental plastic contamination. However, the environmental fate of biodegradable plastics and the products of their incomplete biodegradation, especially micro-biodegradable plastics (MBPs), remains largely unexplored. This comprehensive review aims to assess the risks of unintended consequences associated with the introduction of biodegradable plastics into the environment, namely, whether the incomplete mineralization of biodegradable plastics could enhance the risk of MBPs formation and thus, exacerbate the problem of their environmental dispersion, representing a potentially additional environmental hazard due to their presumed ecotoxicity. Initial evidence points towards the potential for incomplete mineralization of biodegradable plastics under both controlled and uncontrolled conditions. Rapid degradation of PLA in thermophilic industrial composting contrasts with the degradation below 50 % of other biodegradables, suggesting MBPs released into the environment through compost. Moreover, degradation rates of <60 % in anaerobic digestion for polymers other than PLA and PHAs suggest a heightened risk of MBPs in digestate, risking their spread into soil and water. This could increase MBPs and adsorbed pollutants' mobilization. The exact behavior and impacts of additive leachates from faster-degrading plastics remain largely unknown. Thus, assessing the environmental fate and impacts of MBPs-laden by-products like compost or digestate is crucial. Moreover, the ecotoxicological consequences of shifting from conventional plastics to biodegradable ones are highly uncertain, as there is insufficient evidence to claim that MBPs have a milder effect on ecosystem health. Indeed, literature shows that the impact may be worse depending on the exposed species, polymer type, and the ecosystem complexity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Antibiotic resistance genes (ARGs) are a major threat to human and environmental health. This study investigated the occurrence and distribution of ARGs in Lake Cajititlán, a hypereutrophic subtropical lake in Mexico contaminated by anthropogenic sources (urban wastewater and runoff from crop and livestock production). ARGs (a total of 475 genes) were detected in 22 bacterial genera, with Pseudomonas (144 genes), Stenotrophomonas (88 genes), Mycobacterium (54 genes), and Rhodococcus (27 genes) displaying the highest frequencies of ARGs. Among these, Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed the highest number of ARGs. The results revealed a diverse array of ARGs, including resistance to macrolides (11.55 %), aminoglycosides (8.22 %), glycopeptides (6.22 %), tetracyclines (4 %), sulfonamides (4 %), carbapenems (1.11 %), phenicols (0.88 %), fluoroquinolones (0.44 %), and lincosamides (0.22 %). The most frequently observed ARGs were associated with multidrug resistance (63.33 %), with MexF (42 genes), MexW (36 genes), smeD (31 genes), mtrA (25 genes), and KHM-1 (22 genes) being the most common. Lake Cajititlán is a recreational area for swimming, fishing, and boating, while also supporting irrigation for agriculture and potentially acting as a drinking water source for some communities. This raises concerns about the potential for exposure to antibiotic-resistant bacteria through these activities. The presence of ARGs in Lake Cajititlán poses a significant threat to both human and environmental health. Developing strategies to mitigate the risks of antibiotic resistance, including improving wastewater treatment, and promoting strategic antibiotic use and disposal, is crucial. This study represents a significant advancement in the understanding of antibiotic resistance dynamics in a hypereutrophic subtropical lake in a developing country, providing valuable insights for the scientific community and policymakers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Microalgal-bacterial aggregates (MBAs) have recently attracted significant attention as a potential replacement for conventional, suspended-growth wastewater treatment processes. This article evaluates MBAs for full-scale implementation from the perspective of oxygen supply, land use, and energy savings. The results suggest that under ideal conditions, photosynthesis and atmospheric diffusion would provide at most only 2.7% of the oxygen demand in a conventionally designed, nitrifying activated sludge process, which is equivalent to approximately 1.5% of typical treatment plant-wide energy requirements. The results also suggest that a wastewater treatment process using MBAs and relying on solar photosynthesis and atmospheric diffusion for oxygen would have nearly the same footprint as an equivalent well-mixed wastewater treatment pond. While photosynthesis and passive atmospheric diffusion are capable of providing significant oxygen for suspended-growth wastewater treatment processes, the tradeoffs between footprint requirements and energy savings should be carefully considered., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nitrate contamination in groundwater poses a significant threat to water quality and public health, especially in regions with limited data availability. This study addresses this challenge by employing machine learning (ML) techniques to predict nitrate (NO 3 - -N) concentrations in Mexico's groundwater. Four ML algorithms-Extreme Gradient Boosting (XGB), Boosted Regression Trees (BRT), Random Forest (RF), and Support Vector Machines (SVM)-were executed to model NO 3 - -N concentrations across the country. Despite data limitations, the ML models achieved robust predictive performances. XGB and BRT algorithms demonstrated superior accuracy (0.80 and 0.78, respectively). Notably, this was achieved using ∼10 times less information than previous large-scale assessments. The novelty lies in the first-ever implementation of the 'Support Points-based Split Approach' during data pre-processing. The models considered initially 68 covariates and identified 13-19 significant predictors of NO 3 - -N concentration spanning from climate, geomorphology, soil, hydrogeology, and human factors. Rainfall, elevation, and slope emerged as key predictors. A validation incorporated nationwide waste disposal sites, yielding an encouraging correlation. Spatial risk mapping unveiled significant pollution hotspots across Mexico. Regions with elevated NO 3 - -N concentrations (>10 mg/L) were identified, particularly in the north-central and northeast parts of the country, associated with agricultural and industrial activities. Approximately 21 million people, accounting for 10 % of Mexico's population, are potentially exposed to elevated NO 3 - -N levels in groundwater. Moreover, the NO 3 - -N hotspots align with reported NO 3 - -N health implications such as gastric and colorectal cancer. This study not only demonstrates the potential of ML in data-scarce regions but also offers actionable insights for policy and management strategies. Our research underscores the urgency of implementing sustainable agricultural practices and comprehensive domestic waste management measures to mitigate NO 3 - -N contamination. Moreover, it advocates for the establishment of effective policies based on real-time monitoring and collaboration among stakeholders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

By community intervention in 14 non-disinfecting municipal water systems, we quantified sporadic acute gastrointestinal illness (AGI) attributable to groundwater. Ultraviolet (UV) disinfection was installed on all supply wells of intervention communities. In control communities, residents continued to drink non-disinfected groundwater. Intervention and control communities switched treatments by moving UV disinfection units at the study midpoint (crossover design). Study participants (n = 1,659) completed weekly health diaries during four 12-week surveillance periods. Water supply wells were analyzed monthly for enteric pathogenic viruses. Using the crossover design, groundwater-borne AGI was not observed. However, virus types and quantity in supply wells changed through the study, suggesting that exposure was not constant. Alternatively, we compared AGI incidence between intervention and control communities within the same surveillance period. During Period 1, norovirus contaminated wells and AGI attributable risk from well water was 19% (95% CI, -4%, 36%) for children <5 years and 15% (95% CI, -9%, 33%) for adults. During Period 3, echovirus 11 contaminated wells and UV disinfection slightly reduced AGI in adults. Estimates of AGI attributable risks from drinking non-disinfected groundwater were highly variable, but appeared greatest during times when supply wells were contaminated with specific AGI-etiologic viruses.

Wastewater-based epidemiology offers a time- and cost-effective way to monitor SARS-CoV-2 spread in communities and therefore represents a complement to clinical testing. WBE applicability has been demonstrated in a number of cases over short-term periods as a method for tracking the prevalence of SARS-CoV-2 and an early-warning tool for predicting outbreaks in the population. This study reports SARS-CoV-2 viral loads from wastewater treatment plants (WWTPs) and hospitals over a 6-month period (June to December 2020). Results show that the overall range of viral load in positive tested samples was between 1.2 × 10 3 and 3.5 × 10 6 gene copies/l, unveiling that secondary-treated wastewaters mirrored the viral load of influents. The interpretation suggests that the viral titers found in three out of four WWTPs were associated to clinical COVID-19 surveillance indicators preceding 2-7 days the rise of reported clinical cases. The median wastewater detection rate of SARS-CoV-2 was one out of 14,300 reported new cases. Preliminary model estimates of prevalence ranged from 0.02 to 4.6% for the studied period. This comprehensive statistical and epidemiological analysis demonstrates that the applied wastewater-based approach to COVID-19 surveillance is in general consistent and feasible, although there is room for improvements., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A literature review was carried out to analyze the current status of microplastic research in Latin America and the Caribbean (LAC). Specifically, this work focused on publications pertaining to (1) occurrence and distribution of microplastics in the environment, including water, sediments, and soil and (2) the environmental impact of MPs, particularly their presence and effects on aquatic and terrestrial organisms. The review included peer-reviewed articles from Scopus, Science Direct, Web of Science, Google Scholar and two iberoamerican open access databases (Redalyc and SciELO). It was found that LAC has only contributed to 5% of the global scientific output on microplastics, and overall the highest contributor within the region was Brazil (52%), followed by Chile (16%) and Mexico (13%). An additional section analyzing the barriers to conducting microplastic research in LAC and their exacerbation by the current COVID-19 pandemic was included to provide additional context behind the relatively low scientific production and improve recommendations encouraging research in this region., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Caplina/Concordia transboundary coastal aquifer system, located in the Atacama Desert, is the primary source of water supply for domestic use and irrigation for La Yarada-Los Palos (Peru) and Concordia (Chile) agriculture districts, and to a lesser extent, for Tacna province public supply use (Peru). Despite the scarce amount of rainfall (<20 mm/year) in the area and the limited recharge coming from the Andean highlands, this transboundary aquifer system has been overexploited mainly for agriculture since before the 2000s on the Peruvian side. Consequently, this has caused groundwater depletion and seawater intrusion. In this study, comprehensive hydrogeological information was integrated to understand the aquifer system's behavior and the effects to which it has been subjected to groundwater overexploitation. To that end, a 3D hydrogeological framework was developed using the LEAPFROG software and a constant-density groundwater flow model with equivalent heads was generated in FEFLOW software, which was adjusted with Monte Carlo analysis and conventional automated calibration. Finally, eight scenarios, considering various water resource management options proposed by the authority and potential climatic trends (CMIP6), were simulated from 2020 to 2040. The results showed that between 2002 and 2020, the increase in the seawater wedge and the average groundwater level decline were 216 hm 3 /year and 7 m, respectively. It is expected that the depletion will continue with a groundwater level decline between 5 and 8 m and an increase in the seawater wedge between 1120 hm 3 /year and 1175 hm 3 /year for the forecast period. The study concludes that the aquifer system will remain unsustainable for the next 20 years, regardless of the selected scenarios, and suggests that any mitigation measure requires the participation of stakeholders from Peru, Chile, and Bolivia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

We previously observed that exposure to a complex mixture of high molecular weight polycyclic aromatic hydrocarbons (PAHs) increased sensitivity of rainbow trout (Oncorhynchus mykiss) to subsequent challenge with Aeromonas salmonicida, the causative agent of furunculosis. In this study, we evaluate potential mechanisms associated with disease susceptibility from combined environmental factors of dietary PAH exposure and pathogen challenge. Rainbow trout were fed a mixture of ten high molecular weight PAHs at an environmentally relevant concentration (7.82μg PAH mixture/g fish/day) or control diet for 50 days. After 50 days of PAH exposure, fish were challenged with either Aeromonas salmonicida at a lethal concentration 30 (LC 30 ) or growth media without the pathogen (mock challenge). Head kidneys were collected 2, 4, 10 and 20 days after challenge and gene expression (q<0.05) was evaluated among treatments. In animals fed the PAH contaminated diet, we observed down-regulation of expression for innate immune system genes in pathways (p<0.05) for the terminal steps of the complement cascade (complement component C6) and other bacteriolytic processes (lysozyme type II) potentially underlying increased disease susceptibility after pathogen challenge. Increased expression of genes associated with hemorrhage/tissue remodeling/inflammation pathways (p<0.05) was likely related to more severe head kidney damage due to infection in PAH-fed compared to control-fed fish. This study is the first to evaluate transcriptional signatures associated with the impact of chronic exposure to an environmentally relevant mixture of PAHs in disease susceptibility and immunity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Acute gastrointestinal illness (AGI) resulting from pathogens directly entering the piping of drinking water distribution systems is insufficiently understood. Here, we estimate AGI incidence from virus intrusions into the distribution systems of 14 nondisinfecting, groundwater-source, community water systems. Water samples for virus quantification were collected monthly at wells and households during four 12-week periods in 2006-2007. Ultraviolet (UV) disinfection was installed on the communities' wellheads during one study year; UV was absent the other year. UV was intended to eliminate virus contributions from the wells and without residual disinfectant present in these systems, any increase in virus concentration downstream at household taps represented virus contributions from the distribution system (Approach 1). During no-UV periods, distribution system viruses were estimated by the difference between well water and household tap virus concentrations (Approach 2). For both approaches, a Monte Carlo risk assessment framework was used to estimate AGI risk from distribution systems using study-specific exposure-response relationships. Depending on the exposure-response relationship selected, AGI risk from the distribution systems was 0.0180-0.0661 and 0.001-0.1047 episodes/person-year estimated by Approaches 1 and 2, respectively. These values represented 0.1-4.9% of AGI risk from all exposure routes, and 1.6-67.8% of risk related to drinking water exposure. Virus intrusions into nondisinfected drinking water distribution systems can contribute to sporadic AGI.

Polyhydroxyalkanoate (PHA) was produced in bench-scale sequencing batch reactors (SBRs) fed olive pomace fermentate containing a mixed microbial consortium. Initial anaerobic fermentation in a sequencing batch fermentor (SBF) produced soluble carbon compounds, mainly organic acids. SBF effluent was centrifuged, removing solids, and fed into a SBR where intracellular PHA was produced. Buffer pre-treatment of diluted olive pomace increased organic acid production 75% in SBF fermentate over no pre-treatment. Hydraulic retention time (HRT), solids retention time (SRT), pomace concentration/dilution, and aerobic operation vs. anoxic/oxic cycling were studied to improve PHA formation. Maximum %PHA achieved was 39% (on a dry-weight cell basis), and the highest volumetric productivity was 0.042 g PHA/L-day under fully aerobic conditions. The highest PHA conversion yield was 0.3625 g PHA/L fermentate., (Copyright © 2012. Published by Elsevier Ltd.)

Background: Groundwater supplies for drinking water are frequently contaminated with low levels of human enteric virus genomes, yet evidence for waterborne disease transmission is lacking., Objectives: We related quantitative polymerase chain reaction (qPCR)-measured enteric viruses in the tap water of 14 Wisconsin communities supplied by nondisinfected groundwater to acute gastrointestinal illness (AGI) incidence., Methods: AGI incidence was estimated from health diaries completed weekly by households within each study community during four 12-week periods. Water samples were collected monthly from five to eight households per community. Viruses were measured by qPCR, and infectivity assessed by cell culture. AGI incidence was related to virus measures using Poisson regression with random effects., Results: Communities and time periods with the highest virus measures had correspondingly high AGI incidence. This association was particularly strong for norovirus genogroup I (NoV-GI) and between adult AGI and enteroviruses when echovirus serotypes predominated. At mean concentrations of 1 and 0.8 genomic copies/L of NoV-GI and enteroviruses, respectively, the AGI incidence rate ratios (i.e., relative risk) increased by 30%. Adenoviruses were common, but tap-water concentrations were low and not positively associated with AGI. The estimated fraction of AGI attributable to tap-water-borne viruses was between 6% and 22%, depending on the virus exposure-AGI incidence model selected, and could have been as high as 63% among children < 5 years of age during the period when NoV-GI was abundant in drinking water., Conclusions: The majority of groundwater-source public water systems in the United States produce water without disinfection, and our findings suggest that populations served by such systems may be exposed to waterborne viruses and consequent health risks.

The health condition of out-migrating juvenile salmonids can influence migration success. Physical damage, pathogenic infection, contaminant exposure, and immune system status can affect survival probability. The present study is part of a wider investigation of out-migration success in juvenile steelhead (Oncorhynchus mykiss) and focuses on the application of molecular profiling to assess sublethal effects of environmental stressors in field-collected fish. We used a suite of genes in O. mykiss to specifically assess responses that could be directly related to steelhead health condition during out-migration. These biomarkers were used on juvenile steelhead captured in the Snake River, a tributary of the Columbia River, in Washington, USA, and were applied on gill and anterior head kidney tissue to assess immune system responses, pathogen-defense (NRAMP, Mx, CXC), general stress (HSP70), metal-binding (metallothionein-A), and xenobiotic metabolism (Cyp1a1) utilizing quantitative polymerase chain reaction (PCR) technology. Upon capture, fish were ranked according to visual external physical conditions into good, fair, poor, and bad categories; gills and kidney tissues were then dissected and preserved for gene analyses. Transcription responses were tissue-specific for gill and anterior head kidney with less significant responses in gill tissue than in kidney. Significant differences between the condition ranks were attributed to NRAMP, MX, CXC, and Cyp1a1 responses. Gene profiling correlated gene expression with pathogen presence, and results indicated that gene profiling can be a useful tool for identifying specific pathogen types responsible for disease. Principal component analysis (PCA) further correlated these responses with specific health condition categories, strongly differentiating good, poor, and bad condition ranks. We conclude that molecular profiling is an informative and useful tool that could be applied to indicate and monitor numerous population-level parameters of management interest.

We tested the association of common events in drinking water distribution systems with contamination of household tap water with human enteric viruses. Viruses were enumerated by qPCR in the tap water of 14 municipal systems that use non-disinfected groundwater. Ultraviolet disinfection was installed at all active wellheads to reduce virus contributions from groundwater to the distribution systems. As no residual disinfectant was added to the water, any increase in virus levels measured downstream at household taps would be indicative of distribution system intrusions. Utility operators reported events through written questionnaires. Virus outcome measures were related to distribution system events using binomial and gamma regression. Virus concentrations were elevated in the wells, reduced or eliminated by ultraviolet disinfection, and elevated again in distribution systems, showing that viruses were, indeed, directly entering the systems. Pipe installation was significantly associated with higher virus levels, whereas hydrant flushing was significantly associated with lower virus levels. Weak positive associations were observed for water tower maintenance, valve exercising, and cutting open a water main. Coliform bacteria detections from routine monitoring were not associated with viruses. Understanding when distribution systems are most vulnerable to virus contamination, and taking precautionary measures, will ensure delivery of safe drinking water.

Four bench-scale sequencing batch reactors (SBRs) seeded with activated sludge were operated under either fully oxic or anoxic/oxic conditions and fed synthetic wastewater containing either peptone or acetate. The function of each reactor was assessed through the measure of (i) soluble chemical oxygen demand, orthophosphate, ammonia, and nitrate; and (ii) biomass concentrations of phosphorus, polyhydroxyalkanoate, guanosine tetraphosphate, adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate. In all four reactors, the biomass concentration of phosphorous was correlated statistically with the biomass concentration of ppGpp. The microbial consortia in all four reactors removed an appreciable quantity of phosphorous from solution (67-99%), and the net quantity of phosphorous removed from solution corresponded to the net increase in the biomass concentration of phosphorous. Hence, the microbial stringent response (MSR) was associated with excess intracellular accumulation of phosphorous in mixed microbial consortia fed synthetic wastewater. With recognition of the potential role of the MSR in the removal of soluble phosphorous from wastewater, additional research may lead to further optimization of treatment technologies and the development of new treatment systems for the biological removal of phosphorus from wastewater., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The operation of the Federal Columbia River Power System (FCRPS) has negatively affected threatened and endangered salmonid populations in the Pacific Northwest. Barging Snake River spring Chinook salmon Oncorhynchus tshawytscha through the FCRPS is one effort to mitigate the effect of the hydrosystem on juvenile salmon out-migration. However, little is known about the occurrence and transmission of infectious agents in barged juvenile salmon relative to juvenile salmon that remain in-river to navigate to the ocean. We conducted a survey of hatchery-reared spring Chinook salmon at various points along their out-migration path as they left their natal hatcheries and either migrated in-river or were barged through the FCRPS. Salmon kidneys were screened by polymerase chain reaction for nine pathogens and one family of water molds. Eight pathogens were detected; the most prevalent were Renibacterium salmoninarum and infectious hematopoietic necrosis virus. Species in the family Saprolegniaceae were also commonly detected. Pathogen prevalence was significantly greater in fish that were barged through the FCRPS than in fish left to out-migrate in-river. These results suggest that the transmission of infectious agents to susceptible juvenile salmon occurs during the barging process. Therefore, management activities that reduce pathogen exposure during barging may increase the survival of juvenile Chinook salmon after they are released.

The purpose of this research was to develop a better understanding of the dynamic effects of anaerobic hydraulic retention time (HRT) on both enhanced biological phosphorus removal (EBPR) performance and enrichment of phosphorus accumulating organisms (PAOs). The research was conducted using laboratory-scale sequencing batch reactors inoculated with mixed microbial consortia and fed real wastewater. Exposing microorganisms to extended anaerobic HRTs is not recommended for EBPR configured systems. In this research, however, longer anaerobic exposure did not negatively affect performance even if volatile fatty acids were depleted. Further, extended anaerobic HRTs may positively affect phosphorus removal through enhanced aerobic uptake. The EBPR consortia also appear to maintain reserve energetic capacity in the form of polyphosphate that can be used to survive and grow under variable operational and environmental conditions. Finally, the tested EBPR systems yield mixed microbial consortia enriched with PAOs (specifically Candidatus Accumulibacter phosphatis) at approximately 7.1 to 21.6% of the total population.

In disinfection systems, incomplete penetration of chlorine into effluent wastewater particles can result in a residual population of viable microorganisms. In this work, a combined experimental and numerical approach was used to quantify inactivation of microorganisms in effluent particles and identify combinations of particle removal and chlorine dose that would result in a reduction of occluded microorganisms for six full-scale facilities in the United States with different nitrification levels. The results reveal that combined chlorine is more effective for inactivating occluded microorganisms than free chlorine; model calibration results suggest that free chlorine is less effective because it is more reactive. However, nitrified effluents appear to have lower effluent particle concentrations, and decreases in particle concentrations significantly reduce the chlorine required. Additionally, in disinfection systems that are designed and operated based on inactivation of indicator organisms, the chlorine dose may be insufficient to inactivate occluded pathogens to levels consistent with current regulations.

Various methods have been developed to mitigate the adverse effects of the Federal Columbia River Power System on juvenile Pacific salmon out-migrating through the Columbia River basin. In this study, we found that hatchery-reared spring Chinook salmon Oncorhynchus tshawytscha in the river are in varying degrees of health, which may affect delayed mortality and the assessment of the effectiveness of management actions to recover listed stocks (e.g., barging fish downstream versus leaving fish in the river). A laboratory disease challenge with Listonella anguillarum was completed on fish from Rapid River Hatchery and Dworshak National Fish Hatchery (NFH) with different out-migration histories: (1) transported by barge, (2) removed from the river before barging, or (3) left to travel in-river. Barged fish from Rapid River Hatchery experienced less mortality than fish from Dworshak NFH. No statistical differences were found between the hatcheries with fish that had in-river out-migration histories. We suggest that the stressors and low survival associated with out-migration through the hydropower system eliminated any differences that could have been present. However, 18-25% of the fish that were barged or collected before barging died in the laboratory before the disease challenge, compared with less than 2% of those that traveled in-river. Owing to disproportionate prechallenge mortality, the disease-challenged populations may have been biased; thus, they were also considered together with the prechallenge mortalities. The synthesis of prechallenge and disease-challenged mortalities and health characteristics evaluated during out-migration indicated that the benefit of barging was not consistent between the hatcheries. This finding agrees with adult survival and delayed mortality estimates for the individual hatcheries determined from adult returns. The results suggest that the health status of fish and their history before entering the hydropower system (hatchery of origin and out-migration path) are critical variables affecting the conclusions drawn from studies that evaluate mitigation strategies.

Juvenile rainbow trout were fed a diet containing an environmentally relevant mixture of 10 high molecular weight polycyclic aromatic hydrocarbons (PAHs) at a dose of 0.66 or 7.82 µg PAH · g fish(-1) · d(-1). At 3, 7, 14, and 28 d, biomarkers of aryl hydrocarbon receptor activation (AHR), hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity, and cytochrome P4501A (CYP1A)-associated staining increased 14- to 26-fold and 6- to 14-fold, respectively, in fish fed 7.82 µg PAH · g fish (-1) · d(-1). Cytochrome P4501A-associated staining increased 2- to 9-fold on days 3, 7, and 28 in fish fed 0.66 µg PAH · g fish(-1) · d(-1). Bile fluorescent aromatic compounds served as a biomarker of exposure and confirmed that PAH exposure was consistent over 50 d. DNA damage in blood cells, protein oxidation, and lipid peroxidation in the kidney were biomarkers of oxidative stress and all increased in fish fed 7.82 µg PAH · g fish(-1) · d(-1). Fish fed 0.66 µg PAH · g fish(-1) · d(-1) had elevated DNA damage in blood cells but increased protein oxidation or lipid peroxidation in the kidney were not observed. Challenge with Aeromonas salmonicida, at lethal concentration (LC) 20, decreased survival in fish previously fed either 0.66 µg PAH · g fish(-1) · d(-1) or 7.82 µg PAH · g fish(-1) · d(-1) relative to fish fed the control diet. In general, biomarkers of both AHR activation and oxidative stress peaked at 3 to 14 d then declined at 28 to 50 d of PAH exposure and an increase in susceptibility to disease was observed at 50 d. These results link PAH exposure to biomarker responses that may be useful as early indicators of population level responses, such as mortality resulting from an increase in disease susceptibility., (Copyright © 2011 SETAC.)

The objective of this research was to interrogate and develop a better understanding for a process to achieve post-anoxic denitrification without exogenous carbon augmentation within enhanced biological phosphorus removal (EBPR). Sequencing batch reactors fed real wastewater and seeded with mixed microbial consortia were operated under variable anaerobic-aerobic-anoxic and organic carbon loading conditions. The process consistently achieved phosphorus and nitrogen removal, while the observed specific denitrification rates were markedly higher than expected for post-anoxic systems operated without exogenous organic carbon addition. Investigations revealed that post-anoxic denitrification was predominantly driven by glycogen, an intracellular carbon storage polymer associated with EBPR; moreover, glycogen reserves can be significantly depleted post-anoxically without compromising EBPR. Success of the proposed process is predicated on providing sufficient organic acids in the influent wastewater, such that residual nitrate carried over from the post-anoxic period is reduced and polyhydroxyalkanoate (PHA) synthesis occurs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The influence of four main process parameters--solids retention time (SRT), hydraulic retention time (HRT), anoxic-oxic cycling, and carbon-to-nitrogen ratio (C/N ratio)--on poly(hydroxyalkanoate) (PHA) production, while treating brewery wastewater, was studied. Two sets of two-level, three-factor experimental designs were implemented to (1) determine the effects and interactions among process parameters, (2) assess their significance to PHA production, and (3) approximate optimal operational conditions. The HRT and SRT were found to be the crucial operational parameters affecting PHA production. The highest PHA content of 55% (on a cell-weight basis) was produced at a 4-day HRT and 4-day SRT, whereas a maximum PHA concentration of 907 mg/L was obtained at a 2-day HRT and 12-day SRT. The effect of anoxic conditions on PHA production was insignificant. The C/N ratio played a more important role in the PHA concentration in the system than in the PHA content in the biomass.

The health effects of the flame retardant polybrominated diphenyl ethers (PBDEs) in fish are not well understood. To determine the potential effects of this ubiquitous contaminant class on fish health, juvenile subyearling Chinook salmon (Oncorhynchus tshawytscha) were fed a diet that reflected the PBDE congeners found in the stomach contents of subyearling Chinook salmon collected from the highly urbanized and industrialized lower Willamette River in the Columbia River Basin of North America. The diet, consisting of five PBDE congeners (BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154), was fed to the salmon at 2% of their body weight in food per day for 40 days. Two concentrations of the diet (1x and 10x PBDE) were fed to the salmon. The 1x PBDE diet reflected the concentration of PBDEs (190 ng PBDEs/g food) found in the stomach contents of juvenile subyearling Chinook salmon; the 10x diet was prepared at 10 times that concentration. The fish were then exposed to the marine bacterial pathogen Listonella anguillarum to assess susceptibility to infectious disease. Juvenile Chinook salmon fed the 1x PBDE diet were more susceptible to L. anguillarum than salmon fed the control diet. This suggests that juvenile salmonids in the lower Willamette River exposed to PBDEs may be at greater risk for disease than nonexposed juvenile salmonids. In contrast, salmon that consumed the 10x PBDE diet were not more susceptible to the pathogen than salmon fed the control diet. The mechanisms for the dichotomous results observed in disease susceptibility between salmon fed the 1x and 10x PBDE diets are currently not known but have also been observed in other species exposed to PBDEs with respect to immune function., (Published by Elsevier B.V.)

The purpose of the research presented herein was to evaluate the effects of solids residence time (SRT) and organic acid augmentation on biological phosphorus removal (BPR), with a focus on how these operational variables affect key metabolisms and the distribution of the microbial population. Using laboratory-scale sequencing batch reactors seeded with a mixed microbial consortium and fed real wastewater, we observed that longer SRTs can improve BPR performance; organic acid augmentation can stabilize BPR, but it is not necessary for process success; and higher volatile suspended solids concentrations correlate with improved phosphorus removal. The results also suggest that organic acids may not be critical in driving anaerobic phosphorus release, but in driving aerobic growth. Finally, given an observed population similarity across all tested bioreactors, BPR variability appears to be less influenced by the presence of specific microbes and more affected by the induction of critical metabolisms.

The transfer of genetic material among bacteria in the environment can occur both in the planktonic and attached state. Given the propensity of organisms to exist in sessile microbial communities in oligotrophic subsurface conditions, and that such conditions typify the subsurface, this study focuses on exploratory modeling of horizontal gene transfer among surface-associated Escherichiacoli in the subsurface. The mathematics so far used to describe the kinetics of conjugation in biofilms are developed largely from experimental observations of planktonic gene transfer, and are absent of lags or plasmid stability that appear experimentally. We develop a model and experimental system to quantify bacterial filtration and gene transfer in the attached state, on granular porous media. We include attachment kinetics described in Nelson et al. (2007) using the filtration theory approach of Nelson and Ginn (2001, 2005) with motility of E. coli described according to Biondi et al. (1998)., (Copyright 2009 Elsevier B.V. All rights reserved.)

Quantifying infectious viruses by cell culture depends on visualizing cytopathic effect, or for integrated cell culture-PCR, attaining confidence a PCR-positive signal is the result of virus growth and not inoculum carryover. This study developed mathematical methods to calculate infectious virus numbers based on viral growth kinetics in cell culture. Poliovirus was inoculated into BGM cell monolayers at 10 concentrations from 0.001 to 1000 PFU/ml. Copy numbers of negative-strand RNA, a marker of infectivity for single-stranded positive RNA viruses, were measured over time by qRT-PCR. Growth data were analyzed by two approaches. First, data were fit with a continuous function to estimate directly the initial virus number, expressed as genomic copies. Such estimates correlated with actual inoculum numbers across all concentrations (R(2)=0.62, n=17). Second, the length of lag phase appeared to vary inversely with inoculum titers; hence, standard curves to predict inoculum virus numbers were derived based on three definitions of lag time: (1) time of first detection of (-)RNA, (2) second derivative maximum of the fitted continuous function, and (3) time when the fitted curve crossed a threshold (-)RNA concentration. All three proxies yielded standard curves with R(2)=0.69-0.90 (n=17). The primary advantage of these growth kinetics approaches is being able to quantify virions that are unambiguously infectious, a particular advantage for viruses that do not produce CPE., (2009 Elsevier B.V. All rights reserved.)

The overall goal of the study reported herein was to use techniques in the field of risk assessment (specifically a state-space population dynamic model of disease transmission within recreational waters) to explore the relative significance of (1) active shedding of microorganisms from bathers themselves, and (2) the type and concentration of etiological agent on the observed heterogeneity of the incidence of illness in epidemiological studies that have been used to develop ambient water quality criteria. The etiological agent and corresponding dose ingested during recreational contact was found to significantly impact the observed incidence of illness in an epidemiological study conducted in recreational water. In addition, the observed incidence of illness was found not to necessarily reflect background concentrations of indicator organisms, but rather microorganisms shed during recreational contact. Future revisions to ambient water quality criteria should address the etiological agent, dose, and the significance of microbial shedding relative to background concentrations of pathogens and indicator organisms in addition to the incidence of illness and concentration of indicator organisms. Without a quantitative assessment of these additional variables, study findings may potentially be site specific and not representative of the health risks associated with specific indicator concentrations in all recreational waters.

Previous research has demonstrated that production of natural fiber reinforced thermoplastic composites (NFRTCs) utilizing bacterially-derived pure polyhydroxybutyrate (PHB) does not yield a product that is cost competitive with synthetic plastic-based NFRTCs. Moreover, the commercial production of pure PHB is not without environmental impacts. To address these issues, we integrated unpurified PHB in NFRTC construction, thereby eliminating a significant energy and cost sink (ca. 30-40%) while concurrently yielding a fully biologically based commodity. PHB-rich biomass synthesized with the microorganism Azotobacter vinelandii UWD was utilized to manufacture NFRTCs with wood flour. Resulting composites exhibited statistically similar bending strength properties despite relatively different PHB contents. Moreover, the presence of microbial cell debris allowed for NFRTC processing at significantly reduced polymer content, relative to pure PHB-based NFRTCs. Results further indicate that current commercial PHB production yields are sufficiently high to produce composites comparable to those manufactured with purified PHB.

Available filtration methods to concentrate waterborne viruses are either too costly for studies requiring large numbers of samples, limited to small sample volumes, or not very portable for routine field applications. Sodocalcic glass wool filtration is a cost-effective and easy-to-use method to retain viruses, but its efficiency and reliability are not adequately understood. This study evaluated glass wool filter performance to concentrate the four viruses on the U.S. Environmental Protection Agency contaminant candidate list, i.e., coxsackievirus, echovirus, norovirus, and adenovirus, as well as poliovirus. Total virus numbers recovered were measured by quantitative reverse transcription-PCR (qRT-PCR); infectious polioviruses were quantified by integrated cell culture (ICC)-qRT-PCR. Recovery efficiencies averaged 70% for poliovirus, 14% for coxsackievirus B5, 19% for echovirus 18, 21% for adenovirus 41, and 29% for norovirus. Virus strain and water matrix affected recovery, with significant interaction between the two variables. Optimal recovery was obtained at pH 6.5. No evidence was found that water volume, filtration rate, and number of viruses seeded influenced recovery. The method was successful in detecting indigenous viruses in municipal wells in Wisconsin. Long-term continuous filtration retained viruses sufficiently for their detection for up to 16 days after seeding for qRT-PCR and up to 30 days for ICC-qRT-PCR. Glass wool filtration is suitable for large-volume samples (1,000 liters) collected at high filtration rates (4 liters min(-1)), and its low cost makes it advantageous for studies requiring large numbers of samples.

Polyhydroxyalkanoate (PHA) production was achieved using tomato cannery waste coupled with a mixed microbial culture during wastewater treatment. The two-stage PHA production process comprised a sequencing batch reactor (SBR), operating under a periodic feast-famine regime, to accomplish simultaneously wastewater treatment and selection of PHA-accumulating microbes, followed by a batch reactor for the production of PHA-rich biomass. The SBRs were efficient at removing soluble carbon (84%), ammonia (100%), and phosphorus (76%). Meanwhile, PHA-accumulating microbes were enriched under the SBR operating conditions, and PHA content on a cell-weight basis was within the range 7 to 11% in nonfiltered wastewater and 2 to 8% in filtered wastewater. Subsequently, batch studies were implemented with varying loading rates, ranging from 0.4 to 3.2 food-to-microorganism ratios. A maximum 20% PHA content on a cell-weight basis was obtained. Based on the experimental results, a PHA biosynthesis-degradation kinetic model was developed to (1) aid in the design of a pilot- or full-scale PHA production process coupled with wastewater treatment and (2) determine optimal conditions for harvest of PHA-rich biomass.

Efficient utilization of carbon inputs is critical to the economic viability of the current forest products sector. Input carbon losses occur in various locations within a pulp mill, including losses as volatile organics and wastewater. Opportunities exist to capture this carbon in the form of value-added products such as biodegradable polymers. Waste-activated sludge from a pulp mill wastewater facility was enriched for 80 days for a methanol-utilizing consortium with the goal of using this consortium to produce biopolymers from methanol-rich pulp mill waste streams. Five enrichment conditions were utilized: three high-methanol streams from the kraft mill foul condensate system, one methanol-amended stream from the mill wastewater plant, and one methanol-only enrichment. Enrichment reactors were operated aerobically in sequencing batch mode at neutral pH and 25 degrees C with a hydraulic residence time and a solids retention time of 4 days. Non-enriched waste activated sludge did not consume methanol or reduce chemical oxygen demand. With enrichment, however, the chemical oxygen demand reduction over 24-h feed/decant cycles ranged from 79 to 89%, and methanol concentrations dropped below method detection limits. Neither the non-enriched waste-activated sludge nor any of the enrichment cultures accumulated polyhydroxyalkanoates (PHAs) under conditions of nitrogen sufficiency. Similarly, the non-enriched waste activated sludge did not accumulate PHAs under nitrogen-limited conditions. By contrast, enriched cultures accumulated PHAs to nearly 14% on a dry weight basis under nitrogen-limited conditions. This indicates that selectively enriched pulp mill waste activated sludge can serve as an inoculum for PHA production from methanol-rich pulp mill effluents.

Biologically derived polyesters known as polyhydroxyalkanoates (PHAs) represent a potentially "sustainable" replacement to fossil-fuel-based thermoplastics. However, current commercial practices that produce PHA with pure microbial cultures grown on renewable, but refined, feedstocks (i.e., glucose) under sterile conditions do not represent a sustainable commodity. Here, we report on PHA production with a mixed microbial consortium indigenous to an activated sludge process on carbon present in municipal wastewaters. Reactors operated under anaerobic/aerobic and aerobic-only mode and fed primary solids fermenter liquor maintained a mixed microbial consortium capable of synthesizing PHA at 10 to 25% (w/w), while reducing soluble COD by approximately 62 to 71%. More critically, an aerobic batch reactor seeded from the anaerobic/aerobic reactor and fed fermenter liquor achieved approximately 53% PHA (w/w). Results presented suggest that environmentally benign production of biodegradable polymers is feasible. We further used PHA-rich biomass to produce a natural fiber-reinforced thermoplastic composite that can be used to offset advanced wastewater treatment costs.

Applied population dynamics modeling is relied upon with increasing frequency to quantify how human activities affect human and non-human populations. Current techniques include variously the population's spatial transport, age, size, and physiology, but typically not the life-histories of exposure to other important things occurring in the ambient environment, such as chemicals, heat, or radiation. Consequently, the effects of such 'abiotic' aspects of an ecosystem on populations are only currently addressed through individual-based modeling approaches that despite broad utility are limited in their applicability to realistic ecosystems [V. Grimm, Ten years of individual-based modeling in ecology: what have we learned and what could we learn in the future? Ecol. Model. 115 (1999) 129-148][1]. We describe a new category of population dynamics modeling, wherein population dynamical states of the biotic phases are structured on dose, and apply this framework to demonstrate how chemical species or other ambient aspects can be included in population dynamics in three separate examples involving growth suppression in fish, inactivation of microorganisms with ultraviolet irradiation, and metabolic lag in population growth. Dose-structuring is based on a kinematic approach that is a simple generalization of age-structuring, views the ecosystem as a multi-component mixture with reacting biotic/abiotic components. The resulting model framework accommodates (a) different memories of exposure as in recovery from toxic ambient conditions, (b) differentiation between exogenous and endogenous sources of variation in population response, and (c) quantification of acute or sub-acute effects on populations arising from life-history exposures to abiotic species. Classical models do not easily address the very important fact that organisms differ and have different experiences over their life cycle. The dose structuring is one approach to incorporate some of these elements into the existing structures of the classical models, while retaining many of the features (and other limitations) of classical models.

Occlusion of microorganisms in wastewater particles often governs the overall performance of a disinfection system, and the associated health risks of post-disinfected effluents. Little is currently known on the penetration of chemical oxidants into particles developed in wastewater treatment. In this work, a reactive transport model that incorporates intra- and extra-particle chemical decay, radial intra-particle diffusion, mass transfer resistance at particle surfaces, and non-linear reaction kinetics within a competitive multi-particle size aqueous environment, was used to analyze the penetration of ozone and chlorine into wastewater particles. Individual characteristics from two secondary wastewater treatment facilities were used in model calibration. Simulations revealed that significant ozone transport within particles greater than 6 microm required large initial concentrations to exhaust the preferential reaction with aqueous soluble matter. Chlorinated samples exhibited apparently slower reactions and thus deeper penetration (22-40 microm). Chlorine penetration was less sensitive to variations in the extra-particle reaction and disinfectant concentration than ozone. Model simulations that considered elevated initial concentrations of chemical disinfectants revealed that complete inactivation of all particle size domains was not possible with current disinfection practices (e.g., contact times). Reduction in the health risks associated with wastewater particles requires treatment that efficiently balances particle removal (filtration) and particle inactivation (disinfection).

Polyhydroxyalkanoates (PHAs) represent an environmentally effective alternative to synthetic thermoplastics; however, current production practices are not sustainable. In this study, PHA production was accomplished in sequencing batch bioreactors utilizing real wastewaters and mixed microbial consortia from municipal activated sludge as inoculum. Polymer production reached 85, 53, and 10% of the cell dry weight from methanol-enriched pulp and paper mill foul condensate, fermented municipal primary solids, and biodiesel wastewater, respectively. Using denaturing gradient gel electrophoresis of 16S-rDNA from polymerase chain reaction-amplified DNA extracts, distinctly different communities were observed between and within wastewaters following enrichment. Most importantly, functional stability was maintained despite differing and contrasting microbial populations.

Various methods have been developed to mitigate the effects of dams on juvenile Pacific salmon Oncorhynchus spp. migrating to the Pacific Ocean through the Columbia River basin. In this study, we examined the health of hatchery Snake River spring and summer Chinook salmon relative to two mitigating strategies: dam bypass and transportation (e.g., barging). The health of out-migrants was assessed in terms of the difference in the incidence of mortality among fish, categorically grouped into no-bypass, bypass, and transportation life histories, in response to challenge with the marine pathogen Listonella anguillarum during seawater holding. These three life histories were defined as follows: (1) fish that were not detected at any of the juvenile bypass systems above Bonneville Dam were classified as having a no-bypass life history; (2) fish that were detected at one or more juvenile bypass systems above Bonneville Dam were classified as having a bypass life history; and (3) fish that were barged were classified as having the transportation life history. Barged fish were found to be less susceptible to L. anguillarum than in-river fish-whether bypassed or not-which suggests that transportation may help mitigate the adverse health effects of the hydropower system of the Columbia River basin on Snake River spring-summer Chinook salmon. The findings of this study are not necessarily transferable to other out-migrant stocks in the Columbia River basin, given that only one evolutionarily significant unit, that is, Snake River spring-summer Chinook salmon, was used in this study.

Direct current applied to a tin oxide anode submersed in water has the potential to generate hydroxyl radicals, a strong chemical oxidant. Tin oxide anode systems, which may represent a viable alternative disinfectant, were investigated for the disinfection of secondary effluents. Coliform bacteria in the effluent were effectively inactivated in a bench-scale tin oxide disinfection system. The number of anodes in the tin oxide disinfection system influenced the steady-state concentration of hydroxyl radicals and, consequently, the contact time necessary to achieve a specified dose. The life expectancy of the anodes was greater than 30 days and no appreciable loss of tin was observed over a 5-day period. A preliminary estimate of the capital and operating costs of a pilot- or full-scale tin oxide disinfection system designed to meet a discharge requirement of 23 most probable number/100 mL at a 3 785-m3/d (1-mgd) facility was comparable to UV and chlorination/dechlorination facilities. Based on this preliminary assessment, tin oxide disinfection systems are a promising alternative disinfectant for secondary effluents.

Infectious disease within outmigrant juvenile salmon in the Columbia River Basin is modulated, in part, by abiotic stressors that influence host-susceptibility. Through the application of a dose-structured population dynamic model, we show that chemical (both in the river and in the estuary) and in-river (e.g., dams and/or predation) stressors influence host-susceptibility, increasing the mean force of infection (defined as the per capita acquisition rate of infection) by a factor of 2.2 and 1.6, respectively. Using Listonella anguillarum as a model pathogen, nonchemical in-river and chemical stressors contribute equally to a cumulative incidence of delayed disease-induced mortalities in Chinook salmon that range from 3% to 18% for estuary residence times of 30-120 days, respectively. Mitigation of environmental stressors that increase host-susceptibility could represent a significant component in future management strategies to recover listed stocks.

Pharmaceutically active compounds (PhACs) in the environment lately have been acknowledged to constitute a health risk for humans and terrestrial and aquatic ecosystems. Human and veterinary applications are the main sources of PhACs in the environment and the major pathways are excretion and discharge to the environment through sewage treatment plants (STPs). In this study, the occurrence and fate of selected human PhACs belonging to different therapeutic classes (non-steroidal anti-inflammatory drugs, lipid regulators, anti-epileptics, antibiotics and beta-blockers) were investigated in a small river in the very south of Sweden. The objectives of the study were to evaluate the impact of a high and rather constant load in sewage influent on downstream concentrations and whether substances that are metabolized to a high degree in humans also show a low persistency in a natural aquatic environment. Water samples were collected from the influent and effluent of the STP, in a series of dammed reservoirs leading to discharge into the Höje River in Sweden, and at several locations in the river downstream of the outfall. After enrichment by solid-phase extraction, the compounds were analyzed using GC-MS (methylated derivatives) or LC-MS/MS. In addition to the targeted pharmaceuticals, GC-MS analysis of the samples revealed the presence of other sewage-related pollutants (triclosan, caffeine, flame-retardants, antioxidants) and these results where included for comparison. Removal efficiencies were calculated in the STP and found to display a wide range with numerous species surviving treatment at greater than half their influent concentrations, including diclofenac, the anti-epileptic carbamazepine, a beta-blocker (propanolol), and antibiotics trimetoprim and sulfamethoxazole. Low removals were also observed for Tris(2-chloroisopropyl)phosphate (flame retardant), BHT-aldehyde (oxidation product of BHT) and synthetic musk (HHCB). The concentrations of chloride (Cl(-)) and boron (B) were used as natural inert tracers to estimate the relative extent of dilution of PhACs measured in the effluent of the STP on concentrations measured further downstream. Based on spatial trends of concentrations (recalculated to reflect a hypothetical scenario with no dilution), ibuprofen, ketoprofen, naproxen and dicofenac were shown to be subject to significant abiotic or biotic transformations or physical sequestration in the river. The beta-blockers atenolol, metoprolol and propanolol, the antibiotics trimetoprim and sulfametoxazole, and carbamazepine demonstrated a high degree of persistence. Fluctuations in the concentration of carbamazepine and gemfibrozil were observed along the series of reservoirs and within the river and are hypothesized to be due to release of parent compound from glucuronides. Several of the investigated substances (metaprolol, propanolol and carbamazepin) that exhibit low excretion rates as parent compounds demonstrate a surprising persistence in the aquatic environment. It is concluded that pharmaceutical substances with a high metabolic rate in humans (low excretion rate) do not necessarily induce a short lifetime in aquatic environments. Results from this study emphasize the need for a broader view on the concept of persistence that accounts for loading rates, in addition to removal mechanisms (e.g., transformation, volatility and physical sequestration by solids), under a variety of spatial and temporal scales.

We describe the protocol for an inexpensive and nondestructive optical reflectance assay for the measurement of biofilm formation. Reflectance data are obtained using an Ocean Optics (Dunedin, Florida) USB 2000 spectrometer with a polychromatic light source. A fiber optic cable is used both for illumination and collection, and Ocean Optics OOIBase32 Platinum software is used for preliminary processing of the data. Differences in reflectance data collected at times ranging from 2 to 24 h distinguish between cell attachment and volume growth for two strains of Enterococci. Confocal scanning laser microscopy imaging is used to confirm these results. Phase contrast microscopy images are also obtained in conjunction with reflectance measurements for several different biofilm specimens. The experiments consider biofilm formation on glass and polystyrene substrata, but the method can be used for many other abiotic substrata of interest, both opaque and nonopaque.

We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.

Polymerase chain reaction (PCR) was used to detect Cryptosporidium parvum oocysts in secondary effluent samples collected from activated-sludge facilities. Serial dilutions of the purified nucleic acid extracts from the samples were made and PCR was conducted to estimate the C. parvum oocyst concentration via a Poisson distribution-based most probable number (MPN). The degree of oocysts associated with wastewater particles was also evaluated. The sensitivity of the MPN-PCR assay was 20 oocysts/PCR unit. The detection limit of the concentration, extraction, and purification protocols in phosphate buffer saline spiked with a known concentration of oocysts ranged from 1.1 to 4.6 oocysts/L; the detection limit for the wastewater samples ranged from 11 to 4200 oocysts/L depending on the extent of inhibition in each sample. The recovery efficiency of the oocysts ranged from 48 to 59% in most samples. Oocysts were found in two out of seven samples with concentrations of 203 and 308 oocysts/L, as estimated by the MPN-PCR method. The oocysts were found only in the filtrate of the grab samples; particle-associated oocysts were not detected. Association of spiked C. parvum oocysts with particles in secondary effluent drawn from wastewater plants with varying operating conditions indicated a weak correlation between the degree of association and the mean cell residence time of the system.

Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.

The diffusion of a chemical disinfectant into wastewater particles may be viewed as a serial two-step process involving transport through a macroporous network of pathways to micropores that lead into dense cellular regions. Previous research reveals that ultraviolet (UV) light penetration into wastewater particles is limited primarily to macropores, resulting in a residual concentration of targeted organisms in post-disinfected effluents that reflects the number of organisms embedded in the dense cellular regions of particles. Conversely, chlorine was demonstrated as part of this research to penetrate into both the macroporous and microporous network of pathways, implying that the application of chlorine may be designed feasibly to achieve a desired level of inactivation of particle-associated organisms. In the short term, a disinfection model previously developed for UV irradiation may be used to assess the inactivation of particle-associated organisms with chlorine. However, in the long-term, a more rigorous and complete understanding of the transport of chemical disinfectants into particles can be explored utilizing existing mathematical expressions commonly used to model mass transport into porous media. The parameters of interest in this modeling approach include the reaction rate of chlorine with particulate material, the diffusion rate of chlorine within a particle, the mass-transfer rate coefficient across the particle's boundary, and the particle porosity.

The relative concentration of pathogens in water samples collected from storm drains and adjacent surfaces was evaluated using established PCR-based protocols. Out of the 58 samples collected from 21 different storm drains, 22% were PCR positive for Escherichia coli ETEC, Salmonella, or adenovirus. The risk of swimming related illnesses associated with detection of E. coli ETEC and Salmonella ranged from 0.39 to 30:100 000 and 0.3-25:1000, respectively. The detection limit corresponding to a negative-PCR result was evaluated in reference to water quality standards developed using a risk-based approach that integrates human dose-response data with acceptable levels of risk promulgated by the U.S. EPA for recreational contact. The percent of samples with an acceptable detection limit ranged from 0% for Giardia lamblia and Shigella to 100% for E. coli ETEC. The principal factor influencing the detection limit of G. lamblia and Shigella was sample volume. The principal factor influencing the detection limit of the remaining bacteria and protozoa, including E. coli O157:H7, Salmonella, and Cryptosporidium parvum, was the presence of inhibitory compounds in the purified nucleic acid extracts. Both recovery and inhibition adversely impacted the detection limit of viruses. Ambient water quality standards based on the occurrence of specific pathogens enumerated with PCR-based assays could serve as a method of evaluating the biological quality of water but only after significant improvements in filtration and purification protocols. The risk-based methodology developed in this study can be used to evaluate future improvements in filtration and purification protocols.