Evaluation of approaches to quantify total residual oxidants in ballast water management systems employing chlorine for disinfection.

With the maturation and certification of several ballast water management systems that employ chlorine as biocide to prevent the spread of invasive species, there is a clear need for accurate and reliable total residual oxidants (TRO) technology to monitor treatment dose and assure the environmental safety of treated water discharged from ships. In this study, instruments used to measure TRO in wastewater and drinking water applications were evaluated for their performance in scenarios mimicking a ballast water treatment application (e.g., diverse hold times, temperatures, and salinities). Parameters chosen for testing these technologies in the past do not reflect conditions expected during ballast water treatment. Salinity, temperature, and oxidant concentration all influenced the response of amperometric sensors. Oxidation reduction potential (ORP) sensors performed more consistently than amperometric sensors under different conditions but it may be difficult to correlate ORP and TRO measurements for the multitude of biogeochemical conditions found naturally in ballast water. N,N-diethyl-p-phenylenediamine (DPD) analyzers and amperometric sensors were also tested under intermittent sampling conditions mimicking a ballasting scenario, with cyclical dosage and discharge operations. When sampling was intermittent, amperometric sensors required excessive response and conditioning times, whereas DPD analyzers provided reasonable estimates of TRO under the ballasting scenario

Performance of a One-Dimensional Model of Wave-Driven Nearshore Alongshore Tracer Transport and Decay with Applications for Dry Weather Coastal Pollution

Dry weather pollution sources cause coastal water quality problems that are not accounted for in existing beach advisory metrics. A 1D wave-driven advection and loss model was developed for a 30 km nearshore domain spanning the United States/Mexico border region. Bathymetric nonuniformities, such as the inlet and shoal near the Tijuana River estuary mouth, were neglected. Nearshore alongshore velocities were estimated by using wave properties at an offshore location. The 1D model was evaluated using the hourly output of a 3D regional hydrodynamic model. The 1D model had high skill in reproducing the spatially averaged alongshore velocities from the 3D model. The 1D and 3D models agreed on tracer exceedance or nonexceedance above a human illness probability threshold for 87% of model time steps. 1D model tracer was well-correlated with targeted water samples tested for DNA-based human fecal indicators. This demonstrates that a simple, computationally fast, 1D nearshore wave-driven advection model can reproduce nearshore tracer evolution from a 3D model over a range of wave conditions ignoring bathymetric nonuniformities at this site and may be applicable to other locations

RNA Viromics of Southern California Wastewater and Detection of SARS-CoV-2 Single-Nucleotide Variants.

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the coronavirus disease 2019 (COVID-19) pandemic to track disease burden through analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the health care system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven wastewater treatment plants (WTPs), collected from August 2020 to January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single-nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater, and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically important SARS-CoV-2 variants along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. IMPORTANCE Wastewater-based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the coronavirus disease 2019 (COVID-19) pandemic to track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without adding burden to health care systems. In this study, we used metatranscriptomics and reverse transcription-droplet digital PCR (RT-ddPCR) to assay RNA viruses across Southern California wastewater from August 2020 to January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single-nucleotide variants through sequencing. Likewise, wastewater treatment plants (WTPs) harbored different viromes, and we detected other human pathogens, such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology

Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically-important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 – August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs and that the relative abundance of many individual AMR genes/variants increased over time and may be connected to antibiotic use during the COVID-19 pandemic. Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population’s wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds

Salmonella risks due to consumption of aquaculture-produced shrimp

The use of aquaculture is increasing to meet the growing global demand for seafood. However, the use of aquaculture for seafood production incurs potential human health risks, especially from enteric bacteria such as Salmonella spp. Salmonella spp. was the most frequently reported cause of outbreaks associated with crustaceans from 1998 to 2004. Among crustacean species, shrimp are the most economically important, internationally traded seafood commodity, and the most commonly aquaculture-raised seafood imported to the United States. To inform safe aquaculture practices, a quantitative microbial risk assessment (QMRA) was performed, incorporating stochastic variability in pathogen growth, industrial shrimp processing, and consumer shrimp preparation. Several scenarios including gamma irradiation and cooking time were considered in order to examine the relative importance of these practices in terms of their impact on risk. Median annual infection risks for all scenarios considered were below 10−4 and median disability adjusted life year (DALY) metrics were below 10−6 DALY per person per year, however, 95th percentile risks were above 10−4 annual probability of infection and 10−6 DALY per person per year for scenarios with improper cooking and lack of gamma irradiation. The greatest difference between microbiological risks for the scenarios tested was observed when comparing proper vs. improper cooking (5–6 orders of magnitude) and gamma irradiation (4–5 orders of magnitude) compared to (up to less than 1 order of magnitude) for peeling and “deveining” (removing the shrimp digestive tract) vs. peeling only. The findings from this research suggest that restriction of Salmonella spp. to low levels (median 5–30 per L aquaculture pond water) may be necessary for scenarios in which proper downstream food handling and processing cannot be guaranteed.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Sanitary Engineerin

SARS-CoV-2 RNA is enriched by orders of magnitude in primary settled solids relative to liquid wastewater at publicly owned treatment works

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases. Crown Copyright (C) 2021 Published by Elsevier B.V.Peer reviewe