Site-specific risk-based threshold (RBT) concentrations for sewage-associated markers in estuarine swimming waters.

This study establishes site-specific risk-based threshold (RBT) concentrations for sewage-associated markers, including Bacteroides HF183 (HF183), Lachnospiraceae Lachno3 (Lachno3), cross-assembly phage (CrAssphage), and pepper mild mottle virus (PMMoV), utilizing quantitative microbial risk assessment (QMRA) for recreational estuarine waters (EW). The QMRA model calculates a RBT concentration corresponding to a selected target illness risk for ingestion of EW contaminated with untreated sewage. RBT concentrations were estimated considering site-specific decay rates and concentrations of markers and reference pathogen (human norovirus; HNoV), aiding in the identification of high-risk days during the swimming season. Results indicated varying RBT concentrations for fresh (Day 0) and aged (Days 1 to 10) sewage contamination scenarios over 10 days. HF183 exhibited the highest RBT concentration (26,600 gene copis (GC)/100 mL) initially but decreased rapidly with aging (2570 to 3120 GC/100 mL on Day 10) depending on the decay rates, while Lachno3 and CrAssphage remained relatively stable. PMMoV, despite lower initial RBT (3920 GC/100 mL), exhibited increased RBT (4700 to 6440 GC/100 mL) with aging due to its slower decay rate compared to HNoV. Sensitivity analysis revealed HNoV concentrations as the most influential parameter. Comparison of marker concentrations in estuarine locations with RBT concentrations showed instances of marker exceedance, suggesting days of potential higher risks. The observed discrepancies between bacterial and viral marker concentrations in EW highlight the need for optimized sample concentration method and simultaneous measurement of multiple markers for enhanced risk predictions. Future research will explore the utility of multiple markers in risk management. Overall, this study contributes to better understanding human health risks in recreational waters, aiding regulators, and water quality managers in effective decision-making for risk prioritization and mitigation strategies.

Sediment Toxicity Tests: A Critical Review of Their use in Environmental Regulations.

Sediments are an integral component of aquatic systems, linking multiple water uses, functions, and services. Contamination of sediments by chemicals is a worldwide problem, with many jurisdictions trying to prevent future pollution (prospective) and manage existing contamination (retrospective). The present review assesses the implementation of sediment toxicity testing in environmental regulations globally. Currently, the incorporation of sediment toxicity testing in regulations is most common in the European Union (EU), North America, and Australasian regions, with some expansion in Asia and non-EU Europe. Employing sediment toxicity testing in prospective assessments (i.e., before chemicals are allowed on the market) is most advanced and harmonized with pesticides. In the retrospective assessment of environmental risks (i.e., chemicals already contaminating sediments), regulatory sediment toxicity testing practices are applied inconsistently on the global scale. International harmonization of sediment toxicity tests is considered an asset and has been successful through the widespread adoption and deployment of Organisation for Economic Co-operation and Development guidelines. On the other hand, retrospective sediment assessments benefit from incorporating regional species and protocols. Currently used toxicity testing species are diverse, with temperate species being applied most often, whereas test protocols are insufficiently flexible to appropriately address the range of environmental contaminants, including nanomaterials, highly hydrophobic contaminants, and ionized chemicals. The ever-increasing and -changing pressures placed on aquatic resources are a challenge for protection and management efforts, calling for continuous sediment toxicity test method improvement to insure effective use in regulatory frameworks. Future developments should focus on including more subtle and specific toxicity endpoints (e.g., incorporating bioavailability-based in vitro tests) and genomic techniques, extending sediment toxicity testing from single to multispecies approaches, and providing a better link with ecological protection goals. Environ Toxicol Chem 2024;00:1-20. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Comparison of adsorption-extraction (AE) workflows for improved measurements of viral and bacterial nucleic acid in untreated wastewater.

The lack of standardized methods and large differences in virus concentration and extraction workflows have hampered Severe Acute Respiratory Syndrome (SARS-CoV-2) wastewater surveillance and data reporting practices. Numerous studies have shown that adsorption-extraction (AE) method holds promise, yet several uncertainties remain regarding the optimal AE workflow. Several procedural components may influence the recovered concentrations of target nucleic acid, including membrane types, homogenization instruments, speed and duration, and lysis buffer. In this study, 42 different AE workflows that varied these components were compared to determine the optimal workflow by quantifying endogenous SARS-CoV-2, human adenovirus 40/41 (HAdV 40/41), and a bacterial marker gene of fecal contamination (Bacteroides HF183). Our findings suggest that the workflow chosen had a significant impact on SARS-CoV-2 concentrations, whereas it had minimal impact on HF183 and no effect on HAdV 40/41 concentrations. When comparing individual components in a workflow, such as membrane type (MF-Millipore™ 0.45 µm MCE vs. Isopore™ 0.40 µm), we found that they had no impact on SARS-CoV-2, HAdV 40/41, and HF183 concentrations. This suggests that at least some consumables and equipment are interchangeable. Buffer PM1 + TRIzol-based workflows yielded higher concentrations of SARS-CoV-2 than other workflows. HF183 concentrations were higher in workflows without chloroform. Similarly, higher homogenization speeds (5000-10,000 rpm) led to increased concentrations of SARS-CoV-2 and HF183 but had no effect on HAdV 40/41. Our findings indicate that minor enhancements to the AE workflow can improve the recovery of viruses and bacteria from the wastewater, leading to improved outcomes from wastewater surveillance efforts.

Has the Rio Doce "time bomb" been defused? Using a weight-of-evidence approach to determine sediment quality.

The Fundão mine tailings dam rupture of 2015, in the Rio Doce basin, Brazil, resulted in the deposition of tailings downstream of the dam. It has yet to be determined if metals associated with the tailings have contributed toxicity to organisms, burying a time bomb that could be ticking. Currently the data on toxicity to benthic and aquatic organisms have not been assessed sufficiently to allow an informed assessment using an approach based on weight-of-evidence. This study was conducted to ascertain if sediments at "hot spots" that received Fundão tailings reflected elevated concentrations of metals and if these concentrations were sufficient to result in toxicity to freshwater organisms. The lines-of-evidence considered included assessing metals concentrations in relation to sediment quality criteria, establishing biogeochemical characterizations, completing an evaluation of potential metal release upon resuspension to provide information on bioavailability, and identifying acute and chronic toxicity effects using sensitive native species for waters (water flea, Daphnia similis) and sediments (burrowing midge larvae, Chironomus sancticaroli). Only porewater concentrations of iron and manganese exceeded Brazilian surface water criteria, whereas most trace elements exhibited no enrichment or elevated environmental indexes. The concentrations of bioavailable metals were assessed to be low, and metal concentrations did not increase in the overlying water upon resuspension; rather, they decreased through time. Toxicity testing in resuspended waters and bulk sediments resulted in no acute or chronic toxicity to either benthic or aquatic species. The low metal bioavailability and absence of toxicity of the tailings-enriched sediments was attributed to the strong binding and rapid removal of potentially toxic metal ions caused by oxyhydroxides and particles in the presence of iron-rich particulates. The findings of these sediment hot-spot studies indicate the Fundão dam release of tailings more than six years ago is not causing the current release of toxic concentrations of metals into the freshwaters of the Rio Doce. Integr Environ Assess Manag 2024;20:148-158. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Enhancing Sediment Bioaccumulation Predictions: Isotopically Modified Bioassay and Biodynamic Modeling for Nickel Assessment.

Quantifying metal bioaccumulation in a sedimentary environment is a valuable line of evidence when evaluating the ecological risks associated with metal-contaminated sediments. However, the precision of bioaccumulation predictions has been hindered by the challenges in accurately modeling metal influx processes. This study focuses on nickel bioaccumulation from sediment and introduces an innovative approach using the isotopically modified bioassay to directly measure nickel assimilation rates in sediment. Tested in sediments spiked with two distinct nickel concentrations, the measured Ni assimilation rates ranged from 35 to 78 ng g-1 h-1 in the Low-Ni treatment and from 96 to 320 ng g-1 h-1 in the High-Ni treatment. Integrating these rates into a biodynamic model yielded predictions of nickel bioaccumulation closely matching the measured results, demonstrating high accuracy with predictions within a factor of 3 for the Low-Ni treatment and within a factor of 1 for the High-Ni treatment. By eliminating the need to model metal uptake from various sources, this streamlined approach provides a reliable method for predicting nickel bioaccumulation in contaminated sediments. This advancement holds promise for linking bioaccumulation with metal toxicity risks in sedimentary environments, enhancing our understanding of metal-contaminated sediment risks and providing valuable insights to support informed decision-making in ecological risk assessment and management.

Improving toxicity prediction of metal-contaminated sediments by incorporating sediment properties.

For the purpose of sediment quality assessment, the prediction of toxicity risk-levels for aquatic organisms based on simple environmental measurements is desirable. One commonly used approach is the comparison of total contaminant concentrations with corresponding water and sediment quality guideline values, serving as a Line of Evidence (LoE) based on chemistry-toxicity effects relationships. However, the accuracy of toxicity predictions can be improved by considering the factors that modify contaminant bioavailability. In this study we used paired chemistry-ecotoxicity data sets for sediments to evaluate the improvement in toxicity risk predictions using bioavailability-modified guidelines. The sediments were predominantly contaminated with metals, and measurements of sediment particle size, total organic carbon (TOC) and acid volatile sulfide (AVS) were used to modify hazard quotients (HQ). To further assess the predictive efficacy of the bioavailability-modified guideline models, sediments with differing contamination levels were tested for toxicity to a benthic amphipod's reproduction. To account for differences between laboratory exposure and field exposure scenarios, where the latter creates greater dilution, both static-renewal and flow-through test procedures were employed, and flow-through resulted in lower dissolved metal concentrations in the overlying waters. We also investigated how lower AVS concentration by oxidation modified the toxicity. This study reaffirmed that consideration of factors that influence contaminant bioavailability improves toxicity risk predictions, however the improvements may be modest. The sediment particle size data had the greatest influence on the modified HQ, indicating that higher percentage of fine particle size (<63 µm) contributed most to a lower predicted toxicity. The comparison of the static-renewal and flow-through test results continue to raise important questions about the relevance of static or static-renewal toxicity test results for risk assessment decisions, as both these test designs may cause unrealistically high contributions of dissolved metals in overlying waters to toxicity. Overall, this study underscores the value of incorporating outcomes from simple and routine sediment analysis (e.g., particle size, TOC, and consideration of AVS) to enhance the predictive efficacy of toxicity risk assessments in the context of sediment quality risk assessment.

Comparison of concentration and extraction workflows for qPCR quantification of intI1 and vanA in untreated wastewater.

Quantitative polymerase chain reaction (qPCR) measurement of antibiotic resistance genes (ARGs) in untreated municipal wastewater may prove useful in combating the antimicrobial resistance crisis. However, harmonizing and optimizing qPCR-based workflows is essential to facilitate comparisons across studies, and includes achieving highly-effective ARG capture through efficient concentration and extraction procedures. In the current study, combinations of sample volume, membrane types and DNA extraction kits within filtration and centrifugation-based workflows were used to quantify 16S ribosomal RNA (16S rRNA), class 1 integron-integrase gene (intI1) and an ARG encoding resistance to vancomycin (vanA) in untreated wastewater sampled from three wastewater treatment plants (WWTPs). Highly abundant 16S rRNA and intI1 were detected in 100 % of samples from all three WWTPs using both 2 and 20 mL sample volumes, while lower prevalence vanA was only detected when using the 20 mL volume. When filtering 2 mL of wastewater, workflows with 0.20-/0.40-µm polycarbonate (PC) membranes generally yielded greater concentrations of the three targets than workflows with 0.22-/0.45-µm mixed cellulose ester (MCE) membranes. The improved performance was diminished when the sample volume was increased to 20 mL. Consistently greater concentrations of 16S rRNA, intI1 and vanA were yielded by filtration-based workflows using PC membranes combined with a DNeasy PowerWater (DPW) Kit, regardless of the sample volume used, and centrifugation-based workflows with DNeasy Blood & Tissue Kit for 2-mL wastewater extractions. Within the filtration-based workflows, the DPW kit yielded more detection and quantifiable results for less abundant vanA than the DNeasy PowerSoil Pro Kit and FastDNA™ SPIN Kit for Soil. These findings indicate that the performance of qPCR-based workflows for surveillance of ARGs in wastewater varies across targets, sample volumes, concentration methods and extraction kits. Workflows must be carefully considered and validated considering the target ARGs to be monitored.

The distribution of metal and petroleum-derived contaminants within sediments around oil and gas infrastructure in the Gippsland Basin, Australia.

As oil and gas infrastructure comes to the end of its working life, a decommissioning decision must be made: should the infrastructure be abandoned in situ, repurposed, partially removed, or fully removed? Environmental contaminants around oil and gas infrastructure could influence these decisions because contaminants in sediments could degrade the value of the infrastructure as habitat, enter the seafood supply if the area is re-opened for commercial and/or recreational fishing, or be made biologically available as sediment is resuspended when the structures are moved. An initial risk hypothesis, however, may postulate that these concerns are only relevant if contaminant concentrations are above screening values that predict the possibility of environmental harm or contaminant bioaccumulation. To determine whether a substantive contaminants-based risk assessment is needed for infrastructure in the Gippsland Basin (South-eastern Australia), we measured the concentration of metals and polycyclic aromatic hydrocarbons (PAHs) in benthic sediments collected around eight platforms earmarked for decommissioning. The measurements were compared to preset screening values and to background contaminant concentrations in reference sites. Lead (Pb), zinc (Zn), PAHs and other contaminants were occasionally measured at concentrations that exceeded reference values, most often within 150 m of the platforms. The exceedance of a few screening values by contaminants at some platforms indicates that these platforms require further analysis to determine the contaminant risks associated with any decommissioning option.

Unveiling indicator, enteric, and respiratory viruses in aircraft lavatory wastewater using adsorption-extraction and Nanotrap® Microbiome A Particles workflows.

The effective detection of viruses in aircraft wastewater is crucial to establish surveillance programs for monitoring virus spread via aircraft passengers. This study aimed to compare the performance of two virus concentration workflows, adsorption-extraction (AE) and Nanotrap® Microbiome A Particles (NMAP), in detecting the prevalence and concentrations of 15 endogenous viruses including ssDNA, dsDNA, ssRNA in 24 aircraft lavatory wastewater samples. The viruses tested included two indicator viruses, four enteric viruses, and nine respiratory viruses. The results showed that cross-assembly phage (crAssphage), human polyomavirus (HPyV), rhinovirus A (RhV A), and rhinovirus B (RhV B) were detected in all wastewater samples using both workflows. However, enterovirus (EV), human norovirus GII (HNoV GII), human adenovirus (HAdV), bocavirus (BoV), parechovirus (PeV), epstein-barr virus (EBV). Influenza A virus (IAV), and respiratory syncytial virus B (RsV B) were infrequently detected by both workflows, and hepatitis A virus (HAV), influenza B virus (IBV), and respiratory syncytial virus B (RsV A) were not detected in any samples. The NMAP workflow had greater detection rates of RNA viruses (EV, PeV, and RsV B) than the AE workflow, while the AE workflow had greater detection rates of DNA viruses (HAdV, BoV, and EBV) than the NMAP workflow. The concentration of each virus was also analyzed, and the results showed that crAssphage had the highest mean concentration (6.76 log10 GC/12.5 mL) followed by HPyV (5.46 log10 GC/12.5 mL using the AE workflow, while the mean concentrations of enteric and respiratory viruses ranged from 2.48 to 3.63 log10 GC/12.5 mL. Using the NMAP workflow, the mean concentration of crAssphage was 5.18 log10 GC/12.5 mL and the mean concentration of HPyV was 4.20 log10 GC/12.5 mL, while mean concentrations of enteric and respiratory viruses ranged from 2.55 to 3.74 log10 GC/12.5 mL. Significantly higher (p < 0.05) mean concentrations of crAssphage and HPyV were observed when employing the AE workflow in comparison to the NMAP workflow. Conversely, the NMAP workflow yielded significantly greater (p < 0.05) concentrations of RhV A, and RhV B compared to the AE workflow. The findings of this study can aid in the selection of an appropriate concentration workflow for virus surveillance studies and contribute to the development of efficient virus detection methods.

Influence of membrane pore-size on the recovery of endogenous viruses from wastewater using an adsorption-extraction method.

The ongoing COVID-19 pandemic has emphasized the significance of wastewater surveillance in monitoring and tracking the spread of infectious diseases, including SARS-CoV-2. The wastewater surveillance approach detects genetic fragments from viruses in wastewater, which could provide an early warning of outbreaks in communities. In this study, we determined the concentrations of four types of endogenous viruses, including non-enveloped DNA (crAssphage and human adenovirus 40/41), non-enveloped RNA (enterovirus), and enveloped RNA (SARS-CoV-2) viruses, from wastewater samples using the adsorption-extraction (AE) method with electronegative HA membranes of different pore sizes (0.22, 0.45, and 0.80 µm). Our findings showed that the membrane with a pore size of 0.80 µm performed comparably to the membrane with a pore size of 0.45 µm for virus detection/quantitation (repeated measurement one-way ANOVA; p > 0.05). We also determined the recovery efficiencies of indigenous crAssphage and pepper mild mottle virus, which showed recovery efficiencies ranging from 50% to 94% and from 20% to 62%, respectively. Our results suggest that the use of larger pore size membranes may be beneficial for processing larger sample volumes, particularly for environmental waters containing low concentrations of viruses. This study offers valuable insights into the application of the AE method for virus recovery from wastewater, which is essential for monitoring and tracking infectious diseases in communities.

Occurrence of multiple respiratory viruses in wastewater in Queensland, Australia: Potential for community disease surveillance.

The early warning and tracking of COVID-19 prevalence in the community provided by wastewater surveillance has highlighted its potential for much broader viral disease surveillance. In this proof-of-concept study, 46 wastewater samples from four wastewater treatment plants (WWTPs) in Queensland, Australia, were analyzed for the presence and abundance of 13 respiratory viruses, and the results were compared with reported clinical cases. The viruses were concentrated using the adsorption-extraction (AE) method, and extracted nucleic acids were analyzed using qPCR and RT-qPCR. Among the viruses tested, bocavirus (BoV), parechovirus (PeV), rhinovirus A (RhV A) and rhinovirus B (RhV B) were detected in all wastewater samples. All the tested viruses except influenza B virus (IBV) were detected in wastewater sample from at least one WWTP. BoV was detected with the greatest concentration (4.96-7.22 log10 GC/L), followed by Epstein-Barr virus (EBV) (4.08-6.46 log10 GC/L), RhV A (3.95-5.63 log10 GC/L), RhV B (3.74-5.61 log10 GC/L), and PeV (3.17-5.32 log10 GC/L). Influenza viruses and respiratory syncytial virus (RSV) are notifiable conditions in Queensland, allowing the gene copy (GC) concentrations to be compared with reported clinical cases. Significant correlations (ρ = 0.60, p < 0.01 for IAV and ρ = 0.53, p < 0.01 for RSV) were observed when pooled wastewater influenza A virus (IAV) and RSV log10 GC/L concentrations were compared to log10 clinical cases among the four WWTP catchments. The positive predictive value for the presence of IAV and RSV in wastewater was 97 % for both IAV and RSV clinical cases within the four WWTP catchments. The overall accuracy of wastewater analysis for predicting clinical cases of IAV and RSV was 97 and 90 %, respectively. This paper lends credibility to the application of wastewater surveillance to monitor respiratory viruses of various genomic characteristics, with potential uses for increased surveillance capabilities and as a tool in understanding the dynamics of disease circulation in the communities.

Contaminant pulse following wildfire is associated with shifts in estuarine benthic communities.

Novel combinations of climatic conditions due to climate change and prolonged fire seasons have contributed to an increased occurrence of "megafires". Such large-scale fires pose an unknown threat to biodiversity due to the increased extent and severity of burn. Assessments of wildfires often focus on terrestrial ecosystems and effects on aquatic habitats are less documented, particularly in coastal environments. In a novel application of eDNA techniques, we assessed the impacts of the 2019-2020 Australian wildfires on the diversity of estuarine benthic sediment communities in six estuaries in NSW, Australia, before and after the fires. Estuaries differed in area of catchment burnt (0-92%) and amount of vegetative buffer that remained post-fire between burnt areas and waterways. We found greater dissimilarities in the composition and abundance of eukaryotic and bacterial sediment communities in estuaries from burnt catchments with no buffer compared to those with an intact buffer or from unburnt catchments. Shifts in composition in highly burnt catchments were associated with increased concentrations of nutrients, carbon, including fire-derived pyrogenic carbon, and copper, which was representative of multiple highly correlated trace metals. Changes in the relative abundances of certain taxonomic groups, such as sulfate-reducing and nitrifying bacterial groups, in the most impacted estuaries indicate potential consequences for the functioning of sediment communities. These results provide a unique demonstration of the use of eDNA to identify wildfire impacts on ecological communities and emphasize the importance of vegetative buffers in limiting wildfire-associated impacts.

Comparative analysis of Adsorption-Extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) workflows for the recovery of endogenous enveloped and non-enveloped viruses in wastewater.

In this study, two virus concentration methods, namely Adsorption-Extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) along with commercially available extraction kits were used to quantify endogenous pepper mild mottle virus (PMMoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in nucleic acid extracted from 48 wastewater samples collected over six events from eight wastewater treatment plants (WWTPs). The main aim was to determine which workflow (i.e., concentration and extraction methods) produces greater concentrations of endogenous PMMoV and SARS-CoV-2 gene copies (GC) in comparison with each other. Turbidity and total suspended solids (TSS) of wastewater samples within and among the eight WWTPs were highly variable (41-385 NTU and 77-668 mg/L TSS). In 58 % of individual wastewater samples, the log10 GC concentrations of PMMoV were greater by NMVP workflow compared to AE workflow. Paired measurements of PMMoV GC/10 mL from AE and NMVP across all 48 wastewater samples were weakly correlated (r = 0.455, p = 0.001) and demonstrated a poor linear relationship (r2 = 0.207). The log10 GC concentrations of SARS-CoV-2 in 69 % of individual samples were greater by AE workflow compared to NMVP workflow. In contrast to PMMoV, the AE and NMVP derived SARS-CoV-2 GC counts were strongly correlated (r = 0.859, p < 0.001) and demonstrated a strong linear relationship (r2 = 0.738). In general, the PMMoV GC achieved by the NMVP workflow decreased with increasing turbidity, but the PMMoV GC by the AE workflow did not appear to be as sensitive to either turbidity or TSS levels. These findings suggest that wastewater sample turbidity or suspended solids concentration, and the intended target for analysis should be considered when validating an optimal workflow for wastewater surveillance of viruses.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater.

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2-5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

Isotopically Modified Bioassay Bridges the Bioavailability and Toxicity Risk Assessment of Metals in Bedded Sediments.

The application of bioavailability-based risk assessment for the management of contaminated sediments requires new techniques to rapidly and accurately determine metal bioavailability. Here, we designed a multimetal isotopically modified bioassay to directly measure the bioavailability of different metals by tracing the change in their isotopic composition within organisms following sediment exposure. With a 24 h sediment exposure, the bioassay sensed significant bioavailability of nickel and lead within the sediment and determined that cadmium and copper exhibited low bioavailable concentrations and risk profiles. We further tested whether the metal bioavailability sensed by this new bioassay would predict the toxicity risk of metals by examining the relationship between metal bioavailability and metal toxicity to chironomid larvae emergence. A strong dose-toxicity relationship between nickel bioavailability (nickel assimilation rate) and toxicity (22 days emergence ratio) indicated exposure to bioavailable nickel in the sediment induced toxic effects to the chironomids. Overall, our study demonstrated that the isotopically modified bioassay successfully determined metal bioavailability in sediments within a relatively short period of exposure. Because of its speed of measurement, it may be used at the initial screening stage to rapidly diagnose the bioavailable contamination status of a site.

A generic environmental risk assessment framework for deep-sea tailings placement.

Deep-sea tailings placement (DSTP) involves the oceanic discharge of tailings at depth (usually >100 m), with the intent of ultimate deposition of tailings solids on the deep-sea bed (>1000 m), well below the euphotic zone. DSTP discharges consist of a slurry of mine tailings solids (finely crushed rock) and residual process liquor containing low concentrations of metals, metalloids, flotation agents and flocculants. This slurry can potentially affect both pelagic and benthic biota inhabiting coastal waters, the continental slope and the deep-sea bed. Building on a conceptual model of DSTP exposure pathways and receptors, we developed a stressor-driven environmental risk assessment (ERA) framework using causal pathways/causal networks for each of eight pelagic and benthic impact zones. For the risk characterisation, each link in each causal pathway in each zone was scored using four levels of likelihood (not possible, possible, likely and certain) and two levels of consequence (not material, material) to give final risk rankings of low, potential, high or very high risk. Of the 246 individual causal pathways scored, 11 and 18 pathways were considered to be of very high risk and high risk respectively. These were confined to the benthic zones in the mixing zone (continental slope) and the primary and secondary deposition zones. The new risk framework was then tested using a case study of the Batu Hijau copper mine in Indonesia, the largest DSTP operation globally. The major risk of DSTP is smothering of benthic biota, even outside the predicted deposition zones. Timescales for recovery are slow and may lead to different communities than those that existed prior to tailings deposition. We make several recommendations for monitoring programs for existing, proposed and legacy DSTP operations and illustrate how georeferenced causal networks are valuable tools for ERA in DSTP.

RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance.

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting variants of concern (VOC) while also providing virus quantitation data. However, detection sensitivities of both RT-qPCR and sequencing can be impacted through losses occurring during sample handling, virus concentration, nucleic acid extraction, and RT-qPCR. Therefore, process limit of detection (PLOD) assessments are required to estimate the gene copies of target molecule to attain specific probability of detection. In this study, we compare the PLOD of four RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab) for detection of SARS-CoV-2 to that of ATOPlex sequencing by seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in greater detection rates than individual assays, suggesting that application of multiple assays is better suited for the trace detection of SARS-CoV-2 from wastewater samples. Furthermore, while ATOPlex offers a promising approach to SARS-CoV-2 wastewater surveillance, this approach appears to be less sensitive compared to RT-qPCR under the experimental conditions of this study, and may require further refinements. Nonetheless, the combination of RT-qPCR and ATOPlex may be a powerful tool to simultaneously detect/quantify SARS-CoV-2 RNA and monitor emerging VOC in wastewater samples.

Wildfires cause rapid changes to estuarine benthic habitat.

Estuaries are one of the most valuable biomes on earth. Although humans are highly dependent on these ecosystems, anthropogenic activities have impacted estuaries worldwide, altering their ecological functions and ability to provide a variety of important ecosystem services. Many anthropogenic stressors combine to affect the soft sedimentary habitats that dominate estuarine ecosystems. Now, due to climate change, estuaries and other marine areas might be increasingly exposed to the emerging threat of megafires. Here, by sampling estuaries before and after a megafire, we describe impacts of wildfires on estuarine benthic habitats and justify why megafires are a new and concerning threat to coastal ecosystems. We (1) show that wildfires change the fundamental characteristics of estuarine benthic habitat, (2) identify the factors (burnt intensity and proximity to water's edge) that influence the consequences of fires on estuaries, and (3) identify relevant indicators of wildfire impact: metals, nutrients, and pyrogenic carbon. We then discuss how fires can impact estuaries globally, regardless of local variability and differences in catchment. In the first empirical assessment of the impact of wildfires on estuarine condition, our results highlight indicators that may assist waterway managers to empirically detect wildfire impacts in estuaries and identify catchment factors that should be included in fire risk assessments for estuaries. Overall, this study highlights the importance of considering fire threats in current and future estuarine and coastal management.

Application of digital PCR for public health-related water quality monitoring.

Digital polymerase chain reaction (dPCR) is emerging as a reliable platform for quantifying microorganisms in the field of water microbiology. This paper reviews the fundamental principles of dPCR and its application for health-related water microbiology. The relevant literature indicates increasing adoption of dPCR for measuring fecal indicator bacteria, microbial source tracking marker genes, and pathogens in various aquatic environments. The adoption of dPCR has accelerated recently due to increasing use for wastewater surveillance of Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) - the virus that causes Coronavirus Disease 2019 (COVID-19). The collective experience in the scientific literature indicates that well-optimized dPCR assays can quantify genetic material from microorganisms without the need for a calibration curve and often with superior analytical performance (i.e., greater sensitivity, precision, and reproducibility) than quantitative polymerase chain reaction (qPCR). Nonetheless, dPCR should not be viewed as a panacea for the fundamental uncertainties and limitations associated with measuring microorganisms in water microbiology. With dPCR platforms, the sample analysis cost and processing time are typically greater than qPCR. However, if improved analytical performance (i.e., sensitivity and accuracy) is critical, dPCR can be an alternative option for quantifying microorganisms, including pathogens, in aquatic environments.

Monitoring of SARS-CoV-2 in sewersheds with low COVID-19 cases using a passive sampling technique.

Monitoring SARS-CoV-2 RNA in sewer systems, upstream of a wastewater treatment plant, is an effective approach for understanding potential COVID-19 transmission in communities with higher spatial resolutions. Passive sampling devices provide a practical solution for frequent sampling within sewer networks where the use of autosamplers is not feasible. Currently, the design of upstream sampling is impeded by limited understanding of the fate of SARS-CoV-2 RNA in sewers and the sensitivity of passive samplers for the number of infected individuals in a catchment. In this study, passive samplers containing electronegative membranes were applied for at least 24-h continuous sampling in sewer systems. When monitoring SARS-CoV-2 along a trunk sewer pipe, we found RNA signals decreased proportionally to increasing dilutions, with non-detects occurring at the end of pipe. The passive sampling membranes were able to detect SARS-CoV-2 shed by >2 COVID-19 infection cases in 10,000 people. Moreover, upstream monitoring in multiple sewersheds using passive samplers identified the emergence of SARS-CoV-2 in wastewater one week ahead of clinical reporting and reflected the spatiotemporal spread of a COVID-19 cluster within a city. This study provides important information to guide the development of wastewater surveillance strategies at catchment and subcatchment levels using different sampling techniques.