Assessment of nucleic acid extraction protocols for antibiotic resistance genes (ARGs) quantification in aircraft wastewater.

This study evaluated ten nucleic acid extraction protocols (EP1 to EP10) for measuring five endogenous antibiotic resistance genes (ARGs) in four aircraft wastewater samples (AWW1 to AWW4). The targeted ARGs, including blaCTX-M, blaNDM-1, ermB, qnrS, and tetA, encompassed highly and minimally abundant ARGs. TetA and ermB were consistently detected across four aircraft wastewater samples using the DNeasy Blood and Tissue Kit and the AllPrep PowerViral DNA/RNA kit. QnrS displayed high detection rates with specific extraction protocols and aliquot volumes. Concentrations of ARGs varied across aircraft wastewater samples, with differing extraction protocols influencing quantitative results. The concentrations of tetA, ermB, and qnrS in AWW1 were distinct, while AWW2 to AWW4 exhibited a broader range for tetA, ermB, qnrS, blaCTX-M, and blaNDM-1. EP1 consistently produced the highest concentrations for several ARGs. Collective data analysis revealed varying ARG concentrations across the ten extraction protocols, suggesting the importance of careful extraction protocol selection in ARG monitoring in aircraft wastewater samples. Based on the results, we suggest that a small sample volume (as low as 0.2 mL) may be sufficient for ARG characterization in aircraft wastewater samples. The findings also emphasize the need for considering toilet paper removal without compromising nucleic acid extraction efficiency. The study highlights promising prospects for aircraft wastewater monitoring of ARGs, calling for further investigation into the import and spread of unique ARGs through transport hubs.

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.

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.

Application of a Multi-Metal Stable-Isotope-Enriched Bioassay to Assess Changes to Metal Bioavailability in Suspended Sediments.

The direct measurement of particulate contaminant bioavailability is a challenging aspect for the environmental risk assessment of contaminated sites. Here, we demonstrated a multi-metal stable-isotope-enriched bioassay to simultaneously measure the bioavailability of Cd, Cu, and Zn in naturally contaminated sediments following differing periods of resuspension treatment. Freshwater filter-feeding clams were pre-labeled with the isotopes 114Cd, 65Cu, and 68Zn to elevate isotope abundances in their tissues and then exposed to metal-contaminated suspended sediments. The assimilation of sediment-associated metals by clams would decrease the isotope ratios (Cd114/111, Cu65/63, and Zn68/64) in tissues, providing a direct measurement of metal bioavailability. For the sediments tested here, the method revealed bioavailable cadmium and non-bioavailable copper in sediments but was inconclusive for zinc. With a longer resuspension time, the bioavailability of particulate cadmium increased, but that of copper was unaffected. Metal bioavailability predicted using traditional wet-chemical extraction methods was inconsistent with these findings. The study indicated that multi-metal stable-isotope-enriched bioassay provides a new tool for directly assessing metal bioavailability in sediments, and this method is amenable for use in in situ assessments.

In Situ DGT Sensing of Bioavailable Metal Fluxes to Improve Toxicity Predictions for Sediments.

An increased risk of adverse biological effects of metals in sediments may be accompanied by high labile metal fluxes as measured by the diffusive gradients in thin films (DGT) technique. To improve the usefulness of the DGT technique for sediment quality risk assessments, we used the simpler and more cost-effective piston DGTs rather than planar DGT probes to measure bioavailable metal fluxes in naturally contaminated sediments with widely varying composition (properties, metals and concentrations) and assessed their prediction of toxicity to amphipod reproduction in a flow-through microcosm. DGT pistons were deployed in sediments under different conditions, both in the field (in situ) and in the laboratory in sediment cores (lab-equilibrated) and in homogenized sediments (lab-homogenized). We demonstrated that the metal flux toxic units, DGTTU, measured in situ best predicted the magnitude of toxicity to amphipod reproduction. For sediments that had been highly disturbed before testing, DGTTU were less predictive for observed toxicity, but the copper flux alone (DGTTU-Cu) was effective, indicating copper was the primary cause of toxicity in these highly perturbed sediments. Overall, our study highlighted that the adverse effects induced by excessive bioavailable metals in contaminated sediments can be consistently sensed by the DGT pistons.

Intraday variability of indicator and pathogenic viruses in 1-h and 24-h composite wastewater samples: Implications for wastewater-based epidemiology.

We monitored the concentration of indicator viruses crAssphage and pepper mild mottle virus (PMMoV) and human pathogen adenovirus (HAdV) in influent from a wastewater treatment plant in Brisbane, Australia in 1-h and 24-h composite samples. Over three days of sampling, the mean concentration of crAssphage gene copies (GC)/mL in 24-h composite samples did not differ significantly (p = 0.72-0.92), while for PMMoV GC/mL (p value range: 0.0002-0.0321) and HAdV GC/mL (p value range: 0.0028-0.0068) significant differences in concentrations were observed on one day of sampling compared to the other two. For all three viruses, the variation observed in 1-h composite samples was greater than the variation observed in 24-h composite samples. For crAssphage, in 54.1% of 1-h composite samples, the concentration was less than that observed in 24-h composite samples; whereas for PMMoV and HAdV the concentration was less in 79.2 and 70.9% of 1-h composite samples, respectively, compared to the relevant 24-h composite samples. Similarly, the concentration of crAssphage in 1-h compared to 24-h composite samples did not differ (p = 0.1082) while the concentrations of PMMoV (p < 0.0001) and HAdV (p < 0.0001) in 1-h composite samples were significantly different from 24-h composite samples. These results suggest that 24-h composite samples offer increased analytical sensitivity and decreased variability compared to 1-h composite samples when monitoring wastewater, especially for pathogenic viruses with low infection rates within a community. Thus, for wastewater-based epidemiology applications, 24-h composite samples are less likely to produce false negative results and erroneous public health information.

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T90 (time required for 1-log10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58-43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.

Predicting chronic algal toxicity from 1- to 48-h pulsed exposures to mine site waters using time-averaged concentrations.

Despite concentrations often fluctuating in aquatic systems that receive contaminant inputs, there has only been a relatively small number of studies investigating the toxicity of intermittent exposures. This is particularly the case for industrial and mine effluents that may contain complex mixtures of contaminants and other stressors. The lack of information is impeding the regulation of such contaminant exposures, whose risk is often assessed by comparison to continuous exposures in whole effluent toxicity (direct toxicity assessment) testing. The current study compared the toxicity from continuous (72-h) and pulsed (1- to 48-h) exposures of two neutralised mine waters (NMWs) to the freshwater algae, Chlorella sp. When the algal toxicity of the different exposures was related to the time-averaged concentration (TAC) of contaminants, it was found that the TAC was a good predictor of toxicity in any given test, with variability in toxicity between tests mainly related to differences in contaminant concentrations from the neutralisation of the acidic mine waters. When the data from tests on two samples were combined on a whole-effluent TAC basis, the EC50 values (95% confidence intervals) for the continuous and pulsed exposures were 0.68% (0.36-1.3) and 0.63% (0.38-1.1) respectively, for NMW sample one, while the corresponding EC50 values for NMW sample two were 1.3% (1.0-1.7) and 1.9% (1.6-2.2), respectively. The toxicity of the second water was strongly influenced by the zinc, and probably copper, concentrations, while the toxicity of the first appeared to be related to additive or synergistic toxicity from Al, Cd, Mn and Pb. The findings are discussed in relation to using a contaminant TAC-approach to revise water quality guideline values derived for continuous exposures for application to pulsed exposures, where higher concentrations may be permissible for short durations.

Using meta-omics of contaminated sediments to monitor changes in pathways relevant to climate regulation.

Microbially mediated biogeochemical processes are crucial for climate regulation and may be disrupted by anthropogenic contaminants. To better manage contaminants, we need tools that make real-time causal links between stressors and altered microbial functions, and the potential consequences for ecosystem services such as climate regulation. In a manipulative field experiment, we used metatranscriptomics to investigate the impact of excess organic enrichment and metal contamination on the gene expression of nitrogen and sulfur metabolisms in coastal sediments. Our gene expression data suggest that excess organic enrichment results in (i) higher transcript levels of genes involved in the production of toxic ammonia and hydrogen sulfide and (ii) lower transcript levels associated with the degradation of a greenhouse gas (nitrous oxide). However, metal contamination did not have any significant impact on gene expression. We reveal the genetic mechanisms that may lead to altered productivity and greenhouse gas production in coastal sediments due to anthropogenic contaminants. Our data highlight the applicability of metatranscriptomics as a management tool that provides an immense breadth of information and can identify potentially impacted process measurements that need further investigation.

Application of diffusive gradients in thin films (DGT) and simultaneously extracted metals (SEM) for evaluating bioavailability of metal contaminants in the sediments of Taihu Lake, China.

The toxicities of heavy metals in sediments are related to their bioavailability, which is critical for deriving reliable sediment quality guidelines. To evaluate the bioavailability of the metals (Cd, Cu, Ni, Pb and Zn), sediments were collected from Taihu Lake, one of the largest and most important freshwater lakes in China. Concentrations of simultaneously extracted metals (1-M HCl extraction, CSEM) in the sediments, metals released from sediment to pore waters and accumulated by diffusive gradients in thin films (DGT, CDGT), and dissolved metals in the overlying water (COLW) were measured separately. Sediment toxicity was assessed with tubificids (Monopylephorus limosus) and chironomids (Chironomus kiiensis and Chironomus tentans). Significant relationships existed between the total metal concentrations and CSEM, CDGT, and COLW measurements (r2 = 0.43-0.95, n = 27, p < 0.001), with stronger relationships with CSEM (r2 = 0.91-0.95) than CDGT (r2 = 0.56-0.85) and COLW (r2 = 0.43-0.71). Risk quotients were derived by dividing CSEM by sediment quality guideline values (SQGVs), and by dividing both CDGT and COLW by water quality criteria (WQC). Toxicity of the sediments to the three species was better explained by the CSEM-based risk quotient than those derived from CDGT and COLW. The study indicated that DGT piston probes deployed face down in sediments did not accumulate metals in proportion to the bioavailable metal fraction that caused toxicity to these freshwater benthic organisms, and that single measurements of metals in overlying waters are not adequate for predicting risks of toxicity from sediments. The measurement of CSEM was determined to be effective for assessing the risk posed by the metals in the Taihu Lake sediments, but offered limited improvement over measurement of total metal concentrations.

Decay of sewage-associated bacterial communities in fresh and marine environmental waters and sediment.

Understanding the microbial quality of recreational waters is critical to effectively managing human health risks. In recent years, the development of new molecular methods has provided scientists with alternatives to the use of culture-based fecal indicator methods for investigating sewage contamination in recreational waters. Before these methods can be formalized into guidelines, however, we must investigate their utility, including strengths and weaknesses in different environmental media. In this study, we investigated the decay of sewage-associated bacterial communities in water and sediment from three recreational areas in Southeast Queensland, Australia. Outdoor mesocosms with water and sediment samples from two marine and one freshwater sites were inoculated with untreated sewage and sampled on days 0, 1, 4, 8, 14, 28, and 50. Amplicon sequencing was performed on the DNA extracted from water and sediment samples, and SourceTracker was used to determine the decay of sewage-associated bacterial communities and how they change following a contamination event. No sewage-associated operational taxonomic units (OTUs) were detected in water and sediment samples after day 4; however, the bacterial communities remained changed from their background measures, prior to sewage amendment. Following untreated sewage inoculation, the mesocosm that had the most diverse starting bacterial community recovered to about 60% of its initial community composition, whereas the least diverse bacterial community only recovered to about 30% of its initial community composition. This suggests that a more diverse bacterial community may play an important role in water quality outcomes after sewage contamination events. Further investigation into potential links between bacterial communities and measures of fecal indicators, pathogens, and microbial source tracking (MST) markers is warranted and may provide insight for recreational water decision-makers.

Metal Transfer among Organs Following Short- and Long-Term Exposures Using Autoradiography: Cadmium Bioaccumulation by the Freshwater Prawn Macrobrachium australiense.

The uptake, depuration, and organ distribution of the radioisotope 109Cd were used to explore the internal kinetics of this nonessential metal following accumulation from waterborne cadmium by the freshwater decapod crustacean Macrobrachium australiense. Short- (6 h) and long-term (7 to 14 days) exposures to the radioisotope in solutions of 0.56 µg Cd/L were followed by depuration in metal- and isotope-free water for up to 21 days. The anatomical distribution of the radionuclide was visualized using autoradiography at predefined time points. The gills did not become saturated with cadmium after 14 days of exposure and demonstrated a greater rate of cadmium uptake relative to the hepatopancreas. Cadmium concentrations decreased rapidly during depuration from both gills and hepatopancreas after short exposures but slowly following long-term exposures. This suggests that the duration of cadmium exposure influences the depuration rate for this organism. The study demonstrates the complex behavior of cadmium accumulated by M. australiense and improves our understanding of how exposure duration will influence the internal location and potential toxicity of metals.

Bioavailability and Chronic Toxicity of Metal Sulfide Minerals to Benthic Marine Invertebrates: Implications for Deep Sea Exploration, Mining and Tailings Disposal.

The exploration and proposed mining of sulfide massive deposits in deep-sea environments and increased use deep-sea tailings placement (DSTP) in coastal zones has highlighted the need to better understand the fate and effects of mine-derived materials in marine environments. Metal sulfide ores contain high concentrations of metal(loid)s, of which a large portion exist in highly mineralized or sulfidised forms and are predicted to exhibit low bioavailability. In this study, sediments were spiked with a range of natural sulfide minerals (including chalcopyrite, chalcocite, galena, sphalerite) to assess the bioavailability and toxicity to benthic invertebrates (bivalve survival and amphipod survival and reproduction). The metal sulfide phases were considerably less bioavailable than metal contaminants introduced to sediment in dissolved forms, or in urban estuarine sediments contaminated with mixtures of metal(loid)s. Compared to total concentrations, the dilute-acid extractable metal(loid) (AEM) concentrations, which are intended to represent the more oxidized and labile forms, were more effective for predicting the toxicity of the sulfide mineral contaminated sediments. The study indicates that sediment quality guidelines based on AEM concentrations provide a useful tool for assessing and monitoring the risk posed by sediments impacted by mine-derived materials in marine environments.

Assessing the Effects of Bioturbation on Metal Bioavailability in Contaminated Sediments by Diffusive Gradients in Thin Films (DGT).

The burrowing and feeding activities of benthic organisms can alter metal speciation in sediments and affect an organisms' exposure to metals. Recently, the performance of the in situ technique of diffusive gradients in thin films (DGT) for predicting metal bioavailability has been investigated in response to the increasing demand of considering contaminant bioavailability in sediment quality assessments. In this study, we test the ability of the DGT technique for predicting the metal bioavailability in clean and contaminated sediments that are being subjected to varying degrees of sediments disturbance: low bioturbation (bivalve Tellina deltoidalis alone) and high bioturbation (bivalve and actively burrowing amphipod, Victoriopisa australiensis). Significant release of DGT-labile Cd, Ni, Pb, and Zn, but lower Cu and Fe, occurred in the pore and overlying waters of sediments exposed to high bioturbation conditions, resulting in higher bioaccumulation of zinc in bivalves. Strong relationships were found between bioaccumulation of Pb and Zn and time-integrated DGT-metal fluxes, whereas poor relationships were obtained using total or dilute-acid extractable metal concentrations. This results demonstrate that DGT is a useful tool for assessing metal bioavailability in sediments and can provide useful predictions of metal bioavailable to benthic organisms in dynamic sediment environments.

Bioaccumulation kinetics and organ distribution of cadmium and zinc in the freshwater decapod crustacean Macrobrachium australiense.

This study used the radioisotopes (109)Cd and (65)Zn to explore the uptake, retention and organ distribution of these nonessential and essential metals from solution by the freshwater decapod crustacean Macrobrachium australiense. Three treatments consisting of cadmium alone, zinc alone, and a mixture of cadmium and zinc were used to determine the differences in uptake and efflux rates of each metal individually and in the metal mixture over a three-week period, followed by depuration for 2 weeks in metal-free water using live-animal gamma-spectrometry. Following exposure, prawns were cryosectioned and the spatial distribution of radionuclides visualized using autoradiography. Metal uptake and efflux rates were the same in the individual and mixed-metal exposures, and efflux rates were close to zero. The majority of cadmium uptake was localized within the gills and hepatopancreas, while zinc accumulated in the antennal gland at concentrations orders of magnitude greater than in other organs. This suggested that M. australiense may process zinc much faster than cadmium by internally transporting the accumulated zinc to the antennal gland. The combination of uptake studies and autoradiography greatly increases our understanding of how metal transport kinetics and internal processing may influence the toxicity of essential and nonessential metals in the environment.

Importance of subcellular metal partitioning and kinetics to predicting sublethal effects of copper in two deposit-feeding organisms.

The role of subcellular partitioning of copper on the sublethal effects to two deposit-feeding organisms (41-day growth in the bivalve Tellina deltoidalis and 11-day reproduction in the amphipod Melita plumulosa) was assessed for copper-spiked sediments with different geochemical properties. Large differences in bioaccumulation and detoxification strategies were observed. The bivalve accumulated copper faster than the amphipod, and can be considered a relatively strong net bioaccumulator. The bivalve, however, appears to regulate the metabolically available fraction (MAF) of the total metal pool by increasing the net accumulation rate of copper in the biologically detoxified metal pool (BDM), where most of the copper is stored. In the amphipod, BDM concentration remained constant with increasing copper exposures and it can be considered a very weak net bioaccumulator of copper. This regulation of copper, with relatively little stored in detoxified forms, appears to best describe the strategy applied by the amphipod to minimize the potential toxic effects of copper. When the EC50 values for growth and reproduction are expressed based on the MAF of copper, the sensitivity of the two species appears similar, however when expressed based on the net accumulation rate of copper in the metabolically available fraction (MAFrate), the bivalve appears more sensitive to copper. These results indicate that describing the causality of metal effects in terms of kinetics of uptake, detoxification, and excretion rather than threshold metal body concentrations is more effective in predicting the toxic effects of copper. Although the expression of metal toxicity in terms of the rate at which the metal is bioaccumulated into metabolically available forms may not be feasible for routine assessments, a deeper understanding of uptake rates from all exposure routes may improve our ability to assess the risk posed by metal-contaminated sediments.

Metal Fluxes from Porewaters and Labile Sediment Phases for Predicting Metal Exposure and Bioaccumulation in Benthic Invertebrates.

The use of diffusive gradients in thin films (DGT) for predicting metal bioavailability was investigated by exposing the bivalve Tellina deltoidalis to an identical series of metal-contaminated sediments deployed simultaneously in the field and laboratory. To understand the differences in metal exposure occurring between laboratory- and field-based bioassays, we investigated changes in metal fluxes to DGT probes in sediments and in metal concentrations and partitioning to porewaters and overlying waters. DGT-metal fluxes (Cu, Pb, and Zn) were lower in the overlying waters of most field bioassays compared to the laboratory, causing differences in Pb and Zn bioaccumulation between bivalves exposed to laboratory and field conditions. Overall, DGT-metal fluxes provided predictions of metal bioaccumulation similar to those obtained using dilute-acid extractable metal measurements. This study demonstrates that, irrespective of the physicochemical properties of the sediment and type of exposure (laboratory or field), sediments pose a significant risk of bioaccumulation by T. deltoidalis when the Cu, Pb, and Zn DGT flux exceeds 3.5, 1.3, and 156 µg/h/m(2), respectively. The results presented here support the use of the DGT technique for sediment quality assessment and the hypothesis that DGT-metal fluxes may potentially be useful surrogates for the lability of metals for all exposure routes.

Diffusive gradients in thin films technique provide robust prediction of metal bioavailability and toxicity in estuarine sediments.

Many sediment quality assessment frameworks incorporate contaminant bioavailability as a critical factor regulating toxicity in aquatic ecosystems. However, current approaches do not always adequately predict metal bioavailability to organisms living in the oxidized sediment surface layers. The deployment of the diffusive gradients in thin films (DGT) probes in sediments allows labile metals present in pore waters and weakly bound to the particulate phase to be assessed in a time-integrated manner in situ. In this study, relationships between DGT-labile metal fluxes within 5 mm of the sediment-water interface and lethal and sublethal effects to the amphipod Melita plumulosa were assessed in a range of contaminated estuarine sediments during 10-day laboratory-based bioassays. To account for differing toxicities of metals, DGT fluxes were normalized to water (WQG) or sediment quality guidelines or toxicity thresholds specific for the amphipod. The better dose-response relationship appeared to be the one based on WQG-normalized DGT fluxes, which successfully predicted toxicity despite the wide range of metals and large variations in sediment properties. The study indicated that the labile fraction of metals measured by DGT is useful for predicting metal toxicity to benthic invertebrates, supporting the applicability of this technique as a rapid monitoring tool for sediments quality assessments.

Dissolved and particulate copper exposure induces differing gene expression profiles and mechanisms of toxicity in the deposit feeding amphipod Melita plumulosa.

Uptake of metals via ingestion is an important route of exposure for many invertebrates, and it has been suggested that the toxic response to metals accumulated via food differs from that of metals accumulated via the dissolved phase. To test this hypothesis, the deposit-feeding epibenthic amphipod Melita plumulosa was exposed to nontoxic or reproductively toxic concentrations of copper via the overlying water, via ingestion of sediment, or via a combination of the two. Rates of copper uptake from the two exposure routes were predicted using a biokinetic model. Gene expression profiles were measured via microarray analysis and confirmed via quantitative polymerase chain reaction. Differences in expression profiles were related to the exposure route more than to individual or combined rates of copper uptake. Chitinase and digestive protease transcript expression levels correlated to the copper uptake rate from sediment, rather than from the dissolved phase or combined total uptake rate. Overall, this study supports the hypothesis that metals accumulated via ingestion have a different mode of toxic action than metals taken up from water. Consequently, guidelines that only consider dissolved metal exposure, including equilibrium-partitioning-based guidelines, may underestimate the potential effects from deposited or resuspended metal-contaminated sediments.

Development of a triplex RT-qPCR assay for simultaneous quantification of Japanese encephalitis, Murray Valley encephalitis, and West Nile viruses for environmental surveillance.

The co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) has impacted human and animal health in multiple countries worldwide. To facilitate early warnings and surveillance of the presence of these viral infectious agents in the environment, a triplex reverse transcription-quantitative PCR (RT-qPCR) was developed for simultaneous quantification of JEV, MVEV, and WNV in potential hotspots such as piggery and urban wastewater and environmental water samples. The performance of the developed triplex RT-qPCR assay was compared with that of simplex counterparts, all using the same primer and probe sequences. The quantifiable results showed a concordance rate of 93.9%-100% (Cohen's kappa) between the triplex and simplex assays. The mean concentrations of exogenous JEV, MVEV, and WNV using the triplex and simplex RT-qPCR assays were remarkably similar in piggery/urban wastewater and environmental water samples. However, the impacts of the matrix effects (i.e., sample composition and PCR inhibition) of environmental water samples on the accurate quantification of these viruses need to be considered. Taken together, this newly developed triplex RT-qPCR assay of JEV, MVEV, and WNV will allow for a more rapid and cost-efficient sample analysis and data interpretation. The application of the triplex assay for environmental surveillance may be a valuable tool to complement the existing disease and mosquito surveillance approaches used to safeguard the health of both humans and animals.IMPORTANCEThe co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) poses significant threats to human and animal health globally. In this study, a triplex RT-qPCR assay was developed for simultaneous quantification of these viruses in wastewater and environmental water samples. Results demonstrated high concordance and sensitivity of the newly developed triplex RT-qPCR assay compared to simplex assays, indicating its efficacy for environmental surveillance. This cost-effective and rapid assay offers a vital tool for timely monitoring of mosquito-borne viruses in environmental samples, enhancing our ability to mitigate potential outbreaks and safeguard public health.