Application of in vitro bioassays to monitor pharmaceuticals in water: A synthesis of chronological analysis, mode of action, and practical insights.

Pharmaceutical compounds in wastewater have emerged as a significant concern for the aquatic environment. The use of in vitro bioassays represents a sustainable and cost-effective approach for assessing the potential toxicological risks of these biologically active compounds in wastewater and aligns with ethical considerations in research. It facilitates high-throughput analysis, captures mixture effects, integrates impacts of both known and unknown chemicals, and reduces reliance on animal testing. The core aim of the current review was to explore the practical application of in vitro bioassays in evaluating the environmental impacts of pharmaceuticals in wastewater. This comprehensive review strives to achieve several key objectives. First, it provides a summary categorisation of pharmaceuticals based on their mode of action, providing a structured framework for understanding their ecological significance. Second, a chronological analysis of pharmaceutical research aims to document their prevalence and trends over time, shedding light on evolving environmental challenges. Third, the review critically analyses existing bioassay applications in wastewater, while also examining bioassay coverage of representative compounds within major pharmaceutical classes. Finally, it explores the potential for developing innovative bioassays tailored for water quality monitoring of pharmaceuticals, paving the way for more robust environmental monitoring and risk assessment. Overall, adopting effect-based methods for pharmaceutical monitoring in water holds significant promise. It encompasses a broad spectrum of biological impacts, promotes standardized protocols, and supports a bioassay test battery approach indicative of different endpoints, thereby enhancing the effectiveness of environmental risk assessment.

Polyethylene microplastics induced lipidomic responses in Chironomus tepperi: A two-generational exploration.

Microplastics (MPs) accumulating in freshwater sediment have raised concerns about potential risks to benthic dwelling organisms, yet few studies have examined the long-term impacts caused by MP exposure. This study investigated alterations to lipid profiles in an Australian freshwater invertebrate, Chironomus tepperi, induced by polyethylene MP fragments (1-45 µm) at environmentally relevant concentrations (125, 250, 500 and 1000 MPs/kg sediment), using a two-generational experimental design. In the parental generation, the relative abundance of triacylglycerols, total fatty acids and unsaturated fatty acids exhibited apparent hormetic patterns, with low-concentration stimulation and high-concentration inhibition observed. The overall trend in these lipid classes is consistent with previously observed changes to polar metabolite profiles, indicating that ingestion of MPs could inhibit nutrient assimilation from food leading to disruption of energy availability. In the first filial generation continuously exposed to MPs, however, abundance of cholesterol and total fatty acids increased with increasing exposure concentrations, suggesting different effects on energy metabolism between the parental generation and offspring. No differences in the lipidome were observed in first filial larvae that were not exposed, implying that MPs pose negligible carry-over effects. Overall, the combined results of this study together with a preceding metabolomics study provide evidence of a physical effect of MPs with subsequent impacts to bioenergetics. Nevertheless, future research is required to explore the potential long-term impacts caused by MPs, and to unravel the impacts of the surfactant control as a potential contributor to the observed hormetic response, particularly for studies exploring sub-lethal effects of MP exposure using sensitive omics techniques.

High-Throughput Transcriptomics of Water Extracts Detects Reductions in Biological Activity with Water Treatment Processes.

The presence of numerous chemical contaminants from industrial, agricultural, and pharmaceutical sources in water supplies poses a potential risk to human and ecological health. Current chemical analyses suffer from limitations, including chemical coverage and high cost, and broad-coverage in vitro assays such as transcriptomics may further improve water quality monitoring by assessing a large range of possible effects. Here, we used high-throughput transcriptomics to assess the activity induced by field-derived water extracts in MCF7 breast carcinoma cells. Wastewater and surface water extracts induced the largest changes in expression among cell proliferation-related genes and neurological, estrogenic, and antibiotic pathways, whereas drinking and reclaimed water extracts that underwent advanced treatment showed substantially reduced bioactivity on both gene and pathway levels. Importantly, reclaimed water extracts induced fewer changes in gene expression than laboratory blanks, which reinforces previous conclusions based on targeted assays and improves confidence in bioassay-based monitoring of water quality.

Effect-based monitoring of two rivers under urban and agricultural influence reveals a range of biological activities in sediment and water extracts.

Chemical contaminants, such as pesticides, pharmaceuticals and industrial compounds are ubiquitous in surface water and sediment in areas subject to human activity. While targeted chemical analysis is typically used for water and sediment quality monitoring, there is growing interest in applying effect-based methods with in vitro bioassays to capture the effects of all active contaminants in a sample. The current study evaluated the biological effects in surface water and sediment from two contrasting catchments in Aotearoa New Zealand, the highly urbanised Whau River catchment in Tamaki Makaurau (Auckland) and the urban and mixed agricultural Koreti (New River) Estuary catchment. Two complementary passive sampling devices, Chemcatcher for polar chemicals and polyethylene (PED) for non-polar chemicals, were applied to capture a wide range of contaminants in water, while composite sediment samples were collected at each sampling site. Bioassays indicative of induction of xenobiotic metabolism, receptor-mediated effects, genotoxicity, cytotoxicity and apical effects were applied to the water and sediment extracts. Most sediment extracts induced moderate to strong estrogenic and aryl hydrocarbon (AhR) activity, along with moderate toxicity to bacteria. The water extracts showed similar patterns to the sediment extracts, but with lower activity. Generally, the polar Chemcatcher extracts showed greater estrogenic activity, photosynthesis inhibition and algal growth inhibition than the non-polar PED extracts, though the PED extracts showed greater AhR activity. The observed effects in the water extracts were compared to available ecological effect-based trigger values (EBT) to evaluate the potential risk. For the polar extracts, most sites in both catchments exceeded the EBT for estrogenicity, with many sites exceeding the EBTs for AhR activity and photosynthesis inhibition. Of the wide range of endpoints considered, estrogenic activity, AhR activity and herbicidal activity appear to be the primary risk drivers in both the Whau and Koreti Estuary catchments.

ECHIDNA (Emerging CHemIcals Database for National Awareness): a framework to prioritise contaminants of emerging concern in water.

The widespread presence of contaminants of emerging concern (CEC) in surface waters, treated wastewater and drinking water is an ongoing issue for the water industry. The absence of regulatory guidance and limited occurrence, toxicity and removal data are defining criteria of CEC and make it difficult to prioritise which CEC pose the greatest risk. The online Emerging CHemIcals Database for National Awareness (ECHIDNA) aims to classify and prioritise CEC based on their potential risk, with the information presented in an easily accessible and intuitive manner. A candidate list of almost 1,800 potential CEC, including pesticides, pharmaceuticals and industrial compounds, was compiled using both Australian and international resources. These were ranked based on in silico assessment of their persistent, bioaccumulative and toxic (PBT) properties, as well as potential chronic toxicity hazard, yielding 247 CEC for further prioritisation. Risk Quotients (RQ) identified between 5 and 87 CEC posing a risk to human and ecosystem health, respectively, across drinking water, surface water, treated wastewater and raw wastewater. While the ability of the water industry to effectively prioritise CEC is limited by candidate identification and data availability, ECHIDNA can provide valuable information for better decision-making surrounding CEC management.

A Pesticide Decision Support Tool to guide the selection of less environmentally harmful pesticides for the sugar cane industry.

Pesticides applied to agricultural land have been shown to decrease the quality of water entering the Great Barrier Reef lagoon. This issue is addressed by the Reef 2050 Water Quality Improvement Plan which includes a pesticide reduction target. As part of a wider educational strategy, one method that could help meet the target is to provide stakeholders with information that assists in the selection and use of pesticide active ingredients (PAIs) that pose a lower risk to aquatic environments compared to those currently used. This study developed a Pesticide Decision Support Tool (PDST) in collaboration with stakeholders for the sugar cane industry. The PDST covers all PAIs registered and applied to sugar cane in Australia and four additional PAIs registered for use on crops grown in rotation with sugar cane. The PDST incorporates both the measure of mobility and persistence of a PAI and the measure of effect, which is based on the PAI application rate and ecotoxicity threshold value. The aquatic risk, which is the product of the measure of effect and the measure of mobility and persistence, is a measure of the likelihood that a PAI will reach the aquatic environment and cause harmful effects. Insecticide active ingredients (e.g., cadusafos, chlorpyrifos) posed the greatest aquatic risk, followed by herbicide active ingredients (e.g., MSMA, metolachlor), while fungicide AIs typically had a lower aquatic risk. An interactive spreadsheet allows characteristics, including application rate and tank mixes, to be considered when assessing the potential risk. While focusing on sugar cane, the results are equally appropriate to other crops that use the same PAIs provided the application rates are corrected to the new crop. In addition, the approach used in the PDST can be applied internationally and to any PAIs with sufficient toxicity, mobility, and persistence data.

Metabolomic responses in freshwater benthic invertebrate, Chironomus tepperi, exposed to polyethylene microplastics: A two-generational investigation.

The accumulation of microplastics (MPs) in sediments could pose risks to benthic organisms and their progeny. Here, we examined effects on traditional apical endpoints along with changes to whole body metabolite profiles induced by irregular shaped polyethylene MPs (1-45 µm) at environmentally relevant concentrations (125, 250, 500 and 1000 MPs/kg sediment) in Chironomus tepperi using a two-generation exposure regime. Survival and emergence of C. tepperi were negatively affected in the parental generation at the two highest concentrations, whereas endpoints associated with growth were only impacted at 1000 MPs/kg sediment. Metabolites associated with several amino acid and energy metabolism pathways were present at lower abundances at the highest exposure concentration suggesting an overall impact on bioenergetics which relates to the inhibition of food acquisition or nutrient assimilation caused by ingestion of MPs, rather than a traditional receptor-mediated toxicity response. In contrast, no significant effects on apical endpoints were observed in the continuous exposure of first filial generation, and lactic acid was the only metabolite that differed significantly between groups. Larvae in unexposed conditions showed no differences in survival or metabolite profiles suggesting that effects in the parental generation do not carry over to the next filial generation. The findings provide evidence on the underlying impacts of MP ingestion and potential adaption to MP exposure of C. tepperi.

Application of Effect-Based Methods to Water Quality Monitoring: Answering Frequently Asked Questions by Water Quality Managers, Regulators, and Policy Makers.

Effect-based methods (EBM) have great potential for water quality monitoring as they can detect the mixture effects of all active known and unknown chemicals in a sample, which cannot be addressed by chemical analysis alone. To date, EBM have primarily been applied in a research context, with a lower level of uptake by the water sector and regulators. This is partly due to concerns regarding the reliability and interpretation of EBM. Using evidence from the peer-reviewed literature, this work aims to answer frequently asked questions about EBM. The questions were identified through consultation with the water industry and regulators and cover topics related to the basis for using EBM, practical considerations regarding reliability, sampling for EBM and quality control, and what to do with the information provided by EBM. The information provided in this work aims to give confidence to regulators and the water sector to stimulate the application of EBM for water quality monitoring.

Analysis of the literature shows a remarkably consistent relationship between size and abundance of microplastics across different environmental matrices.

Microplastics come in a variety of shapes, polymer types and sizes. Due to the lack of a harmonised approach to analyse and quantify microplastics, there are huge disparities in size detection limits and size classifications used in the literature. This has caused large variations in reported microplastic data and has made comparing microplastic abundance between studies extremely challenging. Herein, we applied a simple mathematical approach that allows for a meaningful comparison between size and abundance (number of particles) of microplastics irrespective of the size classifications used. This method was validated using two separate datasets (microplastics in air and sediment) and applied to re-analyse 127 publications reporting microplastics in various environmental matrices. We demonstrate a strong negative linear relationship between microplastic concentrations and their sizes with comparable slopes across all matrices. Using this method, it is possible to compare the concentration of microplastics of various sizes between studies. It also allows estimation of the abundance of microplastics of a specific size where data are not available. This enables researchers to predict environmentally relevant concentrations of microplastics (particularly for smaller microplastics) and provide realistic exposure scenarios in future toxicity studies, which will greatly improve our understanding of the risks that microplastics pose to living organisms.

Effect-Based Trigger Values Are Essential for the Uptake of Effect-Based Methods in Water Safety Planning.

Effect-based methods (EBMs) using in vitro bioassays and well plate-based in vivo assays are recommended for water quality monitoring because they can capture the mixture effects of the many chemicals present in water. Many in vitro bioassays are highly sensitive, so an effect in a bioassay does not necessarily indicate poor chemical water quality. Consequently, effect-based trigger values (EBTs) have been introduced to differentiate between acceptable and unacceptable chemical water quality and are required for the wider acceptance of EBMs by the water sector and regulatory bodies. These EBTs have been derived for both drinking water and surface water to protect human and ecological health, respectively, and are available for assays indicative of specific receptor-mediated effects, as well as assays indicative of adaptive stress responses, apical effects, and receptor-mediated effects triggered by many chemicals. An overview of currently available EBTs is provided, and a simple approach is proposed to predict interim EBTs for assays currently without an EBT based on the effect concentration of the assay reference compound. There was good agreement between EBTs predicted using this simplistic approach and EBTs from the literature derived using more robust methods. Finally, an interpretation framework that outlines the steps to take if the effect of a sample exceeds the EBT was developed to help facilitate the uptake of EBMs in routine water quality monitoring and water safety planning for drinking water production. Environ Toxicol Chem 2023;42:714-726. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effect-based monitoring to integrate the mixture hazards of chemicals into water safety plans.

Water safety plans (WSPs) are intended to assure safe drinking water (DW). WSPs involve assessing and managing risks associated with microbial, chemical, physical and radiological hazards from the catchment to the consumer. Currently, chemical hazards in WSPs are assessed by targeted chemical analysis, but this approach fails to account for the mixture effects of the many chemicals potentially present in water supplies and omits the possible effects of non-targeted chemicals. Consequently, effect-based monitoring (EBM) using in vitro bioassays and well plate-based in vivo assays are proposed as a complementary tool to targeted chemical analysis to support risk analysis, risk management and water quality verification within the WSP framework. EBM is frequently applied to DW and surface water and can be utilised in all defined monitoring categories within the WSP framework (including 'system assessment', 'validation', 'operational' and 'verification'). Examples of how EBM can be applied within the different WSP modules are provided, along with guidance on where to apply EBM and how frequently. Since this is a new area, guidance documents, standard operating procedures (SOPs) and decision-making frameworks are required for both bioassay operators and WSP teams to facilitate the integration of EBM into WSPs, with these resources being developed currently.

Microplastics profile in constructed wetlands: Distribution, retention and implications.

Wastewater and stormwater are both considered as critical pathways contributing microplastics (MPs) to the aquatic environment. However, there is little information in the literature about the potential influence of constructed wetlands (CWs), a commonly used wastewater and stormwater treatment system. This study was conducted to investigate the abundance and distribution of MPs in water and sediment at five CWs with different influent sources, namely stormwater and wastewater. The MP abundance in the water samples ranged between 0.4 ± 0.3 and 3.8 ± 2.3 MP/L at the inlet and from 0.1 ± 0.0 to 1.3 ± 1.0 MP/L at the outlet. In the sediment, abundance of MPs was generally higher at the inlet, ranging from 736 ± 335 to 3480 ± 4330 MP/kg dry sediment and decreased to between 19.0 ± 16.4 and 1060 ± 326 MP/kg dry sediment at the outlet. Although no significant differences were observed in sediment cores at different depth across the five CWs, more MPs were recorded in silt compared to sandy sediment which indicated sediment grain size could be an environmental factor contributing to the distribution of MPs. Polyethylene terephthalate (PET) fibres were the dominant polymer type found in the water samples while polyethylene (PE) and polypropylene (PP) fragments were predominantly recorded in the sediment. While the size of MPs in water varied across the studied CWs, between 51% and 64% of MPs in the sediment were smaller than 300 µm, which raises concerns about the bioavailability of MPs to a wider range of wetland biota and their potential ecotoxicological effects. This study shows that CWs can not only retain MPs in the treated water, but also become sinks accumulating MPs over time.

Chiral Inversion of 2-Arylpropionic Acid Enantiomers under Anaerobic Conditions.

This work examined the chiral inversion of 2-arylpropionic acids (2-APAs) under anaerobic conditions and the associated microbial community. The anaerobic condition was simulated by two identical anaerobic digesters. Each digester was fed with the substrate containing 11 either pure (R)- or pure (S)-2-APA enantiomers. Chiral inversion was evidenced by the concentration increase of the other enantiomer in the digestate and the changes in the enantiomeric fraction between the two enantiomers. Both digesters showed similar and poor removal of 2-APAs (≤30%, except for naproxen) and diverse chiral inversion behaviors under anaerobic conditions. Four compounds exhibited (S → R) unidirectional inversion [flurbiprofen, ketoprofen, naproxen, and 2-(4-tert-butylphenyl)propionic acid], and the remaining seven compounds showed bidirectional inversion. Several aerobic and facultative anaerobic bacterial genera (Candidatus Microthrix, Rhodococcus, Mycobacterium, Gordonia, and Sphingobium) were identified in both digesters and predicted to harbor the 2-arylpropionyl-CoA epimerase (enzyme involved in chiral inversion) encoding gene. These genera presented at low abundances, <0.5% in the digester dosed with (R)-2-APAs and <0.2% in the digester dosed with (S)-2-APAs. The low abundances of these genera explain the limited extent of chiral inversion observed in this study.

Chiral inversion of 2-arylpropionoic acid (2-APA) enantiomers during simulated biological wastewater treatment.

This study examined the removal and enantio­specific fate of a suite of eleven chiral 2-arylpropionic acids (2-APAs) during biological wastewater treatment simulated in a laboratory-scale membrane bioreactor (MBR). Using pure (R)- and (S)- enantiomers in the MBR influent, chiral inversion was determined through the increase in the concentration of the non-dominant enantiomer and changes in the enantiomeric fraction (EF) between the two enantiomers during the treatment process. Effective (>90%) and similar removal rates between (R)- and (S)- enantiomers were confirmed for eight 2-APAs. In this study, 2-APAs exhibited diverse and distinctive chiral inversion behaviours: two 2-APAs showed (R→S) unidirectional inversion, three 2-APAs showed (S→R) unidirectional inversion, and six 2-APAs showed bidirectional inversion. This is the first study to report chiral inversion behaviours of a comprehensive suite of 2-APAs with a variety of functional groups substituted onto the aryl ring. A decrease in effluent EF over time was observed for two 2-APAs. This study shows that chiral inversion of 2-APAs varies significantly from compound to compound, despite the high similarity in their chemical structures.

Terrestrial dissolved organic matter source affects disinfection by-product formation during water treatment and subsequent toxicity.

Restoring woody vegetation to riparian zones helps to protect waterways from excessive sediment and nutrient inputs. However, the associated leaf litter can be a major source of dissolved organic matter (DOM) leached into surface waters. DOM can lead to the formation of disinfection by-products (DBPs) during drinking water treatment. This study investigated the DBPs formed during chlorination of DOM leached from leaf litter and assessed the potential toxicity of DBPs generated. We compared the leachate of two native Australian riparian trees, Casuarina cunninghamiana and Eucalyptus tereticornis, and a reservoir water source from a catchment dominated by Eucalyptus species. Leachates were diluted to dissolved organic carbon concentrations equivalent to the reservoir (~9 mg L-1). E. tereticornis leachates produced more trihalomethanes (THMs), haloacetic acids (HAAs), and haloketones after chlorination, while C. cunninghamiana produced more chloral hydrate and haloacetonitriles. Leachate from both species produced less THMs and more HAAs per mole of carbon than reservoir water. This may be because reservoir water had more aromatic, humic characteristics while leaf leachates had relatively more protein-like components. Using in vitro bioassays to test the mixture effects of all chemicals, chlorinated E. tereticornis leachate induced oxidative stress in HepG2 liver cells and bacterial toxicity more frequently and at lower concentrations than C. cunninghamiana and reservoir water. Overall, this study has shown that the DOM leached from litter of these species has the potential to generate DBPs and each species has a unique DBP profile with differing bioassay responses. E. tereticornis may pose a relatively greater risk to drinking water than C. cunninghamiana as it showed greater toxicity in bioassays. This implies tree species should be considered when planning riparian zones to ensure the benefits of vegetation to waterways are not offset by unintended increased DBP production and associated toxicity following chlorination at downstream drinking water intakes.

Combined toxicity of therapeutic pharmaceuticals to duckweed, Lemna minor.

Pharmaceuticals, which are designed to be biologically active at low concentrations, are found in surface waters, meaning aquatic organisms can be exposed to complex mixtures of pharmaceuticals. In this study, the adverse effects of four pharmaceuticals, 17α-ethynylestradiol (synthetic estrogen), methotrexate (anticancer drug), diclofenac (nonsteroidal anti-inflammatory drug) and fluoxetine (antidepressant), and their binary mixtures at mg/L concentrations were assessed using the 7-day Lemna minor test, with both apical and biochemical markers evaluated. The studied biochemical markers included chlorophyll a, chlorophyll b, carotenoids and oxidative stress enzymes catalase, glutathione-S-transferase and glutathione reductase, with effects compared to solvent controls. The adverse effects on Lemna minor were dose-dependent for frond number, surface area, relative chlorophyll content and activity of glutathione S-transferase for both individual pharmaceuticals and binary mixtures. According to the individual toxicity values, all tested pharmaceuticals can be considered as toxic or harmful to aquatic organisms, with methotrexate considered highly toxic. The most sensitive endpoints for the binary mixtures were photosynthetic pigments and frond surface area, with effects observed in the low mg/L concentration range. The concentration addition model and toxic unit approach gave similar mixture toxicity predictions, with binary mixtures of methotrexate and fluoxetine or methotrexate and 17α-ethynylestradiol exhibiting synergistic effects. In contrast, mixtures of diclofenac with fluoxetine, 17α-ethynylestradiol or methotrexate mostly showed additive effects. While low concentrations of methotrexate are expected in surface water, chronic ecotoxicological data for invertebrates and fish are lacking, but this is required to better assess the environmental risk of methotrexate.

Effect-Based Trigger Values for Mixtures of Chemicals in Surface Water Detected with In Vitro Bioassays.

Effect-based trigger (EBT) values for in vitro bioassays are important for surface water quality monitoring because they define the threshold between acceptable and poor water quality. They have been derived for highly specific bioassays, such as hormone-receptor activation in reporter gene bioassays, by reading across from existing chemical guideline values. This read-across method is not easily applicable to bioassays indicative of adaptive stress responses, which are triggered by many different chemicals, and activation of nuclear receptors for xenobiotic metabolism, to which many chemicals bind with rather low specificity. We propose an alternative approach to define the EBT from the distribution of specificity ratios of all active chemicals. The specificity ratio is the ratio between the predicted baseline toxicity of a chemical in a given bioassay and its measured specific endpoint. Unlike many previous read-across methods to derive EBTs, the proposed method accounts for mixture effects and includes all chemicals, not only high-potency chemicals. The EBTs were derived from a cytotoxicity EBT that was defined as equivalent to 1% of cytotoxicity in a native surface water sample. The cytotoxicity EBT was scaled by the median of the log-normal distribution of specificity ratios to derive the EBT for effects specific for each bioassay. We illustrate the new approach using the example of the AREc32 assay, indicative of the oxidative stress response, and 2 nuclear receptor assays targeting the peroxisome proliferator-activated receptor gamma and the arylhydrocarbon receptor. The EBTs were less conservative than previously proposed but were able to differentiate untreated and insufficiently treated wastewater from wastewater treatment plant effluent with secondary or tertiary treatment and surface water. Environ Toxicol Chem 2021;40:487-499. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

An audit of microplastic abundance throughout three Australian wastewater treatment plants.

Wastewater treatment plants (WWTPs) have been identified as an important pathway of microplastics to the environment. Most studies have focused on wastewater effluent, but generally only a small fraction of microplastics entering WWTPs are present in treated effluent. Instead, the majority of microplastics are expected to be retained in the sludge. To our knowledge, there is limited information on microplastics in sludge/biosolids from Australian WWTPs, despite 75% of biosolids produced in Australia being used for agriculture. This study evaluated the abundance of microplastics throughout the treatment trains of three WWTPs in Australia. The fate of microplastics >25 µm during treatment and their release to the environment was evaluated using an audit approach. The highest microplastic concentrations were detected in the influent, with fibres the dominant form of microplastic found. The screening and grit removal process preceding primary treatment removed 69-79% of microplastics, with these microplastics transported to landfill. Only 0.2-1.8% of the total microplastics in the influent were present in the final effluent, while 8-16% were retained in biosolids. This equates to between 22.1 × 106 to 133 × 106 microplastic particles per day released in effluent, between 864 × 106 to 1020 × 106 microplastic particles per day in biosolids, and between 4100 × 106 to 9100 × 106 microplastic particles per day transported to landfill. This study shows for the first time that most microplastics are retained during the initial screening and grit removal process with the load of microplastics going to landfill an order of magnitude greater than that in biosolids. Landfills may thus be an important sink (and potential future source) of microplastics from wastewater.

Using bioanalytical tools to detect and track organic micropollutants in the Ganga River near two major cities.

Major rivers in India are subject to ongoing impacts from urban drain discharges, most of which contain high levels of domestic and industrial wastewater and stormwater. The aim of the present study was to determine the levels of bioactive organic micropollutants at the discharge points of major urban drains in comparison to upstream and downstream sites. To achieve this, we employed a panel of in vitro bioanalytical tools to quantify estrogenic, androgenic, progestogenic, glucocorticoid and peroxisome proliferator-like activity in water extracts collected from two Indian cities in the Ganga Basin. Cytotoxicity of the water extracts in a human-derived cell line and the potential to cause oxidative stress in a fish cell line were also investigated. We found high levels of activity for all endpoints in samples directly receiving urban drain discharge and low levels at sites upstream from drain discharges. Estrogenicity was detected at levels equivalent to 10 ng/L 17ß-estradiol, representing a high likelihood of biomarker effects in fish. Sites located downstream from drain discharges exhibited low to intermediate activity in all assays. This study demonstrates the importance of managing urban drain discharges and the utility of applying bioanalytical tools to assess water quality.