Effect-based Trigger Research Articles

Effect of tunnel wash water treatment processes on trace elements, organic micropollutants, and biological effects

Tunnel wash water (TWW) contains high levels of trace elements and organic micropollutants, especially in the dissolved fraction. Discharge poses significant environmental risks. This field study aimed at improving conventional sedimentation treatment by addition of novel secondary treatments: bag filtration, ceramic microfiltration, or granular activated carbon (GAC) filtration. Removal of nine trace elements, 16 polycyclic aromatic hydrocarbons (PAHs), 38 per- and polyfluoroalkyl substances (PFASs), seven benzothiazoles (BTHs), seven benzotriazoles (BTRs), five bisphenols (BPs), and five benzophenones was investigated. Primary sedimentation significantly reduced particles and associated contaminants, achieving over 73 % average removal for trace elements, 65 % for PAHs, and 71 % for PFASs. Subsequent GAC removed over 70 % of dissolved Cr, Cu, Pb, and Zn and over 92 % of dissolved PFASs, BTHs, BTRs, and BPs, including several persistent, mobile and toxic compounds. Following GAC filtration, Cr, Ni, Pb, anthracene, fluoranthene, perfluorooctanesulfonic acid, and bisphenol-A were below environmental quality standards (EQS). GAC consistently reduced responses in in vitro bioassays with endpoints activation of the aryl hydrocarbon receptor, oxidative stress response, and neurotoxicity below effect-based trigger values for surface water. GAC filtration is thus recommended for future TWW treatment. Assessing water quality remains a challenging task due to lack of EQSs for many chemicals.

The use of in vitro bioassays and chemical screening to assess the impact of a minimally processed vegetable facility on wastewater quality.

Fruit- and vegetable-processing facilities may contaminate wastewater via contaminants found in the produce and disinfecting chemicals used. These contaminants may include agrochemicals, pesticides, and disinfectants such as chlorine and quaternary ammonium compounds (QACs). Some compounds may exhibit harmful endocrine-disrupting activity. This study investigated the impact of a minimally processed vegetable facility on wastewater quality via in vitro bioassays and chemical screening. Estrogen activity was assessed via a yeast estrogen screen (YES), and (anti-)androgenic and glucocorticoid activities were evaluated via an MDA-kb2 reporter gene assay. The samples were screened via gas and liquid chromatography-tandem mass spectrometry (GC-MS/MS and LC-MS/MS) to identify target compounds, and GC coupled with time-of-flight mass spectrometry (GC-TOFMS) was used for non-targeted screening. Sample complexity and chemical profiles were assessed using GC-TOFMS. Estrogenic activity was detected in 16 samples (n = 24) with an upper limit of 595 ± 37ng/L estradiol equivalents (EEqs). The final wastewater before discharge had an EEq of 0.23ng/L, which is within the ecological effect-based trigger value range for the estrogenic activity of wastewater (0.2-0.4ng/L EEq). Androgenic activity was detected in one sample with a dihydrotestosterone equivalent (DHTEq) value of 10 ± 2.7ng/L. No antiandrogenic activity was detected. The GC-MS/MS and LC-MS/MS results indicated the presence of multiple pesticides, nonylphenols, triclocarban, and triclosan. Many of these compounds exhibit estrogenic activity, which may explain the positive YES assay findings. These findings showed that wastewater from the facility contained detergents, disinfectants, and pesticides and displayed hormonal activity. Food-processing facilities release large volumes of wastewater, which may affect the quality of the water eventually being discharged into the environment. We recommend expanding conventional water quality monitoring efforts to include additional factors like endocrine activity and disinfectant byproducts.

Priority screening of contaminants of emerging concern (CECs) in surface water: Comparing cell-based bioassays and exposure-activity ratios (EARs)

In this study, we compared a wide range of cell-based bioassays to the use of chemical analysis followed by exposure-activity ratio (EAR) and Toxicological Prioritization index (ToxPi) for prioritizing chemicals, sites, and hazard concerns in water samples. Surface water samples were collected from nine sites in three Central Pennsylvania streams and analyzed for a forty-six contaminants of emerging concern (CECs), including pesticides, personal care products, and pharmaceuticals. Cell-based reporter assays evaluated human and zebrafish molecular initiating events (MIEs) in endocrine and metabolic disruption, altered lipid metabolism, and oxidative stress. Bioassays showed that 12 out of 40 assays had at least one site with activity over the effect-based trigger (EBT) values. The receptors that exhibited the highest number of samples above the EBT that would be expected to cause toxicity were Aryl hydrocarbon receptor (AhR, human and zebrafish), Pregnane X Receptor (PXR), Estrogen Receptor-beta (ERB), and Androgen Receptor (AR). Characterizing the collection sites by their bioactivity aligned closely with the stream in which samples were collected. The sum of all EARs for each chemical indicated that the pharmaceutical Carbamazepine and the pesticides Carbaryl and Atrazine posed the greatest concern. However, predicted activity and site prioritization based on individual chemical analysis and calculated EAR were different than those measured by bioassay, indicating that biologically active chemicals are present in the samples that were not included in the targeted analytes. Taken together, these data show that chemical analysis and EAR analysis are beneficial for prioritization of chemicals, whereas mechanism-based bioassays are more inclusive of known as well as unknown chemical contaminants and thus of more use for overall water quality analysis and site prioritization.

Bioassay predictive values for chemical health risks in drinking water

Bioanalytical tools can be used for assessment of the chemical quality of drinking water and its sources. For water managers it is important to know the probability that a bioassay response above an established health-based ‘effect-based trigger value’ (EBT) indeed implies a harmful chemical (mixture) concentration. This study presents and applies a framework, based on Bayes’ theorem, to derive such risk probabilities for bioassay responses. These were evaluated under varying (in silico) chemical mixture concentrations relevant to drinking water (sources), with toxicity data for six in vitro assays from the ToxCast database. For single chemicals and in silico mixtures, the negative predictive value (NPV) was 100 % for all assays. For water managers, this means that when a bioassay response is below the EBT, a chemical risk is reliably absent, and no further action is required. The positive predictive value (PPV) increased with increasing chemical concentrations (2 µg/L) up to 40–80 %, depending on the assay. For in silico mixtures of increasing numbers of chemicals, the PPV did not increase until higher sum concentrations (>2–10 µg/L). Hence, the ability to accurately signal a harmful chemical (mixture) using bioassays will be lowest for highly diverse, low-concentration chemical mixtures. For water managers, this means in practice that further investigations after an EBT exceedance will, in many cases, not reveal chemicals at harmful concentrations. A solution offered is to increase the trigger value for positive responses to achieve a higher PPV and maintain the EBT for negative responses to ensure an optimal NPV.

Beyond the Drinking Water Directive: The use of reporter gene assays as an added tool for effect-based monitoring of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in drinking water sources

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) are legacy organic micropollutants (OMPs) that are sporadically detected in drinking water (DW) sources. The European Drinking Water Directive requires EU member states to monitor 5 PAHs in DW and its sources. The Dutch national regulations require 6 additional PAHs to be monitored and 7 polychlorinated biphenyls (PCBs). These indicator compounds act as representatives for large compound classes. PCBs alone comprise 209 congeners, it is evident that conventional chemical target analysis (GC-tQ-MS) alone is not sufficient to monitor these entire compound classes. This study investigated the application of reporter gene assays as effect-based methods (EBMs) to monitor PAHs and PCBs in DW sources. Herein, it was assessed what added value the bioassays can bring compared to the current approach of chemical target analysis for PCBs and PAHs. Regulated and non-regulated PAHs and PCBs were tested in four bioassays to determine the relative potency factors (RPFs) for these compounds. Non-regulated congeners were found to be active in the PAH-CALUX and anti-AR CALUX. An assessment of surface water (SW) spiked with standard mixtures containing PAHs and PCBs confirmed the predictable behavior of the PAH-CALUX. Moreover, the bioassay was able to detect AhR-mediated activity caused by non-regulated PAHs and PCBs, whereas this would have been missed by conventional chemical target analysis. Last, a field study was conducted in Dutch DW sources at six sampling moments. The PAH-CALUX detected AhR-mediated activity at all sampling moments and an ecological effect-based trigger (EBT) value was exceeded on multiple accounts. Combined application of GC-tQ-MS and the PAH-CALUX ensures compliancy with monitoring legislation and provides additional insights into potential hazards to humans and the environment.

Endocrine disrupting chemicals in Italian drinking water systems: Insights from a three-year investigation combining chemical and effect-based tools

Drinking water quality can be compromised by endocrine-disrupting chemicals (EDCs). Three phenolic compounds [bisphenol A (BPA), nonylphenol (NP), and 4-octylphenol (OP)] and three hormones [17β-estradiol (E2), estrone (E1), and 17α-ethinylestradiol (EE2)] were analyzed as EDCs potentially occurring in source and drinking water from three full-scale drinking water treatment plants (DWTPs) in the Romagna area (Italy) by a combined approach of HPLC-MS/MS target analysis and effect-based tests for estrogenicity and genotoxicity. The EDC removal efficiency was evaluated at different steps along the treatment process in the most advanced DWTP. NP prevailed in all samples, followed by BPA. Sporadic contamination by OP and E1/E2 appeared only in the source waters; EE2 was never detected. No estrogenic or genotoxic activity was found, except for two samples showing estrogenicity well below the effect-based trigger value suggested for drinking water safety (0.9 ng/L EEQ). BPA and NP levels were largely below the threshold value; however, increases were observed after the intermediate steps of the treatment chain. The good quality of the water relied on the last step, i.e. the activated carbon filtration. DWTPs may represent an extra source of EDCs and monitoring chemical occurrence at all steps of the process is advisable to improve efficiency.

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 Tāmaki 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.

Assessment of bioactive chemicals in wastewater effluents and surface waters using in vitro bioassays in the Nakdong River basin, Korea

Organic micropollutants present in effluents of wastewater treatment plants (WWTPs) can negatively affect the quality of receiving waters or drinking water sources. The present work monitored the concentration of bioactive chemicals using a battery of in vitro bioassays in 14 WWTP effluents, 2 effluent-dominant streams, and 5 river waters in the Nakdong River basin, Korea, for a two-year period. The WWTP effluents showed AR/ERα/TRβ (androgen/estrogen/thyroid hormone) activities at a few to tens ng/L, PAH/PPARγ/p53 (polycyclic-aromatic-hydrocarbon/lipid metabolism/genotoxicity) activities at hundreds ng/L, and PXR/Nrf2 (xenobiotic metabolism/oxidative stress) activities at tens to hundreds μg/L as bioanalytical equivalent concentrations. The concentration level and type of bioactivities were statistically not affected by the source, season, or treatment processes of WWTPs for most endpoints. The effluent-dominant streams showed similar levels of AR/ERα/PAH/PXR/Nrf2 activities compared to the upstream WWTP effluents. The river waters showed lower levels of AR/ERα activities (by factors of 6 or 7) but had only slightly lower PAH/PXR/Nrf2 activities (within factors of 2) than the WWTP effluents when compared based on median concentration. Cytotoxicity was below the quantification limit (0.3 μg/L) in most effluent and river samples. For ERα/PAH/PXR/Nrf2, the median bioactivity levels of the river waters were higher than at least one of the effect-based trigger (EBT) values proposed in the literature. Further monitoring work and reliable/realistic EBT derivation are needed to determine possible ecological risks posed by the observed bioactivities.

Potential risk of estrogenic compounds produced by water blooms to aquatic environment

Some freshwater phytoplankton species have been suggested to produce estrogenic compounds in concentrations which could cause adverse effects to aquatic biota, while other studies showed no estrogenic effects after exposure to phytoplankton extracts or pointed out possible sources of the overestimation of the estrogenic activity. This study aimed to clarify these research inconsistencies by investigating estrogenicity of biomass extracts from both environmental freshwater blooms and laboratory cyanobacterial and algae cultures by in vitro reporter bioassay. Biomasses of 8 cyanobacterial and 3 algal species from 7 taxonomic orders were extracted and tested. Next to this, samples of environmental water blooms collected from 8 independent water bodies dominated by phytoplankton species previously assessed as laboratory cultures were tested. The results showed undetectable or low estrogenicity of both freshwater blooms and laboratory cultures with E2 equivalent concentration (EEQ) in a range from LOQ up to 4.5 ng EEQ/g of dry mass. Moreover, the co-exposure of biomass extracts with environmentally relevant concentration of model estrogen (steroid hormone 17β-estradiol; E2), commonly occurring in surface waters, showed simple additive interaction. However, some of the biomass extracts elicited partially anti-estrogenic effects in co-exposure with higher E2 concentration. In conclusion, our study documents undetectable or relatively low estrogenic potential of biomass extracts from both environmental freshwater blooms and studied laboratory cultured cyanobacterial and algae species. Nevertheless, in case of very high-density water blooms, even this low estrogenicity (detected for two cyanobacterial species) could lead to EEQ content in biomass reaching effect-based trigger values indicating potential risk, if recalculated per water volume at field sites. However, these levels would not occur in water under realistic environmental scenarios and the potential estrogenic effects would be most probably minor compared to other toxic effects caused by massive freshwater blooms of such high densities.

Bioanalytical and chemical characterization of organic micropollutant mixtures in long-term exposed passive samplers from the Joint Danube Survey 4: Setting a baseline for water quality monitoring

Monitoring methodologies reflecting the long-term quality and contamination of surface waters are needed to obtain a representative picture of pollution and identify risk drivers. This study sets a baseline for characterizing chemical pollution in the Danube River using an innovative approach, combining continuous three-months use of passive sampling technology with comprehensive chemical (747 chemicals) and bioanalytical (seven in vitro bioassays) assessment during the Joint Danube Survey (JDS4). This is one of the world's largest investigative surface-water monitoring efforts in the longest river in the European Union, which water after riverbank filtration is broadly used for drinking water production. Two types of passive samplers, silicone rubber (SR) sheets for hydrophobic compounds and AttractSPETM HLB disks for hydrophilic compounds, were deployed at nine sites for approximately 100 days. The Danube River pollution was dominated by industrial compounds in SR samplers and by industrial compounds together with pharmaceuticals and personal care products in HLB samplers. Comparison of the Estimated Environmental Concentrations with Predicted No-Effect Concentrations revealed that at the studied sites, at least one (SR) and 4–7 (HLB) compound(s) exceeded the risk quotient of 1. We also detected AhR-mediated activity, oxidative stress response, peroxisome proliferator-activated receptor gamma-mediated activity, estrogenic, androgenic, and anti-androgenic activities using in vitro bioassays. A significant portion of the AhR-mediated and estrogenic activities could be explained by detected analytes at several sites, while for the other bioassays and other sites, much of the activity remained unexplained. The effect-based trigger values for estrogenic and anti-androgenic activities were exceeded at some sites. The identified drivers of mixture in vitro effects deserve further attention in ecotoxicological and environmental pollution research. This novel approach using long-term passive sampling provides a representative benchmark of pollution and effect potentials of chemical mixtures for future water quality monitoring of the Danube River and other large water bodies.

Impact of a megacity on the water quality of a tropical estuary assessed by a combination of chemical analysis and in-vitro bioassays

Tropical estuaries are threatened by rapid urbanization, which leads to the spread of thousands of micropollutants and poses an environmental risk to such sensitive aqueous ecosystems. In the present study, a combination of chemical and bioanalytical water characterization was applied to investigate the impact of Ho Chi Minh megacity (HCMC, 9.2 million inhabitants in 2021) on the Saigon River and its estuary and provide a comprehensive water quality assessment. Water samples were collected along a 140-km stretch integrating the river-estuary continuum from upstream HCMC down to the estuary mouth in the East Sea. Additional water samples were collected at the mouth of the four main canals of the city center. Chemical analysis was performed targeting up to 217 micropollutants (pharmaceuticals, plasticizers, PFASs, flame retardants, hormones, pesticides). Bioanalysis was performed using six in-vitro bioassays for hormone receptor-mediated effects, xenobiotic metabolism pathways and oxidative stress response, respectively, all accompanied by cytotoxicity measurement. A total of 120 micropollutants were detected and displayed high variability along the river continuum with total concentration ranging from 0.25 to 78 μg L−1. Among them, 59 micropollutants were ubiquitous (detection frequency ≥ 80 %). An attenuation was observed in concentration and effect profiles towards the estuary. The urban canals were identified as major sources of micropollutants and bioactivity to the river, and one canal (Bến Nghé) exceeded the effect-based trigger values derived for estrogenicity and xenobiotic metabolism. Iceberg modelling apportioned the contribution of the quantified and the unknown chemicals to the measured effects. Diuron, metolachlor, chlorpyrifos, daidzein, genistein, climbazole, mebendazole and telmisartan were identified as main risk drivers of the oxidative stress response and xenobiotic metabolism pathway activation. Our study reinforced the need for improved wastewater management and deeper evaluations of the occurrence and fate of micropollutants in urbanized tropical estuarine environments.

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.

Battery of In Vitro Bioassays: A Case Study for the Cost-Effective and Effect-Based Evaluation of Wastewater Effluent Quality

Wastewater treatment plants (WWTPs) represent an important input of contaminants in the environment. Therefore, it is critical to continuously monitor the performance of WWTPs to take appropriate action and avoid an influx of contaminants in the environment. In this study, a battery of seven in vitro bioassays covering a selected spectrum of toxicity effects is proposed for quality control of wastewater effluents. The bioassays address mixture toxicity, which is the combined adverse effect of multiple contaminants and can act as an early warning system. The proposed battery was applied to samples from 11 WWTPs of representative technology from the Danube River Basin (DRB). The order of toxic effects in terms of extent of exceedance of effect-based trigger values (EBTs) was PAH (PAH activity) > PXR (xenobiotic metabolism) > ERα (estrogenic activity) > PPARγ > Nrf2 (oxidative stress) > anti-AR > GR. A mitigation plan for WWTP operators based on EBT exceedance is proposed. This study demonstrates that the proposed effect-based monitoring battery is a complementary tool to the chemical analysis approach. A regular application of such time- and cost-effective bioanalytical tools in the WWTPs of the DRB is proposed to provide a ‘safety net’ for aquatic ecosystems.

How effective are water treatment processes in removing toxic effects of micropollutants? A literature review of effect-based monitoring data

Abstract Over the past decade, effect-based monitoring (EBM) has been increasingly applied for water quality monitoring. Despite being recommended as a monitoring tool in several guideline documents, the use of EBM remains limited to research projects. This study aimed to review the bioanalytical data published from studies conducted on wastewater, drinking water or reuse and to identify knowledge gaps and priorities for action. The results provide an overview of the biological effects associated with raw and treated waters, the reduction of these effects by treatment and a comparison of the detected response with effect-based trigger values. This review highlights a lack of data for many biological effects and the need to more thoroughly investigate effects such as aryl hydrocarbon receptor agonism, genotoxicity and oxidative stress. The results show that most drinking water schemes effectively eliminate the biological effects associated with environmental micropollutants. However, the oxidative stress response and genotoxicity, likely related to formed disinfection by-products, deserve closer attention since they seem to represent a higher concern in drinking water than any other effect. Overall, existing wastewater treatment schemes are less effective in removing biological effects, and consequently, priority should be given to the improvement of wastewater treatment for the better protection of the environment.

Water quality monitoring with in vitro bioassays to compare untreated oil sands process-affected water with unimpacted rivers

The selected battery of in vitro bioassays may be used to monitor exceedances of effect-based trigger (EBT) values in environments potentially receiving treated oil sands process-affected water.

Endocrine disrupting chemicals entering European rivers: Occurrence and adverse mixture effects in treated wastewater

In the present study on endocrine disrupting chemicals (EDCs) in treated wastewater, we used chemical and effect-based tools to analyse 56 wastewater treatment plant (WWTP) effluents from 15 European countries. The main objectives were (i) to compare three different receptor-based estrogenicity assays (ERα-GeneBLAzer, p-YES, ERα-CALUX®), and (ii) to investigate a combined approach of chemical target analysis and receptor-based testing for estrogenicity, glucocorticogenic activity, androgenicity and progestagenic activity (ERα-, GR-, AR- and PR-GeneBLAzer assays, respectively) in treated wastewater. A total of 56 steroids and phenols were detected at concentrations ranging from 25 pg/L (estriol, E3) up to 2.4 μg/L (cortisone). WWTP effluents, which passed an advanced treatment via ozonation or via activated carbon, were found to be less contaminated, in terms of lower or no detection of steroids and phenols, as well as hormone receptor-mediated effects. This result was confirmed by the effect screening, including the three ERα-bioassays. In the GeneBLAzer assays, ERα-activity was detected in 82 %, and GR-activity in 73 % of the samples, while AR- and PR-activity were only measured in 14 % and 21 % of the samples, respectively. 17β-estradiol was confirmed as the estrogen dominating the observed estrogenic mixture effect and triamcinolone acetonide was the dominant driver of glucocorticogenic activity. The comparison of bioanalytical equivalent concentrations (BEQ) predicted from the detected concentrations and the relative effect potency (BEQchem) with measured BEQ (BEQbio) demonstrated good correlations of chemical target analysis and receptor-based testing results with deviations mostly within a factor of 10. Bioassay-specific effect-based trigger values (EBTs) from the literature, but also newly calculated EBTs based on previously proposed derivation options, were applied and allowed a preliminary assessment of the water quality of the tested WWTP effluent samples. Overall, this study demonstrates the high potential of linking chemical with effect-based analysis in water quality assessment with regard to EDC contamination.

In vitro bioanalytical assessment of the occurrence and removal of bioactive chemicals in municipal wastewater treatment plants in Korea

Effluents of wastewater treatment plants (WWTPs) contain various organic micropollutants, some of which can exert negative effects on the quality of receiving waters or drinking water sources. This study monitored two full-scale WWTPs in Korea for the occurrence and removal of bioactive chemicals for a one-year period using a battery of in vitro bioassays as a complementary approach to chemical analysis. Bioassays covering different endpoints were employed, such as hormone receptor activation (AR and ERα), xenobiotic metabolism (PAH and PXR), oxidative stress response (Nrf2), and cytotoxicity. The WWTP influents showed AR, ERα, and PAH activities at ng/L - μg/L and PXR and Nrf2 activities at μg/L - mg/L as bioanalytical equivalent concentrations of a reference compound for each bioassay. These bioactivities decreased along with the WWTP treatment train, with significant removals achieved by the secondary biological treatment processes. Cytotoxicity was observed only for some municipal wastewater (M-WWTP) influents but was below the limit of quantification for most cases. The influent and effluent bioactivities observed in this study were mostly comparable to those reported in other WWTPs in the literature. Comparison of the bioactivities with the effect-based trigger (EBT) values indicates that the impact of WWTP effluents on receiving water quality was low for most endpoints. For Nrf2, however, further investigation is required to evaluate the observed high bioactivities compared with the current EBT. The observed ERα activity could partly be explained by the presence of some steroid estrogens. Overall, our results contribute to an important database for the concentrations and removal efficiencies of bioactive chemicals in WWTPs and demonstrate bioassays as a useful tool for urban water quality monitoring.

Estrogenic, androgenic, and glucocorticoid activities and major causative compounds in river waters from three Asian countries.

Estrogen, androgen, and glucocorticoid receptors (ER, AR, and GR) agonist activities in river water samples from Chennai and Bangalore (India), Jakarta (Indonesia), and Hanoi (Vietnam) were evaluated using a panel of chemical-activated luciferase gene expression (CALUX) assays and were detected mainly in the dissolved phase. The ER agonist activity levels were 0.011-55ng estradiol (E2)-equivalent/l, higher than the proposed effect-based trigger (EBT) value of 0.5ng/l in most of the samples. The AR agonist activity levels were < 2.1-110ng dihydrotestosterone (DHT)-equivalent/l, and all levels above the limit of quantification exceeded the EBT value of 3.4ng/l. GR agonist activities were detected in only Bangalore and Hanoi samples at dexamethasone (Dex)-equivalent levels of < 16-150ng/l and exceeded the EBT value of 100ng/l in only two Bangalore samples. Major compounds contributing to the ER, AR, and GR agonist activities were identified for water samples from Bangalore and Hanoi, which had substantially higher activities in all assays, by using a combination of fractionation, CALUX measurement, and non-target and target chemical analysis. The results for pooled samples showed that the major ER agonists were the endogenous estrogens E2 and estriol, and the major GR agonists were the synthetic glucocorticoids Dex and clobetasol propionate. The only AR agonist identified in major androgenic water extract fractions was DHT, but several unidentified compounds with the same molecular formulae as endogenous androgens were also found.

Evaluation of Three ISO Estrogen Receptor Transactivation Assays Applied to 52 Domestic Effluent Samples.

Estrogens are released to the aquatic environment by wastewater treatment plant (WWTP) effluents and can affect wildlife. In the last three decades, many in vitro assay platforms have been developed to detect and quantify estrogenicity in water. In 2018, the International Organization for Standardization (ISO) standardized protocols became available for three types of in vitro estrogen receptor transactivation assays (ERTAs) detecting estrogenicity in 96-well plates (ISO19040 1-3). Two ERTAs-lyticase Yeast Estrogen Screen (L-YES) and Arxula YES (A-YES)-use genetically modified yeast strains, whereas the third utilizes stably transfected human cells. One human cell based assay is ERα-CALUX, which is based on a genetically modified human bone osteosarcoma cell line. In the present study, we characterized the performance, comparability, and effectiveness of these three ERTAs, including an evaluation involving proposed water quality thresholds (effect-based trigger values [EBTs]). For a robust evaluation, we collected 52 effluent samples over three sampling campaigns at 15 different WWTPs in Switzerland. Estrogen receptor transactivation assay results were correlated and compared with results from chemical analysis targeting known estrogens. The three ERTAs showed comparable data over all campaigns. However, the selection of EBTs plays a significant role in the interpretation and comparison of bioassay resultsto distinguish between acceptable and unacceptable water quality. Applying a fixed cross-assay EBT for effluent of 4 ng L-1 resulted in varying numbers of threshold exceedances ranging between zero and four samples depending on the ERTA used. Using assay-specific EBTs showed exceedances in eight samples (ERα-CALUX) and in one sample (A-YES), respectively. Thus, proposed EBTs do not produce similar risk profiles across samples and further refinement of assay-specific EBTs is needed to account for assay-specific differences and to enable the application of ERTAs as effect-based methods in environmental monitoring. Environ Toxicol Chem 2022;41:2512-2526. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Estrogenic and retinoid-like activity in stagnant waters with mass occurrence of water blooms

Stagnant freshwaters can be affected by anthropogenic pollution and eutrophication that leads to massive growth of cyanobacteria and microalgae forming complex water blooms. These can produce various types of bioactive compounds, some of which may cause embryotoxicity, teratogenicity, endocrine disruption and impair animal or human health. This study focused on potential co-occurrence of estrogenic and retinoid-like activities in diverse stagnant freshwaters affected by phytoplankton blooms with varying taxonomic composition. Samples of phytoplankton bloom biomass and its surrounding water were collected from 17 independent stagnant water bodies in the Czech Republic and Hungary. Total estrogenic equivalents (EEQ) of the most potent samples reached up to 4.9 ng·g−1 dry mass (dm) of biomass extract and 2.99 ng·L−1 in surrounding water. Retinoic acid equivalent (REQ) measured by in vitro assay reached up to 3043 ng·g−1 dm in phytoplankton biomass and 1202 ng·L−1in surrounding water. Retinoid-like and estrogenic activities at some sites exceeded their PNEC and effect-based trigger values, respectively. The observed effects were not associated with any particular species of cyanobacteria or algae dominating the water blooms nor related to phytoplankton density. We found that taxonomically diverse phytoplankton communities can produce and release retinoid-like compounds to surrounding water, while estrogenic potency is likely related to estrogens of anthropogenic origin adsorbed to phytoplankton biomass. Retinoids occurring in water blooms are ubiquitous signalling molecules, which can affect development and neurogenesis. Selected water bloom samples (both water and biomass extracts) with retinoid-like activity caused effects on neurodifferentiation in vitro corresponding to those of equivalent all-trans-retinoic acid concentrations. Co-occurrence of estrogenic and retinoid-like activities in stagnant water bodies as well as the potential of compounds produced by water blooms to interfere with neural differentiation should be considered in the assessment of risks associated with water blooms, which can comprise complex mixtures of natural and anthropogenic bioactive compounds.