Development of a framework to derive effect-based trigger values to interpret CALUX data for drinking water quality.

Bioassays are increasingly being implemented for water quality monitoring as targeted chemical analyses are not always sufficient for the detection of all emerging chemicals or transformation products. However, the interpretation of bioassay results remains challenging, in particular because a positive response does not necessarily indicate that there may be an increased risk. For this purpose, effect-based trigger (EBT) values have been introduced as thresholds above which action needs to be undertaken to determine the cause of the response. The goals of this study were to (i) evaluate various approaches used to determine EBT values and (ii) based on the findings, derive human health EBT values for Chemical Activated LUciferase gene eXpression (CALUX) in vitro bioassays used for routine monitoring of water quality in the Netherlands. Finally, (iii) an uncertainty analysis was carried out to determine the protective power of the derived EBT values and the chance that potentially harmful substances might not be detected. EBT values that can be implemented in routine monitoring could be determined for four of eight selected bioassays. These EBT were compared to bioassay results from routine water quality monitoring carried out in the Netherlands. Furthermore, a framework for the calculation and evaluation of derived EBT values for routine application to monitor drinking water and its sources is proposed.

Comparative field study on bioassay responses and micropollutant uptake of POCIS, Speedisk and SorbiCell polar passive samplers.

Routine water quality monitoring is generally performed with chemical analyses of grab samples, which has major limitations. First, snapshot samples will not give a good representation of the water quality. Second, it is not sufficient to analyze only a limited number of (priority) pollutants. These limitations can be circumvented by an alternative environmental risk assessment that combines time-integrated passive sampling (PS) with effect-based methods. This study aimed to select which of three polar PS devices was best suited for effect-based monitoring strategies. In the first part of this study, Speedisk, SorbiCell and POCIS polar PS devices were compared by simultaneous deployment at five sites. Chemical analyses of 108 moderately polar compounds (-1.82 < log D < 6.28) revealed that highest number of compounds, with the widest range of log KOW, log D and pKa, were detected in extracts of POCIS, followed by Speedisk. SorbiCell samplers accumulated the lowest numbers and concentrations of compounds, so they were not further investigated. In a follow-up study, bioassay responses were compared in extracts of POCIS and Speedisk devices deployed at eight sites. The passive sampler extracts were subjected to bioassays for non-specific toxicity, endocrine disruption, and antibiotics activities. More frequent and higher responses were induced by POCIS extracts, leading to more exceedances of effect-based trigger values for environmental risks. As POCIS outperformed Speedisk, it is better suited as PS device targeting polar compounds for semi-quantitative effect-based water quality monitoring.

Identifying adverse outcome pathways (AOP) for Amsterdam city fish by integrated field monitoring.

The European City Fish project aimed to develop a generic methodology for ecological risk assessment for urban rivers. Since traditional methods only consider a small fraction of substances present in the water cycle, biological effect monitoring is required for a more reliable assessment of the pollution status. A major challenge for environmental risk assessment (ERA) is the application of adverse outcome pathways (AOP), i.e. the linking of pollutant exposure via early molecular and biochemical changes to physiological effects and, ultimately, effects on populations and ecosystems. We investigated the linkage between responses at these different levels. Many AOP aspects were investigated, from external and internal exposure to different classes of micropollutants, via molecular key events (MKE) the impacts on organs and organisms (fish physiology), to changes in the population dynamics of fish. Risk assessment procedures were evaluated by comparing environmental quality standards, bioassay responses, biomarkers in caged and feral fish, and the impact on fish populations. Although no complete AOP was observed, indirect relationships linking pollutant exposure via MKE to impaired locomotion were demonstrated at the most polluted site near a landfill for chemical waste. The pathway indicated that several upstream key events requiring energy for stress responses and toxic defence are likely to converge at a single common MKE: increased metabolic demands. Both fish biomarkers and the bioanalytical SIMONI strategy are valuable indicators for micropollutant risks to fish communities.

Risk-based approach in the revised European Union drinking water legislation: Opportunities for bioanalytical tools.

A plethora of in vitro bioassays are developed in the context of chemical risk assessment and clinical diagnostics to test effects on different biological processes. Such assays can also be implemented in effect-based monitoring (EBM) of (drinking) water quality alongside chemical analyses. Effects-based monitoring can provide insight into risks for the environment and human health associated with exposure to (unknown) complex, low-level mixtures of micropollutants, which fits in the risk-based approach that was recently introduced in the European Drinking Water Directive. Some challenges remain, in particular those related to selection and interpretation of bioassays. For water quality assessment, carcinogenesis, adverse effects on reproduction and development, effects on xenobiotic metabolism, modulation of hormone systems, DNA reactivity, and adaptive stress responses are considered the most relevant toxicological endpoints. An evaluation procedure of the applicability and performance of in vitro bioassays for water quality monitoring, based on existing information, has been developed, which can be expanded with guidelines for experimental evaluations. In addition, a methodology for the interpretation of in vitro monitoring data is required, because the sensitivity of specific in vitro bioassays in combination with sample concentration may lead to responses of chemicals (far) below exposure concentrations that are relevant for human health effects. Different approaches are proposed to derive effect-based trigger values (EBTs), including EBTs based on (1) relative ecotoxicity potency, (2) health-based threshold values for chronic exposure in humans and kinetics of reference chemicals, and (3) read-across from (drinking) water guideline values. Effects-based trigger values need to be chosen carefully in order to be sufficiently but not overly conservative to indicate potential health effects. Consensus on the crucial steps in the selection and interpretation of in vitro bioassay data will facilitate implementation and legal embedding in the context of water quality monitoring of such assays in EBM strategies. Integr Environ Assess Manag 2019;15:126-134. © 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Effect-based trigger values for in vitro and in vivo bioassays performed on surface water extracts supporting the environmental quality standards (EQS) of the European Water Framework Directive.

Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.

SIMONI (Smart Integrated Monitoring) as a novel bioanalytical strategy for water quality assessment: Part II-field feasibility survey.

Because it is impossible to chemically analyze all relevant micropollutants, the implementation of bioanalytical tools is essential to estimate ecological risks of chemical mixtures in regular water-monitoring programs. The first tier of the Smart Integrated Monitoring (SIMONI) strategy, which was described in part I, is based on the combination of passive sampling and bioanalytical measurements. Bioassay responses are compared with effect-based trigger values (EBT), and an overall SIMONI score on all bioassay data was designed to indicate environmental risks. The present study is focused on analyzing the feasibility of the hazard identification tier by evaluating results of 45 field campaigns at sites with different pollution profiles near the city of Amsterdam. A Daphnia assay was performed in situ, while silicon rubber or polar organic chemical integrative sampler (POCIS) extracts were tested with 4 nonspecific (daphnids, algae, bacteria, and cell culture) and 10 specific (9 Chemical Activated Luciferase Gene Expression [CALUX] assays and antibiotics scan) bioassays. Sensitivity analyses demonstrated the relevance of 2 classification variables in the SIMONI score formula on all bioanalytical data. The model indicated increased risks for the ecosystem at surface waters in greenhouse areas and undiluted wastewater-treatment plant (WWTP) effluents. The choice of testing specific bioassays on either polar or nonpolar passive sampling extracts is cost-effective and still provided meaningful insights on micropollutant risks. Statistical analyses revealed that the model provides a relevant overall impact assessment based on bioassay responses. Data analyses on the chemically determined mixture toxic pressure and bioanalytical methods provided similar insights in relative risk ranking of water bodies. The SIMONI combination of passive sampling and bioanalytical testing appears to be a feasible strategy to identify chemical hazards. Environ Toxicol Chem 2017;36:2400-2416. © 2017 SETAC.

SIMONI (smart integrated monitoring) as a novel bioanalytical strategy for water quality assessment: Part i-model design and effect-based trigger values.

It is virtually impossible to reliably assess water quality with target chemical analyses only. Therefore, a complementary effect-based risk assessment by bioanalyses on mixtures of bioavailable micropollutants is proposed: the Smart Integrated Monitoring (SIMONI) strategy. The goal of this strategy is to obtain more reliable information on the water quality to select optimum measures for improvement. The SIMONI strategy is 2-tiered. Tier 1 is a bioanalytical hazard identification of sites. A tier 2 ecological risk assessment is carried out only at a limited number of sites where increased hazards are detected in tier 1. Tier 2 will be customized, based on tier 1 evaluation and additional knowledge of the aquatic system. The present study focuses on the tier 1 bioanalytical hazard identification to distinguish "hot spots" of chemical pollution. First, a selection was made of relevant and cost-effective bioanalytical endpoints to cover a wide spectrum of micropollutant modes of action. Specific endpoints may indicate which classes of chemicals might cause adverse effects. Second, effect-based trigger values (EBT) were derived for these bioassays to indicate potential ecological risks. Comparison of EBT with bioassay responses should discriminate sites exhibiting different chemical hazards. Third, a model was designed to estimate the overall risks for aquatic ecosystems. The associated follow-up for risk management is a "toxicity traffic light" system: green, low hazard (no action required); orange, potential risk (further research needed); and red, high risk (mitigation measures). Thanks to cost-effectiveness, flexibility, and relevance, the SIMONI strategy has the potential to become the first bioanalytical tool to be applied in regular water quality monitoring programs. Environ Toxicol Chem 2017;36:2385-2399. © 2017 SETAC.

Bioassay battery interlaboratory investigation of emerging contaminants in spiked water extracts - Towards the implementation of bioanalytical monitoring tools in water quality assessment and monitoring.

Bioassays are particularly useful tools to link the chemical and ecological assessments in water quality monitoring. Different methods cover a broad range of toxicity mechanisms in diverse organisms, and account for risks posed by non-target compounds and mixtures. Many tests are already applied in chemical and waste assessments, and stakeholders from the science-police interface have recommended their integration in regulatory water quality monitoring. Still, there is a need to address bioassay suitability to evaluate water samples containing emerging pollutants, which are a current priority in water quality monitoring. The presented interlaboratory study (ILS) verified whether a battery of miniaturized bioassays, conducted in 11 different laboratories following their own protocols, would produce comparable results when applied to evaluate blinded samples consisting of a pristine water extract spiked with four emerging pollutants as single chemicals or mixtures, i.e. triclosan, acridine, 17α-ethinylestradiol (EE2) and 3-nitrobenzanthrone (3-NBA). Assays evaluated effects on aquatic organisms from three different trophic levels (algae, daphnids, zebrafish embryos) and mechanism-specific effects using in vitro estrogenicity (ER-Luc, YES) and mutagenicity (Ames fluctuation) assays. The test battery presented complementary sensitivity and specificity to evaluate the different blinded water extract spikes. Aquatic organisms differed in terms of sensitivity to triclosan (algae > daphnids > fish) and acridine (fish > daphnids > algae) spikes, confirming the complementary role of the three taxa for water quality assessment. Estrogenicity and mutagenicity assays identified with high precision the respective mechanism-specific effects of spikes even when non-specific toxicity occurred in mixture. For estrogenicity, although differences were observed between assays and models, EE2 spike relative induction EC50 values were comparable to the literature, and E2/EE2 equivalency factors reliably reflected the sample content. In the Ames, strong revertant induction occurred following 3-NBA spike incubation with the TA98 strain, which was of lower magnitude after metabolic transformation and when compared to TA100. Differences in experimental protocols, model organisms, and data analysis can be sources of variation, indicating that respective harmonized standard procedures should be followed when implementing bioassays in water monitoring. Together with other ongoing activities for the validation of a basic bioassay battery, the present study is an important step towards the implementation of bioanalytical monitoring tools in water quality assessment and monitoring.

Tissue levels and biomarkers of organic contaminants in feral and caged chub (Leuciscus cephalus) from rivers in the West Midlands, UK.

The Birmingham conurbation (West Midlands, UK) has traditionally been a major centre of UK industry and population and consequently has a legacy of pollution, which is reflected in the water quality of local rivers. Three of these rivers, exhibiting good, intermediate and poor overall water quality, were the subject of a study in which the effects of contamination on hepatic biomarkers and tissue contaminant loads in feral and caged chub (Leuciscus cephalus) were investigated. Muscle polychlorinated biphenyls and organochlorine pesticides (PCBs and OCPs), as well as bile pyrene and benzo[a]pyrene-like metabolite levels, were variable in both caged and feral fish, but were generally higher in tissue from the more polluted sites. OCPs were, in most cases, higher in the feral fish than in the caged fish, although the opposite was true of bile PAH metabolites, possibly due to differences in relative accumulation rates. Hepatic CYP1A activity (via ethoxyresorufin-O-deethylase (EROD) activity) in both feral and caged fish was also generally higher at the more polluted sites. EROD activity in feral and caged fish was statistically associated with polycyclic aromatic hydrocarbon (PAH) contamination and specific PCB congeners. Other biomarkers measured (reduced glutathione in liver, and serum aspartate aminotransferase) showed little consistent evidence of relationships with water quality. The assessment of these parameters during different seasons also revealed relatively little evidence of temporal variation. Overall, the caged chub appeared to reflect the pattern of general water quality more accurately than did feral fish, particularly with respect to EROD activity and to some degree bile PAH metabolites, thus supporting their use as sentinel species. However, the fact that muscle OCPs were generally higher in the feral fish suggests that the use of feral fish may be more indicative of exposure to certain classes of contaminant, and so biological monitoring programs should be designed with such considerations in mind.

Comprehensive two-dimensional liquid chromatography coupled to high resolution time of flight mass spectrometry for chemical characterization of sewage treatment plant effluents.

For the first time a comprehensive two-dimensional liquid chromatography (LC×LC) system coupled with a high resolution time-of-flight mass spectrometer (HR-ToF MS) was developed and applied for analysis of emerging toxicants in wastewater effluent. The system was optimized and validated using environmental standard compound mixtures of e.g. carbamate pesticides and polycyclic aromatic hydrocarbons (PAHs), to characterize the chromatographic system, to test the stability of the retention times and orthogonality. Various stationary phases in the second dimension were compared for the LC×LC analysis of silicon rubber passive sampler extracts of a wastewater effluent. A combination of C18 and Pentafluorophenyl (PFP) was found to be most effective. Finally, the hyphenation of LC×LC with HR-ToF MS was optimized, including splitter settings, transfer of data files between the different software packages and background subtraction using instrument software tools, after which tentative identification of 20 environmental contaminants was achieved, including pesticides, pharmaceuticals and food additives. As examples, three pesticides (isoproturon, terbutryn and diazinon) were confirmed by two-dimensional retention alignment.

Assessment of bioavailable PAH, PCB and OCP concentrations in water, using semipermeable membrane devices (SPMDs), sediments and caged carp.

Bioavailable water concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) were assessed at several freshwater sites in and around the city of Amsterdam. Carp (Cyprinus carpio) were caged for 4 weeks at 10 sites, together with semipermeable membrane devices (SPMDs). In addition, sediment samples were taken at each site. SPMDs and sediments were analysed for PAHs, PCBs and OCPs. Carp muscle tissues were analysed for PCBs and OCP, while PAH metabolites were assessed in fish bile. Contaminant concentrations in the water phase were estimated using three different methods: 1. Using fish tissue concentrations and literature bioconcentration factors (BCFs), 2. Using SPMD levels and a kinetic SPMD uptake model, and 3. Using sediment levels and literature sorption coefficients (K(oc)s). Since PAH accumulation in fish is not considered an accurate indicator of PAH exposure, calculated aqueous PAH concentrations from SPMD data were compared with semiquantitatively determined biliary PAH metabolite levels. Contaminant concentrations in the water phase estimated with fish data (Cw(fish)) and SPMD data (Cw(spmd)) were more in line for compounds with lower K(ow) than for compounds with higher K(ow) values. This indicates that the assumption of fish-water sorption equilibrium was not valid. At most sites, sediment-based water levels (Cw(sed)) were comparable with the Cw(spmd), although large differences were observed at certain sites. A significant correlation was observed between biliary PAH metabolite levels in fish and aqueous PAH concentrations estimated with SPMD data, suggesting that both methods may be accurate indicators of PAH exposure in aquatic ecosystems.

Fish bioaccumulation and biomarkers in environmental risk assessment: a review.

In this review, a wide array of bioaccumulation markers and biomarkers, used to demonstrate exposure to and effects of environmental contaminants, has been discussed in relation to their feasibility in environmental risk assessment (ERA). Fish bioaccumulation markers may be applied in order to elucidate the aquatic behavior of environmental contaminants, as bioconcentrators to identify certain substances with low water levels and to assess exposure of aquatic organisms. Since it is virtually impossible to predict the fate of xenobiotic substances with simple partitioning models, the complexity of bioaccumulation should be considered, including toxicokinetics, metabolism, biota-sediment accumulation factors (BSAFs), organ-specific bioaccumulation and bound residues. Since it remains hard to accurately predict bioaccumulation in fish, even with highly sophisticated models, analyses of tissue levels are required. The most promising fish bioaccumulation markers are body burdens of persistent organic pollutants, like PCBs and DDTs. Since PCDD and PCDF levels in fish tissues are very low as compared with the sediment levels, their value as bioaccumulation markers remains questionable. Easily biodegradable compounds, such as PAHs and chlorinated phenols, do not tend to accumulate in fish tissues in quantities that reflect the exposure. Semipermeable membrane devices (SPMDs) have been successfully used to mimic bioaccumulation of hydrophobic organic substances in aquatic organisms. In order to assess exposure to or effects of environmental pollutants on aquatic ecosystems, the following suite of fish biomarkers may be examined: biotransformation enzymes (phase I and II), oxidative stress parameters, biotransformation products, stress proteins, metallothioneins (MTs), MXR proteins, hematological parameters, immunological parameters, reproductive and endocrine parameters, genotoxic parameters, neuromuscular parameters, physiological, histological and morphological parameters. All fish biomarkers are evaluated for their potential use in ERA programs, based upon six criteria that have been proposed in the present paper. This evaluation demonstrates that phase I enzymes (e.g. hepatic EROD and CYP1A), biotransformation products (e.g. biliary PAH metabolites), reproductive parameters (e.g. plasma VTG) and genotoxic parameters (e.g. hepatic DNA adducts) are currently the most valuable fish biomarkers for ERA. The use of biomonitoring methods in the control strategies for chemical pollution has several advantages over chemical monitoring. Many of the biological measurements form the only way of integrating effects on a large number of individual and interactive processes in aquatic organisms. Moreover, biological and biochemical effects may link the bioavailability of the compounds of interest with their concentration at target organs and intrinsic toxicity. The limitations of biomonitoring, such as confounding factors that are not related to pollution, should be carefully considered when interpreting biomarker data. Based upon this overview there is little doubt that measurements of bioaccumulation and biomarker responses in fish from contaminated sites offer great promises for providing information that can contribute to environmental monitoring programs designed for various aspects of ERA.

[The presence of medications in the water cycle]. / Geneesmiddelen in de watercyclus.

Medications and radiographic contrast dyes are sometimes detected in surface waters, ground water and drinking water; these have proven detrimental effects on organisms living in such waters The concentration of medications found in drinking water is at least a thousand times below their minimum therapeutic dosages. In humans, the long-term effects of daily exposure to low dosages of medications and 'mixture toxicity' is not known; based on the concentrations and substance toxicity, it is presumed that the risk is nil.. Physicians can play their part in controlling the problem of medications becoming part of the water cycle by taking this into account when prescribing medications. Users can make a difference by handling their medications with care and by returning all unused portions to the pharmacy. The pharmaceutical industry can also do its part by taking degradability, options for removal and the environmental effects of medications into account during their stages of development.

Validation of the REA bioassay to detect estrogenic activity in the water cycle.

Endocrine disrupting compounds (EDCs) with estrogenic potency contaminate water and might eventually cause adverse effects to the aquatic environment. Many estrogenic compounds are not completely removed by wastewater treatment systems and, together with the run-off from agricultural areas, they enter surface waters. Chemical analytical methods to determine these compounds are usually expensive and laborious. Therefore, screening bioassays which are able to detect compounds based on their effects offer a solution for prior selection of samples that need to be chemically analyzed. In this study, the REA (RIKILT yeast Estrogen bioAssay), which has been developed to detect estrogenic compounds in calf urine and animal feed at RIKILT, is validated at the Water Board Laboratory of Waterproef for water samples. According to EC Decision 2002/657, detection capability CCß, specificity and stability have to be determined for the internal validation of a qualitative screening test. In addition, surface water and effluent samples were analyzed to further demonstrate the applicability of the validated test procedure. Results demonstrate that the REA assay is reproducible and specific for estrogenic compounds in water and meets the criteria as prescribed in EC Decision 2002/657. The assay was sensitive enough to detect estrogenic activity of pollutants in water with a limit of quantification (LOQ) below 1 ng EEQ/L. This means that samples can be compared with preliminary threshold levels for drinking water and surface waters (7 and 1 ng EEQ/L, respectively). The stability of estrogenic activity in water samples is at least 4 weeks, when stored at 4 °C.