Andrew J. Tindall

Future water quality monitoring - Adapting tools to deal with mixtures of pollutants in water resource management

Environmental quality monitoring of water resources is challenged with providing the basis for safeguarding the environment against adverse biological effects of anthropogenic chemical contamination from diffuse and point sources. While current regulatory efforts focus on monitoring and assessing a few legacy chemicals, many more anthropogenic chemicals can be detected simultaneously in our aquatic resources. However, exposure to chemical mixtures does not necessarily translate into adverse biological effects nor clearly shows whether mitigation measures are needed. Thus, the question which mixtures are present and which have associated combined effects becomes central for defining adequate monitoring and assessment strategies. Here we describe the vision of the international, EU-funded project SOLUTIONS, where three routes are explored to link the occurrence of chemical mixtures at specific sites to the assessment of adverse biological combination effects. First of all, multi-residue target and non-target screening techniques covering a broader range of anticipated chemicals co-occurring in the environment are being developed. By improving sensitivity and detection limits for known bioactive compounds of concern, new analytical chemistry data for multiple components can be obtained and used to characterise priority mixtures. This information on chemical occurrence will be used to predict mixture toxicity and to derive combined effect estimates suitable for advancing environmental quality standards. Secondly, bioanalytical tools will be explored to provide aggregate bioactivity measures integrating all components that produce common (adverse) outcomes even for mixtures of varying compositions. The ambition is to provide comprehensive arrays of effect-based tools and trait-based field observations that link multiple chemical exposures to various environmental protection goals more directly and to provide improved in situ observations for impact assessment of mixtures. Thirdly, effect-directed analysis (EDA) will be applied to identify major drivers of mixture toxicity. Refinements of EDA include the use of statistical approaches with monitoring information for guidance of experimental EDA studies. These three approaches will be explored using case studies at the Danube and Rhine river basins as well as rivers of the Iberian Peninsula. The synthesis of findings will be organised to provide guidance for future solution-oriented environmental monitoring and explore more systematic ways to assess mixture exposures and combination effects in future water quality monitoring.

Effect-directed analysis supporting monitoring of aquatic environments — An in-depth overview

Aquatic environments are often contaminated with complex mixtures of chemicals that may pose a risk to ecosystems and human health. This contamination cannot be addressed with target analysis alone but tools are required to reduce this complexity and identify those chemicals that might cause adverse effects. Effect-directed analysis (EDA) is designed to meet this challenge and faces increasing interest in water and sediment quality monitoring. Thus, the present paper summarizes current experience with the EDA approach and the tools required, and provides practical advice on their application. The paper highlights the need for proper problem formulation and gives general advice for study design. As the EDA approach is directed by toxicity, basic principles for the selection of bioassays are given as well as a comprehensive compilation of appropriate assays, including their strengths and weaknesses. A specific focus is given to strategies for sampling, extraction and bioassay dosing since they strongly impact prioritization of toxicants in EDA. Reduction of sample complexity mainly relies on fractionation procedures, which are discussed in this paper, including quality assurance and quality control. Automated combinations of fractionation, biotesting and chemical analysis using so-called hyphenated tools can enhance the throughput and might reduce the risk of artifacts in laboratory work. The key to determining the chemical structures causing effects is analytical toxicant identification. The latest approaches, tools, software and databases for target-, suspect and non-target screening as well as unknown identification are discussed together with analytical and toxicological confirmation approaches. A better understanding of optimal use and combination of EDA tools will help to design efficient and successful toxicant identification studies in the context of quality monitoring in multiply stressed environments.

European demonstration program on the effect-based and chemical identification and monitoring of organic pollutants in European surface waters

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe.

EDA-EMERGE: an FP7 initial training network to equip the next generation of young scientists with the skills to address the complexity of environmental contamination with emerging pollutants

The initial training network consortium novel tools in effect-directed analysis to support the identification and monitoring of emerging toxicants on a European scale (EDA-EMERGE) was formed in response to the seventh EU framework program call to train a new generation of young scientists (13 PhD fellows and 1 postdoctoral fellow) in the interdisciplinary techniques required to meet the major challenges in the monitoring, assessment, and management of toxic pollution in European river basins. This 4-year project is of particular relevance considering the multidisciplinary analytical chemistry and biology skills required to investigate the enormous complexity of contamination, effects, and cause-effect relationships. By integrating innovative mode-of-action-based biodiagnostic tools including in vitro and in vivo tests, transgenic organisms, and ‘omics’ techniques with powerful fractionation and cutting edge, analytical, and computational structure elucidation tools, powerful new EDA approaches are being developed for the identification of toxicants in European surface and drinking waters. Innovative method development by young researchers at major European universities, research centers, and private companies has been closely interlinked with a joint European demonstration program, higher-tier EDA, and specialized training courses and secondments. Using a simplified protocol based on existing EDA tools, EDA-EMERGE fellows are also being trained to organize and run international and interdisciplinary sampling and monitoring campaigns within selected European river basin sites. Strong networking between academia, the private sector, and leading regulators in the field of river basin management and pollution management ensures the relevance of the research for practice and excellent employment opportunities for the fellows. Additionally, an internationally composed advisory board has been tasked to introduce new perspectives on monitoring, assessment, and management of emerging pollutants within and outside of Europe. The combination of cutting edge science with specialized training in complimentary soft skills is being offered with a strong emphasis on commercial exploitation and media competence which further enhances the employability of the fellows in research, academia, and beyond.

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.

Oestrogen reporter transgenic medaka for non-invasive evaluation of aromatase activity.

Vertebrate reproduction involves complex steroid hormone interplay and inter-conversion. A critical element in maintaining sex steroid levels is the enzyme aromatase (cytochrome P450 19A1) which converts androgens to oestrogens. In turn oestrogen signalling is targeted by numerous chemicals, from pharmaceuticals to agricultural chemicals, both frequent sources of contamination in waste waters and consequently rivers. Although many models are now available to address disruption of oestrogen signalling, there are currently no published protocols allowing discrimination between alterations in testosterone metabolism and in oestrogenic signalling. It was with this limitation in mind that we optimised this protocol. We show using a 48h protocol that pre-feeding fry of the choriogenin h-gfp (chgh-gfp) medaka line are sensitive to 0.05nM EE2 (15ng/L), within the range of the lowest published observable physiological effect concentrations for medaka. In addition, co-treatment with testosterone can reveal potential effects of test substances on aromatase enzymatic activity. As the measurements are visualised in real-time without affecting embryo viability, repeated measures are possible. We demonstrate the ability of this model to detect oestrogen receptor agonists, aromatisable androgens, P450 aromatase activity modulators and selective oestrogen response modulators. Importantly, the range of this assay is physiologically relevant.

Effect-based assessment of toxicity removal during wastewater treatment

Wastewaters contain complex mixtures of chemicals, which can cause adverse toxic effects in the receiving environment. In the present study, the toxicity removal during wastewater treatment at seven municipal wastewater treatment plants (WWTPs) was investigated using an effect-based approach. A battery of eight bioassays was applied comprising of cytotoxicity, genotoxicity, endocrine disruption and fish embryo toxicity assays. Human cell-based CALUX assays, transgenic larval models and the fish embryo toxicity test were particularly sensitive to WWTP effluents. The results indicate that most effects were significantly reduced or completely removed during wastewater treatment (76-100%), while embryo toxicity, estrogenic activity and thyroid disruption were still detectable in the effluents suggesting that some harmful substances remain after treatment. The responsiveness of the bioassays was compared and the human cell-based CALUX assays showed highest responsiveness in the samples. Additionally, the fish embryo toxicity test and the transgenic larval models for endocrine disrupting effects showed high responsiveness at low sample concentrations in nearly all of the effluent samples. The results showed a similar effect pattern among all WWTPs investigated, indicating that the wastewater composition could be rather similar at different locations. There were no considerable differences in the toxicity removal efficiencies of the treatment plants and no correlation was observed with WWTP characteristics, such as process configuration or sludge age. This study demonstrated that a biotest battery comprising of multiple endpoints can serve as a powerful tool when assessing water quality or water treatment efficiency in a holistic manner. Rather than analyzing the concentrations of a few selected chemicals, bioassays can be used to complement traditional methods of monitoring in the future by assessing sum-parameter based effects, such as mixture effects, and tackling chemicals that are present at concentrations below chemical analytical detection limits.

Identification of Unknown Antiandrogenic Compounds in Surface Waters by Effect-Directed Analysis (EDA) Using a Parallel Fractionation Approach

Among all the nuclear-receptor mediated endocrine disruptive effects, antiandrogenicity is frequently observed in aquatic environments and may pose a risk to aquatic organisms. Linking these effects to responsible chemicals is challenging and a great share of antiandrogenic activity detected in the environment has not been explained yet. To identify drivers of this effect at a hot spot of antiandrogenicity in the German river Holtemme, we applied effect-directed analysis (EDA) including a parallel fractionation approach, a downscaled luciferase reporter gene cell-based anti-AR-CALUX assay and LC-HRMS/MS nontarget screening. We identified and confirmed the highly potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and two derivatives in the active fractions. The relative potency of C47 to the reference compound flutamide was over 5.2, whereas the derivatives were less potent. C47 was detected at a concentration of 13.7 μg/L, equal to 71.4 μg flutamide equivalents per liter (FEq/L) in the nonconcentrated water extract that was posing an antiandrogenic activity equal to 45.5 (±13.7 SD) FEq/L. Thus, C47 was quantitatively confirmed as the major cause of the measured effect in vitro. Finally, the antiandrogenic activity of C47 and one derivate was confirmed in vivo in spiggin-gfp Medaka. An endocrine disrupting effect of C47 was observed already at the concentration equal to the concentration in the nonconcentrated water extract, underlining the high risk posed by this compound to the aquatic ecosystem. This is of some concern since C47 is used in a number of consumer products indicating environmental as well as human exposure.

Mixture effects in samples of multiple contaminants – An inter-laboratory study with manifold bioassays

Chemicals in the environment occur in mixtures rather than as individual entities. Environmental quality monitoring thus faces the challenge to comprehensively assess a multitude of contaminants and potential adverse effects. Effect-based methods have been suggested as complements to chemical analytical characterisation of complex pollution patterns. The regularly observed discrepancy between chemical and biological assessments of adverse effects due to contaminants in the field may be either due to unidentified contaminants or result from interactions of compounds in mixtures. Here, we present an interlaboratory study where individual compounds and their mixtures were investigated by extensive concentration-effect analysis using 19 different bioassays. The assay panel consisted of 5 whole organism assays measuring apical effects and 14 cell- and organism-based bioassays with more specific effect observations. Twelve organic water pollutants of diverse structure and unique known modes of action were studied individually and as mixtures mirroring exposure scenarios in freshwaters. We compared the observed mixture effects against component-based mixture effect predictions derived from additivity expectations (assumption of non-interaction). Most of the assays detected the mixture response of the active components as predicted even against a background of other inactive contaminants. When none of the mixture components showed any activity by themselves then the mixture also was without effects. The mixture effects observed using apical endpoints fell in the middle of a prediction window defined by the additivity predictions for concentration addition and independent action, reflecting well the diversity of the anticipated modes of action. In one case, an unexpectedly reduced solubility of one of the mixture components led to mixture responses that fell short of the predictions of both additivity mixture models. The majority of the specific cell- and organism-based endpoints produced mixture responses in agreement with the additivity expectation of concentration addition. Exceptionally, expected (additive) mixture response did not occur due to masking effects such as general toxicity from other compounds. Generally, deviations from an additivity expectation could be explained due to experimental factors, specific limitations of the effect endpoint or masking side effects such as cytotoxicity in in vitro assays. The majority of bioassays were able to quantitatively detect the predicted non-interactive, additive combined effect of the specifically bioactive compounds against a background of complex mixture of other chemicals in the sample. This supports the use of a combination of chemical and bioanalytical monitoring tools for the identification of chemicals that drive a specific mixture effect. Furthermore, we demonstrated that a panel of bioassays can provide a diverse profile of effect responses to a complex contaminated sample. This could be extended towards representing mixture adverse outcome pathways. Our findings support the ongoing development of bioanalytical tools for (i) compiling comprehensive effect-based batteries for water quality assessment, (ii) designing tailored surveillance methods to safeguard specific water uses, and (iii) devising strategies for effect-based diagnosis of complex contamination.

Using short-term bioassays to evaluate the endocrine disrupting capacity of the pesticides linuron and fenoxycarb

Several short-term whole-organism bioassays based on transgenic aquatic models are now under validation by the OECD (Organization for Economic Co-operation and Development) to become standardized test guidelines for the evaluation of the endocrine activity of substances. Evaluation of the endocrine disrupting capacity of pesticides will be a domain of applicability of these future reference tests. The herbicide linuron and the insecticide fenoxycarb are two chemicals commonly used in agricultural practices. While numerous studies indicate that linuron is likely to be an endocrine disruptor, there is little information available on the effect of fenoxycarb on vertebrate endocrine systems. Using whole-organism bioassays based on transgenic Xenopus laevis tadpoles and medaka fry we assessed the potential of fenoxycarb and linuron to disrupt thyroid, androgen and estrogen signaling. In addition we used in silico approach to simulate the affinity of these two pesticides to human hormone receptors. Linuron elicited thyroid hormone-like activity in tadpoles at all concentrations tested and, showed an anti-estrogenic activity in medaka at concentrations 2.5mg/L and higher. Our experiments suggest that, in addition to its previously established anti-androgenic action, linuron exhibits thyroid hormone-like responses, as well as acting at the estrogen receptor level to inhibit estrogen signaling. Fenoxycarb on the other hand, did not cause any changes in thyroid, androgen or estrogen signaling at the concentrations tested.