Effect-based Methods Research Articles

Improved component-based methods for mixture risk assessment are key to characterize complex chemical pollution in surface waters

The present monitoring and assessment of water quality problems fails to characterize the likelihood that complex mixtures of chemicals affect water quality. The European collaborative project SOLUTIONS suggests that this likelihood can be estimated, amongst other methods, with improved component-based methods (CBMs). The use of CBMs is a well-established practice in the WFD, as one of the lines of evidence to evaluate chemical pollution on a per-chemical basis. However, this is currently limited to a pre-selection of 45 and approximately 300 monitored substances (priority substances and river basin-specific pollutants, respectively), of which only a few actually co-occur in relevant concentrations in real-world mixtures. Advanced CBM practices are therefore needed that consider a broader, realistic spectrum of chemicals and thereby improve the assessment of mixture impacts, diagnose the causes of observed impacts and provide more useful water management information. Various CBMs are described and illustrated, often representing improvements of well-established methods. Given the goals of the WFD and expanding on current guidance for risk assessment, these improved CBMs can be applied to predicted or monitored concentrations of chemical pollutants to provide information for management planning. As shown in various examples, the outcomes of the improved CBMs allow for the evaluation of the current likelihood of impacts, of alternative abatement scenarios as well as the expected consequences of future pollution scenarios. The outputs of the improved CBMs are useful to underpin programmes of measures to protect and improve water quality. The combination of CBMs with effect-based methods (EBMs) might be especially powerful to identify as yet underinvestigated emerging pollutants and their importance in a mixture toxicity context. The present paper has been designed as one in a series of policy briefs to support decisions on water quality protection, monitoring, assessment and management under the European Water Framework Directive (WFD).

Mixtures of chemicals are important drivers of impacts on ecological status in European surface waters

The ecological status of European surface waters may be affected by multiple stressors including exposure to chemical mixtures. Currently, two different approaches are used separately to inform water quality management: the diagnosis of the deterioration of aquatic ecosystems caused by nutrient loads and habitat quality, and assessment of chemical pollution based on a small set of chemicals. As integrated assessments would improve the basis for sound water quality management, it is recommended to apply a holistic approach to integrated water quality status assessment and management. This allows for estimating the relative contributions of exposure to mixtures of the chemicals present and of other stressors to impaired ecological status of European water bodies. Improved component- and effect-based methods for chemicals are available to support this. By applying those methods, it was shown that a holistic diagnostic approach is feasible, and that chemical pollution acts as a limiting factor for the ecological status of European surface waters. In a case study on Dutch surface waters, the impact on ecological status could be traced back to chemical pollution affecting individual species. The results are also useful as calibration of the outcomes of component-based mixture assessment (risk quotients or mixture toxic pressures) on ecological impacts. These novel findings provide a basis for a causal and integrated analysis of water quality and improved methods for the identification of the most important stressor groups, including chemical mixtures, to support integrated knowledge-guided management decisions on water quality.

High resolution mass spectrometry-based non-target screening can support regulatory environmental monitoring and chemicals management

Non-target screening (NTS) including suspect screening with high resolution mass spectrometry has already shown its feasibility in detecting and identifying emerging contaminants, which subsequently triggered exposure mitigating measures. NTS has a large potential for tasks such as effective evaluation of regulations for safe marketing of substances and products, prioritization of substances for monitoring programmes and assessment of environmental quality. To achieve this, a further development of NTS methodology is required, including: (i) harmonized protocols and quality requirements, (ii) infrastructures for efficient data management, data evaluation and data sharing and (iii) sufficient resources and appropriately trained personnel in the research and regulatory communities in Europe. Recommendations for achieving these three requirements are outlined in the following discussion paper. In particular, in order to facilitate compound identification it is recommended that the relevant information for interpretation of mass spectra, as well as about the compounds usage and production tonnages, should be made accessible to the scientific community (via open-access databases). For many purposes, NTS should be implemented in combination with effect-based methods to focus on toxic chemicals.

Effect-based methods in combination with state-of-the-art chemical analysis for assessment of water quality as integrated approach.

Complex mixtures of chemicals in waste and finally in surface water may pose a risk to the environment and also to human health. This contamination of surface water cannot be addressed with chemical analysis alone. Tools are required to detect and assess these micropollutants which might cause adverse effects. Effect-directed analysis (EDA) with effect-based methods in combination with state-of-the-art chemical analysis can meet this challenge. The present paper summarizes and outlines current experiences with analytical tools and bioassays as integrated approach for assessment of water quality. The need for a holistic and solution-oriented procedure of water quality monitoring is described. To integrate and evaluate existing information about toxicity pathways, which are essential for the EDA approach, the adverse outcome pathway (AOP) concept is useful and recommended. An integration of AOP concept in water quality assessment and further requirements are discussed.

A contribution towards improving the applicability of the Myriophyllum aquaticum sediment contact test

BackgroundWhole sediment contact tests provide realistic exposure scenarios, but the inherent properties of sediments may play a significant role in organism’s response and interfere with result interpretation. Recently, a sediment contact test with a rooted aquatic macrophyte Myriophyllum aquaticum has been standardized. The present study aims to distinguish between effects of basic sediment properties and sediment-bound pollutants on M. aquaticum growth and evaluate the method as a tool in sediment quality assessment. Tests with artificial sediments with different organic matter, sand and clay content, as well as freshwater sediments with different levels of contamination were conducted.ResultsResults were based on comparison to the standard artificial control sediment. Organic matter content and grain size distribution in different variants of artificial sediments significantly affected M. aquaticum growth. Growth was impaired in formulations with low (1%) and high (10%) organic matter content, while better growth of plants compared to control was recorded in artificial samples with higher fine particles content. Because of the presence of unmeasured pollutants in freshwater sediments and inherent sediment properties, results of the M. aquaticum sediment contact tests were not always in accordance with the results of chemical analysis. Whorl, shoot and root parameters had different variability and showed a particular growth pattern in natural sediments. If the threshold of 20% for sediment toxicity is applied, then about 60% of tested natural sediments may be considered as toxic. As sediment structure may influence plant growth, this interpretation may be false as the physico–chemical properties of the control sediment used for comparison are considerably different from the properties of natural sediments.ConclusionsSince inherent properties showed a significant effect on plant growth, the use of suitable controls more similar to tested natural sediments is advisable. To avoid over-, but also under-estimations, the use of the strict toxicity threshold of 20% or even higher inhibition for this test can be recommended, provided the statistical power of the test is high. The results of this study might contribute to the ongoing process of including effect-based methods in water quality monitoring under the Water Framework Directive.

Toxicological and ecotoxicological evaluation of the water quality in a large and eutrophic freshwater lake of China

Effect-based methods (EBMs) are recommended as holistic approach for diagnosis and monitoring of water quality; however, the application of EBMs is still scare in China. In the present study, water quality of the freshwater lake Taihu (China) was investigated by EBMs. Different types of water samples were collected from three bays of the lake during 2015, 2016 and 2017. A battery of seven effect-based bioassays, including both specific and non-specific toxicity assays, was used. The bioassay battery was recently suggested based on joint activities of the EU project SOLUTIONS and the NORMAN network on emerging pollutants and is also under discussion for being implemented into monitoring activities in the context of the European Water Framework Directive (WFD). Adverse effects were observed towards the primary producer, primary consumer and fish, indicating the potential ecotoxicity of water in Taihu Lake. Mutagenic and estrogenic effects were found in the Ames fluctuation assay and ERα CALUX (Chemically Activated Luciferase Gene-eXpression) assay, respectively, highlighting the potential risks on human health. Algal growth inhibition and mutagenic effects can be observed during each of the three years. Acute toxicity towards Daphnia magna and estrogen receptor agonistic effects were found in at least one of the samples collected in 2016 and 2017, but not in 2015. The endpoints for fish toxicity in the Danio rerio fish embryo test included both lethal and additionally several sublethal effects (only for samples from 2017) and were not compared between years. Algal growth inhibition, fish embryo toxicity, mutagenic effect and estrogenicity were observed in each of the three bays, while Daphnia acute toxicity was only found in Zhushan Bay. Taking together, this study provides a big picture on the water quality of Taihu Lake. The battery of effect-based tools is promising to be a routine for water quality monitoring in China.

Effect-based methods are key. The European Collaborative Project SOLUTIONS recommends integrating effect-based methods for diagnosis and monitoring of water quality

The present monitoring and assessment of the chemical status of water bodies fail to characterize the likelihood that complex mixtures of chemicals affect water quality. The European Collaborative Project SOLUTIONS suggests that this likelihood can be estimated with effect-based methods (EBMs) complemented by chemical screening and/or impact modeling. These methods should be used to identify the causes of impacted water quality and to develop programs of measures to improve water quality. Along this line of reasoning, effect-based methods are recommended for Water Framework Directive (WFD) monitoring to cover the major modes of action in the universe of environmentally relevant chemicals so as to evaluate improvements of water quality upon implementing the measures. To this end, a minimum battery of bioassays has been recommended including short-term toxicity to algae, Daphnia and fish embryos complemented with in vitro and short-term in vivo tests on mode-of-action specific effects as proxies for long-term toxicity. The likelihood of adverse impacts can be established with effect-based trigger values, which differentiate good from poor water quality in close alignment with Environmental Quality Standards for individual chemicals, while taking into account mixture toxicity. The use of EBMs is suggested in the WFD as one avenue to establish the likelihood of adverse effects due to chemical pollution in European water systems. The present paper has been written as one component of a series of policy briefs to support decisions on water quality monitoring and management under the WFD.

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.

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.

Effect-based approach for screening of chemical mixtures in whole blood of green turtles from the Great Barrier Reef

Organisms are exposed to mixtures of both known and unknown chemicals which are diverse and variable, and thus difficult and costly to characterise and monitor using traditional target analyses. The objective of this study was to validate and apply in vitro effect-based methods by which whole blood can be used to screen internal exposure to such complex chemical mixtures. For this study, we used whole blood of green sea turtles (Chelonia mydas). To ensure the chemical mixture in blood is transferred with minimal losses or bias, we tested a modified QuEChERS extraction method specifically developed for multi- and non-target instrument analysis. The extracts were dosed to a battery of in vitro bioassays (AhR-CAFLUX, AREc32, NFκB-bla, VM7Luc4E2, Microtox), each with a different mode of action (e.g., AhR receptor mediated xenobiotics, NrF2-mediated oxidative stress, NFκB mediated response to inflammation, estrogen activity and baseline toxicity oxidative stress, respectively) in order to cover a wide spectrum of chemicals. Results confirmed the absence of interferences of the blood extract with the responses of the different assays, thus indicating the methods' compatibility with effect-based screening approaches. To apply this approach, whole blood samples were collected from green turtles foraging in agricultural, urban and remote areas of the Australian Great Barrier Reef. The effect-based screening revealed significant differences in exposure, with higher induction of AhR-CAFLUX, AREc32 and Microtox assays in turtles from the agricultural foraging ground. Overall, these results corroborated with concurrent health, target and non-target analyses in the same animals performed as part of a larger program. This study provides evidence that the proposed effect-based approach is suitable for screening and evaluating internal exposure of organisms to chemical mixtures. The approach could be valuable for advancing understanding on multiple levels ranging from identification of priority chemicals in effect-directed investigations to exploring relationships between exposure and disease, not only in sea turtles, but in any organism.

Inhibition of lipopolysaccharide induced acute inflammation in lung by chlorination

Lipopolysaccharide (LPS, also called endotoxin) is a pro-inflammatory constituent of gram negative bacteria and cyanobacteria, which causes a potential health risk in the process of routine urban application of reclaimed water, such as car wash, irrigation, scenic water refilling, etc. Previous studies indicated that the common disinfection treatment, chlorination, has little effect on endotoxin activity removal measured by Limulus amebocyte lysate (LAL) assay. However, in this study, significant decrease of acute inflammatory effects was observed in mouse lung, while LAL assay still presented a moderate increase of endotoxin activity. To explore the possible mechanisms, the nuclear magnetic resonance (NMR) results showed the chlorination happened in alkyl chain of LPS molecules, which could affect the interaction between LPS and LPS-binding protein. Also the size of LPS aggregates was found to drop significantly after treatment, which could be another results of chlorination caused polarity change. In conclusion, our observation demonstrated that chlorination is effective to reduce the LPS induced inflammation in lung, and it is recommended to use health effect-based methods to assess risk removal of water treatment technologies.