Developing human biomonitoring as a 21st century toolbox within the European exposure science strategy 2020-2030.

Human biomonitoring (HBM) is a crucial approach for exposure assessment, as emphasised in the European Commission's Chemicals Strategy for Sustainability (CSS). HBM can help to improve chemical policies in five major key areas: (1) assessing internal and aggregate exposure in different target populations; 2) assessing exposure to chemicals across life stages; (3) assessing combined exposure to multiple chemicals (mixtures); (4) bridging regulatory silos on aggregate exposure; and (5) enhancing the effectiveness of risk management measures. In this strategy paper we propose a vision and a strategy for the use of HBM in chemical regulations and public health policy in Europe and beyond. We outline six strategic objectives and a roadmap to further strengthen HBM approaches and increase their implementation in the regulatory risk assessment of chemicals to enhance our understanding of exposure and health impacts, enabling timely and targeted policy interventions and risk management. These strategic objectives are: 1) further development of sampling strategies and sample preparation; 2) further development of chemical-analytical HBM methods; 3) improving harmonisation throughout the HBM research life cycle; 4) further development of quality control / quality assurance throughout the HBM research life cycle; 5) obtain sustained funding and reinforcement by legislation; and 6) extend target-specific communication with scientists, policymakers, citizens and other stakeholders. HBM approaches are essential in risk assessment to address scientific, regulatory and societal challenges. HBM requires full and strong support from the scientific and regulatory domain to reach its full potential in public and occupational health assessment and in regulatory decision-making.

Biological effect and chemical monitoring of Watch List substances in European surface waters: Steroidal estrogens and diclofenac - Effect-based methods for monitoring frameworks.

Three steroidal estrogens, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2), estrone (E1), and the non-steroidal anti-inflammatory drug (NSAID), diclofenac have been included in the first Watch List of the Water Framework Directive (WFD, EU Directive 2000/60/EC, EU Implementing Decision 2015/495). This triggered the need for more EU-wide surface water monitoring data on these micropollutants, before they can be considered for inclusion in the list of priority substances regularly monitored in aquatic ecosystems. The revision of the priority substance list of the WFD offers the opportunity to incorporate more holistic bioanalytical approaches, such as effect-based monitoring, alongside single substance chemical monitoring. Effect-based methods (EBMs) are able to measure total biological activities (e.g., estrogenic activity or cyxlooxygenase [COX]-inhibition) of specific group of substances (such as estrogens and NSAIDs) in the aquatic environment at low concentrations (pg/L). This makes them potential tools for a cost-effective and ecotoxicologically comprehensive water quality assessment. In parallel, the use of such methods could build a bridge from chemical status assessments towards ecological status assessments by adressing mixture effects for relevant modes of action. Our study aimed to assess the suitability of implementing EBMs in the WFD, by conducting a large-scale sampling and analysis campaign of more than 70 surface waters across Europe. This resulted in the generation of high-quality chemical and effect-based monitoring data for the selected Watch List substances. Overall, water samples contained low estrogenicity (0.01-1.3 ng E2-Equivalent/L) and a range of COX-inhibition activity similar to previously reported levels (12-1600 ng Diclofenac-Equivalent/L). Comparison between effect-based and conventional analytical chemical methods showed that the chemical analytical approach for steroidal estrogens resulted in more (76%) non-quantifiable data, i.e., concentrations were below detection limits, compared to the EBMs (28%). These results demonstrate the excellent and sensitive screening capability of EBMs.

A human biomonitoring (HBM) Global Registry Framework: Further advancement of HBM research following the FAIR principles.

Data generated by the rapidly evolving human biomonitoring (HBM) programmes are providing invaluable opportunities to support and advance regulatory risk assessment and management of chemicals in occupational and environmental health domains. However, heterogeneity across studies, in terms of design, terminology, biomarker nomenclature, and data formats, limits our capacity to compare and integrate data sets retrospectively (reuse). Registration of HBM studies is common for clinical trials; however, the study designs and resulting data collections cannot be traced easily. We argue that an HBM Global Registry Framework (HBM GRF) could be the solution to several of challenges hampering the (re)use of HBM (meta)data. The aim is to develop a global, host-independent HBM registry framework based on the use of harmonised open-access protocol templates from designing, undertaking of an HBM study to the use and possible reuse of the resulting HBM (meta)data. This framework should apply FAIR (Findable, Accessible, Interoperable and Reusable) principles as a core data management strategy to enable the (re)use of HBM (meta)data to its full potential through the data value chain. Moreover, we believe that implementation of FAIR principles is a fundamental enabler for digital transformation within environmental health. The HBM GRF would encompass internationally harmonised and agreed open access templates for HBM study protocols, structured web-based functionalities to deposit, find, and access harmonised protocols of HBM studies. Registration of HBM studies using the HBM GRF is anticipated to increase FAIRness of the resulting (meta)data. It is also considered that harmonisation of existing data sets could be performed retrospectively. As a consequence, data wrangling activities to make data ready for analysis will be minimised. In addition, this framework would enable the HBM (inter)national community to trace new HBM studies already in the planning phase and their results once finalised. The HBM GRF could also serve as a platform enhancing communication between scientists, risk assessors, and risk managers/policy makers. The planned European Partnership for the Assessment of Risk from Chemicals (PARC) work along these lines, based on the experience obtained in previous joint European initiatives. Therefore, PARC could very well bring a first demonstration of first essential functionalities within the development of the HBM GRF.

Towards a systematic use of effect biomarkers in population and occupational biomonitoring.

Effect biomarkers can be used to elucidate relationships between exposure to environmental chemicals and their mixtures with associated health outcomes, but they are often underused, as underlying biological mechanisms are not understood. We aim to provide an overview of available effect biomarkers for monitoring chemical exposures in the general and occupational populations, and highlight their potential in monitoring humans exposed to chemical mixtures. We also discuss the role of the adverse outcome pathway (AOP) framework and physiologically based kinetic and dynamic (PBK/D) modelling to strengthen the understanding of the biological mechanism of effect biomarkers, and in particular for use in regulatory risk assessments. An interdisciplinary network of experts from the European chapter of the International Society for Exposure Science (ISES Europe) and the Organization for Economic Co-operation and Development (OECD) Occupational Biomonitoring activity of Working Parties of Hazard and Exposure Assessment group worked together to map the conventional framework of biomarkers and provided recommendations for their systematic use. We summarized the key aspects of this work here, and discussed these in three parts. Part I, we inventory available effect biomarkers and promising new biomarkers for the general population based on the H2020 Human Biomonitoring for Europe (HBM4EU) initiative. Part II, we provide an overview AOP and PBK/D modelling use that improved the selection and interpretation of effect biomarkers. Part III, we describe the collected expertise from the OECD Occupational Biomonitoring subtask effect biomarkers in prioritizing relevant mode of actions (MoAs) and suitable effect biomarkers. Furthermore, we propose a tiered risk assessment approach for occupational biomonitoring. Several effect biomarkers, especially for use in occupational settings, are validated. They offer a direct assessment of the overall health risks associated with exposure to chemicals, chemical mixtures and their transformation products. Promising novel effect biomarkers are emerging for biomonitoring of the general population. Efforts are being dedicated to prioritizing molecular and biochemical effect biomarkers that can provide a causal link in exposure-health outcome associations. This mechanistic approach has great potential in improving human health risk assessment. New techniques such as in silico methods (e.g. QSAR, PBK/D modelling) as well as 'omics data will aid this process. Our multidisciplinary review represents a starting point for enhancing the identification of effect biomarkers and their mechanistic pathways following the AOP framework. This may help in prioritizing the effect biomarker implementation as well as defining threshold limits for chemical mixtures in a more structured way. Several ex vivo biomarkers have been proposed to evaluate combined effects including genotoxicity and xeno-estrogenicity. There is a regulatory need to derive effect-based trigger values using the increasing mechanistic knowledge coming from the AOP framework to address adverse health effects due to exposure to chemical mixtures. Such a mechanistic strategy would reduce the fragmentation observed in different regulations. It could also stimulate a harmonized use of effect biomarkers in a more comparable way, in particular for risk assessments to chemical mixtures.

Biomonitoring as an Underused Exposure Assessment Tool in Occupational Safety and Health Context-Challenges and Way Forward.

Recent advances in analytical chemistry have allowed a greater possibility of using quantitative approaches for measuring human exposure to chemicals. One of these approaches is biomonitoring (BM), which provides unequivocal evidence that both exposure and uptake of a chemical have taken place. BM has been a longstanding practice in occupational health for several reasons. BM integrates exposure from all routes. It can help identify unintentional and unexpected exposures and assess the effectiveness of existing risk-management measures. BM also provides relevant information to support policy development by delivering better evidence of workers' exposure to chemical substances, even within the framework of the present regulations. Thus, BM can allow for both the evaluation of the impact of regulation and identification of further needs for new or improved regulation. However, despite all these well-recognized advantages, BM is currently an underused exposure assessment tool. This paper provides an overview of the key aspects to be considered when using BM in the context of occupational health interventions. Additionally, this paper describes the potential of BM as an exposure assessment tool, distinguishing the role of BM in exposure assessment and health surveillance and clarifies ethical and communication aspects to guarantee that general data protection regulations are followed. In addition, actions and research needs are identified (particularly with reference to the European situation), which aim to encourage the increased use of BM as an exposure assessment tool.

Revising Environmental Quality Standards: Lessons Learned.

An environmental quality standard (EQS) is a threshold value applied in regulatory monitoring for retrospective environmental risk assessment. However, an EQS may vary with time and between countries with shared water bodies, challenging coherent risk management. This study aimed to analyze the underlying reasons for changes in EQS values following a revision of previously derived EQSs for 62 substances. Relevant data were retrieved from publicly accessible databases, available literature, registration dossiers, and, in some cases, provided by manufacturers. Ecotoxicological data were assessed regarding reliability and relevance. As in previous studies, EQS derivation followed the European Union guideline. Overall, 61 annual average EQSs (AA-EQS) and 58 maximum acceptable concentration EQSs (MAC-EQS) were derived. Size and completeness of data sets generally increased due to the revision. AA-EQSs increased in 13 cases and decreased in 21 cases. MAC-EQSs increased in 22 cases and decreased in 11 cases. Most EQSs were derived using the deterministic assessment factor (AF) method. The number of substances for which EQSs were derived probabilistically by reference to the species sensitivity distribution (SSD) method increased from 2 to 5 AA-EQSs and from 6 to 11 MAC-EQSs. For AA-EQS derivation, AFs were reduced in 14 cases and increased in 6 cases. For MAC-EQS derivation, AFs were reduced in 9 cases and increased in 2 cases. Results demonstrate that the revisions did not generally lead to either lower or higher EQSs. The majority of EQSs (>93%) changed less than 10-fold. Clearly, EQSs based on small or incomplete data sets with large AFs were more prone to considerable changes in their numeric values when revised than EQSs based on SSDs. Thus, revision can reduce uncertainty and increase robustness of an EQS. In this study, however, available data continued to be insufficient to construct SSDs for the majority of substances. This was mostly due to a lack of reliable data. Integr Environ Assess Manag 2019;00:1-13. © 2019 SETAC.

Monitoring estrogenic activities of waste and surface waters using a novel in vivo zebrafish embryonic (EASZY) assay: Comparison with in vitro cell-based assays and determination of effect-based trigger values.

This study reports the use of the recently developed EASZY assay that uses transgenic cyp19a1b-GFP zebrafish (Danio rerio) embryos to assess in vivo estrogenic activity of 33 surface (SW) and waste water (WW) samples collected across Europe that were previously well-characterized for estrogen hormones and in vitro estrogenic activity. We showed that 18 out of the 33 SW and WW samples induced estrogenic responses in the EASZY assay leading to a significant and concentration-dependent up-regulation of the ER-regulated cyp19a1b gene expression in the developing brain. The in vivo 17ß-estradiol-equivalents (EEQs) were highly correlated with, both, the chemical analytical risk quotient (RQ) based on steroidal estrogen concentrations and EEQs reported from five different in vitro reporter gene assays. Regression analyses between the vitro and in vivo effect concentrations allowed us to determine an optimal cut-off value for each in vitro assay, above which in vivo responses were observed. These in vitro assay-specific effect-based trigger values (EBTs), ranging from 0.28 to 0.58 ng EEQ/L define the sensitivity and specificity of the individual in vitro assays for predicting a risk associated with substances acting through the same mode of action in water samples. Altogether, this study demonstrates the toxicological relevance of in vitro-based assessment of estrogenic activity and recommends the use of such in vitro/in vivo comparative approach to refine and validate EBTs for mechanism-based bioassays.

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.

Towards the review of the European Union Water Framework Directive: Recommendations for more efficient assessment and management of chemical contamination in European surface water resources.

Water is a vital resource for natural ecosystems and human life, and assuring a high quality of water and protecting it from chemical contamination is a major societal goal in the European Union. The Water Framework Directive (WFD) and its daughter directives are the major body of legislation for the protection and sustainable use of European freshwater resources. The practical implementation of the WFD with regard to chemical pollution has faced some challenges. In support of the upcoming WFD review in 2019 the research project SOLUTIONS and the European monitoring network NORMAN has analyzed these challenges, evaluated the state-of-the-art of the science and suggested possible solutions. We give 10 recommendations to improve monitoring and to strengthen comprehensive prioritization, to foster consistent assessment and to support solution-oriented management of surface waters. The integration of effect-based tools, the application of passive sampling for bioaccumulative chemicals and an integrated strategy for prioritization of contaminants, accounting for knowledge gaps, are seen as important approaches to advance monitoring. Including all relevant chemical contaminants in more holistic "chemical status" assessment, using effect-based trigger values to address priority mixtures of chemicals, to better consider historical burdens accumulated in sediments and to use models to fill data gaps are recommended for a consistent assessment of contamination. Solution-oriented management should apply a tiered approach in investigative monitoring to identify toxicity drivers, strengthen consistent legislative frameworks and apply solutions-oriented approaches that explore risk reduction scenarios before and along with risk assessment.

Criteria for Reporting and Evaluating ecotoxicity Data (CRED): comparison and perception of the Klimisch and CRED methods for evaluating reliability and relevance of ecotoxicity studies.

BACKGROUND: The regulatory evaluation of ecotoxicity studies for environmental risk and/or hazard assessment of chemicals is often performed using the method established by Klimisch and colleagues in 1997. The method was, at that time, an important step toward improved evaluation of study reliability, but lately it has been criticized for lack of detail and guidance, and for not ensuring sufficient consistency among risk assessors. RESULTS: A new evaluation method was thus developed: Criteria for Reporting and Evaluating ecotoxicity Data (CRED). The CRED evaluation method aims at strengthening consistency and transparency of hazard and risk assessment of chemicals by providing criteria and guidance for reliability and relevance evaluation of aquatic ecotoxicity studies. A two-phased ring test was conducted to compare and characterize the differences between the CRED and Klimisch evaluation methods. A total of 75 risk assessors from 12 countries participated. Results show that the CRED evaluation method provides a more detailed and transparent evaluation of reliability and relevance than the Klimisch method. Ring test participants perceived it to be less dependent on expert judgement, more accurate and consistent, and practical regarding the use of criteria and time needed for performing an evaluation. CONCLUSIONS: We conclude that the CRED evaluation method is a suitable replacement for the Klimisch method, and that its use may contribute to an improved harmonization of hazard and risk assessments of chemicals across different regulatory frameworks.

CRED: Criteria for reporting and evaluating ecotoxicity data.

Predicted-no-effect concentrations (PNECs) and environmental quality standards (EQSs) are derived in a large number of legal frameworks worldwide. When deriving these safe concentrations, it is necessary to evaluate the reliability and relevance of ecotoxicity studies. Such evaluation is often subject to expert judgment, which may introduce bias and decrease consistency when risk assessors evaluate the same study. The Criteria for Reporting and Evaluating Ecotoxicity Data (CRED) project attempts to address this problem. It aims to improve the reproducibility, transparency, and consistency of reliability and relevance evaluations of aquatic ecotoxicity studies among regulatory frameworks, countries, institutes, and individual assessors. In the present study, the CRED evaluation method is presented. It includes a set of 20 reliability and 13 relevance criteria, accompanied by extensive guidance. Risk assessors who participated in the CRED ring test evaluated the CRED evaluation method to be more accurate, applicable, consistent, and transparent than the often-used Klimisch method. The CRED evaluation method is accompanied by reporting recommendations for aquatic ecotoxicity studies, with 50 specific criteria divided into 6 categories: general information, test design, test substance, test organism, exposure conditions, and statistical design and biological response. An ecotoxicity study in which all important information is reported is more likely to be considered for regulatory use, and proper reporting may also help in the peer-review process.

Erratum to: Criteria for Reporting and Evaluating ecotoxicity Data (CRED): comparison and perception of the Klimisch and CRED methods for evaluating reliability and relevance of ecotoxicity studies.

[This corrects the article DOI: 10.1186/s12302-016-0073-x.].

Scientific Basis for Regulatory Decision-Making of Nanomaterials Report on the Workshop, 20-21 January 2014, Center of Applied Ecotoxicology, Dübendorf.

The key findings of a workshop jointly organized by the Swiss Centre of Applied Ecotoxicity, the Swiss Centre for Applied Human Toxicology (SCAHT), and the Federal Office of Public Health (FOPH) are summarized and provide a critical analysis of the current regulatory framework for nanomaterials and a snapshot of some hot topics in nanoscience.

In vitro bioassays to screen for endocrine active pharmaceuticals in surface and waste waters.

In the context of the European Water Framework Directive (WFD) it is fully recognized that pharmaceuticals can represent a relevant issue for the achievement of the good chemical and ecological status of European surface water bodies. The recent European Directive on the review of priority substances in surface water bodies has included three pharmaceuticals of widespread use (diclofenac, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2)) in the European monitoring list, the so-called watch list. Endocrine active pharmaceuticals such as EE2 and E2 (also occurring as natural hormone) can cause adverse effects on aquatic ecosystems at very low levels. However, monitoring of these pharmaceuticals within the watch list mechanism of the WFD and national monitoring programs can be difficult because of detection problems of most routine analytical methods. With proposed annual average Environmental Quality Standards (AA-EQS) of 0.035 ng/L and 0.4 ng/L, respectively, the estrogenic pharmaceutical EE2 and the natural hormone E2 are among those substances. Sensitive in vitro bioassays could reduce the current detection problems by measuring the estrogenic activity of environmental samples. In a short review article the application of this approach to screen and assess the risks of endocrine active pharmaceuticals with a focus on estrogenic pharmaceuticals in environmental waters is discussed.

Scientific Basis for Regulatory Decision-Making of Nanomaterials Report on the Workshop, 20-21 January 2014, Center of Applied Ecotoxicology, Dübendorf.

The key findings of a workshop jointly organized by the Swiss Centre of Applied Ecotoxicity, the Swiss Centre for Applied Human Toxicology (SCAHT), and the Federal Office of Public Health (FOPH) are summarized and provide a critical analysis of the current regulatory framework for nanomaterials and a snapshot of some hot topics in nanoscience.

Impact of environmental estrogens on Yfish considering the diversity of estrogen signaling.

Research on endocrine disruption in fish has been dominated by studies on estrogen-active compounds which act as mimics of the natural estrogen, 17ß-estradiol (E2), and generally exert their biological actions by binding to and activation of estrogen receptors (ERs). Estrogens play central roles in reproductive physiology and regulate (female) sexual differentiation. In line with this, most adverse effects reported for fish exposed to environmental estrogens relate to sexual differentiation and reproduction. E2, however, utilizes a variety of signaling mechanisms, has multifaceted functions and targets, and therefore the toxicological and ecological effects of environmental estrogens in fish will extend beyond those associated with the reproduction. This review first describes the diversity of estrogen receptor signaling in fish, including both genomic and non-genomic mechanisms, and receptor crosstalk. It then considers the range of non-reproductive physiological processes in fish that are known to be responsive to estrogens, including sensory systems, the brain, the immune system, growth, specifically through the growth hormone/insulin-like growth factor system, and osmoregulation. The diversity in estrogen responses between fish species is then addressed, framed within evolutionary and ecological contexts, and we make assessments on their relevance for toxicological sensitivity as well as ecological vulnerability. The diversity of estrogen actions raises questions whether current risk assessment strategies, which focus on reproductive endpoints, and a few model fish species only, are protective of the wider potential health effects of estrogens. Available - although limited - evidence nevertheless suggests that quantitative environmental threshold concentrations for environmental protection derived from reproductive tests with model fish species are protective for non-reproductive effects as well. The diversity of actions of estrogens across divergent physiological systems, however, may lead to and underestimation of impacts on fish populations as their effects are generally considered on one functional process only and this may underrepresent the impact on the different physiological processes collectively.

Integral assessment of estrogenic potentials in sediment-associated samples: Part 2: Study of estrogen and anti-estrogen receptor-binding potentials of sediment-associated chemicals under different salinity conditions using the salinity-adapted enzyme-linked receptor assay.

BACKGROUND, AIM, AND SCOPE: The enzyme-linked receptor assay (ELRA) detects estrogenic and anti-estrogenic effects at the molecular level of receptor binding and is a useful tool for the integrative assessment of ecotoxicological potentials caused by hormonally active agents (HAA) and endocrine disrupting compounds (EDC). The main advantage of the ELRA is its high sample throughput and its robustness against cytotoxicity and microbial contamination. After a methodological adaptation to salinity of the ELRA, according to the first part of this study, which increased its salinity tolerance and sensitivity for 17-beta-estradiol, the optimised ELRA was used to investigate 13 native sediments characterised by different levels of salinity and chemical contamination. The applicability of the ELRA for routine analysis in environmental assessment was evaluated. Salinity is often a critical factor for bioassays in ecotoxicological sediment assessment. Therefore, salinity of the samples was additionally adjusted to different levels to characterise its influence on elution and binding processes of receptor-binding substances. MATERIALS AND METHODS: The ELRA was carried out with the human estrogen receptor alpha (ER) in a 96-well microplate format using the experimental setup known from the competitive immunoassay based on ligand-protein interaction. It is an important improvement that a physiologically relevant receptor was used as a linking protein instead of an antibody. The microplates were coated with a 17-beta-estradiol-BSA conjugate, and dilution series of estradiol and of native sediment samples were added and incubated with the ER. After a washing step, a biotinylated mouse anti-ER antibody was added to each well. Receptor binding to estradiol, agonistic and antagonistic receptor binding, were determined by a streptavidin-POD-biotin complex with subsequent measurement of the peroxidase activity at the wavelength of 450 nm using a commercial ELISA multiplate reader. The sediment elutriates and pore water samples of sediments were tested in a dilution series to evaluate at which dilution step the receptor-binding potential ends. In the elution process (see Section 2.1 to 2.2), a method was developed to adjust the salinity to the levels of the reference testings, which offers an appropriate option to adjust the salinity in both directions. Statistical evaluation was made with a combination of the Mann-Whitney U test and the pT-method. RESULTS: This part of the study characterised the environmental factor 'salinity' for prospective applications of the ELRA. Using reference substances such as 17-beta-estradiol, the ELRA showed sigmoid concentration-effect relations over a broad range from 0.05 mug/l to 100 mug/l under physiological conditions. After methodological optimisation, both sensitivity and tolerance of the assay against salinity could be significantly raised, and the ELRA became applicable under salinity conditions up to concentrations of 20.5 per thousand. The mean relative inter-test error (n = 3) was around 11% with reference substances and below 5% for single sediments elutriates in three replicates each. For sediment testings, the pore water and different salinity-adjusted elutriates of 13 sediments were used. A clear differentiation of the receptor-binding potential could be reached by application of the pT-method. Thereby, pT-values from one to six could be assigned to the sediments, and the deviation caused by the different salinity conditions was one pT-value. The mean standard deviation in the salinity adaptation procedure of the elutriates was below 5%. DISCUSSION: Although the ELRA has already been used for assessments of wastewater, sludge and soil, its applicability for samples to different salinity levels has not been investigated so far. Even if the ELRA is not as sensitive as the E-screen or the YES-assay, with regard to reference substances like 17-beta-estradiol, it is a very useful tool for pre-screening, because it is able to integrate both estrogenic as well as anti-estrogenic receptor-binding effects. According to the results of sediment testing, and given the integrative power to detect different directions of effects, the ELRA shows sufficient sensitivity and salinity tolerance to discriminate receptor-binding potentials in environmental samples. CONCLUSIONS: The optimised ELRA assay is a fast, cost-effective, reliable and highly reproducible tool that can be used for high-throughput screening in a microplate format in detecting both estrogenic and anti-estrogenic effects. Additionally, the ELRA is robust against microbial contaminations, and is not susceptible towards cytotoxic interferences like the common cell-culture methods. The general applicability and sufficient sensitivity of the ELRA was shown in freshwater environments. Marine and brackish samples can be measured up to salinity levels of 20.5 per thousand. RECOMMENDATIONS AND PERSPECTIVES: In view of the proven sensitivity, functionality and the fastness of the ELRA, it is recommendable to standardise the test method. At the moment, no adequate in vitro test procedure exists which is standardised to DIN or ISO levels. The E-screen and the yeast estrogen/androgen screens (YES/YAS) sometimes underlie strong cytotoxic effects, as reported in the first part of this study. Further development of an ELRA assay using human androgen receptors appears to be very promising to gain information about androgenic and anti-androgenic effects, too. This would offer a possibility to use the ELRA as a fast and reliable pre-screening tool for the detection of endocrine potentials, thus minimising time and cost-expensive animal experiments.

Integral assessment of estrogenic potentials of sediment-associated samples. Part 1: The influence of salinity on the in vitro tests ELRA, E-Screen and YES.

GOAL, SCOPE AND BACKGROUND: Exogenic endocrine-active substances are also called 'Endocrine Disrupting Chemicals' (EDC). They imitate or hinder the function of natural endogenic hormones or disturb the synthesis or the metabolism of hormones or of hormone receptors. The Enzyme-Linked Receptor Assay (ELRA) can detect estrogenic and anti-estrogenic effects at the level of receptor binding and is a useful tool for the integrative detection of contaminant effects. Although the test system has been used repeatedly in sediment assessments, the questions have remained concerning how it responds to variations in the physico-chemical matrix. For some bioassays, the salinity of the sample is a critical factor. This is especially relevant when testing wastewater samples or when sediment-associated samples in the tidal reaches of rivers are tested. Sediments in the tidal reaches of rivers change their salinity several times a day. Against this background, it would be beneficial to have a test procedure of known salinity tolerance. On account of this, the salinity tolerance of the ELRA was tested, assessed with reference substances at several salinity levels, and compared with the E-Screen method and a Yeast Estrogen Screen (YES), which are also frequently applied in environmental testing. The aim of this paper was to explore when the salinity limits within these test procedures are applicable. The trials should reveal the working range to be expected, characterize the salinity-dependent variations in sensitivity of the test, and provide options for methodological adjustments to improve the stability against increased salinity. METHODS: The ELRA was carried out with the human Estrogen Receptor alpha. (ER) using the same principle like a competitive immunoassay based on ligand-protein interaction. However, an essential difference is the use of a physiologically relevant receptor instead of an antibody as a linking protein. The ELRA measures the competition of sample estrogens and anti-estrogens against estradiol supplied as a BSA-coating conjugate for the binding site of dissolved ER. Estradiol or xeno-estrogen binding is quantified by a biotynilated anti-ER antibody and the subsequent measurement of peroxidase activity by a streptavidin-POD-biotin complex. The E-Screen was performed with the human breast cancer cell line MCF-7, which expresses the estrogen receptor constitutively. Cell proliferation depends on binding of estrogens or xeno-estrogens with the receptor. After incubation, estrogen-dependent cell growth was measured by sulforhodamin B staining. The YES was performed with a recombinant yeast strain, transfected with a receptor and a reporter plasmid bearing the estrogen receptor and a vitellogenin gene fused with the reporter gene lacZ. Estrogen or xeno-estrogen-dependent gene induction was measured indirectly by LacZ activity. The salinity levels were simulated in varying concentrations with NaCl from 0 to 40 per thousand or Artificial Sea Water (ASW) from 0 to 32 per thousand. RESULTS: The study characterized the factor 'salinity' for the prospective application fields of the ELRA. With reference substances such as 17-beta-estradiol, the ELRA showed classical sigmoidal concentration-effect relations in a range from 0.05 to 100 microg/l under physiological conditions. After a methodological adjustment to compensate decreasing receptor-binding affinity of estrogens and xeno-estrogens at higher salinity levels, the ELRA became applicable under salinity conditions up to concentrations of 20.5 per thousand. In tests, the ELRA reached under the influence of salinity a mean limit of detection of 0.062 microg/l 17-beta-estradiol. The mean relative inter-test error was around 11%. Above concentrations of 20.5 per thousand there is a risk of false negative assessment. Compared with the E-Screen method using the MCF7 cell line and the yeast estrogen test system (YES), the ELRA shows a lower sensitivity to 17-beta-estradiol. In the E-Screen, the cell proliferation was strongly reduced by sodium chloride induced cytotoxicity. In comparison with the E-Screen, the salinity tolerance of the YES and YAS methods is significantly higher. DISCUSSION: Despite adaption, total salinity tolerance could not be achieved with the ELRA. Freshwater samples were generally appraisable. Higher salinity levels above 20.5 per thousand would tend towards false negative results. The low inter-test error of 11% makes the ELRA suitable for the detection of estrogenic and anti-estrogenic potentials of single substances, substance mixtures, and of environmental samples. CONCLUSIONS: The ELRA is very fast and reproducible, it can be used for high-throughput screening in a microplate format at low cost, it is robust to microbial contamination, and is less susceptible to cytotoxic interferences than cell culture methods. RECOMMENDATIONS AND PERSPECTIVES: In their established form, the YES and the E-Screen methods are not applicable for liquid phase testing at higher salinity conditions. The salinity-adapted test version of the ELRA described here shows a broader working range for samples. Native water samples of more or less brackish origin or high-salinity effluent samples are testable. Results of tests with sediment associated samples of different salinity will be subject of a forthcoming publication.