Integral assessment of estrogenic potentials in sediment-associated samples

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-β-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 α (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-β-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-β-estradiol, the ELRA showed sigmoid concentration-effect relations over a broad range from 0.05 μg/l to 100 μg/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‰. 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-β-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‰. 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

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.     

Identifikation geeigneter Nachweismöglichkeiten von hormonaktiven und reproduktionstoxischen Wirkungen in aquatischen Ökosystemen

Background and objectives There is an urgent need to detect, assess, and reduce effects of hormonally active compounds and endocrine disrupters in aquatic systems, as reflected in national research programs like the Swiss NRP 50 “Endocrine Disruptors” and its consensus platforms. As a medium-term measure, the EU strategy on endocrine disruptors (SEC(2007)1635) uses the Endocrine Disruptor Testing and Assessment (EDTA) Task Force of the Organisation for Economic Co-operation and Development (OECD) along with other research activities. In particular the test methods of the OECD that are currently in validation or already validated may contribute to a better understanding of the extent of endocrine disruption, in particular if they are applied on environmental samples and in the context of risk-assessment strategies, for instance in waste water treatment. This article aims to give an overview and an evaluation on available and validated biological test systems for the detection of endocrine disruptive and reproductive effects in aquatic systems. Based on this a recommendation for a modular ecotoxicological test platform is given. The study focuses on test methods for sex hormone active substances. Material and methods On the basis of an extensive literature search and ongoing international validation efforts by the OECD for methods to detect endocrine disruptive effects, 15 biological test methods (5 in vivo and 10 in vitro) were selected. Comprising, for example, of eight OECD methods and three out of five in-vitro methods mentioned in the Global Water Research Coalition (GWRC) report “Tools to detect estrogenic activity in environmental waters” (Leusch 2008). Experienced users and developers were then asked to rate the test according to given relevant criteria. The resulting criteria profiles were compiled, compared, and evaluated. Results The methods were selected on the basis of validation status, distribution, their suitability for standardisation, and their proven sensitivity for environmental samples. We assumed that specific YES/YAS-procedures and the ER/AR Calux systems achieve the mentioned criteria. In the case that strong cytotoxicity of environmental samples affects the applicability of cellular reporter gene assays, alternatively a molecular receptor binding assay (e.?g. ELRA) could be used. Additional molecular receptor binding assays are currently in validation by the OECD. Modulating effects on steroidgenesis and probably even on aromatase activity can be detected by the OECD validated H295R Steroidgenesis Assay. Its applicability for environmental samples is currently tested. As in-vivo methods different fish assays (e.?g. Fish Screening Assay and the Zebrafish Embryo Test) were evaluated. For the detection of effects on the thyroid-associated hormone system the Amphibian Metamorphosis Assay (XEMA, for Xenopus Metamorphosis Assay) will soon be available as an OECD-Guideline. Because of trends in the international community to avoid tests with vertebrates in the future, only an optional recommendation was given for such tests. Reproduction toxicity, on the other hand, can be sensitively tested by the tests using the gastropod Potamopyrgus antipodarum, which, as invertebrate tests, do not require a permit to perform animal testing procedures. This test is also sensitive for many substances which are able to induce an endocrine disruption in vertebrates. Its validation is currently performed by the OECD. The applicability of the herein proposed test methods of the modular test platform for environmental-sample assessments were previously confirmed by several published scientific studies. Discussion We propose several test methods to be included in an application-oriented modular test platform. These tests should be suitable to efficiently indicate different mechanisms of endocrine disruption and reproduction toxicity, and may be employed in different situations according to their respective advantages. The modular test platform is able to detect impacts on the reproduction relevant effects in invertebrates, amphibians, and fishes. Furthermore the different mode of actions of estrogenic and androgenic receptor binding, steroid genesis and perhaps even the modulation of aromatase are detectable. A research report on the relevance of endocrine substances and pharmaceuticals in aquatic environments (Moltmann et al. 2007) showed that endocrine end-points of in-vivo tests (31 of 71 tested substances) tend to be more sensitive than general ecotoxicological end-points, such as mortality and growth. Consequently, endocrine and general-toxic effects should be detected in an integrative manner by ecotoxicological test platforms. Conclusions By combining literature research with a targeted query for information about the chosen test procedures it was possible to obtain a detailed overview about the current state-of-the-art of science and technology in the detection of hormone-active effects and reproduction toxicity. Ecotoxicological and regulative aspects were considered equally, and the applicability of the test procedures was evaluated. Because of the diversity of endocrine disrupting mechanisms, a modular combination of in-vivo and in-vitro methods in a joint test platform is needed to recognise and differentiate the transitions from hormone-active effects to endocrine disruption. Recommendations and perspectives Our study leads to a proposal of a modular ecotoxicological test platform, which offers an integrative detection of hormone-active and reproduction-relevant effects in the aquatic environment. The modular system presented here allows the switching between test modules according to the continuously developing state-of-the-art of science and technology as well as the incorporation of novel developments. Further standardisation of such methods for regulative applications is recommended.     

Severe leaching of calcium ions from fir needles caused by acid fog

We have measured the components of the throughfall under fir trees (Abies firma) in the field around Mt. Oyama, where the forest appears to be declining, for the period 1994-1998. Exposure experiments of a simulated acid fog to fir twigs were performed under field conditions. There was a similarity between the acid response in the field and that in the laboratory. In both studies, the severe leaching of calcium ions from the needle surface was caused by exposure to acid fog. We also applied acid fog to fir seedlings over 1 year and observed a decrease in the growth of the seedlings due to this application in the dormant season. These results suggest that the severe leaching of calcium ions due to acid fog may cause the deficiency of calcium and be responsible for the decline of the fir trees.