Estrogenic activity of food contact materials-evaluation of 20 chemicals using a yeast estrogen screen on HPTLC or 96-well plates.

Food contact materials (FCM) may contain complex mixtures of estrogenic chemicals. A yeast estrogen screen performed on high performance thin-layer chromatography plates (planar-YES, P-YES) is promising for analysis of such mixtures, as it could allow for better elucidation of effects compared with established methods in microtiter plates. However, the P-YES has not been directly compared with established methods. We compared the performance of a microtiter plate YES (lyticase-YES, L-YES) to P-YES on silica gel HPTLC plates using 17ß-estradiol (E2), 20 chemicals representative of migrants from plastic FCM, and three migrates of coated metal food cans. Effective doses (ED10, ED50) and estradiol equivalencies were calculated for each chemical. Thirteen chemicals had calculable EDs in the L-YES or P-YES, with average EDs 13-fold (range 0.63-36) more potent in P-YES than in the L-YES. Normalized to E2, the median estrogenicity was within 1.5-fold (0.43-8.8) between the assays. Therefore, P-YES was as or more sensitive than L-YES but potencies relative to E2 were comparable between assays. With chromatography, the P-YES detected estrogenicity in coated metal cans, effects that were unmeasurable in L-YES. With the sample preparation methods used in this study, both YES assays are sufficiently sensitive to detect bisphenol A below the specific migration limit for plastic packaging (0.05 mg/kg food). This study demonstrates that P-YES outperforms L-YES because it is more sensitive, provides comparable estradiol equivalents, and circumvents confounding mixture effects. The P-YES will be useful for routine monitoring of FCM and toxicant identification in problematic materials. Graphical abstract.

Solid-phase extraction of estrogens and herbicides from environmental waters for bioassay analysis-effects of sample volume on recoveries.

Ecotoxicological screening of surface waters can involve multiple analyses using multiple bioassay and chemical analytical methods that require enriched samples to reach low concentrations. Such broad screening of the same sample necessitates sufficient sample volume-typically several liters-to produce a sufficient amount of enriched sample. Often, this is achieved by performing parallel solid-phase extractions (SPE) where extracts are combined into a pool-this is a laborious process. In this study, we first validated our existing SPE method for the chemical recovery of an extended set of compounds. We spiked four estrogenic compounds and 11 herbicides to samples from independent rivers (1 L) and wastewater treatment plant effluents (0.5 L). Then, we investigated the effect of increased sample loading of the SPE cartridges on both chemical and biological recoveries by comparing the validated volumes with four times larger sample volumes (i.e., 4 L river water and 2 L effluent). Samples were analyzed by LC-MS/MS and three bioassays: an estrogen receptor transactivation assay (ERα-CALUX), the combined algae test, and a bacterial bioluminescence inhibition assay. Our existing SPE method was found to be suitable for enriching the extended set of estrogens and herbicides in river water and effluents with near to perfect chemical recoveries (~ 100%), except for the herbicide metribuzin (46 ± 19%). In the large volume river and effluent samples, the biological activities and concentrations of the spiked compounds were between 87 and 104% of those measured with the lower sample loading, which is adequate. In addition, the ratio between the large and original volume SPE method for the non-target endpoint (bacterial bioluminescence inhibition) was acceptable (on average 82 ± 9%). Results indicate that our current water extraction method can be applied to up to four times larger sample volumes, resulting in four times more extract volumes, without significant reductions in recoveries for the tested estrogens and herbicides. Graphical abstract ᅟ.

Skyrmions with Attractive Interactions in an Ultrathin Magnetic Film.

We determined the parameters of a classical spin Hamiltonian describing an Fe monolayer on Pd(111) surface with a Pt_{1-x}Ir_{x} alloy overlayer from ab initio calculations. While the ground state of the system is ferromagnetic for x=0.00, it becomes a spin spiral state as Ir is intermixed into the overlayer. Although the Dzyaloshinsky-Moriya interaction is present in the system, we will demonstrate that the frustrated isotropic exchange interactions play a prominent role in creating the spin spiral state, and these frustrated couplings lead to an attractive interaction between Skyrmions at short distances. Using spin dynamics simulations, we show that under these conditions the individual Skyrmions form clusters, and that these clusters remain stable at finite temperature.

Tracing thyroid hormone-disrupting compounds: database compilation and structure-activity evaluation for an effect-directed analysis of sediment.

A variety of anthropogenic compounds has been found to be capable of disrupting the endocrine systems of organisms, in laboratory studies as well as in wildlife. The most widely described endpoint is estrogenicity, but other hormonal disturbances, e.g., thyroid hormone disruption, are gaining more and more attention. Here, we present a review and chemical characterization, using principal component analysis, of organic compounds that have been tested for their capacity to bind competitively to the thyroid hormone transport protein transthyretin (TTR). The database contains 250 individual compounds and technical mixtures, of which 144 compounds are defined as TTR binders. Almost one third of these compounds (n = 52) were even more potent than the natural hormone thyroxine (T4). The database was used as a tool to assist in the identification of thyroid hormone-disrupting compounds (THDCs) in an effect-directed analysis (EDA) study of a sediment sample. Two compounds could be confirmed to contribute to the detected TTR-binding potency in the sediment sample, i.e., triclosan and nonylphenol technical mixture. They constituted less than 1% of the TTR-binding potency of the unfractionated extract. The low rate of explained activity may be attributed to the challenges related to identification of unknown contaminants in combination with the limited knowledge about THDCs in general. This study demonstrates the need for databases containing compound-specific toxicological properties. In the framework of EDA, such a database could be used to assist in the identification and confirmation of causative compounds focusing on thyroid hormone disruption.

Transthyretin-binding activity of contaminants in blood from polar bear (Ursus maritimus) cubs.

We determined the transthyretin (TTR)-binding activity of blood-accumulating contaminants in blood plasma samples of approximately 4-months-old polar bear (Ursus maritimus) cubs from Svalbard sampled in 1998 and 2008. The TTR-binding activity was measured as thyroxine (T4)-like equivalents (T4-EQMeas). Our findings show that the TTR-binding activity related to contaminant levels was significantly lower (45%) in 2008 than in 1998 (mean ± standard error of mean: 1998, 2265 ± 231 nM; 2008, 1258 ± 170 nM). Although we cannot exclude a potential influence of between-year differences in capture location and cub body mass, our findings most likely reflect reductions of TTR-binding contaminants or their precursors in the arctic environment (e.g., polychlorinated biphenyls [PCBs]). The measured TTR-binding activity correlated positively with the cubs' plasma levels of hydroxylated PCBs (OH-PCBs). No such association was found between TTR-binding activity and the plasma levels of perfluoroalkyl substances (PFASs). The OH-PCBs explained 60 ± 7% and 54 ± 4% of the TTR-binding activity in 1998 and 2008, respectively, and PFASs explained ≤1.2% both years. Still, almost half the TTR-binding activity could not be explained by the contaminants we examined. The considerable levels of TTR-binding contaminants warrant further effect directed analysis (EDA) to identify the contaminants responsible for the unexplained part of the observed TTR-binding activity.

Effect-directed analysis to explore the polar bear exposome: identification of thyroid hormone disrupting compounds in plasma.

Compounds with transthyretin (TTR)-binding potency in the blood plasma of polar bear cubs were identified with effect-directed analysis (EDA). This approach contributes to the understanding of the thyroid disrupting exposome of polar bears. The selection of these samples for in-depth EDA was based on the difference between the observed TTR-binding potency on the one hand and the calculated potency (based on known concentrations of TTR-binding compounds and their relative potencies) on the other. A library-based identification was applied to the liquid chromatography-time-of-flight-mass spectrometry (LC-ToF-MS) data by screening for matches between compound lists and the LC-ToF-MS data regarding accurate mass and isotope pattern. Then, isotope cluster analysis (ICA) was applied to the LC-ToF-MS data allowing specific screening for halogen isotope patterns. The presence of linear and branched nonylphenol (NP) was observed for the first time in polar bears. Furthermore, the presence of one di- and two monohydroxylated octachlorinated biphenyls (octaCBs) was revealed in the extracts. Linear and branched NP, 4'-OH-CB201 and 4,4'-OH-CB202 could be successfully confirmed with respect to their retention time in the analytical system. In addition, branched NP, mono- and dihydroxylated-octaCBs showed TTR-binding potencies and could explain another 32 ± 2% of the total measured activities in the extracts.

Pain catastrophizing, pain sensitivity and fear of pain are associated with early life environmental unpredictability: a path model approach.

BACKGROUND: Socioeconomic disadvantages in the childhood environment might strongly influence beliefs and behavior characterizing the adult years. When children experience unpredictable and adverse situations, they develop an unpredictability schema with the core belief that situations are unpredictable. METHODS: In two studies, we examined the association of childhood socioeconomic disadvantages with self-reported pain sensitivity, pain catastrophizing, and pain-related fear. Multidimensional survey measures were used to assess environmental conditions experienced in childhood. In addition, participants completed the Pain Catastrophizing Scale, Pain Sensitivity Questionnaire, Body Awareness Questionnaire, Unpredictability Schema Questionnaire, and Fear of Pain Questionnaire. In Study 1 (N = 252), in separate models, we examined pain sensitivity and pain catastrophizing of a community sample of pain-free young individuals in association with their childhood experiences. In Study 2 (N = 293), in a new sample, but with a wider age range, we examined the association of early life socioeconomic disadvantages with pain-related fear. In both studies, the predictions were tested with Structural Equation Modeling. Our models constituted a path from childhood socioeconomic status and household unpredictability to pain variables via the factors of family resources, unpredictability schemas, and body awareness. RESULTS AND CONCLUSIONS: The findings converged on the conclusion that individuals experiencing disadvantageous early life conditions tended to have an elevated level of pain catastrophizing, higher perceived sensitivity to pain, and higher level of pain-related fear. These associations were mediated by an unpredictability schema and body awareness.

Tuning Ferromagnetism in a Single Layer of Fe above Room Temperature.

The crystallographic and magnetic properties of an Fe monolayer (ML) grown on 2 ML Au/W(110) substrate are studied with spin-polarized low-energy electron microscopy, density functional theory, and relativistic screened Korringa-Kohn-Rostoker calculations. The single layer of iron atoms possesses hexagonal symmetry and reveals a ferromagnetic order at room temperature. We experimentally demonstrate the possibility of tuning the Curie temperature and the magnitude of magnetization of the Fe monolayer by capping with Au. Taking into account several structural models, the calculation results mostly show ferromagnetic states with enhanced magnetic moments of Fe atoms compared to their bulk value and a further increase in their value after covering with Au. The theoretically calculated Curie temperatures are in fair agreement with those obtained in the experiments. The calculations, furthermore, found evidence for the presence of frustrated isotropic Fe-Fe exchange interactions, and a discussion of the structural effects on the magnetic properties is provided herein.

Negative time perspective predicts the self-perceived affective and physical components of pain independently from depression, anxiety, and early life circumstances.

People's attitudes toward time, the time perspectives (TPs) rooted in past experiences, might be crucial for the emotional evaluation of life events, such as painful situations. In a survey-based study (N = 353), we investigated the relationship of TPs with the self-perceived affective and physical aspects of pain. Specifically, we hypothesised that past-negative TP would be associated with a negative emotional appraisal of pain characterised by higher pain-related catastrophizing, fear, and physical sensitivity to pain. We tested this hypothesis with hierarchical regression models, adding depression, anxiety, and variables of early life circumstances to the model stepwise. Early life circumstances (i.e., socioeconomic status, household unpredictability, and family resources) were measured retrospectively using questionnaires. In addition, based on the results of the regression analyses, we built a structural equation model (SEM) and tested whether past-negative mediates the effect of household unpredictability toward emotional and physical appraisal of pain. We found that individuals scoring high on past-negative TP had greater pain catastrophizing, pain-related fear, and sensitivity to pain. Further, there was evidence that these associations are independent from depression, anxiety, and early life circumstances. Finally, SEM analysis suggested that unpredictable early life circumstances were directed toward intense, negatively emotionalised pain perception via the mediation of the past-negative TP. The findings generally support the view that pain behaviour is formed by emotional states and attitudes that are linked to past experiences that are not necessarily specific to the context of pain.

Evaluation of Three ISO Estrogen Receptor Transactivation Assays Applied to 52 Domestic Effluent Samples.

Estrogens are released to the aquatic environment by wastewater treatment plant (WWTP) effluents and can affect wildlife. In the last three decades, many in vitro assay platforms have been developed to detect and quantify estrogenicity in water. In 2018, the International Organization for Standardization (ISO) standardized protocols became available for three types of in vitro estrogen receptor transactivation assays (ERTAs) detecting estrogenicity in 96-well plates (ISO19040 1-3). Two ERTAs-lyticase Yeast Estrogen Screen (L-YES) and Arxula YES (A-YES)-use genetically modified yeast strains, whereas the third utilizes stably transfected human cells. One human cell based assay is ERα-CALUX, which is based on a genetically modified human bone osteosarcoma cell line. In the present study, we characterized the performance, comparability, and effectiveness of these three ERTAs, including an evaluation involving proposed water quality thresholds (effect-based trigger values [EBTs]). For a robust evaluation, we collected 52 effluent samples over three sampling campaigns at 15 different WWTPs in Switzerland. Estrogen receptor transactivation assay results were correlated and compared with results from chemical analysis targeting known estrogens. The three ERTAs showed comparable data over all campaigns. However, the selection of EBTs plays a significant role in the interpretation and comparison of bioassay results to distinguish between acceptable and unacceptable water quality. Applying a fixed cross-assay EBT for effluent of 4 ng L-1 resulted in varying numbers of threshold exceedances ranging between zero and four samples depending on the ERTA used. Using assay-specific EBTs showed exceedances in eight samples (ERα-CALUX) and in one sample (A-YES), respectively. Thus, proposed EBTs do not produce similar risk profiles across samples and further refinement of assay-specific EBTs is needed to account for assay-specific differences and to enable the application of ERTAs as effect-based methods in environmental monitoring. Environ Toxicol Chem 2022;41:2512-2526. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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.

Blood plasma sample preparation method for the assessment of thyroid hormone-disrupting potency in effect-directed analysis.

A sample preparation method combining solid-phase extraction (SPE) and liquid-liquid extraction (LLE) was developed to be used in Effect-Directed Analysis (EDA) of blood plasma. Until now such a method was not available. It can be used for extraction of a broad range of thyroid hormone (TH)-disruptors from plasma with high recoveries. Validation of the method using spiked cow plasma showed good recoveries for hydroxylated polybrominated diphenyl ethers (OH-PBDEs; 93.8 ± 19.5%), hydroxylated polychlorinated biphenyls (OH-PCBs; 93.8 ± 15.5%), other halogenated phenols (OHPs; 107 ± 8.1%), and for short-chain (<8 C-atoms) perfluoroalkyl substances (PFASs; 85.2 ± 24.6%). In the same extracts, the potency of the compound classes spiked to the cow plasma to competitively bind to transthyretin (TTR) was recovered by 84.9 ± 8.8%. Furthermore, the SPE-LLE method efficiently removed endogenous THs from the extracts, thereby eliminating their possible contribution to the binding assay response. The SPE-LLE method was applied to polar bear plasma samples to investigate its applicability in future EDA studies focusing on TH-disrupting compounds in this top predator species that is exposed to relatively high levels of bioaccumulating pollutants. A first screening revealed TTR-binding potency in the polar bear plasma extracts, which could be explained for 60-85% by the presence of OH-PCBs.

Identification strategy for unknown pollutants using high-resolution mass spectrometry: androgen-disrupting compounds identified through effect-directed analysis.

Effect-directed analysis has been applied to a river sediment sample of concern to identify the compounds responsible for the observed effects in an in vitro (anti-)androgenicity assay. For identification after non-target analysis performed on a high-resolution LTQ-Orbitrap, we developed a de novo identification strategy including physico-chemical parameters derived from the effect-directed analysis approach. With this identification strategy, we were able to handle the immense amount of data produced by non-target accurate mass analysis. The effect-directed analysis approach, together with the identification strategy, led to the successful identification of eight androgen-disrupting compounds belonging to very diverse compound classes: an oxygenated polyaromatic hydrocarbon, organophosphates, musks, and steroids. This is one of the first studies in the field of environmental analysis dealing with the difficult task of handling the large amount of data produced from non-target analysis. The combination of bioassay activity assessment, accurate mass measurement, and the identification and confirmation strategy is a promising approach for future identification of environmental key toxicants that are not included as priority pollutants in monitoring programs.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Testing endocrine disruption in biota samples: a method to remove interfering lipids and natural hormones.

A cleanup method was developed to remove coextracted lipids and natural hormones from biota samples in order to test the endocrine-disrupting (ED) capacity of their extracts in in vitro bioassays. Unspiked and spiked fish tissues were cleaned with a combination of dialysis, gel permeation chromatography (GPC), and normal-phase liquid chromatography (NP-HPLC). The spiking mixture consisted of a broad range of environmental pollutants (endocrine disruptors and genotoxic compounds). Chemical recoveries of each test compound, and thyroid-hormone-like and (anti)androgenic activities of the cleaned extracts were investigated. Despite the chemical and toxicological complexity of the spiking mixture and the sequential sample treatment, chemical analysis revealed acceptable recoveries on average: 89 ± 8% after each cleanup step separately and 75 ± 3% after the whole extraction and cleanup procedure in the extracts. In addition, recovered activities in the bioassays were in good agreement with the spiking levels. The developed cleanup method proved to be capable of lipid and natural hormone removal from fish extracts, enabling the measurement of selected endocrine-hormone-like activities in T(4)*-TTR and AR-CALUX bioassays. The method can be used as a sample preparation method of biota samples for toxicity profiling and effect-directed analysis (EDA).

Long-range focusing of magnetic bound states in superconducting lanthanum.

Quantum mechanical systems with long-range interactions between quasiparticles provide a promising platform for coherent quantum information technology. Superconductors are a natural choice for solid-state based quantum devices, while magnetic impurities inside superconductors give rise to quasiparticle excitations of broken Cooper pairs that provide characteristic information about the host superconductor. Here, we reveal that magnetic impurities embedded below a superconducting La(0001) surface interact via quasiparticles extending to very large distances, up to several tens of nanometers. Using low-temperature scanning probe techniques, we observe the corresponding anisotropic and giant oscillations in the LDOS. Theoretical calculations indicate that the quasi-two-dimensional surface states with their strongly anisotropic Fermi surface play a crucial role for the focusing and long-range extension of the magnetic bound states. The quasiparticle focusing mechanism should facilitate the design of versatile magnetic structures with tunable and directed magnetic interactions over large distances, thereby paving the way toward the design of low-dimensional magnet-superconductor hybrid systems exhibiting topologically non-trivial quantum states as possible elements of quantum computation schemes based on Majorana quasiparticles.

Evaluation of a panel of in vitro methods for assessing thyroid receptor ß and transthyretin transporter disrupting activities.

We developed a thyroid testing panel to assess endocrine disrupting chemicals (EDCs) capacities to bind either the thyroid receptor ß (TRß) or the thyroid hormones transporter transthyretin (TTR). We first stably transfected a human U2OS cell line with TRß and a luciferase reporter construct to develop the TRß CALUX® reporter gene assay to assess chemicals' potential to interact with TRß. Secondly, we combined a TTR-binding assay with the TRß CALUX (TTR-TRß CALUX) and optimized the system to evaluate the competitive properties of EDCs towards T4 for TTR binding. Both systems were evaluated with a range of known thyroid-disrupting compounds. The agonistic/antagonistic TRß CALUX successfully predicted 9/9 and 9/12 test compounds, respectively. The TTR-TRß CALUX predicted 9/9 compounds and demonstrated competitive activities when analyzing waste water samples. We concluded that the proposed test battery is a promising screening method able to efficiently generate data on thyroid hormone interferences by chemicals.

Natural estrogens in surface waters of a catchment with intensive livestock farming in Switzerland.

Natural estrogens such as 17α-estradiol (E2α), 17ß-estradiol (E2ß), estrone (E1), and estriol (E3), released to surface waters from both urban and agricultural sources, are endocrine disrupting for fish. Here, we assess the prevalence of livestock farming derived natural estrogens in tributaries and ponds in the agriculturally dominated catchment of Lake Baldegg, Switzerland. Passive samplers were deployed in the main tributary and daily time-proportional water samples were collected in five tributaries for 30 days at the beginning of the vegetation period. Furthermore, we took grab samples of 12 ponds in the catchment. Aqueous samples were liquid-liquid extracted, derivatized, and analysed with LC-MS/MS and stream water samples additionally with ERα-CALUX, a bioassay for assessing total estrogenic activity. Natural estrogens were regularly detected, with mean concentrations ranging from below the limit of detection to 0.55 ng L-1 for E2ß and E1, respectively, and passive sampling and bioassay results largely confirmed these findings. Monte Carlo simulated mean natural estrogen concentrations underestimated measured ones by a factor of three to 11. An agricultural area's hydrological contribution and connectivity to surface waters seemed to be more important for the development of estrogen concentrations in streams than livestock densities in a catchment or the actual loads of slurry applied. Pond water occasionally contained natural estrogens in concentrations up to 8.6 ng L-1 for E2α. The environmental quality standards of the European Union (0.4 ng L-1 for E2ß and 3.6 ng L-1 for E1) were never exceeded for longer than a day in tributaries, but E1 reached critical concentrations for aquatic organisms in ponds.

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.

Validation of Arxula Yeast Estrogen Screen assay for detection of estrogenic activity in water samples: Results of an international interlaboratory study.

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17ß-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8ng/L 17ß-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.