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While many plastic additives show endocrine disrupting properties, this has not been studied for micro- and nanoplastics (MNPs) particles despite their ubiquitous presence in humans. The objective of this study was to determine the effects of various sizes and concentrations of polystyrene (PS)-MNPs (50-10,000 nm, 0.01-100 μg/mL) on estrogen- and androgen receptor (ER and AR) activity and steroidogenesis in vitro. Fluorescent (F)PS-MNPs of ≤1000 nm were internalized in VM7 and H295R cells and FPS-MNPs ≤200 nm in AR-ecoscreen cells. H295R cells displayed the highest uptake and particles were closer to the nucleus than other cell types. None of the sizes and concentrations PS-MNPs tested affected ER or AR activity. In H295R cells, PS-MNPs caused some statistically significant changes in hormone levels, though these showed no apparent concentration or size-dependent patterns. Additionally, PS-MNPs caused a decrease in estriol (E3) with a maximum of 37.5 % (100 μg/mL, 50 nm) and an increase in gene expression of oxidative stress markers GPX1 (1.26-fold) and SOD1 (1.23-fold). Taken together, our data show limited endocrine-disrupting properties of PS-MNPs in vitro. Nevertheless the importance of E3 in the placenta warrants further studies in the potential effects of MNPs during pregnancy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The contamination of freshwater with microplastics (MPs) has been established globally. While the analysis of MPs has predominantly involved spectroscopic methods for revealing particle numbers, the potential of employing spectroscopy for mass estimation has been underutilized. Consequently, there is a need to enhance our understanding of the mass loads of MPs and ensure the complementarity and comparability of various techniques for accurate quantification. This study presents the first comparative results on urban water samples using micro Fourier-transform infrared (μ-FTIR) imaging and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) to identify and quantify MPs in both particle numbers and mass concentration. Two sampling campaigns in summer and winter were conducted at 11 locations within the Amsterdam canal network. An advanced in-situ volume-reducing sampling pump was employed to collect MPs from the surface water within the size fraction of 10-300 μm. The analysis revealed MP concentrations within the range of 16-107 MP/m 3 , estimated to be 2.0-789 μg/m 3 by μ-FTIR imaging and 8.5-754 μg/m 3 by Py-GC-MS. The results of the two analysis techniques showed good comparability in terms of the general trends of MP abundances, with variations in polymer compositions due to the inherent inter-methodological differences. Elevated MP concentrations were observed in the city center compared to the suburban areas. In addition, seasonal differences in MP abundances were noted at the locations with high human activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The integration of untargeted lipidomics approaches in ecotoxicology has emerged as a strategy to enhance the comprehensiveness of environmental risk assessment. Although current toxicity tests with soil microarthropods focus on species performance, that is, growth, reproduction, and survival, understanding the mechanisms of toxicity across all levels of biological organization, from molecule to community is essential for informed decision-making. Our study focused on the impacts of sublethal concentrations of the insecticide teflubenzuron on the springtail Folsomia candida. Untargeted lipidomics was applied to link changes in growth, reproduction, and the overall stress response with lipid profile changes over various exposure durations. The accumulation of teflubenzuron in organisms exposed to the highest test concentration (0.035 mg a.s. kg -1  soil dry wt) significantly impacted reproductive output without compromising growth. The results suggested a resource allocation shift from reproduction to size maintenance. This hypothesis was supported by lipid shifts on day 7, at which point reductions in triacylglycerol and diacylglycerol content corresponded with decreased offspring production on day 21. The hypermetabolism of fatty acids and N-acylethanolamines on days 2 and 7 of exposure indicated oxidative stress and inflammation in the animals in response to teflubenzuron bioaccumulation, as measured using high-performance liquid chromatography-tandem mass spectrometry. Overall, the changes in lipid profiles in comparison with phenotypic adverse outcomes highlight the potential of lipid analysis as an early-warning tool for reproductive disturbances caused by pesticides in F. candida. Environ Toxicol Chem 2024;43:1149-1160. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Only a fraction of the total number of per- and polyfluoroalkyl substances (PFAS) are monitored on a routine basis using targeted chemical analyses. We report on an approach toward identifying bioactive substances in environmental samples using effect-directed analysis by combining toxicity testing, targeted chemical analyses, and suspect screening. PFAS compete with the thyroid hormone thyroxin (T 4 ) for binding to its distributor protein transthyretin (TTR). Therefore, a TTR-binding bioassay was used to prioritize unknown features for chemical identification in a PFAS-contaminated sediment sample collected downstream of a factory producing PFAS-coated paper. First, the TTR-binding potencies of 31 analytical PFAS standards were determined. Potencies varied between PFAS depending on carbon chain length, functional group, and, for precursors to perfluoroalkyl sulfonic acids (PFSA), the size or number of atoms in the group(s) attached to the nitrogen. The most potent PFAS were the seven- and eight-carbon PFSA, perfluoroheptane sulfonic acid (PFHpS) and perfluorooctane sulfonic acid (PFOS), and the eight-carbon perfluoroalkyl carboxylic acid (PFCA), perfluorooctanoic acid (PFOA), which showed approximately four- and five-times weaker potencies, respectively, compared with the native ligand T 4 . For some of the other PFAS tested, TTR-binding potencies were weak or not observed at all. For the environmental sediment sample, not all of the bioactivity observed in the TTR-binding assay could be assigned to the PFAS quantified using targeted chemical analyses. Therefore, suspect screening was applied to the retention times corresponding to observed TTR binding, and five candidates were identified. Targeted analyses showed that the sediment was dominated by the di-substituted phosphate ester of N-ethyl perfluorooctane sulfonamido ethanol (SAmPAP diester), whereas it was not bioactive in the assay. SAmPAP diester has the potential for (bio)transformation into smaller PFAS, including PFOS. Therefore, when it comes to TTR binding, the hazard associated with this substance is likely through (bio)transformation into more potent transformation products. Environ Toxicol Chem 2024;43:245-258. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Comprehensive reference data for steroid hormones are lacking in rat models, particularly for early developmental stages and unconventional matrices as the liver. Therefore, we developed and validated an enzymatic, solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify a panel of 23 steroid hormones in liver and plasma from adult and neonatal rats. Our approach tackles methodological challenges, focusing on undesired byproducts associated with specific enzymatic treatment, and enables a thorough assessment of potential interferences in complex matrices by utilizing unstripped plasma and liver. We propose an optimized enzymatic hydrolysis protocol using a recombinant β-glucuronidase/sulfatase mix (BGS mix) to efficiently deconjugate steroid phase II conjugates. The streamlined sample preparation and high-throughput solid phase extraction in a 96-well plate significantly accelerate sample processing for complex matrices and alarge number of samples. We were able to achieve the necessary sensitivity for accurately measuring the target analytes, particularly estrogens, in small sample sizes of 5-20 mg of liver tissue and 100 μL of plasma. Through the analysis of liver and plasma samples from adult and neonatal rats, including both sexes, our study showed a novel set of steroid hormone reference intervals. This study provides a reliable diagnostic tool for the quantification of steroids in rat models and gives insight in liver and plasma-related steroid hormone dynamics at early developmental stages. In addition, the method covers several pathway intermediates and extend the list of steroid hormones to be investigated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Per- and polyfluoroalkyl substances (PFAS) are a group of xenobiotics that are widely distributed throughout the aquatic environment. Many PFAS are possible thyroid hormone (TH) system disrupting compounds, because they have the capacity to -amongst other- inhibit the TH thyroxine (T 4 ) from binding to its transport protein transthyretin (TTR). This study investigated the occurrence of TH-displacing activity in the Dutch water cycle, and more specifically, the contribution of PFAS to this effect. Over one year of monitoring data of 29 PFAS (linear and branched) showed the continuous presence of PFAS in drinking waters and their surface water sources. Secondly, the FITC-T 4 and TTR-TRβ-CALUX bioassays were mutually compared using positive (HPLC-grade water spiked with PFOA) and negative control samples (HPLC-grade water), as well as relative potency factors (RPFs) of up to 20 PFAS congeners. Both assays were found to be suitable for measuring TH-displacing activity in water samples. As a third aim, a field study was performed in the Dutch water cycle that was comprised of samples from drinking water, surface water, PFAS contaminated sites, and 2 wastewater treatment plants. All samples were analyzed with 1. chemical analysis for 29 PFAS, 2. the FITC-T 4 bioassay, and 3. the TTR-TRβ-CALUX bioassay. The bioassays mutually showed good correlation (R 2 0.85). Bioanalytical equivalent concentrations (BEQ) based on chemically-determined concentrations and RPFs (BEQ chem ) revealed that analyzed PFAS only explained ≤4.1 % of their activity in water extracts measured by both bioassays (BEQ bio ). This indicated that as yet unknown compounds contribute to the majority of the measured TH-displacing activity. Moreover, water treatment processes (e.g. DW production from SW) showed a larger contribution of target PFAS to the BEQ bio . This could be a first lead to identify unknown compounds that contribute to this activity, and as such, enable the assessment of possible risks associated by the occurrence of TH-displacing activity in water., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pharmaceuticals, such as glucocorticoids and antibiotics, are inadequately removed from wastewater and may cause unwanted toxic effects in the receiving environment. This study aimed to identify contaminants of emerging concern in wastewater effluent with antimicrobial or glucocorticoid activity by applying effect-directed analysis (EDA). Effluent samples from six wastewater treatment plants (WWTPs) in the Netherlands were collected and analyzed with unfractionated and fractionated bioassay testing. Per sample, 80 fractions were collected and in parallel high-resolution mass spectrometry (HRMS) data were recorded for suspect and nontarget screening. The antimicrobial activity of the effluents was determined with an antibiotics assay and ranged from 298 to 711 ng azithromycin equivalents·L -1 . Macrolide antibiotics were identified in each effluent and found to significantly contribute to the antimicrobial activity of each sample. Agonistic glucocorticoid activity determined with the GR-CALUX assay ranged from 98.1 to 286 ng dexamethasone equivalents·L -1 . Bioassay testing of several tentatively identified compounds to confirm their activity revealed inactivity in the assay or the incorrect identification of a feature. Effluent concentrations of glucocorticoid active compounds were estimated from the fractionated GR-CALUX bioassay response. Subsequently, the biological and chemical detection limits were compared and a sensitivity gap between the two monitoring approaches was identified. Overall, these results emphasize that combining sensitive effect-based testing with chemical analysis can more accurately reflect environmental exposure and risk than chemical analysis alone., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to identify antimicrobial contaminants in the aquatic environment with effect-directed analysis. Wastewater influent, effluent, and surface water (up- and downstream of the discharge location) were sampled at two study sites. The samples were enriched, subjected to high-resolution fractionation, and the resulting 80 fractions were tested in an antibiotics bioassay. The resulting bioactive fractions guided the suspect and nontargeted identification strategy in the high-resolution mass spectrometry data that was recorded in parallel. Chemical features were annotated with reference databases, assessed on annotation quality, and assigned identification confidence levels. To identify antibiotic metabolites, Phase I metabolites were predicted in silico for over 500 antibiotics and included as a suspect list. Predicted retention times and fragmentation patterns reduced the number of annotations to consider for confirmation testing. Overall, the bioactivity of three fractions could be explained by the identified antibiotics (clarithromycin and azithromycin) and an antibiotic metabolite (14-OH(R) clarithromycin), explaining 78% of the bioactivity measured at one study site. The applied identification strategy successfully identified antibiotic metabolites in the aquatic environment, emphasizing the need to include the toxic effects of bioactive metabolites in environmental risk assessments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

It has been suggested that domestic animals can serve as sentinels for human exposures. In this study our objectives were to demonstrate that i) silicone collars can be used to measure environmental exposures of (domestic) animals, and that ii) domestic animals can be used as sentinels for human residential exposure. For this, we simultaneously measured polycyclic aromatic hydrocarbons (PAHs) using silicone bands worn by 30 pet cats (collar) and their owner (wristband). Collars and wristbands were worn for 7 days and analyzed via targeted Gas Chromatography-Mass Spectrometry (GC-MS). Demographics and daily routines were collected for humans and cats. Out of 16 PAHs, 9 were frequently detected (>50% of samples) in both wristbands and collars, of which Phenanthrene and Fluorene were detected in all samples. Concentrations of wristbands and collars were moderately correlated for these 9 PAHs (Median Spearman's r = 0.51 (range 0.16-0.68)). Determinants of PAH concentrations of cats and humans showed considerable overlap, with vacuum cleaning resulting in higher exposures and frequent changing of bed sheets in lower exposures. This study adds proof-of-principle data for the use of silicone collars to measure (domestic) animal exposure and shows that cats can be used as sentinels for human residential exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A novel method was developed and optimized for the fast-screening analysis of additives in electronics and plastic consumer products using atmospheric pressure matrix-assisted laser desorption ionization (AP-MALDI) coupled with a high-resolution quadrupole time-of-flight (qTOF) mass spectrometer (MS). To simplify sample preparation and increase sample throughput, an innovative 48 well graphene nanoplatelets (GNP) doped AP-MALDI target plate was developed. The GNP incorporated in the target plate fulfilled the role of the MALDI matrix and, therefore, sample extracts could be directly transferred to the AP-MALDI 48 well target plate and analyzed without a subsequent matrix addition. The homogeneously dispersed and immobilized GNP target plates also provided increased signal intensity and reproducibility. Furthermore, analytical standards of various plastic additives and plastic products with known concentrations of additives were studied to assess the AP-MALDI ionization mechanisms and method capability. The analysis time was 15 s per measurement using an automated sequence. The GNP-doped target plates exhibited high desorption/ionization of low molecular weight molecules (<1000 Da) and can be used in both positive and negative ionization modes. The AP-MALDI-qTOF-MS method was applied to screen for additives in various electronics and plastic consumer products. Suspect screening was performed using a database containing 1366 compounds. A total of 56 additives including antioxidants, flame retardants, plasticizers, UV-stabilizers, and UV-filters were identified (confidence level 4). Identification of certain plastic additives in plastic children's toys may indicate that they are recycled from waste electronic and electronic equipment (WEEE).

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are formed by metabolism from the flame retardants polybrominated diphenyl ethers (PBDEs). In the aquatic environment, they are also produced naturally. OH-PBDEs are known for their potential to disrupt energy metabolism, the endocrine system, and the nervous system. This is the first study focusing on the effects of OH-PBDEs at the metabolite level in vivo. The aim of the current study was to investigate the metabolic effects of exposure to OH-PBDEs using metabolomics, and to identify potential biomarker(s) for energy disruption of OH-PBDEs. Zebrafish (Danio rerio) embryos were exposed to two different concentrations of 6-OH-BDE47 and 6-OH-BDE85 and a mixture of these two compounds. In total, 342 metabolites were annotated and 79 metabolites were affected in at least one exposure. Several affected metabolites, e.g. succinic acid, glutamic acid, glutamine, tyrosine, tryptophan, adenine, and several fatty acids, could be connected to known toxic mechanisms of OH-PBDEs. Several phospholipids were strongly up-regulated with up to a six-fold increase after exposure to 6-OH-BDE47, a scarcely described effect of OH-PBDEs. Based on the observed metabolic effects, a possible connection between disruption of the energy metabolism, neurotoxicity and potential immunotoxicity of OH-PBDEs was suggested. Single compound exposures to 6-OH-BDE47 and 6-OH-BDE85 showed little overlap in the affected metabolites. This shows that compounds of similar chemical structure can induce different metabolic effects, possibly relating to their different toxic mechanisms. There were inter-concentration differences in the metabolic profiles, indicating that the metabolic effects were concentration dependent. After exposure to the mixture of 6-OH-BDE47 and 6-OH-BDE85, a new metabolic profile distinct from the profiles obtained from the single compounds was observed. Succinic acid was up-regulated at the highest, but still environmentally relevant, concentration of 6-OH-BDE47, 6-OH-BDE85, and the mixture. Therefore, succinic acid is suggested as a potential biomarker for energy disruption of OH-PBDEs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Plastic particles are ubiquitous pollutants in the living environment and food chain but no study to date has reported on the internal exposure of plastic particles in human blood. This study's goal was to develop a robust and sensitive sampling and analytical method with double shot pyrolysis - gas chromatography/mass spectrometry and apply it to measure plastic particles ≥700 nm in human whole blood from 22 healthy volunteers. Four high production volume polymers applied in plastic were identified and quantified for the first time in blood. Polyethylene terephthalate, polyethylene and polymers of styrene (a sum parameter of polystyrene, expanded polystyrene, acetonitrile butadiene styrene etc.) were the most widely encountered, followed by poly(methyl methacrylate). Polypropylene was analysed but values were under the limits of quantification. In this study of a small set of donors, the mean of the sum quantifiable concentration of plastic particles in blood was 1.6 µg/ml, showing a first measurement of the mass concentration of the polymeric component of plastic in human blood. This pioneering human biomonitoring study demonstrated that plastic particles are bioavailable for uptake into the human bloodstream. An understanding of the exposure of these substances in humans and the associated hazard of such exposure is needed to determine whether or not plastic particle exposure is a public health risk., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Effect-directed analysis (EDA) aims at the detection of bioactive chemicals of emerging concern (CECs) by combining toxicity testing and high-resolution mass spectrometry (HRMS). However, consolidation of toxicological and chemical analysis techniques to identify bioactive CECs remains challenging and laborious. In this study, we incorporate state-of-the-art identification approaches in EDA and propose a robust workflow for the high-throughput screening of CECs in environmental and human samples. Three different sample types were extracted and chemically analyzed using a single high-performance liquid chromatography HRMS method. Chemical features were annotated by suspect screening with several reference databases. Annotation quality was assessed using an automated scoring system. In parallel, the extracts were fractionated into 80 micro-fractions each covering a couple of seconds from the chromatogram run and tested for bioactivity in two bioassays. The EDA workflow prioritized and identified chemical features related to bioactive fractions with varying levels of confidence. Confidence levels were improved with the in silico software tools MetFrag and the retention time indices platform. The toxicological and chemical data quality was comparable between the use of single and multiple technical replicates. The proposed workflow incorporating EDA for feature prioritization in suspect and nontarget screening paves the way for the routine identification of CECs in a high-throughput manner.

Background: The NORMAN Association (https://www.norman-network.com/) initiated the NORMAN Suspect List Exchange (NORMAN-SLE; https://www.norman-network.com/nds/SLE/) in 2015, following the NORMAN collaborative trial on non-target screening of environmental water samples by mass spectrometry. Since then, this exchange of information on chemicals that are expected to occur in the environment, along with the accompanying expert knowledge and references, has become a valuable knowledge base for "suspect screening" lists. The NORMAN-SLE now serves as a FAIR (Findable, Accessible, Interoperable, Reusable) chemical information resource worldwide., Results: The NORMAN-SLE contains 99 separate suspect list collections (as of May 2022) from over 70 contributors around the world, totalling over 100,000 unique substances. The substance classes include per- and polyfluoroalkyl substances (PFAS), pharmaceuticals, pesticides, natural toxins, high production volume substances covered under the European REACH regulation (EC: 1272/2008), priority contaminants of emerging concern (CECs) and regulatory lists from NORMAN partners. Several lists focus on transformation products (TPs) and complex features detected in the environment with various levels of provenance and structural information. Each list is available for separate download. The merged, curated collection is also available as the NORMAN Substance Database (NORMAN SusDat). Both the NORMAN-SLE and NORMAN SusDat are integrated within the NORMAN Database System (NDS). The individual NORMAN-SLE lists receive digital object identifiers (DOIs) and traceable versioning via a Zenodo community (https://zenodo.org/communities/norman-sle), with a total of > 40,000 unique views, > 50,000 unique downloads and 40 citations (May 2022). NORMAN-SLE content is progressively integrated into large open chemical databases such as PubChem (https://pubchem.ncbi.nlm.nih.gov/) and the US EPA's CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard/), enabling further access to these lists, along with the additional functionality and calculated properties these resources offer. PubChem has also integrated significant annotation content from the NORMAN-SLE, including a classification browser (https://pubchem.ncbi.nlm.nih.gov/classification/#hid=101)., Conclusions: The NORMAN-SLE offers a specialized service for hosting suspect screening lists of relevance for the environmental community in an open, FAIR manner that allows integration with other major chemical resources. These efforts foster the exchange of information between scientists and regulators, supporting the paradigm shift to the "one substance, one assessment" approach. New submissions are welcome via the contacts provided on the NORMAN-SLE website (https://www.norman-network.com/nds/SLE/)., Supplementary Information: The online version contains supplementary material available at 10.1186/s12302-022-00680-6., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2022.)

The chemical pollution crisis severely threatens human and environmental health globally. To tackle this challenge the establishment of an overarching international science-policy body has recently been suggested. We strongly support this initiative based on the awareness that humanity has already likely left the safe operating space within planetary boundaries for novel entities including chemical pollution. Immediate action is essential and needs to be informed by sound scientific knowledge and data compiled and critically evaluated by an overarching science-policy interface body. Major challenges for such a body are (i) to foster global knowledge production on exposure, impacts and governance going beyond data-rich regions (e.g., Europe and North America), (ii) to cover the entirety of hazardous chemicals, mixtures and wastes, (iii) to follow a one-health perspective considering the risks posed by chemicals and waste on ecosystem and human health, and (iv) to strive for solution-oriented assessments based on systems thinking. Based on multiple evidence on urgent action on a global scale, we call scientists and practitioners to mobilize their scientific networks and to intensify science-policy interaction with national governments to support the negotiations on the establishment of an intergovernmental body based on scientific knowledge explaining the anticipated benefit for human and environmental health., Competing Interests: Competing interestsThe authors declare that they have no competing interests. HH is Editor-in-Chief of this Journal., (© The Author(s) 2022.)

Per- and polyfluoroalkyl substances (PFASs) are used in a wide range of products and have been found ubiquitously in our indoor environment, and there is evidence that exposure to PFAS can lead to adverse endocrine effects, such as thyroid hormone disruption. Pet cats have a high dust intake due to their grooming behavior and have been shown to be a suitable sentinel species for assessment of toddler's exposure. Here we used paired household dust (n=46) and cat serum (n=27) samples to establish whether dust is a relevant exposure pathway to PFASs. An analytical method for PFAS analysis was optimized using a low volume of cat serum samples, combining solid-phase extraction and online sample cleanup. Dust was extracted with methanol by sonication and cleaned up by addition of active carbon. In total, 27 PFASs were analyzed by liquid chromatography/mass spectrometry analysis. The correlation between PFAS levels in dust and serum, serum lipids and thyroid hormone levels, and PFAS levels in dust between different rooms were statistically evaluated. PFOS and PFDA could be quantified in all cat serum samples (median 2300 pg/mL and 430 pg/mL, respectively), followed by PFOA (median 1100 pg/mL), quantified in 96% of the samples. The levels of 6:2 and 8:2 diPAPs were determined in 65% and 92% of the serum samples, respectively, and were an order of magnitude lower (1.4-160 pg/mL). Household dust on the other hand was dominated by 6:2 and 8:2 diPAPs, with a median of 65 ng/g dust and 49 ng/g dust, respectively. PFOS (median 13 ng/g dust) and PFOA (median 9 ng/g dust) were quantified in 93% of the dust samples. Only eight PFASs were detected (>LOD) in at least 50% of the samples of both matrices and could be paired. Significant correlations between cat serum and dust were found for PFOA (r S =0.32, p<0.049) and PFUnDA (r S =0.55, p<0.001). Significant positive correlations were found between serum total thyroxine (r S =0.11, p<0.05) and PFNA and between serum cholesterol and PFHpA (r S =0.46, p<0.01), PFUnDA (r S =0.40, p<0.05), PFDoDA (r S =0.44, p<0.01), and sum PFAS (r S =0.48, p<0.01). In conclusion, this study confirmed that dust is a relevant exposure pathway for the ingestion of some PFASs for cats, and the serum levels of PFASs could be of relevance for the cat's health., (© 2021. The Author(s).)

The quality of surface waters is threatened by pollution with low concentrations of bioactive chemicals, among which those interfering with steroid hormone systems. Induced by reports of anti-progestogenic activity in surface waters, a two-year four-weekly survey of (anti-)progestogenic activity was performed at three surface water locations in the Netherlands that serve as abstraction points for the production of drinking water. As certain endogenous and synthetic progestogenic compounds are also potent (anti-)androgens, these activities were also investigated. Anti-progestogenic and anti-androgenic activities were detected in the majority of the monitoring samples, sometimes in concentrations exceeding effect-based trigger values, indicating the need for further research. To characterize the compounds responsible for the activities, a high resolution Effect-Directed Analysis (hr-EDA) panel was combined with PR and AR CALUX bioassays, performed in agonistic and antagonistic modes. The influent and effluent of a domestic wastewater treatment plant (WWTP) were included as effluent is a possible emission source of active compounds. As drivers for androgenic and progestogenic activities several native and synthetic steroid hormones were identified in the WWTP samples, namely androstenedione, testosterone, DHT, levonorgestrel and cyproterone acetate. The pesticides metolachlor and cyazofamid were identified as contributors to both the anti-progestogenic and anti-androgenic activities in surface water. In addition, epiconazole contributed to the anti-progestogenic activities in the rivers Rhine and Enclosed Meuse. This study showed the strength of hr-EDA for the identification of bioactive compounds in environmental samples and shed light on the drivers of (anti-)progestogenic and (anti-)androgenic activities in the aquatic environment., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Monitoring of chemical water quality is extremely challenging due to the large variety of compounds and the presence of biologically active compounds with unknown chemical identity. Previously, we developed a high resolution Effect-Directed Analysis (EDA) platform that combines liquid chromatography with high resolution mass spectrometry and parallel bioassay detection. In this study, the platform is combined with CALUX bioassays for (anti)androgenic, estrogenic and glucocorticoid activities, and the performance of the platform is evaluated. It appeared to render very repeatable results, with high recoveries of spiked compounds and high consistency between the mass spectrometric and bioassay results. Application of the platform to wastewater treatment plant effluent and surface water samples led to the identification of several compounds contributing to the measured activities. Eventually, a workflow is proposed for the application of the platform in a routine monitoring context. The workflow divides the platform into four phases, of which one to all can be performed depending on the research question and the results obtained. This allows one to make a balance between the effort put into the platform and the certainty and depth by which active compounds will be identified. The EDA platform is a valuable tool to identify unknown bioactive compounds, both in an academic setting as in the context of legislative, governmental or routine monitoring., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The goal of the present study was to select a Gram-positive (Gram+) and Gram-negative (Gram-) strain to measure antimicrobial activity in environmental samples, allowing high-throughput environmental screening. The sensitivity of eight pre-selected bacterial strains were tested to a training set of ten antibiotics, i.e. three Gram+ Bacillus subtilis strains with different read-outs, and five Gram- strains. The latter group consisted of a bioluminescent Allivibrio fischeri strain and four Escherichia coli strains, i.e. a wild type (WT) and three strains with a modified cell envelope to increase their sensitivity. The WT B. subtilis and an E. coli strain newly developed in this study, were most sensitive to the training set. This E. coli strain carries an open variant of an outer membrane protein combined with an inactivated multidrug efflux transport system. The assay conditions of these two strains were optimized and validated by exposure to a validation set of thirteen antibiotics with clinical and environmental relevance. The assay sensitivity ranged from the ng/mL to μg/mL range. The applicability of the assays for toxicological characterization of aquatic environmental samples was demonstrated for hospital effluent extract. A future application includes effect-directed analysis to identify yet unknown antibiotic contaminants or their transformation products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Effect-directed analysis (EDA) has shown its added value for the detection and identification of compounds with varying toxicological properties in water quality research. However, for routine toxicity assessment of multiple toxicological endpoints, current EDA is considered labor intensive and time consuming. To achieve faster EDA and identification, a high-throughput (HT) EDA platform, coupling a downscaled luminescent Ames and cell-based reporter gene assays with a high-resolution fraction collector and UPLC-QTOF MS, was developed. The applicability of the HT-EDA platform in the analysis of aquatic samples was demonstrated by analysis of extracts from WWTP influent, effluent and surface water. Downscaled assays allowed detection of mutagenicity and androgen, estrogen and glucocorticoid agonism following high-resolution fractionation in 228 fractions. From 8 masses tentatively identified through non-target analysis, 2 masses were further investigated and chemically and biologically confirmed as the mutagen 1,2,3-benzotriazole and the androgen androstenedione. The compatibility of the high-throughput EDA platform with analysis of water samples and the incorporation of mutagenic and endocrine disruption endpoints allow for future application in routine monitoring in drinking water quality control and improved identification of (emerging) mutagens and endocrine disruptors., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We describe the development of a high-resolution, noncontact fraction collector for liquid chromatography (LC) separations, allowing high-resolution fractionation in high-density well plates. The device is based on a low-dead-volume solenoid valve operated at 1-30 Hz for accurate collection of fractions of equal volume. The solenoid valve was implemented in a modified autosampler resulting in the so-called FractioMate fractionator. The influence of the solenoid supply voltage on solvent release was determined and the effect of the frequency, flow rate, and mobile phase composition was studied. For this purpose, droplet release was visually assessed for a wide range of frequencies and flow rates, followed by quantitative evaluation of a selection of promising settings for highly accurate, repeatable, and stable fraction collection. The potential of the new fraction collector for LC-based bioactivity screening was demonstrated by fractionating the LC eluent of a mixture of estrogenic and androgenic compounds, and a surface water sample (blank and spiked with bioactives) combining mass spectrometric detection and two reporter gene assays for bioactivity detection of the fractions. Additionally, a mixture of two compounds was repeatedly LC separated and fractionated to assess the feasibility of the system for analyte isolation followed by nuclear magnetic resonance analysis.

Background: Developmental exposure to di (2-ethylhexyl) phthalate (DEHP) has been implicated in the onset of metabolic syndrome later in life. Alterations in neurobehavior and immune functions are also affected by phthalate exposure and may be linked to the metabolic changes caused by developmental exposure to DEHP. Objectives: Our goal was to study the effects of developmental exposure to DEHP in the context of metabolic syndrome by integrating different parameters to assess metabolic, neurobehavioral, and immune functions in one model. Methods: Female C57BL/6J mice were exposed to DEHP through the diet during gestation and lactation at doses ranging from 3.3 to 100,000 μg/kg body weight/day (μkd). During a 1-year follow-up period, a wide set of metabolic parameters was assessed in the F1 offspring, including weekly body weight measurements, food consumption, physical activity, glucose homeostasis, serum lipids, and endocrine profile. In addition, neurobehavioral and immune functions were assessed by sweet preference test, object recognition test, acute phase protein, and cytokines production. Animals were challenged with a high fat diet (HFD) in the last 9 weeks of the study. Results: Increased free fatty acids (FFA) and, high density lipoprotein (HDL-C) were observed in serum, together with a decrease in glycated hemoglobin levels in blood of 1-year old male DEHP-exposed offspring after HFD challenge. For the most sensitive endpoint measured (FFA), a lower bound of the 90%-confidence interval for benchmark dose (BMD) at a critical effect size of 5% (BMDL) of 2,160 μkd was calculated. No persistent changes in body weight or fat mass were observed. At 33,000 μkd altered performance was found in the object recognition test in males and changes in interferon (IFN)γ production were observed in females. Conclusions: Developmental exposure to DEHP combined with HFD in adulthood led to changes in lipid metabolism and neurobehavior in male offspring and cytokine production in female offspring. Our findings contribute to the evidence that DEHP is a developmental dyslipidemic chemical, however, more research is needed to further characterize adverse health outcomes and the mechanisms of action associated with the observed sex-specific effects.

As many people spend a large part of their life indoors, the quality of the indoor environment is important. Data on contaminants such as flame retardants, pesticides and plasticizers are available for indoor air and dust but are scarce for consumer products such as computers, televisions, furniture, carpets, etc. This review presents information on semi-volatile organic compounds (SVOCs) in consumer products in an attempt to link the information available for chemicals in indoor air and dust with their indoor sources. A number of 256 papers were selected and divided among SVOCs found in consumer products (n = 57), indoor dust (n = 104) and air (n = 95). Concentrations of SVOCs in consumer products, indoor dust and air are reported (e.g. PFASs max: 13.9 μg/g in textiles, 5.8 μg/kg in building materials, 121 ng/g in house dust and 6.4 ng/m 3 in indoor air). Most of the studies show common aims, such as human exposure and risk assessment. The main micro-environments investigated (houses, offices and schools) reflect the relevance of indoor air quality. Most of the studies show a lack of data on concentrations of chemicals in consumer goods and often only the presence of chemicals is reported. At the moment this is the largest obstacle linking chemicals in products to chemicals detected in indoor air and dust., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Effect-directed analysis (EDA) is a commonly used approach for effect-based identification of endocrine disruptive chemicals in complex (environmental) mixtures. However, for routine toxicity assessment of, for example, water samples, current EDA approaches are considered time-consuming and laborious. We achieved faster EDA and identification by downscaling of sensitive cell-based hormone reporter gene assays and increasing fractionation resolution to allow testing of smaller fractions with reduced complexity. The high-resolution EDA approach is demonstrated by analysis of four environmental passive sampler extracts. Downscaling of the assays to a 384-well format allowed analysis of 64 fractions in triplicate (or 192 fractions without technical replicates) without affecting sensitivity compared to the standard 96-well format. Through a parallel exposure method, agonistic and antagonistic androgen and estrogen receptor activity could be measured in a single experiment following a single fractionation. From 16 selected candidate compounds, identified through nontargeted analysis, 13 could be confirmed chemically and 10 were found to be biologically active, of which the most potent nonsteroidal estrogens were identified as oxybenzone and piperine. The increased fractionation resolution and the higher throughput that downscaling provides allow for future application in routine high-resolution screening of large numbers of samples in order to accelerate identification of (emerging) endocrine disruptors.

The Salmonella reversion based Ames test is the most widely used method for mutagenicity testing. For rapid toxicity assessment of e.g. water samples and for effect-directed analysis, however, the Ames test suffers from lack of throughput and is regarded as a laborious, time consuming method. To achieve faster analysis, with increased throughput, a (downscaled) luminescent derivative of the Ames Salmonella/microsome fluctuation test has been developed through expression of the Photorhabdus luminescens luciferase in the Salmonella TA98 and TA100 strains. The applicability of this test is demonstrated by analysis of environmentally relevant compounds, a suspended particulate matter extract and an industrial effluent sample. Use of the luminescent reporter reduced the required detection time from 48 to 28h with a specificity of 84% for responses reported in the literature to a set of 14 mutagens as compared to 72% in the unmodified fluctuation test. Testing of the same compounds in a downscaled luminescent format resulted in an 88% similarity with the response found in the regular luminescent format. The increase in throughput, faster analysis and potential for real-time bacterial quantification that luminescence provides, allows future application in the high-throughput screening of large numbers of samples or sample fractions, as required in effect-directed analysis in order to accelerate the identification of (novel) mutagens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The AR-EcoScreen is a widely used reporter assay for the detection of androgens and anti-androgens. Endogenous expression of glucocorticoid receptors and their affinity for the androgen responsive element that drives reporter expression, however, makes the reporter cells sensitive to interference by glucocorticoids and less specific for (anti-)androgens. To create a glucocorticoid insensitive derivative of the AR-EcoScreen, CRISPR/Cas9 genome editing was used to develop glucocorticoid receptor knockout mutants by targeting various sites in the glucocorticoid gene. Two mutant cell lines were further characterized and validated against the unmodified AR-EcoScreen with a set of 19 environmentally relevant chemicals and a series of environmental passive sampler extracts with (anti-)androgenic activity. Sequencing of the targeted sites revealed premature stop codons following frame-shift mutations, leading to an absence of functional glucocorticoid receptor expression. The introduced mutations rendered cell lines insensitive to glucocorticoid activation and caused no significant difference in the responsiveness towards (anti-)androgens, compared to the unmodified AR-EcoScreen cells, allowing the selective, GR-independent, determination of (anti-)androgenicity in environmental passive sampler extracts. The increase in selectivity for (anti-)androgens improves reliability of the AR-EcoScreen and will provide higher accuracy in determining (anti-)androgenic potential when applied in toxicity screening and environmental monitoring of both single compounds and mixtures., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

BackgroundVarious studies have reported interactions between thyroid hormones and early life chemical exposure. Our objective was to analyze the associations between markers of endocrine-disrupting chemical exposure and thyroid function in newborns, determined through heel prick blood spots.MethodsThree European mother-child cohorts (FLEHSI-Belgium, HUMIS-Norway, and the PCB cohort-Slovakia. Total n=1,784) were pooled for the purpose of this study. Data on thyroid-stimulating hormone (TSH) were obtained from national neonatal screening registries, and samples of cord plasma and/or breast milk were collected to determine exposure to various chemicals. Multiple regression models were composed with exposure and cohort as fixed factors, and adjustments were made for a priori defined covariates.ResultsMedian TSH concentrations were 1, 1.10, and 2.76 mU/l for the Belgian, Norwegian, and Slovak cohorts, respectively. For polychlorinated biphenyl (PCB)-153 and dichlorodiphenyldichloroethylene (p,p'-DDE), children in the third exposure quartile had a 12-15% lower TSH at birth. Results remained unchanged after additional adjustment for birth weight and gestational weight gain. No effect on TSH was observed for the other compounds.ConclusionEarly life exposure to PCB-153 and p,p'-DDE impacts newborn TSH levels. Higher exposure levels were associated with 12-15% lower TSH levels.

We describe the development and evaluation of a GC-MS fractionation platform that combines high-resolution fraction collection of full chromatograms with parallel MS detection. A y-split at the column divides the effluent towards the MS detector and towards an inverted y-piece where vaporized trap solvent is infused. The latter flow is directed outside the GC oven allowing subsequent condensation and stepwise collection of liquid fractions with trapped analytes on a 384-well plate. For study and optimization of the effluent split ratio, restriction capillaries of different lengths and diameters were evaluated. For a wide range of settings, local pressures were monitored during fractionation to assess the influence of MS vacuum and trap solvent infusion on the GC system stability. The platform performance was evaluated by GC-MS analysis and continuous fractionation of an n-alkane mixture followed by GC analysis of each fraction. Comparison of the on-line recorded and fraction-reconstructed chromatogram showed the GC separation is maintained during fractionation. Multiple fractionation cycles of the n-alkane sample on the same 384-well plate yielded a reconstructed chromatogram which was highly similar to that of a single analysis, demonstrating the high repeatability. The applicability of the GC-MS-fractionation platform for bioactivity screening was investigated by applying the AR-Ecoscreen reporter gene bioassay on fractions obtained after analysis of standard solutions and dust samples containing the anti-androgenic compounds vinclozolin and p,p'-DDE., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In a recent study, we showed that the springtail Folsomia candida was quite sensitive the neonicotinoid insecticides imidacloprid and thiacloprid. This study aimed at determining the toxicity of both compounds to F. candida following exposure over three generations, in natural LUFA 2.2 standard soil. In the first generation, imidacloprid was more toxic than thiacloprid, with LC 50 s of 0.44 and 9.0 mg/kg dry soil, respectively and EC 50 s of 0.29 and 1.5 mg/kg dry soil, respectively. The higher LC 50 /EC 50 ratio suggests that thiacloprid has more effects on reproduction, while imidacloprid shows lethal toxicity to the springtails. In the multigeneration tests, using soil spiked at the start of the first generation exposures, imidacloprid had a consistent effect on survival and reproduction in all three generations, with LC 50 s and EC 50 s of 0.21-0.44 and 0.12-0.29 mg/kg dry soil, respectively, while thiacloprid-exposed animals showed clear recovery in the second and third generations (LC 50 and EC 50  > 3.33 mg/kg dry soil). The latter finding is in agreement with the persistence of imidacloprid and the fast degradation of thiacloprid in the test soil.

Thyroid hormone (TH) disrupting compounds are potentially important environmental contaminants due to their possible adverse neurological and developmental effects on both humans and wildlife. Currently, the most successful bio-analytical method to detect and evaluate TH disruptors, which target the plasma transport of TH in environmental samples, is the radio-ligand thyroxine-transthyretin (T 4 -TTR) binding assay. Yet, costly materials and tedious handling procedures prevent the use of this assay in high throughput analysis that is nowadays urgently demanded in environmental quality assessment. For the first time a miniaturized fluorescence T 4 -TTR binding assay was developed in a 96 well microplate and tested with eight TH disrupting compounds. For most of the compounds, the sensitivity of the newly developed assay was slightly lower than the radio-ligand binding assay, however, throughput was enhanced at least 100-fold, while using much cheaper materials. The TH disrupting potency of 22 herring gull (Larus argentatus) egg extracts, collected from two different locations (Musvær and Reiaren) in Norway, was evaluated to demonstrate the applicability of the assay for environmental samples., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Household dust and laundry dryer lint are important indoor environmental matrices that may have notable health effects on humans due to chronic exposure. However, due to the sample complexity the studies conducted on these sample matrices until now were almost exclusively on the basis of target analysis. In this study, comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry (LC × LC-ToF MS) was applied, to enable non-target analysis of household dust as well as laundry dryer lint for the first time. The higher peak capacity and good orthogonality of LC × LC, together with reduced ion suppression in the MS enabled rapid identification of environmental contaminants in these complex sample matrices. A number of environmental contaminants were tentatively identified based on their accurate masses and isotopic patterns, including plasticizers, flame retardants, pesticides, drug metabolites, etc. The identity of seven compounds: tris(2-butoxyethyl) phosphate, tris(2-chloropropyl) phosphate, n-benzyl butyl phthalate, dibutyl phthalate, tributyl phosphate, triethyl phosphate and N, N-diethyl-meta-toluamide was confirmed using two-dimensional retention alignment and their concentrations in the samples were semi-quantitatively determined., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The chemical safety of consumer products is an issue of emerging concern. Plastics are widely used, e.g. as casings of consumer electronics (TVs, computers, routers, etc.), which are present in houses and offices in continuously increasing numbers. In this study, we investigate the estrogenic activity of components of plastics coming from electronics' casings. A recently developed fractionation platform for effect-directed analysis (EDA) was used. This platform combines reversed-phase liquid chromatography in parallel with bioassay detection via nanofractionation and with online high-resolution time-of-flight mass spectrometry (TOFMS) for the identification of bioactives. Four out of eight of the analyzed plastics samples showed the presence of estrogenic compounds. Based on the MS results these were assigned to bisphenol A (BPA), 2,4-di-tert-butylphenol, and a possible bisphenol A analog. All samples contained flame retardants, but these did not show any estrogenicity. The observed BPA, however, could be an impurity of tetrabromo-BPA (TBBPA) or TBBPA-based flame retardants. Due to the plausible migration of additives from plastics into the environment, plastics from consumer electronics likely constitute a source of estrogenic compound contamination in the indoor environment.

To determine demographic, reproductive, and maternal dietary factors that predict perfluoroalkyl substance (PFAS) concentrations in breast milk, we measured perfluorooctane sulfonic (PFOS) and perfluorooctanoic acid (PFOA) concentrations, using liquid chromatography-mass spectrometry, in 184 colostrum samples collected from women participating in a cohort study in Eastern Slovakia between 2002 and 2004. During their hospital delivery stay, mothers completed a food frequency questionnaire, and demographic and reproductive data were also collected. PFOS and PFOA predictors were identified by optimizing multiple linear regression models using Akaike's information criterion (AIC). The geometric mean concentration in colostrum was 35.3 pg/mL for PFOS and 32.8 pg/mL for PFOA. In multivariable models, parous women had 40% lower PFOS (95% CI: -56 to -17%) and 40% lower PFOA (95% CI: -54 to -23%) concentrations compared with nulliparous women. Moreover, fresh/frozen fish consumption, longer birth intervals, and Slovak ethnicity were associated with higher PFOS and PFOA concentrations in colostrum. These results will help guide the design of future epidemiologic studies examining milk PFAS concentrations in relation to health end points in children.

Many chemicals in use end up in the aquatic environment. The toxicity of water samples can be tested with bioassays, but a metabolomic approach has the advantage that multiple end points can be measured simultaneously and the affected metabolic pathways can be revealed. A current challenge in metabolomics is the study of mixture effects. This study aims at investigating the toxicity of an environmental extract and its most abundant chemicals identified by target chemical analysis of >100 organic micropollutants and effect-directed analysis (EDA) using the acetylcholinesterase (AChE) bioassay and metabolomics. Surface water from an agricultural area was sampled with a large volume solid phase extraction (LVSPE) device using three cartridges containing neutral, anionic, and cationic sorbents able to trap several pollutants classes like pharmaceuticals, pesticides, PAHs, PCBs, and perfluorinated surfactants. Targeted chemical analysis and AChE bioassay were performed on the cartridge extracts. The extract of the neutral sorbent cartridge contained most of the targeted chemicals, mainly imidacloprid, thiacloprid, and pirimicarb, and was the most potent AChE inhibitor. Using an EDA approach, other AChE inhibiting candidates were identified in the neutral extract, such as carbendazim and esprocarb. Additionally, a metabolomics experiment on the central nervous system (CNS) of the freshwater snail Lymnaea stagnalis was conducted. The snails were exposed to the extract, the three most abundant chemicals individually, and a mixture of these. The extract disturbed more metabolic pathways than the three most abundant chemicals individually, indicating the contribution of other chemicals. Most pathways perturbed by the extract exposure overlapped with those related to exposure to neonicotinoids, like the polyamine metabolism involved in CNS injuries. Metabolomics for the straightforward comparison between a complex mixture and single compound toxicity is still challenging but, compared to traditional biotesting, is a promising tool due to its increased sensitivity.

Effect-directed analysis (EDA) is a useful tool to identify bioactive compounds in complex samples. However, identification in EDA is usually challenging, mainly due to limited separation power of the liquid chromatography based fractionation. In this study, comprehensive two-dimensional liquid chromatography (LC × LC) based microfractionation combined with parallel high resolution time of flight (HR-ToF) mass spectrometric detection and a high throughput acetylcholinesterase (AChE) assay was developed. The LC × LC fractionation method was validated using analytical standards and a C18 and pentafluorophenyl (PFP) stationary phase combination was selected for the two-dimensional separation and fractionation in four 96-well plates. The method was successfully applied to identify AChE inhibitors in a wastewater treatment plant (WWTP) effluent. Good orthogonality (>0.9) separation was achieved and three AChE inhibitors (tiapride, amisulpride, and lamotrigine), used as antipsychotic medicines, were identified and confirmed by two-dimensional retention alignment as well as their AChE inhibition activity.

Modern toxicology is seeking new testing methods to better understand toxicological effects. One of the most concerning chemicals is the neonicotinoid pesticide imidacloprid. Although imidacloprid is designed to target insects, recent studies have shown adverse effects on nontarget species. Metabolomics was applied to investigate imidacloprid-induced sublethal toxicity in the central nervous system of the freshwater snail Lymnaea stagnalis. The snails (n = 10 snails) were exposed for 10 days to increasing imidacloprid concentrations (0.1, 1, 10, and 100 μg/L). The comparison between control and exposure groups highlighted the involvement and perturbation of many biological pathways. The levels of several metabolites belonging to different metabolite classes were significantly changed by imidacloprid exposure. A change in the amino acids and nucleotide metabolites like tryptophan, proline, phenylalanine, uridine, and guanosine was found. Many fatty acids were down-regulated, and the levels of the polyamines, spermidine and putrescine, were found to be increased which is an indication of neuron cell injury. A turnover increase between choline and acetylcholine led us to hypothesize an increase in cholinergic gene expression to overcome imidacloprid binding to the nicotinic acetylcholine receptors. Metabolomics revealed imidacloprid induced metabolic changes at low and environmentally relevant concentration in a nontarget species and generated a novel mechanistic hypothesis.

In this study we developed a new LC nanofractionation platform that combines a human cell (BG1.Luc) gene reporter assay with a high resolution mass spectrometer for the detection and identification of estrogenic and anti-estrogenic compounds in environmental waters. The selection of this assay was based on its high sensitivity and selectivity, which is required for environmental trace level detection. We modified an autosampler and controlled it with in-house developed software to collect fractions in the low second range in microtiter plates. This ensured that chromatographic separation was maintained and allowed straightforward hyphenation with the bioassay. After bioassay testing, bioassay chromatograms were reconstructed and directly correlated with MS chromatograms that were obtained in parallel. This enabled to pinpoint bioactives in the MS chromatogram within a single fractionation cycle and results in a significant increase in throughput compared to traditional EDA studies. The sensitivity of the platform was low enough for environmental waters (80nM for bisphenol A and 320pM and 3.2nM for estradiol and estriol, respectively). In addition, the ability of the platform to detect anti-estrogens was successfully demonstrated as well. Finally, real samples were analysed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.