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Cyanobacterial blooms represent a worldwide problem in freshwater as well as marine ecosystems as producers of various toxic compounds. This study provides environmentally important information about the common presence of mixtures of retinoids in various water bodies associated with the occurrence of cyanobacterial blooms dominated by many different species. The study documents, for the first time, that retinoids are produced by environmental cyanobacterial blooms dominated by species belonging to different genera such as Microcystis, Dolichospermum, Planktothrix, Woronichinia, Pseudanabaena and others. Samples of biomass of cyanobacterial blooms and their surrounding water were collected from seventeen independent freshwater bodies across the Czech Republic during summer 2015. Retinoid-like activity was detected by an in vitro reporter gene bioassay in water samples from 8 out of 17 localities with a maximal activity of 263 ng all-trans retinoic acid equivalent (REQ)/L. In comparison, in vitro assessment of biomass extracts documented retinoid-like activity at 11 out of 17 localities with a maximal retinoid-like activity of 867 ng REQ/g dry mass (dm). Individual retinoids were detected by chemical analyses in all water samples and in 16 out of 17 biomass samples with 4keto-retinal and all-trans 5,6epoxy retinoic acid being detected in aquatic ecosystems for the first time. Further, all-trans 4keto retinoic acid and retinal were the most commonly detected compounds in both types of samples. With respect to retinoid-like activity, a large proportion was explained in some samples by contributions of individual detected retinoids calculated from their concentrations and relative potencies. However, results also indicate that other unknown compounds with a retinoic acid receptor-mediated mode of action were present. The revealed widespread production of retinoids by cyanobacterial blooms dominated by diverse species across various aquatic ecosystems and their common presence in both biomass and surrounding water raises concern namely because some retinoids belong to the most potent teratogens. These compounds need to be taken into consideration in the assessment of risks associated with massive cyanobacterial blooms.

Pollution of indoor environment, where people spend much of their time, comprises complex mixtures of compounds with vastly understudied hazard potential. This study examined several important specific toxic effects and pollutant levels (177 compounds) of indoor samples (air gas phase, PM10 and dust) from different microenvironments after two extractions with focus on their gas/particle/dust distribution and polarity. The endocrine disruptive (ED) potential was assessed by human cell-based in vitro bioassays addressing anti-/estrogenicity, anti-/androgenicity, aryl hydrocarbon, thyroid and peroxisome proliferator-activated receptor-mediated activities. Potential toxicity to respiratory tract tissue was assessed using human bronchial cell line. The toxicological analyses pointed out the relevance of both inhalation and ingestion exposure, with significant effects detected after exposure to extracts from all three studied matrices with distinct gas/particle distribution patterns. Chemical analyses document the high complexity of indoor pollutant mixtures with greatest levels of phthalates, their emerging alternatives, and PAHs in dust. Despite the detection of up to 108 chemicals, effects were explained only to low extent. This emphasizes data gaps regarding ED potencies of many detected abundant indoor contaminants, but also potential presence of other unidentified ED compounds. The omnipresent ED potentials in indoor environment rise concern regarding associated human health risk.

Regulatory hazard and risk assessment of endocrine-active substances currently specifies four modes of action: interference with sex hormone (oestrogen, androgen) pathways, steroidogenesis, and thyroid hormone signalling. This does not encompass the full complexity of the endocrine system and its extended interfaces with environmental pollutants that can potentially disrupt the carefully maintained balance. Here we take the retinoid signalling pathway as a European case study for both, under- and unregulated endocrine pathways and outline the different levels of interference, discuss their adversity, and indicate crosstalk to other signalling pathways. Retinoid compounds already exist in drinking water sources, occur naturally in cyanobacterial blooms and/or enter surface waters via wastewater discharge, where they pose a potential hazard to the environment and human health - a situation that can be expected to worsen due to water shortages induced by climate-change and population growth. We briefly review relevant aspects of current endocrine disruptor (ED) testing for regulatory purposes and then expand upon the needs for inclusion of disruption of retinoid signalling in (ED) regulatory safety assessment contributing to adverse health outcomes that include cognitive function and neurological disease. An overview of developmental effects of retinoid signalling disruption across species highlights critical processes and potential crosstalk with other signalling pathways. A focused weight of evidence-based evaluation of the biologically plausible associations between neurological disorders and altered retinoid signalling highlights the evidence gaps. We show that monitoring only a limited number of anthropogenic priority chemicals in water is insufficient to address the environmental risks of retinoid signalling disruption. To comprehensively assess impacts on the endpoints, processes, and pathways of the endocrine system that are most vulnerable to chemical interference we need further investigation of the true mixture composition in environmental matrices. On a weight of evidence-basis this information can then be integrated into a reliable, inclusive, quantitative approach that ultimately accommodates all the critical pathways. By focusing on the retinoid signalling pathway, we intend to improve the scope and relevance of an integrated approach for the risk assessment of endocrine disruptors.

In the last decade, it has become evident that complex mixtures of cyanobacterial bioactive substances, simultaneously present in blooms, often exert adverse effects that are different from those of pure cyanotoxins, and awareness has been raised on the importance of studying complex mixtures and chemical interactions. We aimed to investigate cytotoxic and genotoxic effects of complex extracts from laboratory cultures of cyanobacterial species from different orders (Cylindrospermopsis raciborskii, Aphanizomenon gracile, Microcystis aeruginosa, M. viridis, M. ichtyoblabe, Planktothrix agardhii, Limnothrix redekei) and algae (Desmodesmus quadricauda), and examine possible relationships between the observed effects and toxin and retinoic acid (RA) content in the extracts. The cytotoxic and genotoxic effects of the extracts were studied in the human hepatocellular carcinoma HepG2 cell line, using the MTT assay, and the comet and cytokinesis-block micronucleus (cytome) assays, respectively. Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) was used to detect toxins (microcystins (MC-LR, MC-RR, MC-YR) and cylindrospermopsin) and RAs (ATRA and 9cis-RA) in the extracts. Six out of eight extracts were cytotoxic (0.04–2 mgDM/mL), and five induced DNA strand breaks at non-cytotoxic concentrations (0.2–2 mgDM/mL). The extracts with genotoxic activity also had the highest content of RAs and there was a linear association between RA content and genotoxicity, indicating their possible involvement, however further research is needed to identify and confirm the compounds involved and to elucidate possible genotoxic effects of RAs.

Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L−1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.

International audience; ERGO (EndocRine Guideline Optimization) is the acronym of a European Union-funded research and innovation action, that aims to break down the wall between mammalian and non-mammalian vertebrate regulatory testing of endocrine disruptors (EDs), by identifying, developing and aligning thyroid-related biomarkers and endpoints (B/E) for the linkage of effects between vertebrate classes. To achieve this, an adverse outcome pathway (AOP) network covering various modes of thyroid hormone disruption (THD) in multiple vertebrate classes will be developed. The AOP development will be based on existing and new data from in vitro and in vivo experiments with fish, amphibians and mammals, using a battery of different THDs. This will provide the scientifically plausible and evidence-based foundation for the selection of B/E and assays in lower vertebrates, predictive of human health outcomes. These assays will be prioritized for validation at OECD (Organization for Economic Cooperation and Development) level. ERGO will re-think ED testing strategies from in silico methods to in vivo testing and develop, optimize and validate existing in vivo and early life-stage OECD guidelines, as well as new in vitro protocols for THD. This strategy will reduce requirements for animal testing by preventing duplication of testing in mammals and non-mammalian vertebrates and increase the screening capacity to enable more chemicals to be tested for ED properties.

© The Author(s) 2020., The Partnership for Chemicals Risk Assessment (PARC) is currently under development as a joint research and innovation programme to strengthen the scientific basis for chemical risk assessment in the EU. The plan is to bring chemical risk assessors and managers together with scientists to accelerate method development and the production of necessary data and knowledge, and to facilitate the transition to next-generation evidence-based risk assessment, a non-toxic environment and the European Green Deal. The NORMAN Network is an independent, well-established and competent network of more than 80 organisations in the field of emerging substances and has enormous potential to contribute to the implementation of the PARC partnership. NORMAN stands ready to provide expert advice to PARC, drawing on its long experience in the development, harmonisation and testing of advanced tools in relation to chemicals of emerging concern and in support of a European Early Warning System to unravel the risks of contaminants of emerging concern (CECs) and close the gap between research and innovation and regulatory processes. In this commentary we highlight the tools developed by NORMAN that we consider most relevant to supporting the PARC initiative: (i) joint data space and cutting-edge research tools for risk assessment of contaminants of emerging concern; (ii) collaborative European framework to improve data quality and comparability; (iii) advanced data analysis tools for a European early warning system and (iv) support to national and European chemical risk assessment thanks to harnessing, combining and sharing evidence and expertise on CECs. By combining the extensive knowledge and experience of the NORMAN network with the financial and policy-related strengths of the PARC initiative, a large step towards the goal of a non-toxic environment can be taken.

Passive sampling is a method that can conveniently be used for monitoring of organic contaminants in the aquatic environment comprising its various compartments. The time-integrating properties of passive sampling in combination with the application of a sampling matrix (polymers) with well-defined and constant properties make it possible to achieve a lower inherent variability of exposure information compared to traditional whole water grab sampling. This chapter provides a practical guidance on the selection and use of passive samplers for monitoring organic contaminants in the aquatic environment that should assist users of passive samplers who wish to implement passive sampling methods in their research or monitoring work according to the state of the art. It outlines principles of passive sampling, sampler preparation, field deployment, laboratory processing for chemical and toxicological analysis, calculation of aqueous concentrations and associated uncertainty considerations. Aspects of quality assurance are also addressed.

Stagnant water bodies have generally received little attention regarding the presence of endocrine disruptive compounds, although they can integrate diverse pollutants from multiple different sources. Many compounds of anthropogenic as well as natural origin can contribute to the overall estrogenicity of surface waters and some of them can exhibit adverse effects on aquatic biota even in very low concentrations. This study focused on freshwater ponds and reservoirs affected by water blooms and determined the estrogenic activity of water by in vitro bioassay as well as concentrations of several important groups of estrogenic compounds (estrogenic hormones, alkylphenols, and phytoestrogens) by LC-MS/MS analyses. Estrogenic hormones were found at concentrations up to 7.1 ng.L−1, similarly to flavonoids, whose concentrations did not exceed 12.5 ng.L−1. Among alkylphenols, only bisphenol A and 4-tert-octylphenol were detected in levels reaching 100 ng.L−1 at maximum. Estrogenic activity of water samples varied from below the quantification limit to 1.95 ng.L−1. There does not seem to be any general causal link of the massive phytoplankton occurrence with the estrogenicity of water or concentration of phytoestrogens, since they showed no direct relationship with the phytoplankton abundance or composition across sites. The contribution of the analysed compounds to the estrogenic activity was calculated in three scenarios. In minimum scenario, just the compounds above quantification limit (LOQ) were taken into account and for most samples, only minor part (

Lead nanoparticles (NPs) are released into air from metal processing, road transport or combustion processes. Inhalation exposure is therefore very likely to occur. However, even though the effects of bulk lead are well known, there is limited knowledge regarding impact of Pb NPs inhalation. This study focused on acute and subchronic exposures to lead oxide nanoparticles (PbO NPs). Mice were exposed to PbO NPs in whole body inhalation chambers for 4-72 h in acute experiment (4.05 × 10

Some phytoplankton species were shown to produce teratogenic retinoids. This study assessed the variability in the extracellular production of compounds with retinoid-like activity for 50 independent cultivations of wide spectra of species including 12 cyanobacteria (15 strains) and 4 algae of different orders. Extracellular retinoid-like activity was detected for repeated cultivations of six cyanobacteria. The results were consistent for some species including Microcystis aeruginosa and Aphanizomenon gracile. The detected retinoid-like activities ranged from below the limit of quantification of 16 ng/L to over 6 µg all-trans retinoic acid (ATRA) equivalent/L. Nontargeted virtual fractionation together with suspect screening approach enabled to identify some retinoid-like compounds in exudates, including ATRA, 9/13-cis retinoic acid, all-trans 5,6-epoxy retinoic acid, 4keto-ATRA, 4keto-retinal, 4hydroxy-ATRA, and retinal. Most of them were for the first time repeatedly detected in exudates of all studied algae (at ng/L levels) and cyanobacteria. Their relative potencies ranged from 0.018 (retinal) to 1 compared to ATRA. They accounted for less than 0.1–50% of total detected retinoid-like activity. The high detected activities and concentrations of retinoids in some samples and their direct accessibility from exudates document potential risk of developmental toxicity for organisms in proximity of massive water blooms.

Herbicides are the most widely used group of pesticides but after reaching water bodies they are able to cause adverse effects on non-target organisms. Different formulations using the same active ingredient are frequently available, which raises the issue of potential influence of different formulation types on herbicide toxicity. The present study evaluated the toxicity and teratogenic effects of the active ingredient clomazone and its two formulations (Rampa ® EC and GAT Cenit 36 CS, both containing 360 g a.i./l of clomazone) on zebrafish embryos. The crucial difference between the two formulation types is the way of active substance release. This investigation is the first report on zebrafish embryotoxicity of both clomazone and its formulations. The technical active ingredient and formulations caused mortality and diverse teratogenic effects, showing different levels of toxicity. The LC 50 values for the technical ingredient, Rampa ® EC and GAT Cenit 36 CS were 61.4, 9.6 and 92.5 mg a.i./l, respectively. Spontaneous movements in 22 hpf embryos decreased under exposure to both the technical ingredient and formulations. A significant number of underdeveloped embryos was detected after exposure to clomazone and Rampa ® EC, while no underdevelopment was noted in embryos exposed to GAT Cenit 36 CS. Exposure to the technical ingredient and formulations led also to a series of morphological changes and interfered with the growth of zebrafish embryos. The EC 50 based on detection of edemas, spine and tail tip deformations and gas bladder absence (120 hpf) was 12.1, 10.1 and 24.1 mg/l for technical clomazone, Rampa ® EC and GAT Cenit 36 CS, while teratogenicity index (TI) based on LC 50 /EC 50 ratio was 5.1, 1 and 3.8, respectively. The data in this study showed that the emulsifiable concentrate formulation (Rampa ® EC) caused statistically significantly higher toxicity, and the aqueous capsule suspension (GAT Cenit 36 CS) lower toxicity than technical clomazone. It indicates that different formulations with the same active ingredient may have different environmental impacts, which is why risk assessment based only on active ingredient toxicity might not be sufficient in terms of preventing formulation effects on the environment.

BACKGROUND The reduction of toxicity in secondary effluents by solar photo-Fenton has not been widely studied. In this present work, a strategy combining monitoring of micropollutants and application of a selected battery of bioassays has been applied to evaluate the effectiveness of the solar photo-Fenton process for secondary effluent treatment at neutral pH in a raceway pond reactor (RPR) following two operating strategies: sequential iron dosage and the use of Fe(III)–EDDS complex at pilot plant scale. The applied battery of bioassays comprises in vivo (algal growth inhibition test) and in vitro, endocrine disruptor tests (androgenic/glucocorticoid activity and estrogenicity) and cytotoxicity tests. RESULTS The results obtained revealed high efficiency of micropollutant removal (>99%) by both strategies assayed. Regarding toxicity, androgenic/glucocorticoid activity and estrogenicity were efficiently removed in a similar fashion, and the relatively low potential phytotoxicity and cytotoxicity was also attenuated. Both RPR strategies are able to enhance the quality of municipal effluents from chemical and toxicological points of view. CONCLUSION To enable water reuse, the feasibility of solar photo-Fenton reaction at neutral pH in open reactors for ecotoxicity and micropollutant removal in secondary municipal wastewater has been demonstrated for the first time. © 2017 Society of Chemical Industry

Although the production of engineered nanoparticles increases our knowledge of toxicity and mechanisms of bioactivity during relevant exposures is lacking. In the present study mice were exposed to PbO nanoparticles (PbONP; 192.5 mu g/m(3); 1.93 x 10(6) particles/cm(3)) for 2, 5 and 13 weeks through continuous inhalation. The analyses addressed Pb and PbONP distribution in organs (lung, liver, kidney, brain) using electrothermal atomic absorption spectrometry and transmission electron microscopy, as well as histopathology and analyses of oxidative stress biomarkers. New LC-MS/MS methods were validated for biomarkers of lipid damage F2-isoprostanes (8-iso-prostaglandins F2-alpha and E-2) and hydroxylated deoxoguanosine (8-OHdG, marker of DNA oxidation). Commonly studied malondialdehyde was also measured as TBARS by HPLC-DAD. The study revealed fast blood transport and distribution of Pb from the lung to the kidney and liver. A different Pb accumulation trend was observed in the brain, suggesting transfer of NP along the nasal nerve to the olfactory bulbs. Long-term inhalation of PbONP caused lipid peroxidation in animal brains (increased levels of TBARS and both isoprostanes). Membrane lipid damage was also detected in the kidney after shorter exposures, but not in the liver or lung. On the contrary, longer exposures to PbONP increased levels of 8-OHdG in the lung and temporarily increased lung weight after 2 and 5 weeks of exposure. The histopathological changes observed mainly in the lung and liver indicated inflammation and general toxicity responses. The present long-term inhalation study indicates risks of PbONP to both human health and the environment.

To meet the United Nations (UN) sustainable development goals and the European Union (EU) strategy for a non-toxic environment, water resources and ecosystems management require cost-efficient solutions for prevailing complex contamination and multiple stressor exposures. For the protection of water resources under global change conditions, specific research needs for prediction, monitoring, assessment and abatement of multiple stressors emerge with respect to maintaining human needs, biodiversity, and ecosystem services. Collaborative European research seems an ideal instrument to mobilize the required transdisciplinary scientific support and tackle the large-scale dimension and develop options required for implementation of European policies. Calls for research on minimizing society’s chemical footprints in the water–food–energy–security nexus are required. European research should be complemented with targeted national scientific funding to address specific transformation pathways and support the evaluation, demonstration and implementation of novel approaches on regional scales. The foreseeable pressure developments due to demographic, economic and climate changes require solution-oriented thinking, focusing on the assessment of sustainable abatement options and transformation pathways rather than on status evaluation. Stakeholder involvement is a key success factor in collaborative projects as it allows capturing added value, to address other levels of complexity, and find smarter solutions by synthesizing scientific evidence, integrating governance issues, and addressing transition pathways. This increases the chances of closing the value chain by implementing novel solutions. For the water quality topic, the interacting European collaborative projects SOLUTIONS, MARS and GLOBAQUA and the NORMAN network provide best practice examples for successful applied collaborative research including multi-stakeholder involvement. They provided innovative conceptual, modelling and instrumental options for future monitoring and management of chemical mixtures and multiple stressors in European water resources. Advancement of EU water framework directive-related policies has therefore become an option. © 2019, The Author(s)., Funding text #1 This article has been prepared as an outcome of the projects SOLUTIONS (European Union’s Seventh Framework Programme for research, technological development and demonstration under Grant Agreement No. 603437), GLOBAQUA (European Union’s Seventh Framework Programme for research, technological development and demonstration under Grant Agreement No. 603629) and MARS (European Union’s Seventh Framework Programme for research, technological development and demonstration under Grant Agreement No. 603378). Funding text #2 The authors declare that they have no competing interests. CIS Common Implementation Strategy DG Directorate General DPSIR Drivers–Pressure–State–Impacts–Response ECHA European Chemical Agency EEA European Environmental Agency EFSA European Food Safety Authority EU European Union ICPDR International Commission for the Protection of the Danube River ICPR International Commission for the Protection of the Rhine NGO Non-Governmental Organisation SDG sustainable development goals UN United Nations WFD Water Framework Directive

Cyanobacteria routinely release potentially harmful bioactive compounds into the aquatic environment. Several recent studies suggested a potential link between the teratogenicity of effects caused by cyanobacteria and production of retinoids. To investigate this relationship, we analysed the teratogenicity of field-collected cyanobacterial bloom samples by means of an in vivo zebrafish embryo test, an in vitro reporter gene bioassay and by the chemical analysis of retinoids. Extracts of biomass from cyanobacterial blooms with the dominance of Microcystis aeruginosa and Aphanizomenon klebahnii were collected from water bodies in the Czech Republic and showed significant retinoid-like activity in vitro, as well as high degrees of teratogenicity in vivo. Chemical analysis was then used to identify a set of retinoids in ng per gram of dry weight concentration range. Subsequent fractionation and bioassay-based characterization identified two fractions with significant in vitro retinoid-like activity. Moreover, in most of the retinoids eluted from these fractions, teratogenicity with malformations typical for retinoid signalling disruption was observed in zebrafish embryos after exposure to the total extracts and these in vitro effective fractions. The zebrafish embryo test proved to be a sensitive toxicity indicator of the biomass extracts, as the teratogenic effects occurred at even lower concentrations than those expected from the activity detected in vitro. In fact, teratogenicity with retinoid-like activity was detected at concentrations that are commonly found in biomasses and even in bulk water surrounding cyanobacterial blooms. Overall, these results provide evidence of a link between retinoid-like activity, teratogenicity and the retinoids produced by cyanobacterial water blooms in the surrounding environment.

Cyanobacterial blooms occur with increasing frequency in freshwater ecosystems, posing a hazard to human and environmental health. Exposure of human to cyanobacterial metabolites occurs mostly via accidental ingestion through contaminated drinking water or during recreational activities and, most frequently, results in gastrointestinal symptoms. Despite the clinical manifestation, cyanobacterial metabolites are rather investigated for their toxicity towards specific organs or tissues, especially hepato-, nephro- and neurotoxicity, then for effects on the gastrointestinal tract and the associated lymphoid tissue. The aim of this review was to systematically summarize available literature on the effects on the gastrointestinal tract and the mucosal innate immune system and compile the data from both, in vitro and in vivo studies, focusing on human-health relevant models. Our systematic literature review revealed significant data gaps in the understanding on metabolites breaching the gastrointestinal barrier and the role of the immune system in the establishment of clinical symptoms. Microcystins and cylindrospermopsin were linked to gastrointestinal symptoms, immune system effects or both. Furthermore, implications for cyanobacterial bloom lipopolysaccharides in gastrointestinal inflammation were reported in several cases, while other metabolites received only minor attention. The collected data indicate the need for a reassessment of potential enterotoxicity of microcystins and cylindrospermopsin. Additionally, the carcinogenic potential of cyanotoxins, especially microcystins, has to be clarified, as an increasing amount of epidemiological studies show correlations between cyanobacterial blooms and gastrointestinal cancer incidence. Furthermore, other, often highly abundant bioactive metabolites like aeruginosins, have to be toxicologically evaluated at levels also accounting for (sub-)chronic exposure to low concentrations and in combination with naturally co-occurring metabolites, as can be expected in drinking water supplies.studies, focusing on human-health relevant models. Our systematic literature review revealed significant data gaps in the understanding on metabolites breaching the gastrointestinal barrier and the role of the immune system in the establishment of clinical symptoms. Microcystins and cylindrospermopsin were linked to gastrointestinal symptoms, immune system effects or both. Furthermore, implications for cyanobacterial bloom lipopolysaccharides in gastrointestinal inflammation were reported in several cases, while other metabolites received only minor attention. The collected data indicate the need for a reassessment of potential enterotoxicity of microcystins and cylindrospermopsin. Additionally, the carcinogenic potential of cyanotoxins, especially microcystins, has to be clarified, as an increasing amount of epidemiological studies show correlations between cyanobacterial blooms and gastrointestinal cancer incidence. Furthermore, other, often highly abundant bioactive metabolites like aeruginosins, have to be toxicologically evaluated at levels also accounting for (sub-)chronic exposure to low concentrations and in combination with naturally co-occurring metabolites, as can be expected in drinking water supplies.

Environmental water quality monitoring aims to provide the data required for safeguarding the environment against adverse biological effects from multiple chemical contamination arising from anthropogenic diffuse emissions and point sources. Here, we integrate the experience of the international EU-funded project SOLUTIONS to shift the focus of water monitoring from a few legacy chemicals to complex chemical mixtures, and to identify relevant drivers of toxic effects. Monitoring serves a range of purposes, from control of chemical and ecological status compliance to safeguarding specific water uses, such as drinking water abstraction. Various water sampling techniques, chemical target, suspect and non-target analyses as well as an array of in vitro, in vivo and in situ bioanalytical methods were advanced to improve monitoring of water contamination. Major improvements for broader applicability include tailored sampling techniques, screening and identification techniques for a broader and more diverse set of chemicals, higher detection sensitivity, standardized protocols for chemical, toxicological, and ecological assessments combined with systematic evidence evaluation techniques. No single method or combination of methods is able to meet all divergent monitoring purposes. Current monitoring approaches tend to emphasize either targeted exposure or effect detection. Here, we argue that, irrespective of the specific purpose, assessment of monitoring results would benefit substantially from obtaining and linking information on the occurrence of both chemicals and potentially adverse biological effects. In this paper, we specify the information required to: (1) identify relevant contaminants, (2) assess the impact of contamination in aquatic ecosystems, or (3) quantify cause–effect relationships between contaminants and adverse effects. Specific strategies to link chemical and bioanalytical information are outlined for each of these distinct goals. These strategies have been developed and explored using case studies in the Danube and Rhine river basins as well as for rivers of the Iberian Peninsula. Current water quality assessment suffers from biases resulting from differences in approaches and associated uncertainty analyses. While exposure approaches tend to ignore data gaps (i.e., missing contaminants), effect-based approaches penalize data gaps with increased uncertainty factors. This integrated work suggests systematic ways to deal with mixture exposures and combined effects in a more balanced way, and thus provides guidance for future tailored environmental monitoring. © 2019, The Author(s)., Funding text #1 1 UFZ‑Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig, Germany. 2 Institute for Environmental Research, RWTH Aachen University, 52074 Aachen, Germany. 3 Office of Research and Development, Atlantic Ecology Division, United States Environmental Protection Agency, Narragansett, RI, USA. 4 Center for Applied Geoscience, Eberhard Karls Uni‑ versity Tübingen, 72074 Tübingen, Germany. 5 Alterra, Wageningen University and Research Centre, P.O. Box 47, 6700 AA Wageningen, The Netherlands. 6 Environment and Climate Change Canada, Burlington, ON, Canada. 7 Sophus Bauditz Vej 19 B, 2800 Kgs. Lyngby, Denmark. 8 Water and Soil Quality Research Group, Institute of Environmental Assessment and Water Research (IDAEA‑ CSIC), Jordi Girona 18‑26, 08034 Barcelona, Spain. 9 Unité d’Ecotoxicologie Funding text #2 The SOLUTIONS Project is supported by the Seventh Framework Programme (FP7‑ENV‑2013) of the European Union under Grant Agreement No. 603437. G.A. Umbuzeiro thanks FAPESP Projects 2013/16956‑6 and 2015/24758‑5. We like to thank all partners for their continued efforts in making this project a success story. Funding text #3 The SOLUTIONS Project is supported by the Seventh Framework Programme (FP7‑ENV‑704 2013) of the European Union under Grant Agreement No. 603437. G.A. Umbuzeiro thanks 705 FAPESP Projects 2013/16956‑6 and 2015/24758‑5.

Anthropogenic eutrophication of freshwater bodies increases the occurrence of toxic cyanobacterial blooms. The cyanobacterial toxin cylindrospermopsin (CYN) is detected in the environment with increasing frequency, driving the scientific effort to assess emerging health risks from CYN-producing blooms. Oral exposure to CYN results primarily in hepatotoxicity. Nevertheless, extrahepatic manifestations of CYN toxicity have been reported. Furthermore, cyanotoxins have been detected in aerosols and dust particles, suggesting potential toxic effects in the respiratory tract. To assess the susceptibility of airway epithelia towards cyanotoxins, monolayers of immortalized human bronchial epithelial cells HBE1 and 16HBE14o- were exposed to a concentration range of 0.1–10 μM CYN. Cytotoxic endpoints were assessed as morphologic alterations, resazurin reduction capacity, esterase activity, neutral red uptake, and by impedimetric real-time cell analysis. Depending on the endpoint assessed, EC50 values ranged between 0.7 and 1.8 μM (HBE1) and 1.6–4.8 μM (16HBE14o-). To evaluate alterations of other cellular events by subcytotoxic concentration of CYN (1 μM), phosphorylation of mitogen-activated protein kinases ERK and p38 was determined. Only a slight increase in p38 phosphorylation was induced by CYN in HBE1 cell line after 48 h, while activities of both ERK1/2 and p38 gradually and significantly increased in 16HBE14o- cells during 8–48 h exposure. This study suggests possible hazards of inhalation CYN exposures, which may severely impact the integrity of airway epithelia and epithelial cell signaling. Further research of CYN-induced toxicity and underlying mechanisms is needed, as well as more data on environmental concentrations of cyanotoxins in aerosols for exposure assessment.

© 2019, The Author(s). The present monitoring and assessment of the chemical status of water bodies fail to characterize the likelihood that complex mixtures of chemicals affect water quality. The European Collaborative Project SOLUTIONS suggests that this likelihood can be estimated with effect-based methods (EBMs) complemented by chemical screening and/or impact modeling. These methods should be used to identify the causes of impacted water quality and to develop programs of measures to improve water quality. Along this line of reasoning, effect-based methods are recommended for Water Framework Directive (WFD) monitoring to cover the major modes of action in the universe of environmentally relevant chemicals so as to evaluate improvements of water quality upon implementing the measures. To this end, a minimum battery of bioassays has been recommended including short-term toxicity to algae, Daphnia and fish embryos complemented with in vitro and short-term in vivo tests on mode-of-action specific effects as proxies for long-term toxicity. The likelihood of adverse impacts can be established with effect-based trigger values, which differentiate good from poor water quality in close alignment with Environmental Quality Standards for individual chemicals, while taking into account mixture toxicity. The use of EBMs is suggested in the WFD as one avenue to establish the likelihood of adverse effects due to chemical pollution in European water systems. The present paper has been written as one component of a series of policy briefs to support decisions on water quality monitoring and management under the WFD.

Recently, the Guardian published an article entitled “EU clean water laws under attack from industry lobbyists” by Arthur Neslen (https://www.theguardian.com/environment/2019/may/15/eu-clean-water-laws-under-attack-from-industry-lobbyists) expressing concerns regarding a roll back in European clean water regulations. As principal investigators of the large EU-funded project “SOLUTIONS for present and future emerging pollutants in land and water resources management”, we appreciate such an open debate on water quality protection in Europe, which we would like to enrich with conclusions from 5 years of extensive research and stakeholder dialogue within SOLUTIONS and other large EU projects., This article has been prepared as an outcome of the SOLUTIONS project (European Union’s Seventh Framework Programme for research, technological development and demonstration under Grant Agreement No. 603437).

Cadmium nanoparticles can represent a risk in both industrial and environmental settings, but there is little knowledge on the impacts of their inhalation, especially concerning longer-term exposures. In this study, mice were exposed to cadmium oxide (CdO) nanoparticles in whole body inhalation chambers for 4 to 72 h in acute and 1 to 13 weeks (24 h/day, 7 days/week) in chronic exposure to investigate the dynamics of nanoparticle uptake and effects. In the acute experiment, mice were exposed to 2.95 × 106 particles/cm3 (31.7 μg CdO/m3). The same concentration and a lower one (1.18 × 106 particles/cm3, 12.7 μg CdO/m3) were used for the chronic exposure. Transmission electron microscopy documented distribution of nanoparticles into all studied organs. Major portion of nanoparticles was retained in the lung, but longer exposure led to a greater relative redistribution into secondary organs, namely the kidney, and also the liver and spleen. Accumulation of Cd in the lung and liver occurred already after 24 h and in the brain, kidney, and spleen after 72 h of exposure, and a further increase of Cd levels was observed throughout the chronic exposure. There were significant differences in both Cd accumulation and effects between the two exposure doses. Lung weight in the higher exposure group increased up to 2-fold compared to the control. Histological analyses showed dose-dependent alterations in lung and liver morphology and damage to their tissue. Modulation of oxidative stress parameters including glutathione levels and increased lipid peroxidation occurred mainly after the greater chronic exposure. The results emphasize risk of longer-term inhalation of cadmium nanoparticles, since adverse effects occurring after shorter exposures gradually progressed with a longer exposure duration.

Yeast biosensors have become suitable tools for the screening and detection of environmental pollutants because of their various advantages compared to other sensing technologies. On the other hand, many limitations remain with regard to their optimal performance and applicability in several contexts, such as low-concentration samples and on-site testing. This review summarizes the current state of yeast biosensors, with special focus on screening and assessment of environmental contaminants, discusses both pros and cons, and suggests steps towards their further development and effective use in the environmental assessment.

Recombinant yeast assays (RYAs) have been proved to be a suitable tool for the fast screening of compounds with endocrine disrupting activities. However, ready-to-use versions more accessible to less equipped laboratories and field studies are scarce and far from optimal throughputs. Here, we have applied freeze-drying technology to optimize RYA for the fast assessment of environmental compounds with estrogenic and androgenic potencies. The effects of different cryoprotectants, initial optical density and long-term storage were evaluated. The study included detailed characterization of sensitivity, robustness and reproducibility of the new ready-to-use versions, as well as comparison with the standard assays. Freeze-dried RYAs showed similar dose-responses curves to their homolog standard assays, with Lowest Observed Effect Concentration (LOEC) and Median effective Concentration (EC50) of 1 nM and 7.5 nM for testosterone, and 0.05 nM and 0.5 nM for 17β-estradiol, respectively. Freeze-dried cells stored at 4 °C retained maximum sensitivity up to 2 months, while cells stored at -18 °C showed no decrease in sensitivity throughout the study (10 months). This ready-to-use RYA is easily accessible and may be potentially used for on-site applications.

The goal of this study was to contribute to understanding of the mechanisms behind sensitivity differences between early and late instar larvae of Chironomus riparius and to address the influence of the differences in standard testing approaches on the toxicity evaluation. A 10-day contact sediment toxicity test was carried out to assess sensitivity to cadmium exposure in relation to different age and laboratory culture line origin of test organisms. Chironomid larvae of early (OECD 218 method) and late instar (US-EPA600/R-99/064 method) differed substantially in sensitivity of traditional endpoints (OECD: LOEC 50 and 10 μg Cd/g dry weight (dw); US-EPA: LOEC > 1000 and 100 μg Cd/g dw for survival and growth, respectively). Bioaccumulated cadmium and metallothioneins (MTs) concentrations were analyzed to investigate the role of MTs in reduced sensitivity to cadmium in late instar larvae. Metallothioneins were induced after treatment to greater Cd concentrations, but their levels in relation to cadmium body burdens did not fully explain low sensitivity of late instars to cadmium, which indicates some other effective way of detoxification in late instars. This study brings new information related to the role of MTs in age-dependent toxicant sensitivity and discusses the implications of divergence in data generated by chironomid sediment toxicity tests by standardized methods using different instars.

Bioassays are particularly useful tools to link the chemical and ecological assessments in water quality monitoring. Different methods cover a broad range of toxicity mechanisms in diverse organisms, and account for risks posed by non-target compounds and mixtures. Many tests are already applied in chemical and waste assessments, and stakeholders from the science-police interface have recommended their integration in regulatory water quality monitoring. Still, there is a need to address bioassay suitability to evaluate water samples containing emerging pollutants, which are a current priority in water quality monitoring. The presented interlaboratory study (ILS) verified whether a battery of miniaturized bioassays, conducted in 11 different laboratories following their own protocols, would produce comparable results when applied to evaluate blinded samples consisting of a pristine water extract spiked with four emerging pollutants as single chemicals or mixtures, i.e. triclosan, acridine, 17 alpha-ethinylestradiol (EE2) and 3-nitrobenzanthrone (3-NBA). Assays evaluated effects on aquatic organisms from three different trophic levels (algae, daphnids, zebrafish embryos) and mechanism-specific effects using in vitro estrogenicity (ER-Luc, YES) and mutagenicity (Ames fluctuation) assays. The test battery presented complementary sensitivity and specificity to evaluate the different blinded water extract spikes. Aquatic organisms differed in terms of sensitivity to triclosan (algae > daphnids > fish) and acridine (fish > daphnids > algae) spikes, confirming the complementary role of the three taxa for water quality assessment. Estrogenicity and mutagenicity assays identified with high precision the respective mechanism-specific effects of spikes even when non-specific toxicity occurred in mixture. For estrogenicity, although differences were observed between assays and models, EE2 spike relative induction EC50 values were comparable to the literature, and E2/EE2 equivalency factors reliably reflected the sample content. In the Ames, strong revertant induction occurred following 3-NBA spike incubation with the TA98 strain, which was of lower magnitude after metabolic transformation and when compared to TA100. Differences in experimental protocols, model organisms, and data analysis can be sources of variation, indicating that respective harmonized standard procedures should be followed when implementing bioassays in water monitoring. Together with other ongoing activities for the validation of a basic bioassay battery, the present study is an important step towards the implementation of bioanalytical monitoring tools in water quality assessment and monitoring. (C) 2016 Elsevier Ltd. All rights reserved.

Ambient air pollution, namely exposure to air particulate matter (PM), has been shown to be connected with a number of adverse health effects. At least part of the effects can be caused by organic pollutant mixtures associated with PM, which can elicit a wide range of specific toxic potentials. These potentials could be affected by seasonal variation of pollutant mixtures and PM size fraction. To examine this, six size subfractions of PM10 were collected at rural and urban site in the Czech Republic in a year-long sampling campaign. The samples were assessed for aryl hydrocarbon (AhR)-mediated activity, estrogenicity and anti-androgenicity using mammalian cell models. The concentrations of detected toxic potentials differed among seasons. The greatest levels were observed in samples collected during winter when AhR-mediated effects and estrogenicity were at least 10-times greater than in summer. While the observed potentials were mostly less pronounced in samples from rural area, during winter, their AhR-mediated activity was twice as great as at the urban site. This was probably caused by the low-quality of fuel used for heating at the rural site. Assessed toxic potentials were associated mainly with PM size fractions with lesser aerodynamic diameters (

In animals, retinoic acids (RAs), one of the main derivatives of vitamin A, are crucial for a variety of physiological processes. RAs, including all-trans-RA, 9-cis-RA, 13-cis-RA, and their corresponding metabolites (i.e., all-trans-4-oxo-RA, 9-cis-4-oxo-RA and 13-cis-4-oxo-RA) can be excreted through urination from humans and animals. Sewage treatment plants (STPs) are a significant source of RAs and 4-oxo-RAs into aquatic environments. RAs and 4-oxo-RAs can be identified and quantified by use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). RAs and 4-oxo-RAs have been reported in various environmental matrices including rivers, lakes, reservoirs and coastal marine environments as well as in sewage effluents discharged from STPs. Greater concentrations of RAs and 4-oxo-RAs have been observed during blooms of cyanobacteria and microalgae, suggesting that cyanobacteria and microalgae are natural sources of RAs and 4-oxo-RAs in aquatic environments. These potential sources of RAs and 4-oxo-RAs raise concerns about their concentrations and risks in aquatic environments because excessive intake of these chemicals can result in abnormal morphological development in animals. Teratogenic effects were observed in amphibians, fish embryos, gastropods, mammals and birds when exposed to RAs. This review summarizes sources, concentrations, adverse effects and ecological risks of RAs and 4-oxo-RAs in aquatic environments. An interim, predicted no-effect concentration (PNEC) of RAs (in terms of at-RA) for freshwater environments was determined to be 3.93 ng/L at-RA equivalents. Based on limited data on concentrations of RAs in freshwater ecosystems, their hazard quotients were found to range from zero to 16.41, depending on the environmental conditions of receiving waters. Ecological risks of RAs in marine environments are yet to be explored due to the paucity of data related to both their concentrations in marine environment and toxic potencies to marine species. This review updates current knowledge of RAs and 4-oxo-RAs in aquatic environments and calls for more studies on their concentrations and fate in aquatic environments, especially estuarine and coastal marine environments with a view to enabling a comprehensive assessment of their ecological risks around the globe. Keywords: Retinoids, Sewage, Cyanobacteria, Aquatic environments, Ecological risks, Marine

Complex mixtures of contaminants from multiple sources, including agriculture, industry or wastewater enter aquatic environments and might pose hazards or risks to humans or wildlife. Targeted analyses of a few priority substances provide limited information about water quality. In this study, a combined chemical and effect screening of water quality in the River Bosna, in Bosnia and Herzegovina was carried out, with focus on occurrence and effects of contaminants of emerging concern. Chemicals in water were sampled at 10 sites along the Bosna River by use of passive sampling. The combination of semipermeable membrane devices (SPMDs) and polar organic chemical integrative samplers (POCIS) enabled sampling of a broad range of contaminants from hydrophobic (PAHs, PCBs, OCPs) to hydrophilic compounds (pesticides, pharmaceuticals and hormones), which were determined by use of GC-MS and LC-MS (MS). In vitro, cell-based bioassays were applied to assess (anti)androgenic, estrogenic and dioxin-like potencies of extracts of the samplers. Of a total of 168 targeted compounds, 107 were detected at least once. Cumulative pollutant concentrations decreased downstream from the city of Sarajevo, which was identified as the major source of organic pollutants in the area. Responses in all bioassays were observed for samples from all sites. In general, estrogenicity could be well explained by analysis of target estrogens, while the drivers of the other observed effects remained largely unknown. Profiling of hazard quotients identified two sites downstream of Sarajevo as hotspots of biological potency. Risk assessment of detected compounds revealed, that 7 compounds (diazinon, diclofenac, 17β-estradiol, estrone, benzo[k]fluoranthene, fluoranthene and benzo[k]fluoranthene) might pose risks to aquatic biota in the Bosna River. The study brings unique results of a complex water quality assessment in a region with an insufficient water treatment infrastructure.

Hearing loss is an injury that can develop over time, and people may not even be aware of it until it becomes a severe disability. Ototoxicants are substances that may damage the inner ear by either affecting the structures in the ear itself or by affecting the nervous system. We have examined the possibility that ototoxicants may present a health hazard in association with environmental exposures, adding to existing knowledge of their proven hazards under medical therapeutic conditions or occupational activities. In addition to the already described human environmental ototoxicants, mainly organochlorines such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), hexachlorocyclohexane (HCH) and hexachlorobenzene (HCB), we have examined the ubiquitous chemical stressors phthalates, bisphenol A/S/F/, PFCs, flame retardants (FRs) and cadmium for potential ototoxic properties, both as single substances or as chemical mixtures. Our literature review confirmed that these chemicals may disturb thyroid hormones homeostasis, activate aryl hydrocarbon receptor (AhR), and induce oxidative stress, which in turn may initiate a chain of events resulting in impairment of cochlea and hearing loss. With regard to auditory plasticity, diagnostics of a mixture of effects of ototoxicants, potential interactions of chemical and physical agents with effects on hearing, parallel deterioration of hearing due to chemical exposures and ageing, metabolic diseases or obesity, even using specific methods as brainstem auditory evoked potentials (BAEP) or otoacoustic emissions (OAEs) registration, may be difficult, and establishment of concentration-response relationships problematic. This paper suggests the establishment of a class of environmental oxotoxicants next to the established classes of occupational and drug ototoxicants. This will help to properly manage risks associated with human exposure to chemical stressors with ototoxic properties and adequate regulatory measures.

Growth inhibition of freshwater microalga Pseudokirchneriella subcapitata caused by a waste water treatment plant (WWTP) effluent extract was investigated using an effect directed analysis (EDA) approach. The objective was to identify compounds responsible for the toxicity by combining state-of-the-art sampling, bioanalytical, fractionation and non-target screening techniques. Three fractionation steps of the whole extract were performed and bioactive fractions were analysed with GC (xGC)-MS and LC-HRMS. In total, 383 compounds were tentatively identified, and their toxicity was characterized using US EPA Ecotox database, open scientific literature or modelled by ECOSAR. Among the top-ranking drivers of toxicity were pesticides and their transformation products, pharmaceuticals (barbiturate derivatives and macrolide antibiotics e.g. azithromycin), industrial compounds or caffeine and its metabolites. Several of the top-ranking pesticides are no longer registered for use in plant protection products or biocides in the Czech Republic (e.g. prometryn, atrazine, acetochlor, resmethrin) and some are approved only for use in biocides (e.g. terbutryn, carbendazim, phenothrin), which indicates that their non-agricultural input into aquatic environment via WWTPs should be carefully considered. The study demonstrated a functional strategy of combining biotesting, fractionation and non-target screening techniques in the EDA study focused on the identification of algal growth inhibitors in WWTP effluent.

Embargo until 15 September 2020 A “dynamic” passive sampling (DPS) device, consisting of an electrically driven large volume water pumping device coupled to a passive sampler exposure cell, was designed to enhance the sampling rate of trace organic compounds. The purpose of enhancing the sampling rate was to achieve sufficient method sensitivity, when the period available for sampling is limited to a few days. Because the uptake principle in the DPS remains the same as for conventionally-deployed passive samplers, free dissolved concentrations can be derived from the compound uptake using available passive sampler calibration parameters. This was confirmed by good agreement between aqueous concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and hexachlorobenzene (HCB) derived from DPS and conventional caged passive sampler. The DPS device enhanced sampling rates of compounds that are accumulated in samplers under water boundary layer control (WBL) more than five times compared with the conventionally deployed samplers. The DPS device was deployed from a ship cruising downstream the Danube River to provide temporally and spatially integrated concentrations. A DPS-deployed sampler with surface area of 400 cm2 can reach sampling rates up to 83 L d−1. The comparison of three passive samplers made of different sorbents and co-deployed in the DPS device, namely silicone rubber (SR), low density polyethylene (LDPE) and SDB-RPS Empore™ disks showed a good correlation of surface specific uptake for compounds that were sampled integratively during the entire exposure period. This provided a good basis for a cross-calibration between the samplers. The good correlation of free dissolved PAHs, PCBs and HCB concentration estimates obtained using SR and LDPE confirmed that both samplers are suitable for the identification of concentration gradients and trends in the water column. We showed that the differences in calculated aqueous concentrations between sampler types are mainly associated with different applied uptake models.

Embargo until 10 March 2020 Many aquatic pollutants can be present at low concentrations, but their mixtures can still affect health or behavior of exposed organisms. In this study, toxicological and chemical analyses were combined for spatial contamination profiling using an innovative passive sampling approach. A novel Dynamic Passive Sampler (DPS) was employed as a mobile sampler from a ship cruising along 2130 km of the Danube river during the Joint Danube Survey 3 (JDS3). The sampling was performed in eight subsequent river stretches with two types of complementary passive samplers: silicone rubber sheets (SR) used for non-polar chemicals and SDB-RPS Empore™ disks (ED) for more hydrophilic compounds. Besides extensive chemical analyses, the bioactivity of samples was characterized by a battery of reporter gene bioassays. Cross-calibration of the employed passive samplers enabled robust estimation of water concentrations applicable for compounds with a wide range of physicochemical properties. DPS was suitable for sampling of water contaminants even at pg L−1 levels, with 209 of 267 analyzed compounds detected in the samples. Biological effects were detected in both ED and SR extracts across all river stretches by bioassays focused on xenobiotic metabolism mediated by the aryl hydrocarbon and pregnane X receptors, endocrine disruptive potential mediated by estrogen and androgen receptors and the oxidative stress response. The bioassay responses expressed as bioanalytical equivalent concentrations (BEQbio) were comparable with data obtained from large volume active sampling. The extracts of the ED samplers were more biologically active than extracts of SR samplers. Except of estrogenicity, where the analyzed chemicals explained on average 62% of the effects in ED samples, the detected chemicals explained

International audience; Background : The European Commission Implementing Decision EU 2015/495 included three steroidal estrogens, namely 17α-ethinyl estradiol, 17β-estradiol, and estrone, in the so-called “watch list” of the EU Water Framework Directive (WFD). The monitoring of these compounds is difficult because the detection limits of the majority of the available analytical methods cannot achieve the very low target concentrations required to meet proposed environmental quality criteria. In 2014, a combined Science-Policy Interface/Chemical Monitoring of Emerging Pollutants project was launched to meet this monitoring challenge. The project involved 24 research organizations and environmental agencies from 12 different countries.Methods : Sixteen surface water (SW) and 17 wastewater (WW) samples were collected across Europe and analysed using five in vitro effect-based and three chemical analytical methods. A general description of the project and data evaluation is provided by Könemann and colleagues in the companion publication 2018. In our study, we compared bioanalytical and chemical analytical results with regard to their application for aquatic status assessment. Therefore we considered the potential to predict population-relevant risks for aquatic organisms and the specificity and sensitivity of the various methods used in both approaches. Finally, we tested and discussed the applicability and relevance of previously suggested effect-based trigger values (EBT).Results and discussion : Results of the risk assessment based on chemical analytical data correlated highly with estrogenic activities (expressed as 17β-estradiol equivalents (EEQ) determined using effect-based methods), demonstrating the ability of the bioassays to predict the mixture risk caused by steroidal estrogens. For about 15% of SW and 40% of WW samples detection limits of chemical-analytical methods were too high to allow a status assessment, while detection limits of all effect-based methods were below proposed EBT. This demonstrates that effect-based methods are suitable for status assessment of surface waters. The in vitro effect-based methods were quite specific for steroidal estrogens and highly sensitive, meaning they have a low probability to detect false positive or negative results. After testing of three alternative EBT proposals, we concluded to use preliminary 400 pg/L EEQ as screening EBT corresponding to the AA-EQS of E2. Further test specific refinements in the application of this value are not excluded.Conclusions : We conclude that water quality assessment can progress from a purely analytical approach to effect-based monitoring, from single substance to known and unknown mixture assessment and from in vitro screening to population-relevant risk assessment. Despite a few limitations, effect-based in vitro methods are recommendable for monitoring steroidal estrogens under the WFD because they, a) are able to sensitively quantify the activity of steroidal estrogens in all surface and wastewater samples, b) are able to detect the combined effect of estrogen mixtures including unknown chemicals with estrogen receptor activating properties, c) allow an ecotoxicological status assessment using EBT to calculate risk quotients. This approach is similar to the risk ratio used in regulatory environmental risk assessments, but allows for an integrated mixture assessment. Outlook : The results of this study support the introduction of a holistic approach for the regulation of chemicals in the aquatic environment under the EU WFD, as proposed recently by EU Water Directors. An ecotoxicological status assessment for one of the most relevant modes of action of endocrine disruption will allow authorities responsible for water quality assessment to focus available monitoring resources and to improve the ecological status of EU waterbodies.

Environment international 114, 95-106 (2018). doi:10.1016/j.envint.2018.02.013, Published by Elsevier Science, Amsterdam [u.a.]

The European Decision EU 2015/495 included three steroidal estrogens, estrone, 17 beta-estradiol and 17 alpha-ethinyl estradiol, in the "watch-list" of the Water Framework Directive (WFD). As consequence, these substances have to be chemically monitored at the level of their environmental quality standards, which can be challenging. This project aimed to identify reliable effect-based methods (EBMs) for screening of endocrine disrupting compounds, to harmonise monitoring and data interpretation methods, and to contribute to the current WFD review process. Water and wastewater samples were collected across Europe and analysed using chemical analyses and EBMs. The results showed that 17 beta-estradiol equivalents were comparable among methods, while results can vary between methods based on the relative potencies for individual substances. Further, derived 17 beta-estradiol equivalents were highly correlated with LC-MS/MS analyses. This study shows that the inclusion of effect-based screening methods into monitoring programmes for estrogens in surface waterbodies would be a valuable complement to chemical analysis. (C) 2018 Elsevier B.V. All rights reserved.

Detection of endocrine disrupting compounds in water and sediment samples has gained much importance since the evidence of their effects was reported in aquatic ecosystems in the 1990s. The aim of this review is to highlight the advances made in the field of in vitro analysis for the detection of hormonally active compounds with estrogenic, androgenic and progestogenic effects in water and sediment samples. In vitro assays have been developed from yeast, mammalian and in a few cases from fish cells. These assays are based either on the hormone-mediated proliferation of sensitive cell lines or on the hormone-mediated expression of reporter genes. In vitro assays in combination with various sample enrichment methods have been used with limits of detection as low as 0.0027 ng L-1 in water, and 0.0026 ng g-1 in sediments for estrogenicity, 0.1 ng L-1 in water, and 0.5 ng g-1 in sediments for androgenicity, and 5 ng L-1 in water for progestogenicity expressed as equivalent concentrations of standard reference compounds of 17β-estradiol, dihydrotestosterone and progesterone, respectively. The experimental results and limits of quantification, however, are influenced by the methods of sample collection, preparation, and individual laboratory practices.

Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.

Cyanobacterial species produce wide range of bioactive compounds. This study characterized production of retinoid-like compounds with embryotoxic and teratogenic potential by commonly occurring cyanobacterial species with tendency to form massive water blooms. The major goal was to simultaneously assess the intracellular and extracellular retinoid-like activity from several independent cultivations of one coccal (Microcystis aeruginosa) and four filamentous cyanobacteria (Aphanizomenon gracile, Cylindrospermopsis raciborskii, Limnothrix redekeii, and Planktothrix agardhii) and characterize the variability in its production among cultivations. The retinoid-like activity was evaluated by in vitro assay along with chemical analyses of nine retinoids: all-trans retinoic acid (ATRA), 9-cis retinoic acid (9cis-RA), 13cis-RA, 13cis-RA methyl ester, 5,6 epoxy-RA, 4keto-ATRA, 4keto-retinal, 4hydoxy-retinoic acid (4OH-ATRA), retinal and retinol. The production of retinoid-like compounds was recalculated per volume, per biomass dry weight and per cell to provide relevant data for risk assessment in relation to occurrence of massive water blooms in the environment. Total produced retinoid-like activity of five selected species ranged from 170 to 25,600ng ATRA-equivalents (REQ)/g dm corresponding to 0.001-0.392ng REQ/106 cyanobacterial cells. Results from chemical analyses showed that all tested extracts contained 4keto-ATRA and retinal. All-trans retinoic acid, 9/13cis-retinoic acid and 5,6 epoxy-retinoic acid were detected in most exudate and extract samples. The reported results of recalculated total retinoid-like activity enable potential predictions of its production by the studied species in water blooms of known cell densities relevant for risk assessment.

Surface waters can contain a diverse range of organic pollutants, including pesticides, pharmaceuticals and industrial compounds. While bioassays have been used for water quality monitoring, there is limited knowledge regarding the effects of individual micropollutants and their relationship to the overall mixture effect in water samples. In this study, a battery of in vitro bioassays based on human and fish cell lines and whole organism assays using bacteria, algae, daphnids and fish embryos was assembled for use in water quality monitoring. The selection of bioassays was guided by the principles of adverse outcome pathways in order to cover relevant steps in toxicity pathways known to be triggered by environmental water samples. The effects of 34 water pollutants, which were selected based on hazard quotients, available environmental quality standards and mode of action information, were fingerprinted in the bioassay test battery. There was a relatively good agreement between the experimental results and available literature effect data. The majority of the chemicals were active in the assays indicative of apical effects, while fewer chemicals had a response in the specific reporter gene assays, but these effects were typically triggered at lower concentrations. The single chemical effect data were used to improve published mixture toxicity modeling of water samples from the Danube River. While there was a slight increase in the fraction of the bioanalytical equivalents explained for the Danube River samples, for some endpoints less than 1% of the observed effect could be explained by the studied chemicals. The new mixture models essentially confirmed previous findings from many studies monitoring water quality using both chemical analysis and bioanalytical tools. In short, our results indicate that many more chemicals contribute to the biological effect than those that are typically quantified by chemical monitoring programs or those regulated by environmental quality standards. This study not only demonstrates the utility of fingerprinting single chemicals for an improved understanding of the biological effect of pollutants, but also highlights the need to apply bioassays for water quality monitoring in order to prevent underestimation of the overall biological effect.

Exposure to particulate matter (PM) in ambient air has been shown to lead to adverse health consequences. Six size fractions of PM with aerodynamic diameter smaller than 10 μm (PM10) and gas phase were collected at six localities with different major pollution sources. Extracts of samples were assessed for AhR-mediated toxicity, (anti-)estrogenicity, (anti-)androgenicity and genotoxicity. The biological responses were interpreted relative to chemical characterization. Historically, for regulatory purposes, evaluation of air pollution was based mainly on assessment of the sum of PM10. In the case of AhR-mediated activity, PM1 was responsible for more than 75% of the activity of the particulate fraction from all localities. The assessed effects were correlated with concentrations of polycyclic aromatic hydrocarbons (PAH), organic carbon content and specific surface area of the PM. A significant proportion of biologically active chemicals seems to be present in the gas phase of air. The results suggest that an average daily exposure based just on the concentrations of contaminants contained in PM10, as regulated in EU legislation so far, is not a sufficient indicator of contaminants in air particulates and adoption of standards more similar to other countries and inclusion of other parameters besides mass should be considered. Keywords: PM size fractions, Dioxin-like toxicity, Estrogenicity, Antiandrogenicity, Genotoxicity

Water is a vital resource for natural ecosystems and human life, and assuring a high quality of water and protecting it from chemical contamination is a major societal goal in the European Union. The Water Framework Directive (WFD) and its daughter directives are the major body of legislation for the protection and sustainable use of European freshwater resources. The practical implementation of the WFD with regard to chemical pollution has faced some challenges. In support of the upcoming WFD review in 2019 the research project SOLUTIONS and the European monitoring network NORMAN has analyzed these challenges, evaluated the state-of-the-art of the science and suggested possible solutions. We give 10 recommendations to improve monitoring and to strengthen comprehensive prioritization, to foster consistent assessment and to support solution-oriented management of surface waters. The integration of effect-based tools, the application of passive sampling for bioaccumulative chemicals and an integrated strategy for prioritization of contaminants, accounting for knowledge gaps, are seen as important approaches to advance monitoring. Including all relevant chemical contaminants in more holistic “chemical status” assessment, using effect-based trigger values to address priority mixtures of chemicals, to better consider historical burdens accumulated in sediments and to use models to fill data gaps are recommended for a consistent assessment of contamination. Solution-oriented management should apply a tiered approach in investigative monitoring to identify toxicity drivers, strengthen consistent legislative frameworks and apply solutions-oriented approaches that explore risk reduction scenarios before and along with risk assessment. This article has been supported by, and prepared as a common out- come of the NORMAN network ( www.norman-network.net ) on emerg- ing pollutants and the SOLUTIONS project (European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 603437). Andreas Kortenkamp, University of Brunel, is acknowledged for proof reading. The views expressed herein do not necessarily re fl ect the views or pol- icies of the authors' agencies. Bo N. Jacobsen and Giordano Giorgi are ac- knowledged for their comments.

Teratogenic effects, which were remarkably similar to those induced by retinoic acids, have been seen in wild frogs indicating possible source of retinoids in the environment. Recent studies indicate that some cyanobacterial species can contain teratogenic retinoic acids (RAs) and their analogues. Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis. The study investigated the effects of exudates (extracellular compounds) of two cyanobacteria species with retinoic-like activity and one algae species on embryonic development of amphibians. The retinoid-like activity determined by in vitro reporter gene assay reached 528 ng retinoid equivalents (REQ)/L and 1000 ng REQ/L in exudates of Cylindrospermopsis raciborskii and Microcystis aeruginosa, respectively, while algal exudates showed no detectable activity. Total mean of retinoid-like copounds into exudate was 35.6 ng ATRA/mil.cells for M.aeruginosa and 6.71 ng ATRA/mil.cells for C.raciborskii, respectively. Toxicity tests with amphibian embryos up to 96 h of development were carried out according to the standard guide for the Frog Embryo Teratogenesis Assay Xenopus. Lowest observed effect concentrations (LOEC) of malformations (2.5–2.6 µg/L REQ) were two times lower than LOEC for ATRA (5 µg/L). The exudates of both cyanobacteria were indeed provoking diverse teratogenic effects (e.g. tail, gut and eyes deformation) and interference with growth in frogs embryos, while such effects were not observed for the algae. Xenopus embryos were also exposed to all-trans retinoic acid (ATRA) in concentration range (1−40 µg/L) equivalent to the REQs detected in cyanobacterial exudates. ATRA (10 µg/L) caused similar teratogenic phenotypes at corresponding REQs as cyanobacterial exudates. The study confirms the ability of some species of cyanobacteria to produce retinoids naturally and excrete them directly into the environment at concentrations which might have adverse influence on the development of amphibians.

Compounds with estrogenic potencies and their adverse effects in surface waters have received much attention. Both anthropogenic and natural compounds contribute to overall estrogenic activity in freshwaters. Recently, estrogenic potencies were also found to be associated with cyanobacteria and their blooms in surface waters. The present study developed and compared the solid phase extraction and LC-MS/MS analytical approaches for determination of phytoestrogens (8 flavonoids - biochanin A, coumestrol, daidzein, equol, formononetin, genistein, naringenin, apigenin and 5 sterols - ergosterol, (beta-sitosterol, stigmasterol, campesterol, brassicasterol) and cholesterol in water. The method was used for analyses of samples collected in stagnant water bodies dominated by different cyanobacterial species. Concentrations of individual flavonoids ranged from below the limit of detection to 3.58 ng/L. Sterols were present in higher amounts up to 2.25 mu g/L. Biological potencies of these phytoestrogens in vitro were characterized using the hER alpha-HeLa-9903 cell line. The relative estrogenic potencies (compared to model estrogen - 17 beta-estradiol) of flavonoids ranged from 2.25E-05 to 1.26E-03 with coumestrol being the most potent. None of the sterols elicited estrogenic response in the used bioassay. Estrogenic activity was detected in collected field water samples (maximum effect corresponding to 2.07 ng/L of 17 beta-estradiol equivalents, transcriptional assay). At maximum phytoestrogens accounted for only 1.56 pg/L of 17 beta-estradiol equivalents, contributing maximally 8.5% of the total estrogenicity of the water samples. Other compounds therefore, most likely of anthropogenic origin such as steroid estrogens, are probably the major drivers of total estrogenic effects in these surface waters.

Complex mixtures of micropollutants, including pesticides, pharmaceuticals and industrial chemicals emitted by wastewater effluents to European rivers may compromise the quality of these water resources and may pose a risk to ecosystem health and abstraction of drinking water. In the present study, an integrated analytical and bioanalytical approach was applied to investigate the impact of untreated wastewater effluents from the city of Novi Sad, Serbia, into the River Danube. The study was based on three on-site large volume solid phase extracted water samples collected upstream and downstream of the untreated wastewater discharge. Chemical screening with liquid chromatography high resolution mass spectrometry (LC-HRMS) was applied together with a battery of in vitro cell-based bioassays covering important steps of the cellular toxicity pathway to evaluate effects on the activation of metabolism (arylhydrocarbon receptor AhR, peroxisome proliferator activated receptor gamma PPARγ), specific modes of action (estrogen receptor ERα, androgen receptor AR) and adaptive stress responses (oxidative stress, inflammation). Increased effects, significantly changed contamination patterns and higher chemical concentrations were observed downstream of the wastewater discharge. A mass balance approach showed that enhanced endocrine disruption was in good agreement with concentrations of detected hormones, while only a smaller fraction of the effects on xenobiotic metabolism (

Phytoplankton can produce various bioactive metabolites, which may affect other organisms in the aquatic environment. This study provides the first information on the total retinoid-like activity associated with both intracellular and extracellular metabolites produced by selected phytoplankton species that could play a role in teratogenic effects and developmental disruption in exposed organisms. The studied species included a coccoid cyanobacteria (Microcystis aeruginosa), a filamentous cyanobacteria (Aphanizomenon gracile) and a green alga (Desmodesmus quadricauda), all of which commonly occur in freshwater bodies in Europe. Methanolic extracts from cellular material and extracellular exudates were prepared from cultures cultivated in two light-intensity variants with five replicates for each species. The retinoid-like activity was evaluated by in vitro assays along with chemical analyses of two potent retinoic acids (all-trans retinoic acid (ATRA) and 9cis-RA). The mean total retinoid-like activity of metabolites produced by the three studied species representing different phytoplankton taxonomic groups ranged from 705 to 5572 ng ATRA equivalent/g dry matter corresponding to 0.064–0.234 ng ATRA/106 cells. Retinoid-like activity was found in the cellular extracts of all species, while only the extracellular exudates of cyanobacteria exhibited detectable activity (41–1081 ng ATRA/L). The greatest extracellular as well as total (extra- and intra- cellular together) retinoid-like activity was detected for Microcystis aeruginosa. The two potent retinoic acids studied were more frequently detected in cellular extracts than in extracellular exudates of all species. Their contribution to observed in vitro effects was relatively low for all tested samples (

Aquatic environments are often contaminated with complex mixtures of chemicals that may pose a risk to ecosystems and human health. This contamination cannot be addressed with target analysis alone but tools are required to reduce this complexity and identify those chemicals that might cause adverse effects. Effect-directed analysis (EDA) is designed to meet this challenge and faces increasing interest in water and sediment quality monitoring. Thus, the present paper summarizes current experience with the EDA approach and the tools required, and provides practical advice on their application. The paper highlights the need for proper problem formulation and gives general advice for study design. As the EDA approach is directed by toxicity, basic principles for the selection of bioassays are given as well as a comprehensive compilation of appropriate assays, including their strengths and weaknesses. A specific focus is given to strategies for sampling, extraction and bioassay dosing since they strongly impact prioritization of toxicants in EDA. Reduction of sample complexity mainly relies on fractionation procedures, which are discussed in this paper, including quality assurance and quality control. Automated combinations of fractionation, biotesting and chemical analysis using so-called hyphenated tools can enhance the throughput and might reduce the risk of artifacts in laboratory work. The key to determining the chemical structures causing effects is analytical toxicant identification. The latest approaches, tools, software and databases for target-, suspect and non-target screening as well as unknown identification are discussed together with analytical and toxicological confirmation approaches. A better understanding of optimal use and combination of EDA tools will help to design efficient and successful toxicant identification studies in the context of quality monitoring in multiply stressed environments.

Endocrine-disruptive potential and concentrations of polar organic contaminants were measured in seven headwaters flowing through relatively unpolluted areas of the Czech Republic. Towns with Wastewater Treatment Plant (WWTP) discharges were the first known sources of anthropogenic pollution in the areas. River water was sampled several kilometers upstream (US) and several tens of meters downstream (DS) of the WWTP discharges, by use of Pesticide and Pharmaceutical Polar Organic Integrative Samplers (POCIS-Pest, POCIS-Pharm). Extracts of passive samplers were tested by use of a battery of in vitro bioassays to determine overall non-specific cytotoxicity, endocrine-disruptive (ED) potential and dioxin-like toxicity. The extracts were also used for quantification of polar organics. There was little toxicity to cells caused by most extracts of POCIS. Estrogenicity was detected in all types of samples even though US locations are considered to be background. At US locations, concentrations of estrogen equivalents (EEq) ranged from less than the detection limits (LOD) to 0.5 ng EEq/POCIS. Downstream concentrations of EEqs ranged from less than LOD to 4.8 ng EEq/POCIS. Concentrations of EEq in POCIS extracts from all DS locations were 1 to 14 times greater than those at US locations. Concentrations of EEq measured in extracts of POCIS-Pest and POCIS-Pharm were in a good agreement. Neither antiestrogenic nor anti/androgenic activities were detected. Concentrations of 2,3,7,8-TCDD equivalents (TEqbio) were detected in both types of POCIS at concentrations ranging from less than the LOD to 0.39 ng TEqbio/POCIS. Nearly all extracts of POCIS-Pharm contained greater concentrations of TEqbio activity than extracts of POCIS-Pest. Concentrations of pesticides and pharmaceuticals in extracts of POCIS were generally small at all sampling sites, but levels of some pharmaceuticals were significantly greater in both types of POCIS from DS locations. Chemical analyses along with the results of bioassays documented impacts of small towns with WWTPs on headwaters. Keywords: Androgen, Dioxin-like activity, Estrogen, In vitro assay, POCIS, Waste Water Treatment Plant

Here is presented some of the first information on interactions of compounds produced by cyanobacteria and green algae with estrogen receptor signaling. Estrogenic potency of aqueous extracts and exudates (culture spent media with extracellular products) of seven species of cyanobacteria (10 different laboratory strains) and two algal species were assessed by use of in vitro trans-activation assays. Compounds produced by cyanobacteria and algae, and in particular those excreted from the cells, were estrogenic. Most exudates were estrogenic with potencies expressed at 50% of the maximum response under control of the estrogen receptor ranging from 0.2 to 7.2 ng 17β-estradiol (E2) equivalents (EEQ)/L. The greatest estrogenic potency was observed for exudates of Microcystis aerigunosa, a common species that forms water blooms. Aqueous extracts of both green algae, but only one species of cyanobacteria (Aphanizomenon gracile) elicited significant estrogenicity with EEQ ranging from 15 to 280 ng 17β-estradiol (E2)/g dry weight. Scenedesmus quadricauda exudates and extracts of Aphanizomenon flos-aquae were antagonistic to the ER when coexposed to E2. The EEQ potency was not correlated with concentrations of cyanotoxins, such as microcystin and cylindrospermopsin, which suggests that the EEQ was comprised of other compounds. The study demonstrates some differences between the estrogenic potency of aqueous extracts prepared from the same species, but of different origin, while the effects of exudates were comparable within species. The observed estrogenic potencies are important namely in relation to the possible mass expansion of cyanobacteria and release of the active compounds into surrounding water. Keywords: Cyanobacteria, Eutrophication, Endocrine disruption, Estrogenicity, Algae, Phytoplankton

We studied three cores from a well-dated sediment archive from an oxbow lake of the River Morava, Czech Republic, using a high-resolution (1 cm) multiproxy stratigraphic analysis and a concentration of selected heavy metals. The objective was to investigate how the vertical distribution of pollutants could be affected by episodic flood sedimentation in archives with high sedimentation rates. Stratigraphic proxies (magnetic susceptibility/MS/, X-ray densitometry and visible-light diffuse reflectance spectrometry), supported by grain-size analyses, enabled us to identify relatively coarse-grained flood layers and correlate them in the proximal-to-distal direction in the oxbow-lake delta. A correlation of the flood layers with the time series of the river discharge was used to improve the age model based primarily on 137Cs dating. The refined age model is nonlinear and shows periods of lower and higher sediment accumulation rates, which coincided with periods of lower and higher frequency of floods in the river, respectively. Concentrations of most heavy metals, normalized to lithogenic aluminium, revealed vigorous short-term variation related to floods, whose magnitude exceeds that of long-term trends. There is a substantial risk that temporal contamination trends, even if normalized to lithogenic elements, can be strongly influenced by facies, which may lead to potential misinterpretations. Element normalizing to MS, which proved to be a good proxy of grain size, provided smoother long-term trends whose interpretation was less affected by short-term variations. The long-term trends exhibit decreasing concentrations of most heavy metals for the last ~ 25 years, most notably between ~ 1987 and ~ 1992. These historical patterns correspond well to the significant improvement in environmental pollution reported elsewhere in Central Europe. In contrast, concentrations of Na have systematically increased up until the present, partly being driven by the widespread use of de-icing salt for winter road treatment.