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Continuously increasing plastic production causes a constant accumulation of microplastic particles (MPs) in the aquatic environment, especially in industrialized and urbanized areas with elevated wastewater discharges. This coincides with the release of persistent organic pollutants (polycyclic aromatic hydrocarbons (PAHs), pesticides) entering limnic ecosystems. Although the assessment of potential effects of environmental pollutants sorbed to MPs under chronic exposure scenarios seems vital, data on potential hazards and risk by combined exposure to pollutants and microplastics for aquatic vertebrates is still limited. Therefore, zebrafish (Danio rerio) were exposed over 21 days to the organophosphate insecticide chlorpyrifos (CPF; 10 and 100 ng/L) and the PAH benzo(k)fluoranthene (BkF; 0.78 and 50 µg/L) either dissolved directly in water or sorbed to different MPs (irregular polystyrene, spherical polymethyl methacrylate; ≤ 100 µm), where CPF was sorbed to polystyrene MPs and BkF was sorbed to polymethyl methacrylate MPs. Contaminant sorption to MPs and leaching were documented using GC-EI-MS; potential accumulation was studied in cryosections of the gastrointestinal tract. Enzymatic biomarkers and biotransformation were measured in liver and brain. Overall, exposure to non-contaminated MPs did not induce any adverse effects. Results of fluorescence tracking, CYP1A modulation by BkF as well as changes in acetylcholinesterase activity (AChE) by CPF were less pronounced when contaminants were sorbed to MPs, indicating reduced bioavailability of pollutants. Overall, following exposure to waterborne BkF, only minor amounts of parent BkF and biotransformation products were detected in zebrafish liver. Even high loads of MPs and sorbed contaminants did not induce adverse effects in zebrafish; thus, the potential threat of MPs as vectors for contaminant transfer in limnic ecosystems can be considered limited.

In microplastics (MPs) research, there is an urgent need to critically reconsider methodological approaches and results published, since public opinion and political decisions might be based on studies using debatable methods and reporting questionable results. For instance, recent studies claim that MPs induce intestinal damage and that relatively large MPs are transferred to, e.g., livers in fish. However, there is methodological criticism and considerable concern whether MP transfer to surrounding tissues is plausible. Likewise, there is an ongoing discussion in MP research if MPs act as vectors for adsorbed hazardous chemicals. In this study, effects of very small (4–6 μm) and very large (125–500 μm) benzo(a) pyrene (BaP)-spiked polyethylene (PE) particles administered via different uptake routes (food chain vs. direct uptake) were compared in a 21-day zebrafish (Danio rerio) feeding experiment. Particular care was taken to prevent cross-contamination of MPs during dissection and histological sample preparation. In contrast to numerous reports in literature describing similar approaches, independent of exposure route and MP size, no adverse effects could be detected. Likewise, no BaP accumulation could be documented, and MPs were exclusively seen in the lumen of the intestinal tract, which, however, did not induce any histopathological effects. Results indicate that in fish MPs are taken up, pass along the intestinal lumen and are excreted without any symptoms of adverse effects., Funding Agencies:German Federal Ministry for Science and Research (BMBF) within the "Joint Programming Initiative Healthy and Productive Seas and Oceans" (JPI Oceans) project EPHEMARE ("Ecotoxicological Effects of Microplastics in Marine Ecosystems") 03F0735ABelgian BELSPO within the "Joint Programming Initiative Healthy and Productive Seas and Oceans" (JPI Oceans) project EPHEMARE ("Ecotoxicological Effects of Microplastics in Marine Ecosystems") BR/154/A1/EPHEMARESwedish FORMAS within the "Joint Programming Initiative Healthy and Productive Seas and Oceans" (JPI Oceans) project EPHEMARE ("Ecotoxicological Effects of Microplastics in Marine Ecosystems") 2015-01865French IdEx grant from University of Bordeaux within the "Joint Programming Initiative Healthy and Productive Seas and Oceans" (JPI Oceans) project EPHEMARE ("Ecotoxicological Effects of Microplastics in Marine Ecosystems")

Information about acute fish toxicity is routinely required in many jurisdictions for environmental risk assessment of chem­icals. This information is typically obtained using a 96-hour juvenile fish test for lethality according to OECD test guideline (TG) 203 or equivalent regional guidelines. However, TG 203 has never been validated using the criteria currently required for new test methods including alternative methods. Characterization of the practicality and validity of TG 203 is important to provide a benchmark for alternative methods. This contribution systematically summarizes the available knowledge on limitations and uncertainties of TG 203, based on methodological, statistical, and biological consider­ations. Uncertainties stem from the historic flexibility (e.g., use of a broad range of species) and constraints of the basic test design (e.g., no replication). Other sources of uncertainty arise from environmental safety extrapolation based on TG 203 data. Environmental extrapolation models, combined with data from alternative methods, including mechanistic indicators of toxicity, may provide at least the same level of environmental protection. Yet, most importantly, the 3R advan­tages of alternative methods allow a better standardization, characterization, and an improved basic study design. This can enhance data reliability and thus facilitate the comparison of chemical toxicity, as well as the environmental classifi­cations and prediction of no-effect concentrations of chemicals. Combined with the 3R gains and the potential for higher throughput, a reliable assessment of more chemicals can be achieved, leading to improved environmental protection.

The purpose of the present study was to assess the hazard potentials of contaminated suspended particulate matter (SPM) sampled during a flood event for floodplain soils using in vitro bioassays and chemical analysis. Sediment-contact tests were performed to evaluate the direct exposure of organisms to native soils and SPM at two different trophic levels. For comparison, acetonic extracts were tested using both contact tests and additionally two cell-based biotests for cytotoxicity and Ah receptor-mediated activity (EROD-Assay). The sediment-contact tests were carried out with the dehydrogenase assay with Arthrobacter globiformis and the fish embryo assay with Danio rerio. The results of this study clearly document that native samples may well be significantly more effective than corresponding extracts in the bacteria contact assay or the fish embryo test. These results question the commonly accepted concept that acetonic extracts are likely to overestimate the toxicity of soil and SPM samples. Likewise, the priority organic compounds analyzed failed to fully explain the toxic potential of the samples. The outcomes of this study revealed the insufficient knowledge regarding the relationship between the different exposure pathways. Finally, there is concern about adverse effects by settling suspended particulate matter and remobilized sediments in frequently inundated floodplain soils due to an increase of the hazard potential, if compared with infrequently inundated floodplain soils. We showed that the settling of SPM and sediments revealed a significant impact on the dioxin-like potencies of riparian soils.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96 h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96 h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV < 30%) for most chemicals and laboratories. The reproducibility was lower (CV > 30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236.Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

This article introduces ‘Tox-Box’, a joint research project designed to develop a holistic approach towards a harmonized testing strategy for exposure- and hazard-based risk management of anthropogenic trace substances in drinking water to secure a long-term drinking water supply. The main task of the Tox-Box consortium is to enhance the existing health-related indicator value concept (German: GOW-Konzept - Gesundheitlicher Orientierungswert) through development and prioritization of additional end point-related testing strategies for genotoxicity, neurotoxicity, germ cell damage, and endocrine effects. In this context, substance-specific modes of action will be identified and characterized. Toxicological data collected by the 12 Tox-Box subprojects will be evaluated and weighted to structure a hierarchical testing strategy for an improved risk assessment. A technical guidance document for exposure and hazard-based risk management of anthropogenic trace substances in drinking water will eventually be prepared.Dieser Artikel stellt das Verbundprojekt “Tox-Box” vor, das einen ganzheitlichen Ansatz für eine harmonisierte Teststrategie eines Expositions-bezogenen und Gefahren-basierten Risikomanagements von anthropogenen Spurenstoffen in Trinkwasser entwickeln und somit einen Beitrag zur langfristigen Sicherung der Trinkwasserversorgung leisten soll. Die Hauptaufgabe des Tox-Box-Konsortiums ist die Weiterentwicklung des bestehenden GOW-Konzeptes (Gesundheitlicher Orientierungswert) durch Erforschung und Priorisierung zusätzlicher Endpunkt-bezogener Teststrategien für Gentoxizität, Neurotoxizität, Keimzellschädigung und endokrine Effekte. In diesem Kontext werden zudem Substanz-spezifische Wirkmechanismen identifiziert und charakterisiert. Im Anschluss werden die toxikologischen Daten aus den 12 Teilprojekten evaluiert und gewichtet um eine hierarchische Teststrategie für eine verbesserte Risikobewertung zu erstellen. Zum Abschluss des Projektes wird eine technische Rich

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance. © 2013 Elsevier Inc.

Worldwide, the zebrafish has become a popular model for biomedical research and (eco)toxicology. Particularly the use of embryos is receiving increasing attention, since they are considered as replacement method for animal experiments. Zebrafish embryos allow the analysis of multiple endpoints ranging from acute and developmental toxicity determination to complex functional genetic and physiological analysis. Particularly the more complex endpoints require the use of post-hatched eleutheroembryo stages. According to the new EU Directive 2010/63/EU on the protection of animals used for scientific purposes, the earliest life-stages of animals are not defined as protected and, therefore, do not fall into the regulatory frameworks dealing with animal experimentation. Independent feeding is considered as the stage from which free-living larvae are subject to regulations for animal experimentation. However, despite this seemingly clear definition, large variations exist in the interpretation of this criterion by national and regional authorities. Since some assays require the use of post-hatched stages up to 120h post fertilization, the literature and available data are reviewed in order to evaluate if this stage could still be considered as non-protected according to the regulatory criterion of independent feeding. Based on our analysis and by including criteria such as yolk consumption, feeding and swimming behavior, we conclude that zebrafish larvae can indeed be regarded as independently feeding from 120h after fertilization. Experiments with zebrafish should thus be subject to regulations for animal experiments from 120h after fertilization onwards.

Previous in vitro studies have reported the potential of perfluorooctane sulfonate (PFOS) to increase the toxicity of other compounds. Given the complex nature of mixtures of environmental pollutants in aquatic systems together with the persistent and bioaccumulative properties of PFOS, this study aimed at evaluating the long-term effects and toxicity-increasing behavior of PFOS in vivo using the zebrafish (Danio rerio). Fish were maintained in flow-through conditions and exposed to single and binary mixtures of PFOS and the endocrine disruptor bisphenol A (BPA) at nominal concentrations of 0.6, 100 and 300 mu g/L and 10, 200 and 400 mu g/L, respectively. F1 and F2 generations were evaluated from 0 to 180 days post-fertilization (dpf) and F3 generation was evaluated from 0 to 14 dpf. Survival was documented in all generations, whereas growth, fecundity, fertilization rate, histological alterations (in liver, thyroid and gonads) and vitellogenin (Vtg) induction in males were evaluated for Fl and F2 generations. Data for growth were collected at 30, 90 and 180 dpf and data for histological evaluations and Vtg induction were analyzed at 90 and 180 dpf. No significant effects on survival were seen in the Fl generation in any treatment following 180 d exposure: however, in the F2 generation, 300 mu g/L PFOS both alone and in combination with BPA (10, 200 and 400 mu g/L) induced 100% mortality within 14 dpf. PFOS (0.6 and 300 mu g/L) did not increase the Vtg-inducing potential of BPA (10, 200 and 400 mu g/L) in a binary mixture. In contrast, binary mixtures with 300 mu g/L PFOS suppressed the Vtg levels in Fl males at 90 dpf when compared to single BPA exposures. Whereas the lowest tested PFOS concentration (0.6 mu g/L) showed an estrogenic potential in terms of significant Vtg induction, Vtg levels were generally found to decrease with increasing PFOS-exposure in both Fl and F2 generations. In Fl generation, BPA-exposure was found to increase Vtg levels in a concentratio

Purpose Traditionally, methods for sediment extractions are characterised using chemical analyses. However, in order to evaluate sediment extracts with regard to biological effects and, thus, bioaccessibility, extraction methods have to be compared to effect data obtained from experiments with in situ exposure scenarios, i.e., sediment contact tests. This study compares four extraction methods and sediment contact test data from a previous project with respect to predictive power in the fish embryo test with zebrafish (Danio rerio). Materials and methods A natural and an artificial sediment spiked with a mixture of six organic pollutants (2,4-dinitrophenol, diuron, fluoranthene, nonylphenol, parathion and pentachlorophenol) were extracted using (a) membrane dialysis extraction (MDE), (b) a Soxhlet procedure, (c) hydroxypropyl-ß-cyclodextrin (HPCD) or (d) Tenax®-TA. Whereas the former two are regarded being exhaustive with respect to non-covalently bound contaminants, the latter two are considered to predict bioaccessibility. Resulting extracts were tested in the fish embryo assay with D. rerio for embryotoxic and teratogenic potential. Results and discussion Mortalities caused by organic extracts from Soxhlet extraction and MDE were high. However, HPCD extracts turned out to be at least as effective as extracts obtained with these two methods. One possible reason might be short ageing time of the spiked sediments. Only Tenax®-TA extracts gave results comparable to the sediment contact assay for natural sediment, but revealed low reproducibility. Significant differences between natural and artificial sediment were found for extracts obtained with techniques using native (i.e., non-freeze-dried) sediments, i.e., HPCD and Tenax®-TA. In contrast, MDE and Soxhlet extracts used freeze-dried sediment and did not differentiate between natural and artificial sediment. Therefore, freeze-drying has likely altered and equalised sediment properties that influence accessibility

Bankside groundwater is widely used as drinking water resource and, therefore, contamination has to be avoided. In the European Union groundwater protection is explicit subject to Water Framework Directive. While groundwater pollution may originate from different sources, this study investigated on impacts via flood events. Groundwater was sampled with increasing distance to the river Rhine near Karlsruhe, Germany. Samples were HPLC–MS–MS analyzed for the river contaminant carbamazepine to indicate river water infiltration, giving permanent presence in 250 m distance to the river (14–47 lg Lÿ1). Following a flood event, concentrations of about 16–20 lg Lÿ1 could also be detected in a distance of 750 m to the river. Furthermore, estrogenic activity as determined with the Yeast Estrogen Screen assay was determined to increase up to a 17b-ethinylestradiol equivalent concentration (E-EQ) = 2.9 ng Lÿ1 near the river, while activity was initially measured following the flood with up to E-EQ = 2.6 ng Lÿ1 in 750 m distance. Detections were delayed with increasing distance to the river indicating river water expansion into the aquifer. Flood suspended matter and floodplain soil were fractionated and analyzed for estrogenic activity in parallel giving up to 1.4 ng gÿ1 and up to 0.7 ng gÿ1, respectively. Target analysis focusing on known estrogenic active substances only explained <1% of measured activities. Nevertheless, river water infiltration was shown deep into bankside groundwater, thus, impacting groundwater quality. Therefore, flood events have to be in the focus when aiming for groundwater and drinking water protection as well as for implementation of Water Framework Directive.

Purpose This study was carried out to determine hazards of particle-bound contaminants in rivers to retention areas close to public well fields in the context of flood events. The focus was on the assessment of soil contamination at a planned retention area. Soil core samples were chemically and biologically analyzed. Samples were fractionated to identify and compare contaminant loads and biological effects of soil and flood suspended particulate matter (SPM). Material and methods Soil cores were sampled at inundated and non-inundated sites at a planned retention area. Soil was analyzed for hexachlorobenzene (HCB), polychlorinated biphenyls (PCBs) as well as for polycyclic aromatic hydrocarbons (PAHs). The highest inducing soil sample was fractionated applying a recently developed automated fractionation method to receive further insight into contaminant loads in soil at inundated sites. Impacts on biological scale were assessed using in vitro biotests for xenometabolic activity (7-ethoxyresorufin-o-deethylase (EROD) assay) as well as for mutagenic activity (Ames fluctuation assay). EROD induction was calculated as biological equivalent concentration (bio-TEQ), and mutagenic potentials were given as no observed effect concentration (NOEC) and maximum induction factor (IFmax). Results and discussion Soil core samples of each site induced EROD activity. However, extracts of soil sampled at a ground swale was by far the highest inducing (topsoil bio-TEQ?=?41,000 pg/g). Further, chemical analysis yielded relative increases in concentration in particular: HCB (0.05 mg/kg), PCBs (0.19 mg/kg), and EPA-PAHs (39 mg/kg). Extracts of soil samples caused no mutagenic effects. PAHs caused the bulk of EROD activity (bio-TEQ?=?32,000 pg/g) with ground swale topsoil fractions. Further, fractions containing moderately polar and polar substances gave elevated effects (bio-TEQ?=?8,200 pg/g). Mutagenic potentials were shown with most fractions. However, highest induction was observed w

Purpose The presented study investigated on contamination of suspended particulate matter (SPM) in rivers that was sampled long-term and with higher frequency during a flood event at the river Rhine. It was conducted to determine in vitro biological effects as well as to identify and quantify compound classes and effective contaminants. Research was part of investigation on hazards of contaminants bound to SPM to inundated sites and retention areas that are inundated during flood events. Material and methods SPM was sampled in 2006 and more frequently in a flood event (August, 2007) at the river Rhine barrage of Iffezheim, Germany. SPM was GC-MS analyzed for hexachlorobenzene (HCB), several polychlorinated biphenyls (PCBs) as well as for polycyclic aromatic hydrocarbons (PAHs). Flood samples were fractionated applying a recently developed automated fractionation method to receive further insight into contaminant loads in flood SPM. Impacts on biological scale were assessed using in vitro biotests for xenometabolic 7-ethoxyresorufin-o-deethylase (EROD) assay as well as for mutagenic activity (Ames fluctuation assay). EROD induction was calculated as biological equivalent concentrations (bio-TEQs) and mutagenic potentials were shown as NOECs and maximum induction factors. Results and discussion Chemical analysis gave low concentrations of PCBs (2006 and 2007) and HCB (2006). HCB concentrations increased during the flood in 2007 (maximum, 110 µg/kg SPM). Concentrations of PCBs were only initially elevated in the flood (maximum, 67 µg/kg SPM). EROD induction bio-TEQs ranged from 1,160 to 6,640 pg/g SPM in 2006 and showed maximum bio-TEQ at the peak discharge in 2007. There was no mutagenic activity with SPM of both years. Fractionation indicated highest EROD induction in PAH fractions with prioritized (EPA-) PAHs contributing to less than 1% to the fractions total bio-TEQ but also fractions containing more polar-to-polar substances were shown to contribute minor. Furthe

Suspended particulate matter (SPM) sampled during a flood event in the year 2004 at the rivers Neckar and Rhine (Southwest Germany) was assessed for aryl hydrocarbon receptor (AhR)-mediated activities using EROD induction in the rainbow trout liver cell line RTL-W1. All EROD inductions were normalized to the positive control TCDD and given as bio-TEQ values. Since all samples indicated elevated AhR-mediated toxicities, an effect-directed analysis (EDA) was applied to identify substances causing the effects. In three primary fractions (F1 to F3) non-polar aliphatics, non-polar aromatic substances and more polar substances were separated. Fraction F2, co-eluting with non-polar polyaromatic substances (PACs) including polycyclic aromatic hydrocarbons (PAHs) gave highest AhR-agonistic effects and, thus, were sub-fractionated into seven secondary fractions (F2-1 to F2-7). Fraction F2-1, co-eluting with PCBs and PCDD/Fs, did not cause AhR-agonist activities. F2-2 to F2-4 containing PACs of less than 16 aromatic C-atoms produced minor activities. Highest inductions were detected with fraction F2-5 to F2-7, containing substances of more than 16 aromatic C-atoms (bio-TEQs up to approximately 4500 pg/g). Concentrations and relative potencies (REPs) of priority EPA-PAHs allowed the calculation of chemical toxicity equivalent concentrations (chem-TEQ values). Based on the chem-TEQs, EPA-PAHs explained between 5 and 58% of crude extract bio-TEQs from both rivers. Whereas fractions F2-1 to F2-4 indicated no biological activities, EPA-PAHs in fraction F2-5 to F2-7 accounted for 2 to 137% of AhR-related activities.

Despite evidence for a conserved role of thyroid-stimulating hormone (TSH) in regulating vertebrate thyroid function, molecular data on thyroid responses to TSH are mainly limited to mammalian species. In this study, we examined histological and molecular changes in the thyroid of Xenopus laevis tadpoles during a 12-day treatment with 20mg/l perchlorate (PER) and 50mg/l ethylenethiourea (ETU). Inhibition of thyroid hormone (TH) synthesis by PER and ETU was evident from developmental retardation, reduced expression of TH-regulated genes and up-regulation of tshb-A mRNA. Thyroid histopathology revealed goiters with strikingly different follicular morphologies following PER and ETU treatment. Using real-time PCR, we analyzed thyroids sampled on day 12 for differential expression of 60 candidate genes. Further temporal analyses were performed for a subset of 14 genes. Relative to the control, PER and ETU treatment modulated the expression of 51 and 49 transcripts, respectively. Particularly genes related to TH synthesis and protein metabolism were similarly affected by PER and ETU. However, several genes were differentially expressed in PER- and ETU-treated tadpoles. Specifically, goiter formation in the PER treatment was associated with low expression of genes related to DNA replication but high expression of negative growth regulators. Results from this work provide for the first time a characterization of gene expression profiles during goitrogenesis in a non-mammalian vertebrate model. Overall, our data suggest that, in addition to TSH over-stimulation, further mechanisms related to the mode of goitrogen action contribute to the regulation of thyroid gene expression., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

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