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The uptake of microplastic particles and the transfer of potential harmful substances along with microplastics has been studied in a variety of organisms, especially invertebrates. However, the potential accumulation of very small microplastic particles along food webs ending with vertebrate models has not been investigated so far. Therefore, a simple artificial food chain with Artemia sp. nauplii and zebrafish (Danio rerio) was established to analyze the transfer of microplastic particles and associated persistent organic pollutants (POPs) between different trophic levels. Very small (1-20 μm) microplastic particles accumulated in Artemia nauplii and were subsequently transferred to fish. Virgin particles not loaded with POPs did not cause any observable physical harm in the intestinal tracts of zebrafish, although parts of the particles were retained within the mucus of intestinal villi and might even have been taken up by epithelial cells. The transfer of associated POPs was tested with the polycyclic aromatic hydrocarbon benzo[a]pyrene and an ethoxyresorufin-O-deethylase (EROD) assay for CYP1A induction in zebrafish liver as well as via fluorescence analyses. Whereas a significant induction in the EROD assay could not be shown, because of high individual variation and low sensitivity regarding substance concentration, the fluorescence tracking of benzo[a]pyrene indicates that food-borne microplastic-associated POPs may actually desorb in the intestine of fish and are thus transferred to the intestinal epithelium and liver. Environ Toxicol Chem 2016;35:1656-1666. © 2016 SETAC.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

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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 96h 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 48/96h 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.

The current paper critically reviews the state-of-the-science on (1) microplastics (MP) types and particle concentrations in freshwater ecosystems, (2) MP and nanoplastics (NP) uptake and tissue translocation, (3) MP/NP-induced effects in freshwater organisms, and (4) capabilities of MP/NP to modulate the toxicity of environmental chemicals. The reviewed literature as well as new data on MP and NP concentrations in the river Elbe and on particle uptake into human cells indicate an environmental relevance of small particles in the low nano-and micrometer range higher than that of larger MP.

Originally designed as an alternative for the acute fish toxicity test according to, e.g., OECD TG 203, the fish embryo test (FET) with the zebrafish (Danio rerio) has been optimized, standardized, and validated during an OECD validation study and adopted as OECD TG 236 as a test to assess toxicity of embryonic forms of fish. Given its excellent correlation with the acute fish toxicity test and the fact that non-feeding developmental stages of fish are not categorized as protected stages according to the new European Directive 2010/63/EU on the protection of animals used for scientific purposes, the FET is ready for use not only for range-finding but also as a true alternative for the acute fish toxicity test, as required for a multitude of national and international regulations. If-for ethical reasons-not accepted as a full alternative, the FET represents at least a refinement in the sense of the 3Rs principle. Objections to the use of the FET have mainly been based on the putative lack of biotransformation capacity and the assumption that highly lipophilic and/or high molecular weight substances might not have access to the embryo due to the protective role of the chorion. With respect to bioactivation, the only substance identified so far as not being activated in the zebrafish embryo is allyl alcohol; all other biotransformation processes that have been studied in more detail so far were found to be present, albeit, in some cases, at lower levels than in adult fish. With respect to larger molecules, the extension of the test duration to 96 h (i.e., beyond hatch) has-at least for the substances tested so far-compensated for the reduced access to the embryo; however, more research is necessary to fully explore the applicability of the FET to substances with a molecular weight >3 kDa as well as substances with a neurotoxic mode of action. An extension of the endpoints to also cover sublethal endpoints makes the FET a powerful tool for the detection of teratogenicity, dioxin-like activity, genotoxicity and mutagenicity, neurotoxicity, as well as various forms of endocrine disruption.

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