Histological, enzymatic and chemical analyses of the potential effects of differently sized microplastic particles upon long-term ingestion in zebrafish (Danio rerio).

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.

Microplastics and sorbed contaminants - Trophic exposure in fish sensitive early life stages.

The present study evaluated very small microplastic particle (MPs) transfer to zebrafish and marine medaka larvae via prey experimentally exposed to MPs from the onset of feeding. Larvae were fed Paramecium or Artemia nauplii loaded with fluorescent 1-5 or 10-20 µm MP. Pollutant accumulation was analyzed by optically tracking of benzo[a]pyrene (BaP) and recording cyp1a transcription. Paramecium transferred 1-5 µm particles only, whereas Artemia efficiently transferred both MPs. Although zebrafish and medaka larvae fed from the onset of active food intake (2-3 dph, respectively) on Paramecium and from days 6-7 post-hatch on Artemia nauplii, neither MP accumulation nor translocation to tissues was detected. MP egestion started within few hours after ingestion. Cyp1a induction and fluorescent analyses proved BaP bioavailability after transfer via Paramecium and Artemia. Unicellular or plankton organisms ingest contaminants via MPS and transfer effectively these to sensitive early life-stages of vertebrates, giving rise to whole-life exposure.

Microplastic accumulation patterns and transfer of benzo[a]pyrene to adult zebrafish (Danio rerio) gills and zebrafish embryos.

Since only a few studies have investigated effects of microplastics (MPs) by routes other than ingestion, this study was designed to analyze the accumulation patterns and transfer of toxic substances associated with microplastic exposure by simple attachment to (1) adult zebrafish (Danio rerio) gills and (2) zebrafish embryos. Two sizes of fluorescently labelled polymers (1-5 and 10-20 µm) loaded with the model polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) were used to analyze fate, accumulation and transfer of microplastic-associated persistent organic pollutants (POPs) on gills and embryos. Results indicate that microplastics did not permanently accumulate at high amounts in adult zebrafish gills after 6 nor 24 h of incubation: Most particles only superficially adhered to the mucus layer on the filaments, which is constantly being excreted. In contrast, the smaller and heavier MPs (1-5 µm) accumulated in high numbers on the surface of zebrafish egg chorions. In both exposure scenarios, transfer of BaP could be visualized with fluorescence microscopy: A prominent BaP signal was visible both in gill filaments and arches after 6 and 24 h incubation and in zebrafish embryos after exposure to BaP-spiked microplastics. Furthermore, the gill EROD (Ethoxyresorufin-O-deethylase) assay showed a clear trend to CYP 1A (Cytochrom P450 1 A) induction via exposure to BaP-spiked microplastics. However, BaP from spiked microplastics did not reach sufficiently high concentrations to be able to induce morphological effects in the fish embryo toxicity test (FET). In contrast, control exposure to waterborne BaP did induce effects in the FET. As a conclusion, microplastics can also transfer POPs not only via ingestion, but also by simple attachment to epithelia or via the water column. However, further studies are needed to clarify if these interactions are of environmental concern relative to waterborne exposure to toxic substances.

Transfer of benzo[a]pyrene from microplastics to Artemia nauplii and further to zebrafish via a trophic food web experiment: CYP1A induction and visual tracking of persistent organic pollutants.

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.

Acetylcholinesterase in zebrafish embryos as a tool to identify neurotoxic effects in sediments.

In order to clarify the suitability of zebrafish (Danio rerio) embryos for the detection of neurotoxic compounds, the acetylcholinesterase assay was adapted and validated with a series of priority pollutants listed as relevant for the European water policy (Aroclor 1254, 2,3-benzofuran, bisphenol A, chlorpyrifos, paraoxon-methyl, quinoline, and methyl mercury chloride) as well as acetonic extracts from three sediments of known contamination. The acute toxicities of the model substances and the sediment extracts were determined by means of the fish embryo test as specified in OECD TG 236, and concentrations as low as the effective concentration at 10% inhibition (EC10) were used as the highest test concentration in the acetylcholinesterase test in order to avoid nonspecific systemic effects mimicking neurotoxicity. Among the model compounds, only the known acetylcholinesterase inhibitors paraoxon-methyl and chlorpyrifos produced a strong inhibition to about 20 and 33%, respectively, of the negative controls. For the sediment extracts, a reduction of acetylcholinesterase activity to about 60% could only be shown for the Vering Canal sediment extracts; this could be correlated to high contents of acetylcholinesterase-inhibiting polycyclic aromatic hydrocarbons (PAHs) as identified by chemical analyses. Co-incubation of the Vering Canal sediment extracts with chlorpyrifos at EC10 concentrations each did not significantly increase the inhibitory effect of chlorpyrifos, indicating that the mode of action of acetylcholinesterase inhibition by the sediment-borne PAHs is different to that of the typical acetylcholinesterase blocker chlorpyrifos. Overall, the study documents that zebrafish embryos represent a suitable model not only to reveal acetylcholinesterase inhibition, but also to investigate various modes of neurotoxic action.

Heterochrony in mandible development of larval shrimp (Decapoda: Caridea)--a comparative morphological SEM study of two carideans.

Mandible development in the larval stages I-V of two palaemonid shrimp species, Palaemon elegans and Macrobrachium amazonicum, was analyzed using scanning electron microscopy, light microscopy, and confocal laser scanning microscopy. In contrast to the zoea I of P. elegans, first-stage larvae of M. amazonicum are nonfeeding. At hatching, the morphology of the mandibles is fully expressed in P. elegans, while it appears underdeveloped in M. amazonicum, presenting only small precursors of typical caridean features. In successive zoeal stages, both species show similar developmental changes, but the mandibular characters of the larvae in M. amazonicum were delayed compared to the equivalent stages in P. elegans, especially in the development of submarginal setae and mandible size. In conclusion, our results indicate heterochrony (postdisplacement) of mandible development in M. amazonicum compared to that in P. elegans, which is related to initial lack of mandible functionality or planktivorous feeding at hatching, respectively. This conclusion is supported by comparison with other palaemonid zoeae exhibiting different feeding modes. Our data suggest that an evolutionary ground pattern of mandible morphology is present even in species with nonfeeding first-stage larvae.