Assessment of urban stream sediment pollutants entering estuaries using chemical analysis and multiple bioassays to characterise biological activities.

Stormwater contaminants are a major source of often neglected environmental stressors because of the emphasis placed on the management of municipal and industrial wastewaters. Stormwater-derived pollutants in sediments from two New Zealand estuaries was characterised by analytical chemistry and bioassays. Contaminants were extracted from sediment using accelerated solvent extraction (ASE), recovered and concentrated by solid phase extraction (SPE), and analysed for polycyclic aromatic hydrocarbons (PAHs), selected metals, and musk fragrances. The concentrations of PAHs were below the ANZECC Interim Sediment Quality Guideline values while those of lead and zinc exceeded them in some samples. The sediment extracts containing organic contaminants exhibited acute toxicity in the zebrafish fish embryo toxicity (FET) and teratogenicity, induction of biotransformation (EROD activity), and genotoxicity (comet assay) in zebrafish. The potential of the extracts to interact with endocrine signalling processes was assessed by GeneBLAzer reporter gene bioassays and they exhibited estrogenic, androgenic, and anti-progestagenic activities.

The fish embryo test (FET): origin, applications, and future.

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.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

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.

Cartilage and bone malformations in the head of zebrafish (Danio rerio) embryos following exposure to disulfiram and acetic acid hydrazide.

In order to investigate teratogenic effects, especially on cartilage and bone formation, zebrafish embryos were exposed for 144h to the dithiocarbamate pesticide disulfiram (20-320µg/L) and acetic acid hydrazide (0.375-12g/L), a degradation product of isoniazid. After fixation and full-mount staining, disulfiram could be shown to induce strong cartilage malformations after exposure to ≥80µg/L, whereas acetic acid hydrazide caused cartilage alterations only from 1.5g/L. Undulating notochords occurred after exposure to disulfiram even at the lowest test concentration of 20µg/L, whereas at the two lowest concentrations of acetic acid hydrazide (0.375 and 0.75g/L) mainly fractures of the notochord were observed. Concentrations of acetic acid hydrazide≥1.5g/L resulted in undulated notochords similar to disulfiram. Cartilages and ossifications of the cranium, including the cleithrum, were individually analyzed assessing the severity of malformation and the degree of ossification in a semi-quantitative approach. Cartilages of the neurocranium such as the ethmoid plate proved to be more stable than cartilages of the pharyngeal skeleton such as Meckel's cartilage. Hence, ossification proved significantly more susceptible than cartilage. The alterations induced in the notochord as well as in the cranium might well be of ecological relevance, since notochord malformation is likely to result in impaired swimming and cranial malformation might compromise regular food uptake.

Developmental effects of coumarin and the anticoagulant coumarin derivative warfarin on zebrafish (Danio rerio) embryos.

Coumarin and warfarin, two substances which are intensively metabolized in animals and humans, were tested for teratogenicity and embryo lethality in a 3-day in vitro assay using zebrafish embryos. Warfarin is a coumarin derivative, but in contrast to the mother substance warfarin has anticoagulant properties. Both substances produced teratogenic and lethal effects in zebrafish embryos. The LC(50) and EC(50) values for coumarin are 855 µM and 314 µM, respectively; the corresponding values for warfarin are 988 µM and 194 µM. For coumarin, three main or fingerprint endpoints (malformation of head, tail and growth retardation) were identified, whereas malformation of tail was the only fingerprint endpoint of warfarin. The analysis of the ratios between the zebrafish embryo effect concentrations of both substances and human therapeutic plasma concentrations confirmed the teratogenic potential of warfarin, as well as the equivocal status of coumarin.

Sediment-contact fish embryo toxicity assay with Danio rerio to assess particle-bound pollutants in the Tietê River Basin (São Paulo, Brazil).

The Tietê River and its tributary Pinheiros River receive a highly complex organic and inorganic pollutants load from sanitary sewage and industrial sources, as well as agricultural and agroindustrial activities. The aim of the present study was to evaluate the embryotoxic and teratogenic effects of sediments from selected locations in the Tietê River Basin by means of the sediment contact embryo toxicity assay with Danio rerio, in order to provide a comprehensive and realistic insight into the bioavailable hazard potential of these sediment samples. Lethal and sub-lethal effects were recorded, and high embryo toxicity could be found in the samples not only in the vicinity of the megacity São Paulo (Billings reservoir and Pinheiros River samples), but also downstream (in the reservoirs Barra Bonita, Promissão and Três Irmãos). Results confirm that most toxicity is due to the discharges of the metropolitan area of São Paulo. However, they also indicate additional sources of pollutants along the river course, probably from industrial, agricultural and agroindustrial residues, which contribute to the degradation of each area. The sediment contact fish embryo test showed to be powerful tool to detect embryo toxicity in sediments, not only by being a sensitive method, but also for taking into account bioavailability. This test provides an ecological highly realistic and relevant exposure scenario, and should therefore be added in ecotoxicological sediment quality assessments.

Zebrafish (Danio rerio) embryos as a model for testing proteratogens.

Zebrafish embryos have been shown to be a useful model for the detection of direct acting teratogens. This communication presents a protocol for a 3-day in vitro zebrafish embryo teratogenicity assay and describes results obtained for 10 proteratogens: 2-acetylaminofluorene, benzo[a]pyrene, aflatoxin B(1), carbamazepine, phenytoin, trimethadione, cyclophosphamide, ifosfamide, tegafur and thio-TEPA. The selection of the test substances accounts for differences in structure, origin, metabolism and water solubility. Apart from 2-acetylaminofluorene, which mainly produces lethal effects, all proteratogens tested were teratogenic in zebrafish embryos exposed for 3 days. The test substances and/or the substance class produced characteristic patterns of fingerprint endpoints. Several substances produced effects that could be identified already at 1 dpf (days post fertilization), whereas the effects of others could only be identified unambiguously after hatching at ≥ 3 dpf. The LC50 and EC50 values were used to calculate the teratogenicity index (TI) for the different substances, and the EC20 values were related to human plasma concentrations. Results lead to the conclusion that zebrafish embryos are able to activate proteratogenic substances without addition of an exogenous metabolic activation system. Moreover, the teratogenic effects were observed at concentrations relevant to human exposure data. Along with other findings, our results indicate that zebrafish embryos are a useful alternative method for traditional teratogenicity testing with mammalian species.

Oxygen requirements of zebrafish (Danio rerio) embryos in embryo toxicity tests with environmental samples.

The zebrafish embryo test is a widely used bioassay for the testing of chemicals, effluents and other types of environmental samples. Oxygen depletion in the testing of sediments and effluents is especially important and may be a confounding factor in the interpretation of apparent toxicity. In order to identify oxygen levels critical to early developmental stages of zebrafish, oxygen consumption of zebrafish embryos between 0 and 96h post-fertilization, minimum oxygen levels required by the embryos for survival as well as the effects of oxygen depletion following exposure to model sediments were determined. No significant effects on zebrafish embryo development were observed for oxygen concentrations between 7.15 and 3.33mg/L, whereas at concentrations between 3.0and 2.0mg/L minor developmental retardations were observed, yet without any pathological consequences. Oxygen concentrations lower than 0.88mg/L were 100% lethal. In the sediment contact tests with zebrafish embryos, native sediments rich in organic materials rapidly developed strongly hypoxic conditions, particularly at the sediment-water interface (0 to 500µm distance to the sediment).