Assessing the toxicity of sediments using the medaka embryo-larval assay and 2 other bioassays.

Sediments are sinks for aquatic pollutants, and analyzing toxicity in such complex matrices is still challenging. To evaluate the toxicity of bioavailable pollutants accumulated in sediments from the Bizerte lagoon (Tunisia), a novel assay, the medaka embryo-larval assay by sediment contact, was applied. Japanese medaka (Oryzias latipes) embryos were incubated in direct contact with sediment samples up to hatching. Lethal and sublethal adverse effects were recorded in embryos and larvae up to 20 d postfertilization. Results from medaka embryo-larval assay were compared with cytotoxicity (Microtox®), genotoxicity (SOS chromotest), and pollutant content of sediments. The results highlight differences in the contamination profile and toxicity pattern between the different studied sediments. A significant correlation was shown between medaka embryo-larval assay by sediment contact and SOS chromotest responses and concentrations of most organic pollutants studied. No correlation was shown between pollutant levels and Microtox. According to the number of sediment samples detected as toxic, medaka embryo-larval assay by sediment contact was more sensitive than Microtox, which in turn was more sensitive than the SOS chromotest; and medaka embryo-larval assay by sediment contact allowed sediment toxicity assessment of moderately polluted sediments without pollutant extraction and using an ecologically realistic exposure scenario. Although medaka embryo-larval assay by sediment contact should be tested on a larger sample set, the results show that it is sensitive and convenient enough to monitor the toxicity of natural sediments. Environ Toxicol Chem 2016;35:2270-2280. © 2016 SETAC.

Transcriptional responses and embryotoxic effects induced by pyrene and methylpyrene in Japanese medaka (Oryzias latipes) early life stages exposed to spiked sediments.

Japanese medaka (Oryzias latipes) embryos were exposed to sediments spiked with environmental concentrations (300 and 3,000 ng/g dry weight) of pyrene (Pyr) and methylpyrene (MePyr) throughout their development. Embryotoxicity, teratogenicity, and transcriptional responses (qRT-PCR) were analyzed in embryos and newly hatched larvae. The genotoxicity of the two polycyclic aromatic hydrocarbons (PAHs) was also tested in prolarvae using the comet assay. Exposure to each compound had a clear impact on embryonic development and resulted in several teratogenic effects, including cardiovascular injuries, reduced absorption of yolk sac reserves, and jaw and spinal deformities. Interestingly, the overall toxic effects of Pyr and MePyr considerably overlapped those induced following dioxin exposure. qRT-PCR analysis revealed the transcriptional induction of genes involved in mitochondrial energetic metabolism (coxI), xenobiotic biotransformation (cyp1a), and cell cycle regulation (wnt1) by the two PAHs. MePyr also activated cell cycle arrest (p53), oxidative DNA damage repair (ogg1), and retinoid-mediated (raldh2 and rarα1) gene transcription. DNA damage was not found to be significantly increased following Pyr and MePyr exposure. The lack of significant genotoxic effect in comparison to the control might be the consequence of the efficient onset of DNA damage repair mechanisms as suggested by ogg1 gene transcription upregulation. Results reported in the present study have brought new insights into the modes of action of Pyr, and the effects of MePyr exposure have been investigated in fish ELS for the first time.

Development of a reference artificial sediment for chemical testing adapted to the MELA sediment contact assay.

Most persistent organic pollutants, due to their hydrophobic properties, accumulate in aquatic sediments and represent a high risk for sediment quality. To assess the toxicity of hydrophobic pollutants, a novel approach was recently proposed as an alternative to replace, refine and reduce animal experimentation: the medaka embryo-larval sediment contact assay (MELAc). This assay is performed with Japanese medaka embryos incubated on a natural sediment spiked with the compound being tested. With the aim of improving this assay, our study developed a reference exposure protocol with an artificial sediment specifically designed to limit natural sediment composition uncertainties and preparation variability. The optimum composition of the new artificial sediment was tested using a model polycyclic aromatic hydrocarbon (PAH), fluoranthene. The sediment was then validated with two other model PAHs, benz[a]anthracene and benzo[a]pyrene. Various developmental end points were recorded, including survival, embryonic heartbeat, hatching delay, hatching success, larval biometry and abnormalities. The final artificial sediment composition was set at 2.5 % dry weight (dw) Sphagnum peat, 5 % dw kaolin clay and 92.5 % dw silica of 0.2- to 0.5-mm grain size. In contrast with natural sediments, the chemical components of this artificial matrix are fully defined and readily identifiable. It is totally safe for fish embryos and presents relatively high sorption capacities for hydrophobic compounds. Studies with other hydrophobic and metallic contaminants and mixtures should be performed to further validate this artificial sediment.

Effects of copper and cadmium spiked-sediments on embryonic development of Japanese medaka (Oryzias latipes).

Because of their high capacity to accumulate contaminants such as persistent organic pollutants and heavy metals, aquatic sediments are considered as a long-term source of contamination for aquatic organisms. In compliance with the increasing interest both for sediment quality evaluation and the use of fish early life stage (ELS) toxicity assays, we proposed an embryo-larval test to evaluate embryotoxicity and genotoxicity of sediment-bound contaminants. Pre-blastula stage medaka (Oryzias latipes) embryos were exposed by static sediment contact to two model heavy metals (cadmium and copper) at environmental concentrations during the whole 10-day embryonic development. Lethal and sub-lethal effects were recorded in both embryos and larvae for 20 days post fertilisation (dpf) using several global toxicity and phenotypic endpoints. The comet assay was also performed on medaka prolarvae to evaluate genotoxic effects of the tested chemicals. Environmental concentrations of cadmium (Cd) and copper (Cu) did not affect embryo and larval survival. However, both heavy metals significantly induced morphological abnormalities, particularly spinal and cardiovascular deformities. Cd but not Cu induced tachycardia. Both heavy metals induced a significant increase in DNA damage at all tested concentrations. Resulting LOEC values for Cd and Cu corresponded to 1.9 and 8.5 µg/g d.w. sediment, respectively. Although metal bioavailability is probably lower for naturally contaminated sediments, the relatively low toxicity thresholds for both Cd and Cu raise the question of possible risk for fish embryos developing in direct contact to sediments. This study demonstrates the applicability, sensitivity and relevance of the Japanese medaka embryo-larval assay (MELA) to evaluate sediment hazardous potency at environmental concentrations of heavy metals.