A new spiked sediment assay using embryos of the Japanese medaka specifically designed for a reliable toxicity assessment of hydrophobic chemicals.

Despite their low water solubility, hydrophobic pollutants are widespread in the aquatic environment and could represent a threat for living organisms. EU regulations on chemicals require accurate and reliable data on chemical toxicity. Current normalised fish toxicity assays, in particular those advocated by OECD guidelines, do not allow reliable toxicity assessment of hydrophobic compounds due to their low water solubility. In order to accurately evaluate the toxicity of this kind of compounds, a new spiked sediment assay using embryos of the Japanese medaka was developed. It consists of directly exposing fertilised eggs, during their entire embryonic development, onto the reference sediment spiked with the test compound. A large set of lethal or sublethal effects in embryos and newly hatched larvae, including non-invasive endpoints is analysed in order to maximise the sensitivity of the test. The approach was validated using four model pollutants with different modes of action: DMBA, PCB126, PCB153 and 4-nonylphenol (NP). All compounds, except PCB153, induced a dose-dependent increase in toxic effects. In fact, lethal effects only occurred at the highest tested concentration. In contrast, sub-lethal effects including skeletal deformations, cardiac activity modulation, body length reduction and hatching delay were observed at low to moderate concentrations of DMBA and PCB126. NP induced subtle effects in embryos, altering cardiac activity and hatching success but only at high concentrations. Although a few more improvements would make it a fully standardised assay, this spiked sediment assay using medaka embryos proves to be sensitive enough to measure hydrophobic chemical toxicity using an environmentally realistic mode of exposure.

Detection of DNA damage in yolk-sac larvae of the Japanese Medaka, Oryzias latipes, by the comet assay.

This study was set up to determine the suitability of the early life stage (ELS) alkaline comet assay for the detection of DNA strand breaks induced by genotoxicants in whole organism. This assay was performed on cells of medaka 2 days posthatch (dph). An efficient procedure for cell dissociation using enzymatic and mechanical digestion was developed. This protocol ensures 80% viability of cells and low DNA damage background. Cells from 2 dph medaka larvae were exposed in vitro to model genotoxicants, hydrogen peroxide, cadmium, and fluoranthene, followed by comet assay analysis. Results show a significant increase in the percentage of DNA damage of dissociated cells by all the tested compounds when compared to controls. The assay was also performed in vivo on medaka larvae (2 dph) exposed for 24 h to waterborne cadmium or fluoranthene. Significant induction of DNA damage levels were observed following larvae exposure to cadmium and fluoranthene at concentrations of 0.1 and 50 µM, respectively. This study demonstrates that cells of embryo life stage medaka respond to known DNA damaging agents and that the ELS comet assay may be a useful biomarker to detect DNA strand breakage in whole body of pluricellular organism induced by a range of agents. This technique may provide a sensitive, nonspecific endpoint of genotoxicity as part of ELS toxicity test.

Partial purification and characterization of acetylcholinesterase (AChE) from the estuarine copepod Eurytemora affinis (Poppe).

Oligohaline copepods such as Eurytemora affinis are widespread in estuaries of northwestern Europe. These minute crustaceans are highly sensitive to contamination and thus serve as useful bioindicators for the monitoring of pollutant effects. The use of decreased cholinesterase (ChE) activity as a sublethal biomarker of exposure to neurotoxic compounds supposes that ChE has been defined in copepods. This study reports the partial purification and characterization of ChE extracted from E. affinis. Analysis by non-denaturing PAGE and by isoelectric focusing indicated that the enzyme is probably a single dimeric form of 140 KDa, with a pI of 6.2. This enzyme is likely an acetylcholinesterase (AChE) since it hydrolyzes acetylthiocholine iodide at a higher rate than other substrates, such as butyrylthiocholine and propionylthiocholine, at pH 7.0 and 25 degrees C, and is inhibited by eserine but not by iso-OMPA. The enzyme exhibited high sensitivity to some of the various pollutants tested. The kinetic properties of this ChE were compared with those of other invertebrate ChEs.