Limitations of waterborne exposure of fish early life stages to BDE-47.

2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is acknowledged as the most abundant congener of all polybrominated diphenyl ethers (PBDEs). Despite its limited residence in the water column, most ecotoxicological research using fish early life stages (ELS) has focused on its waterborne bioavailability. These studies have been supported either by chemical analysis in solutions or in tissues after ≤ 168 h exposures to relatively high waterborne concentrations with dimethyl sulfoxide (DMSO) as solvent carrier (≤ 0.5%). Using noninvasive physiological and anatomical features in medaka ELS, we investigated the viability of waterborne BDE-47 exposures (100-10,000 µg/L; 1% DMSO) and evaluated the developmental effects in relation to the actual BDE-47 present in water. Embryos were exposed for 10 days under semi-static (24-h renewal) conditions and waterborne BDE-47 concentrations (i.e., dissolved) were quantitated daily and their accumulation in eleutheroembryonic tissues was analyzed 4 days after exposures finished. BDE-47 in solution rapidly decreased after each renewal by >50% in 24h. This was confirmed by discernible precipitation occurring at ≥ 5,000 µg/L on the bottom of the container and attached to the chorionic filaments of eggshell. The fast dissipation from water may explain why, besides the subtle, yet significant effects on post-hatching growth (short length at ≥5000µg/L), no other significant deleterious developmental effects were observed despite the fact that BDE-47 accumulated in tissues in response to BDE-47 treatment. Waterborne BDE-47 exposure was unachievable under traditional semi-static exposure conditions, but was achievable in repeated pulse exposures lasting a few hours whenever the medium was renewed. Hence, this research encourages the use of alternate - more realistic - exposure routes (e.g., particulate matter or sediments) when evaluating early developmental toxicity of BDE-47 or any other PBDE sharing similar properties.

Dynamics of BNF-induced in vivo ethoxyresorufin-O-deethylase (EROD) activity during embryonic development of medaka (Oryzias latipes).

This study aimed to characterize quantitatively the temporal basal and induced ethoxyresorufin-O-deethylase (EROD) activity as indicator of cytochrome P4501A (CYP1A) function during embryonic development of medaka (Oryzias latipes). For this purpose, non-invasive methods over fluorescence images of the whole embryo (non-organ-specific [NOS] EROD activity) or specifically of the gallbladder (organ-specific [OS] EROD activity) were used. To induce this EROD activity, embryos were continuously exposed to ß-naphthoflavone (BNF; 0.005, 0.05, 0.5, 5 µg/L). Analytical chemistry suggested no signs of BNF dissipation. Mean fluorescence intensity values for EROD induction increased with BNF concentration throughout embryonic development. Significant increments in the NOS activity were seen from exposures to ≥ 0.5 µg BNF/L as early as 2 days post-fertilization (dpf), and in the OS EROD activity as soon as the gallbladder was conspicuous (i.e. 4 dpf). Morphometric in vivo analysis of the gallbladder during embryonic development did not indicate significant dilation after BNF treatment suggesting normal hepatic bile formation. The conditions optimized in this study using intact embryos should allow the quantitation of EROD activity induced by specific chemicals, mixtures and environmental samples in terms of BNF-equivalents, offering a proper estimation of their potency. These results demonstrate the utility of medaka in a fish embryo test for a non-invasive CYP1A analysis expressed as EROD activity, fitting in the three R principles for the minimization of animal use in ecotoxicology evaluations and that are among the objectives of the European Community regulation for the Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH).