Size does matter - Determination of the critical molecular size for the uptake of chemicals across the chorion of zebrafish (Danio rerio) embryos.

In order to identify the upper limits of the molecular size of chemicals to cross the chorion of zebrafish, Danio rerio, differently sized, non-toxic and chemically inert polyethylene glycols (PEGs; 2000-12,000Da) were applied at concentrations (9.76mM) high enough to provoke osmotic pressure. Whereas small PEGs were expected to be able to cross the chorion, restricted uptake of large PEGs was hypothesized to result in shrinkage of the chorion. Due to a slow, but gradual uptake of PEGs over time, molecular size-dependent equilibration in conjunction with a regain of the spherical chorion shape was observed. Thus, the size of molecules able to cross the chorion could be narrowed down precisely to ≤4000Da, and the time-dependency of the movement across the chorion could be described. To account for associated alterations in embryonic development, fish embryo toxicity tests (FETs) according to OECD test guideline 236 (OECD, 2013) were performed with special emphasis to changes in chorion shape. FETs revealed clear-cut size-effects: the higher the actual molecular weight (=size) of the PEG, the more effects (both acutely toxic and sublethal) were found. No effects were seen with PEGs of 2000 and 3000Da. In contrast, PEG 8000 and PEG 12,000 were found to be most toxic with LC50 values of 16.05 and 16.40g/L, respectively. Likewise, the extent of chorion shrinkage due to increased osmotic pressure strictly depended on PEG molecular weight and duration of exposure. A reflux of water and PEG molecules into the chorion and a resulting re-shaping of the chorion could only be observed for eggs exposed to PEGs ≤4000Da. Results clearly indicate a barrier function of the zebrafish chorion for molecules larger than 3000 to 4,000Da.

Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio).

Prior to hatching, the zebrafish embryo is surrounded by an acellular envelope, the chorion. Despite repeated speculations, it could not be clarified unequivocally whether the chorion represents an effective barrier and, thus, protects the embryo from exposure to distinct chemicals. Potentially, there is a risk of generating false negative results in developmental toxicity studies due to limited permeability of the chorion for some compounds. The simplest way to exclude this is to remove the chorion and expose the "naked" embryo. In the context of ecotoxicity testing, standardized protocols do not exist for fish embryo dechorionation, and survival rates of dechorionated embryos have usually not been subjected to statistical analysis. Since reproducibly high survival rates are of fundamental importance for chemical toxicity assessment, the present study was designed to develop and optimize a dechorionation procedure. With appropriate modifications of the fish embryo test protocol, embryos can be dechorionated at 24h post-fertilization (hpf) with survival rates of ≥90%. However, for fish embryo tests with dechorionated embryos, the standard positive control test substance, 3,4-dichloroaniline, should be replaced by another compound, e.g., acetone, since 3,4-dichloroaniline exerts its effects during the first 24h of development. Dechorionation of younger stages (<24 hpf) is generally possible, however with lower survival rates. The effect of dechorionation was demonstrated with the cationic polymer Luviquat HM 552, which is blocked by the chorion non-dechorionated embryos due to its molecular weight of ~400,000 Dalton, but becomes strongly toxic after dechorionation.