The distribution of different fluorescence dyes in the egg and embryo of the zebrafish (Danio rerio)

The fish embryo test (FET) with Danio rerio is a well-established method for the toxicological evaluation of treated waste water, chemicals and sediments and was standardized at a national level in DIN 38415-6 in 2001. In order to study the distribution of chemicals in the FET, the present study investigated the individual distribution of seven different fluorescent dyes in different compartments of the zebrafish egg (chorion, perivitelline space, different organs of the embryo). For a better assessment of the effects of side chains, rhodamine b and fluorescein were chosen as basic fluorochromes, which were then modified by diverse substituents that differed in their partition coefficient, molecular size and charge. The substances tested were rhodamine b, sulforhodamine b, fluorescein, 2,7-dichlorofluorescein and 5-carboxy-2,7-dichlorofluorescein. The permeability of the chorion was evaluated through the use of dextran fluorescein with different molecular sizes: 3 kDa and 40 kDa. Finally, the distribution of fluorescein was studied with respect to its dependancy on different concentrations of the solvent DMSO (0.01 %, 0.1 % and 1 %). Substances with high water solubility such as rhodamine b and sulforhodamine b, accumulated in the yolk, although sulforhodamine b with its charged substituents did not diffuse through the changing chorion before 48 hours. The smallest molecule, fluorescein, accumulated in the blood and brain as early as after 24 hours, whereas the slightly larger molecules 2,7-dichlorofluorescein and 5-carboxy-2,7-dichlorofluorescein could only pass the chorion after 48 hours. Neither of the two largest molecules, dextran fluorescein with 3 kDa nor 40 kDa, was able to pass the chorion at all, and 3 kDa dextran fluorescein accumulated in the pore canals. The uptake of fluorescein was increased through the use of DMSO concentrations of 1 % and 0.1 %, whereas fluorescein which was dissolved in only 0.01 % was not able to pass the chorion. The study thus demonstrated that the barrier function of the chorion depends on the polarity and partition coefficient of the substances tested and that the ratio of undissociated and dissociated substances might be a crucial parameter for toxicity. Furthermore, this study shows that higher DMSO concentrations decrease the barrier function of the chorion, which might present a new approach for the investigation of the chorion’s relevance for embryo toxicity