For morphological effects observation zebrafish embryo test was carried out according to 31: In each well of a 24well dish 5 zebrafish embryos (4-8-cell stage) were exposed for 48 hr at 26°C to 1 ml test solution containing one NP isomer (2.5-50 ^mol/L) and 1% DMSO resulting in a total number of 60 embryos for each test, either exposure or control. Since all experiments were repeated twice a total of 120 zebrafish embryos were used for each concentration of each chemical. Negative controls were exposed to the 1% DMSO solvent only. EC50 was calculated from concentration-effect curves for the spinal malformations. Measurement was the amount of embryo showing any spinal deformation without respecting a certain degree of the misdevelopment. Four-parameter logistic curves were used to describe the concentration-effect relation. From these, EC50 values were calculated.

For RT-PCR 3-4 hr old zebrafish embryos were placed into 24-well dishes (5 embryos per well). For each experiment, either exposure or control, 30 embryos were used. The embryos were treated with the indicated NP at a final concentration of 10 ^mol/L for 1 hr in water containing DMSO at 26 °C. These parameters were chosen because in preliminary experiments expression effects were observable after 1 hr and the concentration of NP were selected on the basis of our previous study 23. After incubation, the embryos were washed in fresh water and subsequently shock frozen in liquid nitrogen. All experiments -covering 6 NP isomers, the technical NP mixture (Table 1) and a negative control with DMSO only – were repeated 5 times at different days. The concentration of the test substances was 10 ^mol/L, i.e., in the range of EC10 – EC50 of acute morphological effects of NP isomers in zebrafish after 48h exposure 23.

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