Zebrafish embryo collection and exposure

AB line wild-type zebrafish (Danio rerio) were maintained in a recirculating ZebTec housing system (Techniplast) at 28 °C with a 12:12 h (light/dark) photoperiod. Conductivity was maintained at approximately 500 μS/cm, pH at 7.2 and dissolved oxygen at 95% saturation. Fish were fed ad libitum three times daily (twice on flake and once on Artemia nauplii). Males and females were placed together in breeding aquaria the night before spawning in a ratio of 3:2. The next morning, eggs were collected within 30 min of spawning, pooled from several breeding tanks, washed with ZebTec system water to remove debris and then randomly transferred into polypropylene beakers.

Zebrafish embryos were exposed to CuCl₂ (Sigma-Aldrich 751944) at nominal concentrations of 0 and 325 μg/L from <1 hpf (before complete hardening of the protective chorion) to 4 hpf (sphere stage) at either 26.5 °C or 34 °C. The tested treatments were chosen based on previous studies^16,37. Six replicates for each treatment were used and each replicate consisted of a polypropylene beaker containing 150 ml of the respective treatment solutions and 166 ± 21 embryos. After the exposure, the embryos were rinsed thoroughly with fresh ZebTec system water and incubated at 26.5 °C until 96 hpf. Half of the water in each beaker was replaced every day to remove the wastes and dead embryos. Mortality rate and hatching efficiency were recorded daily until 96 hpf. At 96 hpf, larvae were washed, collected and stored at −80 °C until DNA and RNA were isolated.

Total Cu concentration in the exposure water was measured using an Inductively Coupled Plasma-Mass Spectrometer (ICPMS, Elan DRC II). The accuracy of analytical methods was checked using certified reference standards (ICP/MS Multi-Element Standards ICP-MS-QC2-1, ACCUStandard). The mean concentration and standard deviation in the 325 μg/L treatment were 280.4 ± 13.6 μg/L.