Illustration of early life stage zebrafish acute toxicity test (from left to right): breeding, spawning, collection of eggs, selection of eggs using an inverted microscope and distribution of embryos over pre incubated 24 well plates prepared with respective exposure concentrations/control. n = 20/exposure condition + 4 internal negative control / plate; hpf = hours post fertilisation.

Morphology and Development of zebrafish embryos. (A) Microscopic images of teratogenic effects in zebrafish embryos exposed to 0 µM (control), 0.80 µM Cu, 0.25 µM Cd and the Cu+Cd co-exposure at 96 hpf. (B) Illustration of malformations in hatched zebrafish embryos (impaired eye development, tail curvature, oedema, blood accumulation). (C) Visualization of zebrafish embryo hatching at 96 hpf. The colours represent hatching failure in Cu (green), Cd (blue) and Cu+Cd (red). Grey colour represents hatching success. (D) Graphical representation of hatching rate of zebrafish embryos in exposed and control groups at various time points. The data points represent average ± standard error of mean (SEM) of minimum 20 biological replicates. Two independent experiments were performed and the results of two experiments confirm each other. *p < 0.05.

Behavioral analysis of zebrafish larvae: (A) Representative video tracks of zebrafish larvae exposed to 0 µM (control), 0.80 µM Cu, 0.25 µM Cd and Cu+Cd at 96hpf. (B-E) effects of single and combined exposure of Cu and Cd at 96hpf on: (B) movement in surviving embryos (C) swimming speed (mm/s), (D) swimming duration (seconds) and (E) swimming distance (mm). The data points represent average ± standard error of mean (SEM) of minimum 20 biological replicates. Two independent experiments were performed and the results of two experiments confirm each other. *p < 0.05, * *p < 0.01, * ** *p < 0.0001.

Metal accumulation. (A) Cu and Cd accumulation in zebrafish embryos 96 hpf exposed to 0.80 μM Cu, 0.25 μM Cd and Cu+Cd. The values are the average of ± standard error of mean (SEM) of minimum ten biological replicates in each exposure group. (B) Cu and Cd concentration in zebrafish adults exposed to 0.80 μM Cu, 0.25 μM Cd and Cu+Cd. The values are the average ± SEM of minimum ten biological replicates in each exposure group All the metal present in the experimental media were in the dissolved phase and the total measured metal concentration was 94 – 100 % of the desired nominal concentrations. Two independent experiments were performed and the results of two experiments confirm each other. For embryos D′Agostino-Pearson normality test failed, therefore, a non-parametric test (Kruskal-Wallis one way ANOVA) on ranks was performed and Dunn’s method was used for multiple comparison. For adults, a parametric test was performed, and Holm-Sidak’s multiple comparison test was used to compare the treatment and control group. *p < 0.05, * *p < 0.01, * **p < 0.001, * ** *p < 0.0001.

Gene expression analysis of Zebrafish embryo. Relative gene expression levels of genes representing different classes of genes (antioxidant, apoptosis, development, heat-shock, DNA repair, and metal transport) in zebrafish embryos exposed to 0.80 μM Cu, 0.25 μM Cd and Cu+Cd at 96 hpf. The values are the average ± standard error of mean (SEM) of minimum six biological replicates. Two independent experiments were performed and the results of two experiments confirm each other. A non-parametric test was used to compare the treatment and control groups. *p < 0.05, * *p < 0.01, * **p < 0.001.

Gene expression analysis of zebrafish adults. Graphs representing the relative gene expression levels of genes representing different classes of genes (antioxidative, apoptosis, metal transport, heat-shock and DNA repair) in gills, liver and gut of adult zebrafish exposed to 0.80 μM Cu, 0.25 μM Cd and Cu+Cd after 7 days exposure. The values are the average ± standard error of mean (SEM) of minimum six biological replicates in each exposure condition. Two independent experiments were performed and the results of two experiments confirm each other. A non-parametric test based on ranking (Kruskal-Wallis test) was used to compare the treatment and control groups. *p < 0.05, * *p < 0.01, * **p < 0.001.