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Figure 6 The effect of Caf@CDs on early development of the zebrafish embryo. (a) Images of zebrafish embryos after exposure to caffeine (Caf), CDs or Caf@CDs from 4 till 24 hpf. Embryos exposed to Caf@γ-CD show underdeveloped eyes, ears and severely impaired convergent extension. Less severe morphological abnormalities were also present in embryos exposed to 5 mM Caf. Exposure to 5 mM α-CD, 1.25 mM Caf@α-CD, or 5 mM Caf@β-CD was lethal to the embryos. Examples of live embryos at a most advanced developmental stage are shown. Scale bar, 1 mm. (b) Changes in the proportion between the dead, morphologically abnormal (affected), and unaffected (normal) embryos. The percentage of embryos which died upon treatment with Caf or Caf@CDs was significantly higher than that of untreated control. Also, pure α-CD was more toxic than the other two CDs, whereas β-CD and γ-CD had no effect on both the fish survival and their morphology. In order to reduce the number of animals, in the case of treatments which caused 100% rapid mortality, the experiments were repeated twice. Bars present mean values, n is the number of repetitions, each on 24 embryos. Mann-Whitney U test was performed on samples with ≥3 biological replicas. Statistically significant differences (p < 0.01) were marked with different letters.