Figure 1. EFS-Wasp1 (Fish Management Systems Ltd., Carrickfergus, U.K.) chamber for electrical stunning with larvae (Danio rerio) in embryo media.

Figure 2. Results for zebrafish embryo and larva euthanasia, presented per experimental dish (results from <12 h post fertilization, 24 h post fertilization, and 4 days post fertilization pooled, n = 20–202). The dishes in which treatments lead to no surviving embryo or larva were scored as “passed” (green), whereas the dishes with one-or-more surviving embryos or larvae were scored as “failed” (red). For the results per individual time point, see Table 4.

Figure 3. Survival rates for zebrafish following the control treatment, hypothermic shock, or electrical stunning, at 3 different developmental stages (<12 hpf, 24 hpf, and 4 dpf), presented as result per dish. Each dot represents one dish, with the total number of dishes per group indicated at the top of each column (n = 0–77). hpf; hours post fertilization. dpf; days post fertilization.

Figure 4. Survival rates for zebrafish following tricaine, 2-phenoxyethanol (2-PE), or benzocaine treatment, at 3 different developmental stages (<12 hpf, 24 hpf, and 4 dpf), presented as result per dish. Each dot represents one dish, with the total number of dishes per group indicated at the top of each column (n = 9–21). hpf; hours post fertilization. dpf; days post fertilization.

Figure 5. Survival rates for zebrafish following lidocaine hydrochloride (HCl), lidocaine HCl with ethanol (EtOH), or clove oil treatment, at 3 different developmental stages (<12 hpf, 24 hpf, and 4 dpf), presented as result per dish. Each dot represents one dish, with the total number of dishes per group indicated at the top of each column (n = 12–32). Statistical difference between the treatments was assessed by the chi-squared test (p < 0.0001), with post hoc analysis using Fisher’s exact test; ****; p < 0.0001. Note that the statistical analyses were performed on passed/failed dish frequencies (see Table 4 for details) and not on the continuous data presented here. ns; no statistical significance (p = 0.2987). hpf; hours post fertilization. dpf; days post fertilization.

Figure 6. Aversive behavior in zebrafish larvae (4 days post fertilization) during electrical stunning, anesthetic overdose, and hypothermic shock, presented as result per dish. Each experimental dish (n = 3–15 per treatment) contained 10–15 larvae. Dishes were scored as “No larvae”, “Some larvae”, or “All larvae” depending on the proportion of the larvae displaying aversive behavior.