PMC

A-B) Bars represent average mean developmental malformations and mortality for 445–476 embryos analyzed over 4 independent experiments. Significant differences between groups were calculated with a binomial test that calculates lowest effect levels (LELs) for each endpoint. Red bars indicate significant increases in A-B) mortality, and C) any malformations compared to zinc adequate embryos exposed to 0 ppb arsenic. The same statistical approach was used to evaluate any significant effect of arsenic specifically in zinc deficient embryos and no significant difference was found.

Bars indicate the average amount (± SEM) of zinc, as measured by ICP-OES, in A) fish diet B) adult fish and C) embryos produced by the adult fish. B) Data was obtained on fish that were 4–6 months old and had been on the diet for at least 2 months. C) Embryos were 120 hpf at time of analysis. A-C) Data is representative of at least three independent experiments and n = 24 for diet, n = 35 for adults, and n = 34 for embryos. Significant differences between the zinc adequate and zinc deficient samples were calculated using t-tests and *** indicate significant differences between the groups where p < 0.001 respectively.

Bars represent mean (± SEM) mRNA levels of the indicated genes in zinc adequate (white bars), or zinc deficient (grey bars) embryos exposed to 0, 50, or 500 ppb arsenic. RNA was collected at 48 (A-D) and 120 (E-H) hpf. Data are from 2–3 independent experiments where (A-D) n = 9, or (E-H) n = 7–8. Significant differences between samples were calculated using two-way ANOVAs with results detailed in individual tables for each gene and time point. Bonferroni post-tests were used to determine differences between zinc adequate and zinc deficient embryos at a given arsenic dose where * and ** indicate significant differences between the groups where p < 0.05 and p < 0.01 respectively.

A-B) Bars are indicative of the mean (± SEM) amount of A) zinc, or B) calcium, as determined by ICP-OES, in zinc adequate or zinc deficient embryos continuously exposed to 0, 50, or 500 ppb arsenic starting at 6 hpf and collected at 120 hpf. Data is representative of at least three independent experiments and n = 9–12. C-E) Bars represent mean (± SEM) mRNA levels of indicated zinc transporters at 120 hpf in zinc adequate (white bars) or zinc deficient (grey bars) embryos exposed to 0, 50, or 500 ppb arsenic. Data represent an average of 7–8 replicates per treatment group and were obtained from 2 independent experiments. A-E) Significant differences between samples were calculated using two-way ANOVAs or one way ANOVAs as appropriate with Bonferroni post-tests. For post-tests *, **, and *** indicate significant differences between the groups where p < 0.05, p < 0.01, and p < 0.001 respectively.

Embryos were collected from zinc adequate or zinc deficient fish, exposed to 0, 50, or 500 ppb arsenic from 6–120 hpf and analyzed for larval activity. Locomotor activity of embryos at 120 hpf were measured by larval photomotor response assay and all data come from at least four independent experiments n = 228–329. A) Indicates movement (y-axis) over time where light conditions during the assay changed and are indicated by white (light) or black (dark) bars on the x-axis. B) A cumulative distribution plot illustrating the total distance moved over time. C) The overall area under the curve for movement was compared using a Kolmogorov-Smirnov test. The condition was considered significant if the P value was less than 0.01 and the percent change was greater than 10%.

A-G) Bars represent mean (± SEM) mRNA levels of the indicated genes in zinc adequate (white bars), or zinc deficient (grey bars). RNA was collected at the indicated time point and are from 3 independent experiments where n = 7–9. A-B) Bars show the dynamic nature of insulin gene expression over developmental time and ND denotes that transcript level was below detection limit. C) Significant difference between zinc adequate and zinc deficient insa mRNA levels at 0 hpf were calculated by t-test where ** indicates p < 0.01. (D-G) Significant differences between samples exposed to 0, 50, or 500 ppb arsenic were calculated using two-way ANOVAs with results detailed in individual tables for each gene and time point. Bonferroni post-tests were used to determine differences between zinc adequate and zinc deficient embryos at a given arsenic dose where * and ** indicate significant differences between the groups where p < 0.05 and p < 0.01 respectively.