Adverse Outcomes Following Exposure to Perfluorooctanesulfonamide (PFOSA) in Larval Zebrafish (Danio rerio): A Neurotoxic and Behavioral Perspective

Abstract

Toxicity mechanisms of per- and polyfluoroalkyl substances (PFASs), a chemical class present in diverse ecosystems, as well as many of their precursors, have been increasingly characterized in aquatic species. Perfluorooctanesulfonamide (PFOSA, C₈H₂F₁₇NO₂S) is a common precursor of perfluorooctane sulfonic acid (PFOS), a long-chain PFAS. Here, we assessed sub-lethal endpoints related to development, oxidative stress, transcript levels, and distance moved in zebrafish embryos and larvae following continuous exposure to PFOSA beginning at 6 h post-fertilization (hpf). PFOSA decreased survival in fish treated with 1 µg/L PFOSA; however, the effect was modest relative to the controls (difference of 10%). Exposure up to 10 µg/L PFOSA did not affect hatch rate, nor did it induce ROS in 7-day-old larvae fish. The activity of larval fish treated with 100 µg/L PFOSA was reduced relative to the solvent control. Transcripts related to oxidative stress response and apoptosis were measured and BCL2-associated X, apoptosis regulator (bax), cytochrome c, somatic (cycs), catalase (cat), superoxide dismutase 2 (sod2) were induced with high concentrations of PFOSA. Genes related to neurotoxicity were also measured and transcript levels of acetylcholinesterase (ache), elav-like RNA binding protein 3 (elavl3), growth-associated protein 43 (gap43), synapsin II (syn2a), and tubulin 3 (tubb3) were all increased in larval fish with higher PFOSA exposure. These data improve our understanding of the potential sub-lethal toxicity of PFOSA in fish species.

Keywords: aquatic toxicology; behavior; neurotoxicity; perfluorinated chemicals.

Figure 1

Percent of surviving zebrafish following exposure to one of either ERM, 0.1% DMSO or 0.1, 1, 10, or 100 µg/L PFOSA over 7 days. Error bars (S.E.M.) are small and within symbols in some cases.

Figure 2

Total percent of hatched zebrafish embryos/larvae following exposure to one of either ERM, 0.1% DMSO or 0.1, 1, 10, or 100 µg/L PFOSA over 7 days.

Figure 3

Normalized reactive oxygen species (to µg/mL media/protein). Each circle represents a biological replicate; the mean (±S.D.) is represented by the horizontal line (One-Way ANOVA and Dunnett’s multiple comparisons test; n = 5/treatment). ns = not significant.

Figure 4

The distance moved in each of the light and dark zones (10 min bins) of 7-day zebrafish larvae exposed to 0.1% DMSO, ERM or 0.1, 1, 10, or 100 µg/L PFOSA. Graphs are the combined output from three independent VMR runs. Columns depict mean (±S.D.) (Kruskal–Wallis test and Dunn’s multiple comparisons test; n = 8–12 fish/treatment/run). Asterisk indicates difference at ** p < 0.01.

Figure 5

Linear regression for relative expression of (A) bax, (B) cat, and (C) sod2 in 7-day old zebrafish. The DMSO and ERM group were combined (“Cntl”). Each circle indicates a biological replicate or beaker of pooled fish (n = 4 to 6). The solid line indicates the relationship between expression and the region between the two outermost dotted lines is the 95% confidence interval of the X intercept.

Figure 6

Relative expression of (A) bax and (B) elavl3 in 7-day old larval zebrafish exposed to 0.1% DMSO, ERM or 0.1, 1, or 10 µg/L PFOSA. Each circle is a beaker of pooled fish (biological replicate), and the mean (±S.D.) is indicated by the horizontal line (One-Way ANOVA, Dunnett’s multiple comparisons test; n = 4 to 6). Asterisk indicates * p < 0.05 or ** p < 0.01.

Figure 7

Linear regression for relative expression of (A) ache, (B) elavl3, (C) gap43, (D) syn2a, and (E) tubulin (tubb3) in 7-day old zebrafish. The DMSO and ERM group were combined (“Cntl”). Each circle indicates a biological replicate or beaker of pooled fish (n = 4 to 6). The solid line indicates the relationship between expression and the region between the two outermost dotted lines is the 95% confidence interval of the X intercept.