Evaluation of Developmental Toxicity, Developmental Neurotoxicity, and Tissue Dose in Zebrafish Exposed to GenX and Other PFAS

Abstract

Background: Per- and polyfluoroalkyl substances (PFAS) are a diverse class of industrial chemicals with widespread environmental occurrence. Exposure to long-chain PFAS is associated with developmental toxicity, prompting their replacement with short-chain and fluoroether compounds. There is growing public concern over the safety of replacement PFAS.

Objective: We aimed to group PFAS based on shared toxicity phenotypes.

Methods: Zebrafish were developmentally exposed to 4,8-dioxa-3H-perfluorononanoate (ADONA), perfluoro-2-propoxypropanoic acid (GenX Free Acid), perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid (PFESA1), perfluorohexanesulfonic acid (PFHxS), perfluorohexanoic acid (PFHxA), perfluoro-n-octanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), or 0.4% dimethyl sulfoxide (DMSO) daily from 0-5 d post fertilization (dpf). At 6 dpf, developmental toxicity and developmental neurotoxicity assays were performed, and targeted analytical chemistry was used to measure media and tissue doses. To test whether aliphatic sulfonic acid PFAS cause the same toxicity phenotypes, perfluorobutanesulfonic acid (PFBS; 4-carbon), perfluoropentanesulfonic acid (PFPeS; 5-carbon), PFHxS (6-carbon), perfluoroheptanesulfonic acid (PFHpS; 7-carbon), and PFOS (8-carbon) were evaluated.

Results: PFHxS or PFOS exposure caused failed swim bladder inflation, abnormal ventroflexion of the tail, and hyperactivity at nonteratogenic concentrations. Exposure to PFHxA resulted in a unique hyperactivity signature. ADONA, PFESA1, or PFOA exposure resulted in detectable levels of parent compound in larval tissue but yielded negative toxicity results. GenX was unstable in DMSO, but stable and negative for toxicity when diluted in deionized water. Exposure to PFPeS, PFHxS, PFHpS, or PFOS resulted in a shared toxicity phenotype characterized by body axis and swim bladder defects and hyperactivity.

Conclusions: All emerging fluoroether PFAS tested were negative for evaluated outcomes. Two unique toxicity signatures were identified arising from structurally dissimilar PFAS. Among sulfonic acid aliphatic PFAS, chemical potencies were correlated with increasing carbon chain length for developmental neurotoxicity, but not developmental toxicity. This study identified relationships between chemical structures and in vivo phenotypes that may arise from shared mechanisms of PFAS toxicity. These data suggest that developmental neurotoxicity is an important end point to consider for this class of widely occurring environmental chemicals. https://doi.org/10.1289/EHP5843.

Figure 1.

Study design. Zebrafish were semi-statically exposed to test PFAS daily, from $0–5$ dpf. At 6 dpf. developmental toxicity, developmental neurotoxicity, and PFAS tissue concentrations were assessed. Test PFAS included in Study 1, solubilized in DMSO (final concentration 0.4% DMSO), are highlighted in light blue. Because GenX Free Acid was not stable in DMSO, the compound was retested in all three assays using DI water as a diluent in Study 2 (highlighted in blue). In Study 3, a set of sulfonic acid aliphatic PFAS solubilized in DMSO were tested in the DevTox and DNT assays (shown in green). Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; DevTox, developmental toxicity; DI, deionized; DMSO, dimethyl sulfoxide; DNT, developmental neurotoxicity; dpf, days post fertilization; GenX Free Acid, perfluoro-2-propoxypropanoic acid; PFAS, per- and polyfluoroalkyl substances; PFBS, perfluorobutanesulfonic acid; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid; PFPeS, perfluoropentanesulfonic acid.

Figure 2.

Measures of developmental toxicity in zebrafish exposed to PFAS. Zebrafish were semi-statically exposed to $0.04–80.0\mathrm{\mu }\mathrm{M}$ ADONA, GenX Free Acid, PFESA1, PFHxA, PFHxS, PFOA, or PFOS daily, from $0–5$ dpf. At 6 dpf, larvae were assessed for developmental toxicity. Representative images for (A) PFOS and (B) PFHxS are shown. DevTox assay scores for (C) PFOS, (D) PFHxS, (E) PFHxA, (F) PFOA, (G) ADONA, or (H) PFESA1 are shown. Significance relative to the 0.4% DMSO control was determined by a Kruskal-Wallis ANOVA with a Dunn’s multiple comparison test (*$p<0.05$, **$p<0.0001$). If a test for linear trend was significant ($p<0.05$), with developmental toxicity observed at the highest concentration tested, nonlinear regression was performed with Hill slope curve fitting for half-maximal ${\text{EC}}_{50}$ value determinations. $n=6$ larvae per concentration per chemical tested. Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; DevTox, developmental toxicity; DMSO, dimethyl sulfoxide; dpf, days post fertilization; ${\text{EC}}_{50}$, half maximal effective concentration; GenX Free Acid, perfluoro-2-propoxypropanoic acid; PFAS, per- and polyfluoroalkyl substances; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid.

Figure 2.

Measures of developmental toxicity in zebrafish exposed to PFAS. Zebrafish were semi-statically exposed to $0.04–80.0\mathrm{\mu }\mathrm{M}$ ADONA, GenX Free Acid, PFESA1, PFHxA, PFHxS, PFOA, or PFOS daily, from $0–5$ dpf. At 6 dpf, larvae were assessed for developmental toxicity. Representative images for (A) PFOS and (B) PFHxS are shown. DevTox assay scores for (C) PFOS, (D) PFHxS, (E) PFHxA, (F) PFOA, (G) ADONA, or (H) PFESA1 are shown. Significance relative to the 0.4% DMSO control was determined by a Kruskal-Wallis ANOVA with a Dunn’s multiple comparison test (*$p<0.05$, **$p<0.0001$). If a test for linear trend was significant ($p<0.05$), with developmental toxicity observed at the highest concentration tested, nonlinear regression was performed with Hill slope curve fitting for half-maximal ${\text{EC}}_{50}$ value determinations. $n=6$ larvae per concentration per chemical tested. Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; DevTox, developmental toxicity; DMSO, dimethyl sulfoxide; dpf, days post fertilization; ${\text{EC}}_{50}$, half maximal effective concentration; GenX Free Acid, perfluoro-2-propoxypropanoic acid; PFAS, per- and polyfluoroalkyl substances; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid.

Figure 3.

Locomotor activity in zebrafish developmentally exposed to PFAS. Zebrafish were semi-statically exposed to $4.4–80.0\mathrm{\mu }\mathrm{M}$ ADONA, PFESA1, PFHxA, PFHxS, or PFOA, $0.2–3.1\mathrm{\mu }\mathrm{M}$ PFOS, or 0.4% DMSO as a vehicle control daily from $0–5$ dpf. At 6 dpf, larvae were assessed for developmental toxicity. Morphologically normal larvae with inflated swim bladders were subjected to behavioral testing. (A, C, E, G, I, K) Distance moved (cm) each 2-min period over the entire 40-min testing period are shown. (B, D, F, H, J, L) To make statistical comparisons, the mean distance moved during each 10-min light 1 (L1), 10-min light 2 (L2), 10-min dark 1 (D1), or 10-min dark 2 (D2) periods are shown. For all chemicals except PFESA1, 14–23 larvae were tested per chemical concentration and the same DMSO control larvae ($n=394$) were used. PFESA1 was tested separately ($n=35–40$ per chemical per concentration; 339 DMSO control larvae were evaluated). Repeated measures ANOVA models were run separately by period (L1, L2, D1, or D2). If a significant effect of concentration was detected ($p<0.0125$), within-period pairwise comparisons to control were computed using t-tests with a Dunnett adjustment for multiple comparisons (*$p<0.05$, **$p<0.001$). Significance relative to period-specific DMSO controls are shown. Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; ANOVA, analysis of variance; D, dark period; DMSO, dimethyl sulfoxide; dpf, days post fertilization; L, light period; PFAS, per- and polyfluoroalkyl substances; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid.

Figure 3.

Locomotor activity in zebrafish developmentally exposed to PFAS. Zebrafish were semi-statically exposed to $4.4–80.0\mathrm{\mu }\mathrm{M}$ ADONA, PFESA1, PFHxA, PFHxS, or PFOA, $0.2–3.1\mathrm{\mu }\mathrm{M}$ PFOS, or 0.4% DMSO as a vehicle control daily from $0–5$ dpf. At 6 dpf, larvae were assessed for developmental toxicity. Morphologically normal larvae with inflated swim bladders were subjected to behavioral testing. (A, C, E, G, I, K) Distance moved (cm) each 2-min period over the entire 40-min testing period are shown. (B, D, F, H, J, L) To make statistical comparisons, the mean distance moved during each 10-min light 1 (L1), 10-min light 2 (L2), 10-min dark 1 (D1), or 10-min dark 2 (D2) periods are shown. For all chemicals except PFESA1, 14–23 larvae were tested per chemical concentration and the same DMSO control larvae ($n=394$) were used. PFESA1 was tested separately ($n=35–40$ per chemical per concentration; 339 DMSO control larvae were evaluated). Repeated measures ANOVA models were run separately by period (L1, L2, D1, or D2). If a significant effect of concentration was detected ($p<0.0125$), within-period pairwise comparisons to control were computed using t-tests with a Dunnett adjustment for multiple comparisons (*$p<0.05$, **$p<0.001$). Significance relative to period-specific DMSO controls are shown. Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; ANOVA, analysis of variance; D, dark period; DMSO, dimethyl sulfoxide; dpf, days post fertilization; L, light period; PFAS, per- and polyfluoroalkyl substances; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid.

Figure 4.

Media concentrations of test PFAS at 6 dpf. Zebrafish were semi-statically exposed to $4.4–80.0\mathrm{\mu }\mathrm{M}$ PFHxS, PFHxA, PFOA, ADONA, or PFESA1 or $0.2–3.1\mathrm{\mu }\mathrm{M}$ PFOS daily from $0–5$ dpf. At 6 dpf, media was collected for targeted analytical chemistry ($n=3$). Measured media concentrations for (A) PFOS, (B) PFHxS, (C) PFHxA, (D) PFOA, (E) ADONA, and (F) PFESA1 are shown. One observation for PFOA nominal media concentration $14.1\mathrm{\mu }\mathrm{M}$ was $<\text{MDL}$ and therefore not shown on the plot. However, it was included in the regression analysis using the value MDL/sqrt(2). Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; dpf, days post fertilization; MDL, method detection limit; PFAS, per- and polyfluoroalkyl substances; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid; sqrt, square-root.

Figure 5.

Internal tissue doses of test PFAS at 6 dpf. Zebrafish were semi-statically exposed to $25.1–80.0\mathrm{\mu }\mathrm{M}$ ADONA, PFOA, PFESA1, or PFHxA, or $14.0–44.8\mathrm{\mu }\mathrm{M}$ PFHxS, or $1.0–3.1\mathrm{\mu }\mathrm{M}$ PFOS. At 6 dpf, larvae were pooled and flash frozen ($n=4$ biological replicates with 10 pooled larvae per replicate) for targeted analytical chemistry. Measured internal tissue doses for (A) PFOS, (B) PFHxS, (C) PFHxA, (D) PFOA, (E) ADONA, and (F) PFESA1 are shown. Significance was determined by a Welch’s ANOVA followed by a Dunnett T3 test ($p<0.05$). Additional one-sample Student’s t-tests were performed for PFHxA, PFHxS, PFOA, and PFOS ($p<0.05$). Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; ANOVA, analysis of variance; Dil, dilution; dpf, days post fertilization; PFAS, per- and polyfluoroalkyl substances; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid.

Figure 6.

Developmental and behavioral assays and media and tissue concentrations in zebrafish exposed to GenX Free Acid diluted in DI water. (A) Developmental toxicity scores at 6 dpf obtained from zebrafish developmentally exposed to $0.04–80.0\mathrm{\mu }\mathrm{M}$ GenX Free Acid diluted in DI water daily, from $0–5$ dpf. Significance was determined by one-way ANOVA with a Tukey’s multiple comparison test ($p<0.05$). $n=6$ larvae per concentration tested. For the DNT assay, zebrafish were exposed to $4.4–80.0\mathrm{\mu }\mathrm{M}$ GenX Free Acid daily, from $0–5$ dpf. At 6 dpf, locomotor activity was assessed. (B) Distance moved (cm) each 2-min period or (C) mean distance moved during the light 1 (L1), light 2 (L2), dark 1 (D1), or dark 2 (D2) 10-min periods are shown. Repeated measures ANOVA models were run separately by period (L1, L2, D1, or D2). If a significant effect of concentration was detected ($p<0.0125$), within-period pairwise comparisons to control were computed using t-tests with a Dunnett adjustment for multiple comparisons (*$p<0.05$). $n=17–21$ zebrafish per concentration and 161 DI water control larvae were assessed. (D) Media concentrations and (E) internal tissue dose at 6 dpf following daily exposure to $25.1–80.0\mathrm{\mu }\mathrm{M}$ GenX Free Acid. $n=3$ media replicates and $n=4$ biological replicates each comprising 10 pooled larvae. Significance was determined by a Welch’s ANOVA followed by a Dunnett T3 test ($p<0.05$). Additional one-sample Student’s t-tests were performed ($p<0.05$). Note: ANOVA, analysis of variance; D, dark phase; DevTox, developmental toxicity; DI, deionized; Dil, dilution; DNT, developmental neurotoxicity; dpf, days post fertilization; GenX Free Acid, perfluoro-2-propoxypropanoic acid; L, Light period.

Figure 7.

Measures of developmental toxicity in zebrafish exposed to alkyl sulfonic acid PFAS. Zebrafish were semi-statically exposed to $1.7–100.0\mathrm{\mu }\mathrm{M}$ PFBS, PFPeS, PFHxS, PFHpS, or PFOS or 0.4% DMSO daily from $0–5$ dpf. At 6 dpf, larvae were assessed for developmental toxicity. DevTox assay scores for (A) PFBS, (B) PFPeS, (C) PFHxS, (D) PFHpS, or (E) PFOS are shown. Significance relative to the 0.4% DMSO control was determined by a Kruskal-Wallis ANOVA with a Dunn’s multiple comparison test (**$p<0.0001$). If a test for linear trend was significant ($p<0.05$), with developmental toxicity observed at the highest concentration tested, nonlinear regression was performed with Hill slope curve fitting for half-maximal ${\text{EC}}_{50}$ value determinations. $n=8$ larvae per concentration per chemical tested. Note: ANOVA, analysis of variance; DevTox, developmental toxicity; DMSO, dimethyl sulfoxide; dpf, days post fertilization; ${\text{EC}}_{50}$, half maximal effective concentration; PFAS, per- and polyfluoroalkyl substance; PFBS, perfluorobutanesulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFHxS, perfluorohexanesulfonic acid; PFOS, perfluorooctanesulfonic acid; PFPeS, perfluoropentanesulfonic acid.

Figure 8.

Locomotor activity in zebrafish exposed to alkyl sulfonic acid PFAS. Zebrafish were semi-statically exposed to $5.5–100.0\mathrm{\mu }\mathrm{M}$ PFBS, $3.1–5.5\mathrm{\mu }\mathrm{M}$ PFPeS, $3.1–31.4\mathrm{\mu }\mathrm{M}$ PFHxS, $1.7–9.8\mathrm{\mu }\mathrm{M}$ PFHpS, or $0.5–3.1\mathrm{\mu }\mathrm{M}$ PFOS daily from $0–5$ dpf. For all chemicals except PFPeS, 14–25 larvae were tested per chemical concentration and the same DMSO control larvae ($n=327$) were used. PFPeS was tested separately ($n=21–22$ larvae per concentration; 186 DMSO control larvae were evaluated). At 6 dpf, larvae were assessed for developmental toxicity. Morphologically normal larvae with inflated swim bladders were subjected to behavioral testing. (A, C, E, G, I) Distance moved (cm) each 2-min period or (B, D, F, H, J) mean distance moved during the light 1 (L1), light 2 (L2), dark 1 (D1), or dark 2 (D2) 10-min periods are shown. Repeated measures ANOVA models were run separately by period (L1, L2, D1, or D2). If a significant effect of concentration was detected ($p<0.0125$), within-period pairwise comparisons to control were computed using t-tests with a Dunnett adjustment for multiple comparisons (*$p<0.05$). ANOVA, analysis of variance; D, dark phase; DMSO, dimethyl sulfoxide; dpf, days post fertilization; L, light period; PFAS, per- and polyfluoroalkyl substances; PFBS, perfluorobutanesulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFHxS, perfluorohexanesulfonic acid; PFOS, perfluorooctanesulfonic acid; PFPeS, perfluoropentanesulfonic acid.

Figure 8.

Locomotor activity in zebrafish exposed to alkyl sulfonic acid PFAS. Zebrafish were semi-statically exposed to $5.5–100.0\mathrm{\mu }\mathrm{M}$ PFBS, $3.1–5.5\mathrm{\mu }\mathrm{M}$ PFPeS, $3.1–31.4\mathrm{\mu }\mathrm{M}$ PFHxS, $1.7–9.8\mathrm{\mu }\mathrm{M}$ PFHpS, or $0.5–3.1\mathrm{\mu }\mathrm{M}$ PFOS daily from $0–5$ dpf. For all chemicals except PFPeS, 14–25 larvae were tested per chemical concentration and the same DMSO control larvae ($n=327$) were used. PFPeS was tested separately ($n=21–22$ larvae per concentration; 186 DMSO control larvae were evaluated). At 6 dpf, larvae were assessed for developmental toxicity. Morphologically normal larvae with inflated swim bladders were subjected to behavioral testing. (A, C, E, G, I) Distance moved (cm) each 2-min period or (B, D, F, H, J) mean distance moved during the light 1 (L1), light 2 (L2), dark 1 (D1), or dark 2 (D2) 10-min periods are shown. Repeated measures ANOVA models were run separately by period (L1, L2, D1, or D2). If a significant effect of concentration was detected ($p<0.0125$), within-period pairwise comparisons to control were computed using t-tests with a Dunnett adjustment for multiple comparisons (*$p<0.05$). ANOVA, analysis of variance; D, dark phase; DMSO, dimethyl sulfoxide; dpf, days post fertilization; L, light period; PFAS, per- and polyfluoroalkyl substances; PFBS, perfluorobutanesulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFHxS, perfluorohexanesulfonic acid; PFOS, perfluorooctanesulfonic acid; PFPeS, perfluoropentanesulfonic acid.

Figure 9.

Identification of shared phenotypes between structurally similar PFAS. (A) Heatmap depicting LOEC values for the DevTox assay and significant hyperactivity in the L1, L2, D1, and/or D2 periods of the DNT assay (Studies 1, 2, and 3). If chemicals were replicated in Study 1 and Study 2, the lowest observed LOEC value was used. Linear regression of (B) Study 3 DevTox assay ${\text{EC}}_{50}$ or (C) Study 3 DNT assay LOEC values for aliphatic sulfonic acid PFAS. Note: ADONA, 4,8-dioxa-3H-perfluorononanoate; D, dark period; DevTox, developmental toxicity; DNT, developmental neurotoxicity; ${\text{EC}}_{50}$, half maximal effective concentration; GenX Free Acid, perfluoro-2-propoxypropanoic acid; L, light period; LOEC, lowest observed effect concentration; PFAS, per- and polyfluoroalkyl substances; PFBS, perfluorobutanesulfonic acid; PFESA1, perfluoro-3,6-dioxa-4-methyl-7-octene-1-sulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFHxA, perfluorohexanoic acid; PFHxS, perfluorohexanesulfonic acid; PFOA, perfluoro-n-octanoic acid; PFOS, perfluorooctanesulfonic acid; PFPeS, perfluoropentanesulfonic acid.