Survival, Growth, and Reproduction Responses in a Three-Generation Exposure of the Zebrafish (Danio rerio) to Perfluorooctane Sulfonate

Abstract

A prior multigenerational perfluorooctane sulfonic acid (PFOS) exposure investigation in zebrafish reported adverse effects at 0.734 µg/L, among the lowest aquatic effect levels for PFOS reported to date. The present three-generation PFOS exposure quantified survival, growth, reproduction, and vitellogenin (VTG; egg yolk protein) responses in zebrafish, incorporating experimental design and procedural improvements relative to the earlier study. Exposures targeting 0.1, 0.6, 3.2, 20, and 100 µg/L in parental (P) and first filial (F1) generations lasted for 180 days post fertilization (dpf) and the second filial generation (F2) through 16 dpf. Survival decreased significantly in P and F2 generation exposures, but not in F1, at the highest PFOS treatment (100 µg/L nominal, 94-205 µg/L, measured). Significant adverse effects on body weight and length were infrequent, of low magnitude, and occurred predominantly at the highest exposure treatment. Finally, PFOS had no significant effects on P or F1 egg production and survival or whole-body VTG levels in P or F1 male fish. Overall, the predominance and magnitude of adverse PFOS effects at <1 µg/L reported in prior research were largely nonrepeatable in the present study. In contrast, the present study indicated a threshold for ecologically relevant adverse effects in zebrafish at 117 µg/L (SE 8 µg/L, n = 10) for survival and 47 µg/L (SE 11 µg/L, n = 19) for all statistically significant negative effects observed. Environ Toxicol Chem 2024;43:115-131. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Keywords: Growth; Multigenerational exposure; Perfluorooctane sulfonic acid (PFOS); Reproduction; Zebrafish.

FIGURE 1:

Graphical flow chart of the multigenerational exposure of zebrafish to perfluorooctanesulfonic acid (PFOS) with associated exposure methods, exposure times (dpf = days post fertilization), and experimental endpoints that were measured.

FIGURE 2:

(A–D) Relationship between fish weight and number of fish present in each replicate at days 60 and 90 in the P and F1 generations. Analysis of covariance detected significant negative relationships (p < 0.05) between fish weight and fish number at each time point in each generation. Perfluorooctanesulfonic acid (PFOS) values represent cumulative measured concentrations in the exposure water reported as significant figures based on analytical method detection sensitivity. dpf = days post fertilization.

FIGURE 3:

Bar charts represent the mean cumulative measured concentrations of perfluorooctanesulfonic acid (PFOS) in the exposure water for the P, F1, and F2 generation exposures at the completion of each experiment (180, 180, and 16 days post fertilization [dpf], respectively). Values above bars represent mean concentrations reported as significant figures based on analytical method detection sensitivity and error bars represent standard deviations. The n values represent the number of analytical sample replicates contributing to each PFOS measured concentration, including controls (not shown). All measurements of control samples returned values below the limit of detection, which was 12 ng/L.

FIGURE 4:

Zebrafish survival (%) in the P, F1, and F2 generations. Bars represent mean survival (n = 5), and error bars represent standard deviations. Exposure concentrations represent nominal values; the measured perfluorooctanesulfonic acid (PFOS) values are provided in Table 1 to provide analytically supported cumulative exposure concentrations. Asterisks represent statistically significant differences (p ≤ 0.01) relative to controls.

FIGURE 5:

(A–G) Whole-body wet weights in the P, F1, and F2 generations representing the mean and standard deviation (n = 5) of the total fish weights in each replicate (relative to controls). Exposure concentrations represent measured cumulative mean perfluorooctanesulfonic acid (PFOS) concentrations through to the measurement endpoint reported as significant figures based on analytical method detection sensitivity. Asterisks represent statistically significant differences (p < 0.05) relative to controls. dpf = days post fertilization.

FIGURE 6:

(A–D) Zebrafish egg production/female and egg survival (24 h) in the P and F1 generations, with bars representing means and error bars representing standard deviations (n = 5). The measurements represent the summation of 8 weekly breeding trials, and perfluorooctanesulfonic acid (PFOS) values are cumulative measured exposure concentrations reported as significant figures based on analytical method detection sensitivity. There were no statistically significant differences (p < 0.05) relative to controls.

FIGURE 7:

(A and B) Vitellogenin (VTG) concentrations in male zebrafish whole-body tissues collected at 180 days post fertilization (dpf) in both the P and F1 generations represented in a box and whisker chart in which n = 5. The “X” and the solid line within each bar represent the mean and median values, respectively. Perfluorooctanesulfonic acid (PFOS) values represent the cumulative measured water concentrations at 180 dpf reported as significant figures based on analytical method detection sensitivity. There were no statistically significant effects of PFOS relative to controls in either the P or F1 generations (p > 0.05).