Toxicity, uptake kinetics and behavior assessment in zebrafish embryos following exposure to perfluorooctanesulphonicacid (PFOS)

Abstract

Perfluorooctanesulphonicacid (PFOS), a persistent organic contaminant, has been widely detected in the environment, wildlife and humans, but few studies have assessed its effect on aquatic organisms. The present study evaluated the effect of PFOS on zebrafish embryos. Zebrafish embryos exhibited developmental toxicity of bent spine, uninflated swim bladder, decreased heart rate and affected spontaneous movement after exposure to various PFOS concentrations (0-8mg/L) from 6 to 120h post-fertilization (hpf). The LC(50) at 120hpf was 2.20mg/L and the EC(50) at 120hpf was 1.12mg/L. Continuous exposure to PFOS from 1 to 121hpf resulted in a steady accumulation with no evidence of elimination. PFOS induced cell death at 24hpf was consistently found in the brain, eye, and tail region of embryos. PFOS exposure induced lesions in the muscle fibers with histological examination. Behavior assessment of PFOS in zebrafish embryos elevated the basal rate of swimming after 4 days of exposure, and larvae exposed to PFOS (0.25-4mg/L) for only 1h at 6dpf swam faster with increasing PFOS concentration. Embryos/larvae exposed to 8mg/L PFOS for 24h periods from 1 to 121hpf showed the highest incidence of malformations in the 97-121hpf window. This is the first study to define uptake kinetics and to focus on behavioral consequences following PFOS exposure in zebrafish. Our results further the understanding of the toxicity of PFOS to aquatic organisms and suggest the need for additional research to identify the mode of PFOS toxicity.

Fig. 1

The control (a, b) and dominant malformation images of PFOS treated larvae (c, d) at 120 hpf. SB: swim bladder; BS: bent spine;USB: uninflated swim bladder; MT: malformed tail. Scale bars = 0.5 mm.

Fig. 2

Concentration-response curves at 120 hpf for PFOS exposure. Embryos were exposed to various concentrations of PFOS from 6 to 120 hpf.

Fig. 3

Uptake of PFOS (4.0 μM) in zebrafish embryos exposed from 1 to 121 hpf. The values are presented as mean±SEM (n=3).

Fig. 4

The number of heart beats over a 10 second period for zebrafish embryos at 48 and 60 hpf after exposure to PFOS at various concentrations. Asterisks indicate a statistically significant difference from control (*P < 0.05; **P<0.001). Values represent the mean±SEM of three replicates of 10 embryos each.

Fig. 5

Spontaneous movements (number of bends per min) of zebrafish embryos at different times after exposure to PFOS at various concentrations (open circles) vs. controls (filled circles). Each data point represents the average bends per minute of 45 embryos±SEM (15 embryos per treatment with a total of three repeats).

Fig. 6

Apoptotic cells (bright white) in whole mount embryos at 24 hpf after expose to PFOS at 1.0-8.0 μM vs. controls.

Fig. 7

The swimming speed of 5 dpf larvae exposed to 0-2.0 μM PFOS from 6-96 hpf (A) or larvae at 6 dpf after 1h exposure to 0 - 8.0 μM PFOS (B). Asterisks indicate a significant difference from control (P < 0.05). Values represent the mean speed in 60 s intervals±SEM with three replicates of 8-10 larvae each.

Fig. 8

The swimming speed of larvae at 6 dpf when subject to light stimulation after exposure to PFOS at concentrations of 0.5-2.0 μM (open circles) from 6 to 96h vs. controls (filled circles). Speed was analyzed for 120 min using 20 min light (visible light) followed by a 20 min interval of dark (infrared light) in 24-well plates.

Fig. 9

Vascular patterning of 96 hpf fli-1 zebrafish larvae in control (a) and treatment groups (b, c) exposed to 1.0-8.0 μM PFOS from 6 to 96 hpf. Aberrant patterning includes crossing (arrows), split or absent (asterisks), wider or thinner (arrowhead) blood vessels. Scale bar = 50 μm.

Fig. 10

Blood vessels in the eyes of 96 hpf fli-1 zebrafish larvae from control (a, d) and treatment groups (b-c, e-f) exposed to 1.0-8.0 μM PFOS from 6 to 96 hpf. Abnormalities include fusion (bold arrows) or absence (asterisks). Scale bars = 200 μm (a–c) and 50 μm (d-f).

Fig. 11

Percent of total malformation (solid line), malformation of blood vessels in eyes (dash line) or trunks (dash dot line) of fli-1 zebrafish larvae at 96 hpf after expose to 0-8.0 μM PFOS from 6 to 96 hpf. Dots sharing the same letter at the same concentration indicate no significant difference at P = 0.05. The values are presented as mean±SEM (n=3).

Fig. 12

Sensitivities of zebrafish embryos exposed to 16.0 μM PFOS continuous for 24 h at different stages. (A) Percent malformation at 192 hpf; (B) Swimming speed of larvae at 6 dpf; (C) Uptake of PFOS at the end of each 24 h exposure period; (D) Typical malformation found at 144 hpf: opaque head of apparent necrosis (arrows), bent spine (cyphosis), and uninflated swim bladder. Bars sharing the same letter indicate no significant difference at P = 0.05. The values are presented as mean±SEM (n=3). Scale bar = 0.5 mm.