Early life tributyltin exposure has long term physiological effects on the zebrafish (Danio rerio) visual system

Abstract

Tributyltin (TBT) is an antiestrogenic endocrine disrupting compound used in the production of plastic, timber, and aquatic antifouling paints. Previous studies focusing on short-term effects of TBT exposure have identified immediate detrimental effects. Here, we evaluate whether a transient (24 h) exposure to TBT during development can cause persistent effects that remain after removal from treatment. Zebrafish (Danio rerio) larvae were exposed to environmentally relevant concentrations of TBT (0.04 and 0.4 μg/L) when they were either 3- or 7-days post-fertilization (dpf). After exposure, larvae were returned to recovery conditions and assessed 2-weeks, 4-weeks, or > 5 months postexposure. Exposure to 0.4 μg/L TBT at 3 dpf decreased total and distal retinal thicknesses. Adult (>5 month) photopic electroretinograms revealed physiological changes to photoreceptor a-wave and ON-bipolar cell b-wave components, with greater deficits in the 0.4 μg/L group. TBT exposure at 7 dpf significantly increased retinal inner plexiform layer thickness at 2-weeks, an effect that persisted to adulthood. Adult electroretinograms were also altered, with 0.04 μg/L TBT increasing and delaying a-wave and OFF-bipolar d-wave responses and increasing b-wave amplitude. Thus, the impact of TBT exposure depends on both concentration and exposure age, with retinal sequelae characterized by early anatomical and later physiological deficits. These data suggest that TBT exposure during critical periods of visual system development causes persistent age- and concentration-dependent deficits that are specific to the retina, revealing a previously unknown effect of this compound.

Keywords: Electroretinogram; Endocrine disruptor; Estrogen; Optomotor response; Vision.

Figure 1.. Early retinal differences occur after TBT exposure at 3 dpf.

Two weeks after exposure (top row) (A) total retinal thickness was significantly reduced in the 0.4 μg/L TBT exposure group. (B) Inner plexiform layer thickness and (C) inner nuclear layer thickness were not significantly affected by treatment. However (D) thickness of the photoreceptor layer was significantly lower in the 0.4 μg/L group. Four weeks after exposure (middle row) no differences in (E) total retinal, (F) inner plexiform layer, (G) inner nuclear layer, or (H) photoreceptor layer thicknesses were noted. Adult retinas (bottom row) also showed no difference in (I) total retinal thickness, (J) inner plexiform layer, (K) inner nuclear layer, or (L) photoreceptor layer thickness. Box and whisker plots show the median (center line) and mean (x) of the data. Control = solvent control (0.1% ethanol). Measurements were made from 2–4 larvae at each age and treatment group. Significant differences are denoted by asterisks.

Figure 2.. Persistent differences in retinal anatomy after 7 dpf TBT exposure were isolated to the inner retina.

Two weeks after exposure at 7 dpf (top row) showed no difference in (A) total retinal thickness but (B) a significant TBT-induced increase in thickness of the inner plexiform layer. (C) Inner nuclear layer and (D) photoreceptor layer thicknesses were not different. No differences in any retinal measurement (E-H) were noted 4-weeks after exposure (middle row). Measurements of adult retinas (bottom row) revealed a significant increase in inner plexiform layer thickness (J) but no change in (I) total retinal thickness, (K) inner nuclear layer thickness, or (L) photoreceptor layer thickness. Box and whisker plots show the median (center line) and mean (x) of the data. Significant differences are denoted by asterisks. Control = solvent control (0.1% ethanol).

Figure 3.. TBT exposure at 3 dpf alters adult ERG a-wave and b-wave responses.

(A) Average ERG traces from each treatment group show qualitative differences in responses. Red square pulse indicates the duration of light stimulation. During light stimulation, corneal-negative a-waves and positive b-waves were assessed; d-waves, evoked by light OFF, were also examined. Exposure to either 0.04 μg/L TBT or 0.4 μg/L TBT when zebrafish larvae were 3 dpf, significantly affected a-wave (B,E) and b-wave (C,F) components of the ERG, but not d-waves (D,G). The b-wave component is most sensitive to TBT exposure, with 0.04 μg/L TBT increasing the amplitude and delaying the response of this component. TBT at 0.4 μg/L, on the other hand, delayed both the a-wave and b-waves. For panels (A-G), N = 5 (control); N = 6 (0.04 μg/L TBT); N = 6 (0.4 μg/L TBT). Box and whisker plots show the median (center line) and mean (x) of the data. Data includes all technical replicates that meet criteria as described in methods. Control = solvent control (0.1% ethanol).

Figure 4.. Exposure to 0.04 μg/L TBT at 7 dpf affects all adult ERG components.

(A) Average ERG traces from each treatment group show qualitative differences in responses. Red square pulse indicates the duration of light stimulation. During light stimulation, corneal-negative a-waves and positive b-waves were assessed; d-waves, evoked by light OFF, were also examined. Exposure to 0.04 μg/L TBT for 24 hr when zebrafish larvae were 7 dpf, significantly affected a-wave (B,E), b-wave (C,F), and d-wave (D,G) components of the ERG. The amplitudes of all three components increased in the 0.04 μg/L TBT treatment group; 0.04 μg/L TBT also delayed a-wave and d-wave peak responses. For panels (A-G), N = 4 (control); N = 7 (0.04 μg/L TBT); N = 5 (0.4 μg/L TBT). Box and whisker plots show the median (center line) and mean (x) of the data. Data includes all technical replicates that meet criteria as described in methods. Control = solvent control (0.1% ethanol).