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Developmental Exposure to Low Concentrations of Two Brominated Flame Retardants, BDE-47 and BDE-99, Causes Life-Long Behavioral Alterations in Zebrafish

Lilah Glazer, Corinne N. Wells, Meghan Drastal, Kathryn-Ann Odamah, Richard E. Galat, Mamta Behl, Edward D. Levin.
Neurotoxicology (2017). DOI: https://doi.org/10.1016/j.neuro.2017.09.007 PMID: 28935585

Publication

Abstract

BACKGROUND:
Polybrominated diphenyl ethers (PBDEs) were widely used as flame retardants until the early 2000s, mainly in home furnishings and electronics. The persistence of PBDEs in the environment leads to continued ubiquitous exposure to low levels, with infants and children experiencing higher exposures than adults. Accumulating evidence suggest that low-level exposures during early life stages can affect brain development and lead to long-term behavioral impairments. We investigated the effects of zebrafish exposure to low doses of the two prominent PBDEs; 2,2',4,4',5,-Pentabromodiphenyl ether (BDE-99) and 2,2',4,4',-Tetrabromodiphenyl ether (BDE-47), during embryo-development on short- and long-term behavioral endpoints. We included the organophosphate pesticide chlorpyrifos (CPF) due to its well documented neurotoxicity across species from zebrafish to humans.

METHODS:
Zebrafish embryos were exposed to the following individual treatments; 0.1% DMSO (vehicle control); 0.3μM CPF; 0.01, 0.03, 0.1, 0.3μM BDE-47; 0.003, 0.03, 0.3, 1, 3, 10, 20μM BDE-99 from 5 until 120h post fertilization (hpf). Low exposure levels were determined as those not causing immediate overt toxicity, and behavior assays were conducted in the low-level range. At 144 hpf the larvae were tested for locomotor activity. At approximately 6 months of age adult zebrafish were tested in a behavioral battery including assays for anxiety-related behavior, sensorimotor response and habituation, social interaction, and predator avoidance.

RESULTS:
In the short-term, larval locomotor activity was reduced in larvae treated with 0.3μM CPF and 0.1μM BDE-47. BDE-99 treatment caused non-monotonic dose effects, with 0.3μM causing hyperactivity and 1μM or higher causing hypoactivity. In the long-term, adult anxiety-related behavior was reduced in all treatments as measured in both the novel tank dive test and tap test.

DISCUSSION:
We show that exposure of zebrafish embryos to low concentrations of the brominated flame retardants BDE-47 and BDE-99, and the organophosphate pesticide CPF, caused both short- and long-term behavioral impairments. Interestingly, we also found that at very low exposure concentrations, where there were no visible effects on larval activity, adult behavior was still strongly affected.

Figures

Figure 1. The zebrafish startle tap testing apparatus and method.

A) Top view showing the testing apparatus. Fish were tested in eight cylindrical chambers arranged in a 2 × 4 array.
B) Schematic diagram illustrating the testing chambers above the tap solenoids which are computer controlled to deliver one vibrational startle per minute (ten total trials). Modified from Eddins et al. (2010).
C) Representative tracking of eight zebrafish during the startle task. Left image- 5 s before the tap (pre-tap); right image- 5 s after the tap (post-tap).

Figure 2. The MUPET apparatus.

A) Top view showing the tank and bottom 3 × 5 grid.
B) Side view of the experimental set-up. The MUPET is situated on top of a light box with a computer screen on each side of the tank and a video camcorder placed above the tank.
C) Top view showing the experimental set-up for the shoaling and predator avoidance tests, using partitions to create two adjacent lanes across the tank width.

Figure 3. 6 dpf larval locomotor activity in response to alternating light/dark conditions.

6 dpf larval locomotor activity in response to alternating light/dark conditions following developmental exposure to CPF (A), BDE-47 (B), and BDE-99 (C). The fish were recorded for 50 min, starting with 10 min in the dark (Habituation), followed by two cycles of 10 min in the light and 10 min in the dark. Asterisks indicate significant difference from the 0.1% DMSO control group.

Figure 4. Novel tank dive test.

Adult zebrafish that were developmentally exposed to CPF (A,D), BDE-47 (B,E), and BDE-99 (C,F) were individually placed in the testing tank (novel environment) and recorded for 5 min. Total activity (A–C) was calculated as the total distance traveled by the fish in each minute of the trial. Dive response (D–F) was calculated as the average distance of the fish from the bottom of the tank in each minute of the trial. Asterisks indicate significant difference from the 0.1% DMSO control group.

Figure 5. Startle tap test.

Adult zebrafish were individually placed in cylindrical arenas, allowed a short 30 s acclimation period and subjected to a sequence of 10 taps, one tap per minute. A) Average activity of the fish during 5-s before and after each tap. The lower, gradually rising bars (upward dashed line on 0.1% DMSO) show the pre-tap activity, and the higher, gradually decreasing bars (downward dashed line on 0.1% DMSO) show the post-tap activity. B–D) Post-tap linear regression slopes of 0.1% DMSO compared to CPF (B), BDE-47 (C) and BDE-99 (D). Asterisks indicate significant difference from the 0.1% DMSO control group.

Figure 6. Shoaling test.

Adult zebrafish that were developmentally exposed to CPF, BDE-47, and BDE-99 were individually placed in the testing tank (MUPET) and recorded for 7 min. After the first two minutes a video of a zebrafish shoal was played on one of the two flanking monitors for the remaining 5 min of the trial. A) Average distance from the tank side on which the video was played in the 2-min before and the 2-min after the video began playing for each treatment. Asterisk indicates significant difference between the two time-intervals. B) Difference calculated between the two intervals described in A in each treatment.

Figure 7. Predator avoidance test.

Developmentally exposed adult zebrafish were individually placed in the testing tank (MUPET) and recorded for 9 min consisting of one min acclimation followed by alternating minute-long stimulus/no stimulus events. The stimulus was either a blue slow-growing dot or a red fast-growing dot appearing repeatedly on one of the screens. A) Total activity of the fish calculated as the total distance traveled throughout the trial. B) Flee response was calculated as the difference in average distance from the tank side between trial minutes in which the dot stimulus was presented and minutes in which there was no stimulus. Blue bars represent blue dot flee response and red bars represent red dot flee response. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Supplemental Materials

Supplementary Data