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Developmental Exposure to Low Concentrations of Organophosphate Flame Retardants Causes Life-Long Behavioral Alterations in Zebrafish

Lilah Glazer, Andrew B Hawkey, Corinne N Wells, Meghan Drastal, Kathryn-Ann Odamah, Mamta Behl, and Edward D Levin.
Toxicological Sciences (2018) DOI: http://dx.doi.org/10.1093/toxsci/kfy173 PMID: 29982741


Publication


Abstract

As the older class of brominated flame retardants (BFRs) are phased out of commercial use because of findings of neurotoxicity with developmental exposure, a newer class of flame retardants have been introduced, the organophosphate flame retardants (OPFRs). Presently, little is known about the potential for developmental neurotoxicity or the behavioral consequences of OPFR exposure. Our aim was to characterize the life-long neurobehavioral effects of 4 widely used OPFRs using the zebrafish model. Zebrafish embryos were exposed to 0.1% DMSO (vehicle control); or one of the following treatments; isopropylated phenyl phosphate (IPP) (0.01, 0.03, 0.1, 0.3 µM); butylphenyl diphenyl phosphate (BPDP) (0.003, 0.03, 0.3, 3 µM); 2-ethylhexyl diphenyl phosphate (EHDP) (0.03, 0.3, 1 µM); isodecyl diphenyl phosphate (IDDP) (0.1, 0.3, 1, 10 µM) from 0- to 5-days postfertilization. On Day 6, the larvae were tested for motility under alternating dark and light conditions. Finally, at 5–7 months of age the exposed fish and controls were tested on a battery of behavioral tests to assess emotional function, sensorimotor response, social interaction and predator evasion. These tests showed chemical-specific short-term effects of altered motility in larvae in all of the tested compounds, and long-term impairment of anxiety-related behavior in adults following IPP, BPDP, or EHDP exposures. Our results show that OPFRs may not be a safe alternative to the phased-out BFRs and may cause behavioral impacts throughout the lifespan. Further research should evaluate the risk to mammalian experimental models and humans.

Figures


Figure 1. Six dpf larval locomotor activity in response to alternating light/dark conditions following developmental exposure to IPP (A), BPDP (B), EHDP (C), and IDDP (D).

The fish were recorded for 50 min, starting with 10 min in the dark (Habituation), followed by 2 cycles of 10 min in the light and 10 min in the dark.
Asterisks indicate significant difference from the 0.1% DMSO control group.
Sample sizes: 0.1% DMSO—Set 1 n = 92, Set 2 n = 63; IPP—0.01 µM n = 62, 0.03 µM n = 88, 0.1 µM n = 81; BPDP—0.003 µM n = 18, 0.03 µM n = 67, 0.3 µM n = 67; EHDP—0.03 µM n = 64, 0.3 µM n = 57; IDDP—0.1 µM n = 63, 0.3 µM n = 49, 1 µM n = 65, 10 µM n = 15.

Figure 2. Novel tank dive test.

Adult zebrafish that were developmentally exposed to IPP (A, E), BPDP (B, F), EHDP (C, G), and IDDP (D, H) were individually placed in the testing tank (novel environment) and recorded for 5 min. Total activity (A–D) was calculated as the total distance traveled by the fish in each minute of the trial. Dive response (E–H) 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. Sample sizes: 0.1% DMSO—Set 1 n = 20, Set 2 n = 59; IPP—0.01 µM n = 18, 0.03 µM n = 26, 0.1 µM n = 9; BPDP—0.003 µM n = 36, 0.03 µM n = 71, 0.3 µM n = 55; EHDP—0.03 µM n = 58, 0.3 µM n = 40; IDDP—0.1 µM n = 46, 0.3 µM n = 28, 1 µM n = 60, 10 µM n = 31. The same sample sizes were used in all adult behavior assays.

Figure 3. 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, 1 tap per min. Average activity of IPP (A), BPDP (B), EHDP (C), and IDDP (D) exposed fish during 5 s before and after each tap. The lower, gradually rising bars (upward dashed line on 0.1% DMSO) show the pretap activity, and the higher, gradually decreasing bars (downward dashed line on 0.1% DMSO) show the posttap activity. Asterisks indicate significant difference from the 0.1% DMSO control group.

Figure 4. Shoaling test.

Adult zebrafish that were developmentally exposed to IPP (A, E), BPDP (B, F), EHDP (C, G), and IDDP (D, H) were individually placed in the testing tank (MUPET) and recorded for 7 min. After the first 2 min, a video of a zebrafish shoal was played on one of the 2 flanking monitors for the remaining 5 min of the trial. Average distance from the tank side on which the video was played was calculated in the 2-min before (left bar) and the 2-min after (right bar) the video began playing for Set 1 (A; IPP) and Set 2 (B; BPDP, EHDP, IDDP). Asterisk indicates significant difference between the 2 time intervals. Differences were calculated between the 2 intervals described earlier in the Set 1 (C) and Set 2 (D) treatments.

Figure 5. Predator avoidance test.

Developmentally exposed adult zebrafish were individually placed in the testing tank (MUPET) and recorded for 9 min consisting of 1-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. Flee response was calculated for Set 1 (A) and Set 2 (B) treatments 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. Dark bars represent blue dot flee response and light bars represent red dot flee response. Asterisk indicates significant difference between blue and red stimulus response in IPP-exposed fish.

Tables


Table 1. Summary of Behavioral Findings.

Supplemental Materials


Supplementary Data

Raw Data