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, 120 (11), 1585-91

Early Zebrafish Embryogenesis Is Susceptible to Developmental TDCPP Exposure


Early Zebrafish Embryogenesis Is Susceptible to Developmental TDCPP Exposure

Sean P McGee et al. Environ Health Perspect.


Background: Chlorinated phosphate esters (CPEs) are widely used as additive flame retardants for low-density polyurethane foams and have frequently been detected at elevated concentrations within indoor environmental media.

Objectives: To begin characterizing the potential toxicity of CPEs on early vertebrate development, we examined the developmental toxicity of four CPEs used in polyurethane foam: tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), and 2,2-bis(chloromethyl)propane-1,3-diyl tetrakis(2-chlorethyl) bis(phosphate) (V6).

Methods: Using zebrafish as a model for vertebrate embryogenesis, we first screened the potential teratogenic effects of TDCPP, TCEP, TCPP, and V6 using a developmental toxicity assay. Based on these results, we focused on identification of susceptible windows of developmental TDCPP exposure as well as evaluation of uptake and elimination of TDCPP and bis(1,3-dichloro-2-propyl)phosphate (BDCPP, the primary metabolite) within whole embryos. Finally, because TDCPP-specific genotoxicity assays have, for the most part, been negative in vivo and because zygotic genome remethylation is a key biological event during cleavage, we investigated whether TDCPP altered the status of zygotic genome methylation during early zebrafish embryogenesis.

Results: Overall, our findings suggest that the cleavage period during zebrafish embryogenesis is susceptible to TDCPP-induced delays in remethylation of the zygotic genome, a mechanism that may be associated with enhanced developmental toxicity following initiation of TDCPP exposure at the start of cleavage.

Conclusions: Our results suggest that further research is needed to better understand the effects of a widely used and detected CPE within susceptible windows of early vertebrate development.

Conflict of interest statement

The authors declare they have no actual or potential competing financial interests.


Figure 1
Figure 1
Developmental toxicity of CPE-based FRs during zebrafish embryogenesis. (A) Four structurally related CPE-based FRs screened for toxicity during zebrafish embryogenesis, all of which have been detected in polyurethane foam collected from baby products (Stapleton et al. 2011). (B) Mean percent mortality (± SD) at 96 hpf after static exposure to vehicle (0.1% DMSO) or a CPE (50 μM) during 5.25 hpf (50% epiboly) to 96 hpf (n = 2 beakers/treatment). (C) Four-parameter concentration–response curve fit to mean percent mortality (± SD) at 96 hpf following static TDCPP exposure during 5.25–96 hpf (n = 5 beakers/treatment for 3 and 4 μM TDCPP, and 2 beakers/treatment for the remaining TDCPP doses). *p < 0.05 compared with vehicle controls.
Figure 2
Figure 2
Effect of TDCPP exposure during cleavage on developmental toxicity. (A) Brightfield images of normal zebrafish stages used to define exposure windows. (B) Mean percent mortality (± SD) and (C) mean percent malformed (± SD) embryos at 96 hpf following static exposure to TDCPP during 5.25–96 hpf, 0.75–96 hpf, or 0.75–2 hpf. For exposures during 0.75–2 hpf, embryos were incubated in vehicle (0.1% DMSO) during 2–96 hpf. All surviving 96-hpf larvae that exhibited a range of abnormal phenotypes were included in malformation data. n = 3 beakers/treatment. *p < 0.05 for within–exposure window effects compared with vehicle controls. **p < 0.05 for within-treatment effects compared with exposures initiated at 5.25 hpf.
Figure 3
Figure 3
Effect of TDCPP exposure on embryonic phenotypes. Zebrafish embryos were treated with vehicle (0.1% DMSO) or 3 μM TDCPP from 0.75 hpf (2‑cell) to 96 hpf and assessed for gross malformations under transmitted light at 96 hpf. Images are representative of within-treatment phenotypes observed within surviving larvae after developmental TDCPP exposure. Mild = trunk curvature and/or tail malformations; moderate = trunk curvature, tail malformations, craniofacial malformations, and decreased body length; severe = trunk curvature, tail malformations, craniofacial malformations, decreased body length, pericardial edema, and yolk sac edema.
Figure 4
Figure 4
Mean (± SD) TDCPP or BDCPP concentration (ng) detected in homogenates of 20 whole embryos exposed in triplicate to vehicle (0.1% DMSO) from 0.75 hpf (2‑cell) to 24 hpf (prim-5) or to 3 µM TDCPP from 0.75 hpf (2‑cell) to 2 hpf (64‑cell), followed by incubation in vehicle until 24 hpf (prim-5); 0.75 hpf (2‑cell) to 24 hpf (prim-5); or 2.25 hpf (128‑cell) to 24 hpf (prim-5). n = three replicate embryo pools per treatment and time point. TDCPP levels within embryos exposed during 0.75–2 hpf alone were nondetectable by 24 hpf. *p < 0.05 for within–exposure window differences in concentration compared with vehicle controls. **p < 0.05 for within-treatment differences in concentrations relative to 2-hpf embryos.
Figure 5
Figure 5
Effect of TDCPP on cell morphology during cleavage. Brightfield images of six different stages of cleavage in embryos treated with vehicle (0.1% DMSO) or 3 µM TDCPP under static conditions. TDCPP exposure did not appear to have adverse impacts on gross cell morphology (size, shape, viability) or cell cycle progression during cleavage. Images are representative of 60 embryos per treatment.
Figure 6
Figure 6
TDCPP and gDNA methylation during cleavage. gDNA extracted from TDCPP-treated embryos (+)—but not vehicle-treated embryos (–)—at the end of cleavage (2 hpf) was completely digested by methylation-sensitive restriction endonucleases (white arrows), suggesting that normal gDNA methylation at 2 hpf was absent in TDCPP-treated embryos. Gels are representative of three independent gDNA samples per time point and treatment.

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