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Ecotoxicological information

Endocrine disrupter testing in aquatic vertebrates – in vivo

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Administrative data

fish: other
cardiotoxic effects in developing fish embryos
Type of information:
experimental study
Adequacy of study:
supporting study

Data source

Reference Type:
Aryl Phosphate Esters Within a Major PentaBDE Replacement Product Induce Cardiotoxicity in Developing Zebrafish Embryos: Potential Role of the Aryl Hydrocarbon Receptor.
McGee SP, Konstantinov A, Stapleton HM, Volz DC
Bibliographic source:
Toxicological Sciences, 133, 144-156

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Initial screening assays on cardiotoxic effects in developing zebrafish embryos
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) tetrabromophthalate
EC Number:
EC Name:
Bis(2-ethylhexyl) tetrabromophthalate
Cas Number:
Molecular formula:
1,2-bis(2-ethylhexyl) 3,4,5,6-tetrabromobenzene-1,2-dicarboxylate
Specific details on test material used for the study:
Purity (TBPH): 99.5%

Sampling and analysis

Analytical monitoring:
not specified

Test solutions

0.2% DMSO
Details on test solutions:
For initial screening assays, embryos were exposed to vehicle (0.2% DMSO) or water-borne TBPH (0.1–10 μM)

Test organisms

Aquatic vertebrate type:
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Adult wild-type (5D) zebrafish were raised and maintained on a 14-h:10-h light:dark cycle within a five-shelf stand-alone recirculating system
(Aquatic Habitats, Apopka, FL) containing photoperiod enclosures and conditioned reverse osmosis (RO) water (approx. 27–28°C). Originally obtained
from 5D Tropicals Inc. (Plant City, Florida), founder fish for our 5D colony as specific pathogen free for the common fish pathogen Pseudoloma neurophilia (microsporidia). Adult females and males were bred off- or on-system using breeding traps suspended within 1- or 3-L tanks, respectively, to allow spawned eggs to settle to the tank bottom. For all experiments newly fertilized eggs were staged. All fish were handled humanely and treated with regard for alleviation of suffering in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols at the University of South Carolina—Columbia.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
1 h

Test conditions

No data
Test temperature:
28 °C
No data
Dissolved oxygen:
No data
Not applicable
No data
Nominal and measured concentrations:
Concentrations in the range from 0.1 to 10 µM (= 0.071-7.01 mg/L) were tested.
Details on test conditions:
At test initiation, working solutions were prepared fresh by spiking stock solutions into embryo media (EM) (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2, 0.33mM MgSO4). All exposures were conducted using a static exposure protocol within solvent- and RO-rinsed 40-ml glass beakers containing 14 ml of treatment solution. Each treatment group consisted of triplicate beakers, and each replicate beaker consisted of 20 viable wild-type (5D) zebrafish embryos. All embryos were incubated at 28°C under 14-h:10-h light:dark cycle for the entire experiment. For initial screening assays, embryos were exposed to vehicle (0.2% DMSO) or water-borne TBPH (0.1–10μM) from 5.25 hpf (50% epiboly) to 96 hpf (24 h posthatch). To identify developmental windows sensitive to TPP and mono-ITP exposure, embryos were exposed to vehicle (0.2% DMSO), TPP (4μM), or mono-ITP (0.5μM) using the following exposure scenarios: (1) 5.25–96 hpf, (2) 10–96 hpf, (3) 24–96 hpf, and (4) 48–96 hpf. TCDD was also used as a positive control for inducing AHR2-mediated cardiotoxicity within zebrafish embryos. Twenty wild-type (5D) embryos per replicate beaker were exposed in triplicate from 5.25 to 6.25 hpf to 1 ng/ml TCDD with gentle rocking at 28°C. After a 1-h static exposure, embryos were rinsed thrice with clean EM and maintained in clean glass beakers containing vehicle (0.2% DMSO) until 96 hpf.

At 96 hpf, vehicle control and treated larvae (15 per treatment) were anesthetized by transfer to EM containing 50 mg/l MS-222; this concentration of MS-222 does not affect cardiac function within control 96-hpf fish. Heart rates of anesthetized 96-hpf fish were immediately assessed using an Olympus MVX10 MacroView stereomicroscope and timelapsed imaging procedure. For each sample, larvae were oriented in right lateral recumbency, and both heart chambers were focused under 6.3× magnification using transmitted light. Time-lapsed imaging software was programmed to capture 20 frames/s, generating a total of 100 frames over a 5-s analysis period. For heart rate assessments, the average pixel intensity per frame within the ventricle alone was exported from Adobe Photoshop CS4 Extended into Microsoft Excel. Intensity-by-time data were then plotted to visualize temporal heart beat patterns, and heart rates (beats/min) for individual 96-hpf fish were then calculated by (1) counting the number of troughs per 5 s and (2) multiplying this number by twelve. All heart rates from vehicle and treatment groups were reported as mean ± SD.

Reference substance (positive control):

Results and discussion

Effect concentrations
1 h
Dose descriptor:
other: EC0
Effect conc.:
>= 0.071 - <= 7.01 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: body weight, pericardial edema, effects on the developing heart
Details on results:
TBPH did neither affect body weight nor cause pericardial edema (PE) or targeted effects on the developing heart at concentrations which caused no mortality in initial screening assays.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Bis(2-ethylhexyl) tetrabromophthalate (TBPH) did not cause cardiotoxic effects.
Executive summary:

Cardiotoxic effects in developing zebrafish embryos were investigated. Bis(2 -ethylhexyl) tetrabromophthalate (TBPH) was tested at concentrations in the range from 0.1 to 10 µM (0.071-7.01 mg/L) from 5.3 to 96 hpf (= 24 hph). TBPH did neither affect body weight nor cause pericardial edema (PE) or targeted effects on the developing heart at concentrations which caused no mortality in initial screening assays. Concluding, bis(2-ethylhexyl) tetrabromophthalate did not cause cardiotoxic effects in an in vivo study.