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Short-term toxicity to fish

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Reference
Endpoint:
fish embryo acute toxicity (FET)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 236 (Fish embryo acute toxicity (FET) test)
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Specific details on test material used for the study:
GO was synthesized from natural graphite using a modified Hummer's method.
Size distribution (nm): 511.8 ± 25.7
Analytical monitoring:
yes
Details on sampling:
not specified
Vehicle:
no
Details on test solutions:
GO at 6 concentrations (0, 1, 5, 10, 50, and 100 mg/L) dispersed in standard water.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Wild-type zebrafish were purchased from a local commercial source (Gao Feng Aquarium, Beijng, China).
- Method of breeding: The zebrafish were kept in the flow-through feeding equipment (made by Esen Corp.) at 26 °C with a 14-h light/10-h dark cycle and fed on bloodworms, dry flake food, and brine shrimp twice daily. Zebrafish embryos were obtained from spawning adults in groups of male and female zebrafish (ratio=2:1) in spawning boxes overnight. Spawning was induced in the morning when the light was turned on and the embryos were collected after 30 min.
- collected emryos: The collected zebrafish embryos were rinsed for 3 times with standard water. The developmental stage of the embryos was determined according to the method described by Kimmel et al.. Embryos were examined under a dissecting microscope to ensure that normal embryos and embryos at a certain blastula stage were selected for the exposure experiments.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
not specified
Post exposure observation period:
not applicable
Hardness:
no details given
Test temperature:
28 ± 0.5 °C
pH:
no details given
Dissolved oxygen:
no details given
Salinity:
no details given
Conductivity:
no details given
Nominal and measured concentrations:
Nominal: 0, 1, 5, 10, 50, and 100 mg/L
Details on test conditions:
Twenty embryos were selected and exposed to GO at 6 concentrations (0, 1, 5, 10, 50, and 100 mg/L) dispersed in standard water. Subsequently, the embryos were transferred into 24-well multiplates with 1 embryo per well. Three replicates were used for each concentration. The solutions were changed once per day. All 24-well multiplates were kept at 28 ± 0.5 °C with a 14-h light / 10-h dark cycle. The development of zebrafish embryos and larvae was observed at specified times with microscope (OLYMPUS, CX21BIM). Furthermore, the toxicological endpoint corresponding to the embryonic developmental stages were examined at 8 - 96 hpf (hours post-fertilization), including spontaneous movement in 20 s, heart rate, hatching rate, length of larvae, mortality, and malformations.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC0
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
morphology
Remarks on result:
other: highest dose tested
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: highest dose tested
Details on results:
Spontaneous movement in 20 s of zebrafish embryos was examined at 24 hpf. No significant effect (P<0.05) was observed on spontaneous movement of zebrafish embryos exposed to suspension of GO, which indicated that the development of zebrafish embryos treated with GO (1-100 mg/L) at 24 hpf was unaffected.
The heart rates of zebrafish embryos at 48 hpf and larvae at 96 hpf were recorded after exposure to GO at different concentrations (0, 1, 5, 10, 50, and 100 mg/L). The heart rate of embryos treated with GO was slightly (P<0.05) decreased at 100 mg/L at 48 hpf and dropped from 5 to 100 mg/L at 96 hpf. These results indicated that GO had effects on the heart rate of Zebrafish embryos.
GO had no effect on the hatching and no affect on the length of larvae at concentrations ranging from 1 to 100 mg/L. No abnormality such as tail detachment, somite formation, circulatory system, or pericardial cyst pigmentation was observed in the developmental endpoints in the treated embryos. Furthermore, GO did not induce mortality of embryos.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Statistical analysis was performed using SPSS 16.0 (SPSS, Chicago, IL, USA). Data were shown as the mean ± standard error and evaluated by using one-way analysis of variance (ANOVA) with post-hoc least significant difference (LSD) comparisons of means. The differences were considered significant for P<0.05.
Validity criteria fulfilled:
not specified
Conclusions:
GO did not show high toxicity to zebrafish embryos, but had slightly sublethal effects on the heart rate. In these tests, no abnormality or mortality was found, and GO at the concentration ≤ 100 mg/L did not show severe toxicity in the aquatic environment.
Executive summary:

In this study, an embryo-larval test was performed similar to the OECD Guideline for Testing of Chemicals, No. 236 (adopted 2013)

2-h post-fertilization (2 hpf) embryos of zebrafish were used to investigate the toxicity of the test item Graphene oxide (GO) on early stages of development. The test concentrations ranged from 1 mg/L to 100 mg/L. Major endpoints such as spontaneous movement in 20 s, heart rate, hatching rate, length of larvae, mortality, and malformation were examined. GO slightly affected the heart rate of zebrafish embryos, but no obvious morphological malformation or mortality was observed. No abnormality such as tail detachment, somite formation, circulatory system, or pericardial cyst pigmentation was observed in the developmental endpoints in the treated embryos. In these tests, no abnormality or mortality was found, and GO at the concentration 100 mg/L did not show severe toxicity in the aquatic environment.

Description of key information

Key value for chemical safety assessment

Additional information

In this supporting study, an embryo-larval test was performedsimilar to the OECD Guideline for Testing of Chemicals, No. 236 (adopted 2013)

2-h post-fertilization (2 hpf) embryos of zebrafish were used to investigate the toxicity of the test item Graphene oxide (GO) on early stages of development. The test concentrations ranged from 1 mg/L to 100 mg/L. Major endpoints such as spontaneous movement in 20 s, heart rate, hatching rate, length of larvae, mortality, and malformation were examined. GO slightly affected the heart rate of zebrafish embryos, but no obvious morphological malformation or mortality was observed. No abnormality such as tail detachment, somite formation, circulatory system, or pericardial cyst pigmentation was observed in the developmental endpoints in the treated embryos. In these tests, no abnormality or mortality was found, and GO at the concentration 100 mg/L did not show severe toxicity in the aquatic environment.