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Ecotoxicological information

Short-term toxicity to fish

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fish embryo acute toxicity (FET)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September - October 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 236 (Fish embryo acute toxicity (FET) test)
GLP compliance:
Specific details on test material used for the study:
Purity of test item 99.4% (dry basis, HPLC)
Supplier Hayashibara Co., Ltd.
Lot number 161208
The test sample was treated as 100% in purity.

Storage condition
The test sample was put into a bag containing silica gel, sealed and stored in a dark storage place at room temperature.

Identification and stability of test sample under storage condition
The infrared (IR) spectrum of the test sample measured at this test facility was confirmed to be identical to that provided by the sponsor.
In addition, the IR spectrum of the test sample after the completion of the experiment was the same as that before the start of the experiment, and it indicated that the test sample was stable under the storage condition.
Analytical monitoring:
Details on sampling:
The collected test solutions were used as samples for high-performance liquid chromatography (HPLC) analysis without treatment.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The weighed test sample (0.050 g) was mixed with dilution water (0.5 L), stirred and dissolved to prepare the test solution of 100 mg/L (nominal).
On the day before the start of exposure, the prepared test solution of 2 mL was added into each well of test vessel beforehand, and the test vessel was conditioned for 24 hours. On the day of start of exposure, the test solution filled in each well was removed, and the wells were refilled with freshly prepared test solution.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: Zebra fish
- Strain: NIES-R
- Source: A group bred and maintained in this test facility. The group’s ancestor was supplied from National Institute for Environmental Studies, Japan
- Age at study initiation (mean and range, SD): not applicable
- Length at study initiation (length definition, mean, range and SD): not applicable
- Weight at study initiation (mean and range, SD): not applicable
- Method of breeding: Two male and one female fish per breeding tank (approximately 3 L)
- Maintenance of the brood fish: Male and female fish whose histories until the definitive test were clear, and that matured enough to reproduce were selected.

- Acclimation period: not applicable
- Acclimation conditions (same as test or not): not applicable
- Type and amount of food during acclimation: not applicable
- Feeding frequency during acclimation: not applicable
- Health during acclimation (any mortality observed): not applicable

QUARANTINE (wild caught)
- Duration: not applicable
- Health/mortality: not applicable

- Food type: not applicable
- Amount: not applicable
- Frequency: not applicable
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
Post exposure observation period:
52.1-55.1 mg CaCO3/L
Test temperature:
Dissolved oxygen:
8.2-8.4 mg/L
not applicable
18.5-21.6 mS/m
Nominal and measured concentrations:
The measured concentration of the test item in the test solution at the start of exposure was 99.2 mg/L (99.2% of the nominal concentration), and that at the end of exposure was 86.4 mg/L (86.4% of the nominal concentration). The measured concentrations of the test item were maintained within the range of 80-120% of the nominal concentrations.
Details on test conditions:
- Test vessel: Polystyrene 24-well plate (Sumitomo Bakelite, length: 8.6 cm, width: 12.8 cm, depth: 2.0 cm, well base area: 1.8 cm2, well capacity: 3.4 mL)
Cover on test vessel: Transparent plastic lid (the lid was sealed with a paraffin film)
- Renewal rate of test solution: none
- No. of organisms per vessel: one per well
- No. of vessels per concentration (replicates): 20 wells
- No. of vessels per control (replicates): 4 wells for internal plate control, 20 wells for positive control, 24 wells for negative control
- No. of vessels per vehicle control (replicates): see above
- Biomass loading rate: not determined

Chemical characteristics of dilution water (Sampling on July 10, 2018)
Unit Results Determination limit
Total hardness (Ca, Mg) mg/L 20 1
Suspended solid mg/L < 1 1
pH - 7.6 (23.8°C) -
Total organic carbon mg/L < 0.5 0.5
Chemical oxygen demand mg/L < 1 1
Free residual chlorin mg/L < 0.02 0.02
Ammonium ion mg/L < 0.1 0.1
Total cyanide mg/L < 0.05 0.05
Alkalinity mg/L 21 1
Electric conductivity mS/m 8.7 0.1
Total mercury mg/L < 0.0005 0.0005
Cadmium mg/L < 0.001 0.001
Chromium (VI) mg/L < 0.01 0.01
Lead mg/L < 0.001 0.001
Arsenic mg/L < 0.005 0.005
Iron mg/L < 0.01 0.01
Copper mg/L < 0.001 0.001
Cobalt mg/L < 0.001 0.001
Manganese mg/L < 0.005 0.005
Aluminum mg/L < 0.02 0.02
Zinc mg/L < 0.1 0.1
Nickel mg/L < 0.001 0.001
Silver mg/L < 0.0001 0.0001
1,2-dichloropropane mg/L < 0.002 0.002
Chlorothalonil mg/L < 0.001 0.001
Propyzamide mg/L < 0.0008 0.0008
Chlornitrofen mg/L < 0.0001 0.0001
Simazine mg/L < 0.0003 0.0003
Thiobencarb mg/L < 0.001 0.001
Diazinon mg/L < 0.0005 0.0005
Isoxathion mg/L < 0.0008 0.0008
Fenitrothion mg/L < 0.0003 0.0003
EPN mg/L < 0.0006 0.0006
Dichlorvos mg/L < 0.001 0.001
Iprobenfos mg/L < 0.0008 0.0008
PCB mg/L < 0.0005 0.0005
Boron mg/L < 0.1 0.1
Fluorine mg/L 0.1 0.1
Sulfate ion mg/L 6.9 0.5
Chloride ion mg/L 7.8 0.2
Sodium mg/L 8.4 0.2
Potassium mg/L 1.6 0.2
Calcium mg/L 5.7 0.1
Magnesium mg/L 1.3 0.1

- Adjustment of pH: no
- Photoperiod: 16-hour light/8-hour dark
- Light intensity: not stated

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

(1) Coagulation of embryo (observation every 24 hours)
(2) Lack of somite formation (observation every 24 hours)
Lack of somite formation at after 48 hours was considered dead.
(3) Non-detachment of the tail (observation every 24 hours)
(4) Lack of heartbeat (observation after 48, 72 and 96 hours)
Using inverted microscope with a capacity of more than at least 80-fold magnification, embryos showing no heartbeat were observed for one minute were considered dead. Irregular heartbeat or visible heartbeat without circulation in aorta abdominalis were considered non-lethal.
Additionally, hatching of the test organisms in all test levels was observed at 48, 72 and 96 hours. Complete separation of the body of test organism from the chorion was considered hatched.

100 mg/L
Reference substance (positive control):
Key result
96 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
including coagulation, Lack of soimte formation, non-detachment of the tail, Lack of heartbeat
Key result
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
including coagulation, Lack of soimte formation, non-detachment of the tail, Lack of heartbeat
Results with reference substance (positive control):
Number of dead embryo in the positive control at the end of exposure was 20, and the mortality of that was 100 %, which met the criterion for the validity of the test (i.e. more than 30% dead embryo).
Reported statistics and error estimates:
no statistical analysis applied
Validity criteria fulfilled:
Under the conditions of the test, no effect of the test item on zebrafish embryo could be observed. The LC50 (96h) was determined to be > 100 mg/L
Executive summary:

An acute Fish Embryo Toxicity Test according to OECD Guideline 236 was conducted. The test was designed as limit test with one test item concentration: 100 mg/L. No mortality and no other effects of the test substance were observed during the test.

Description of key information

one study on Fish embryo toxicity available.

No effects of the substance were observed hence no LC50 value is given.

Key value for chemical safety assessment

Additional information