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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Conducted according to guideline in effect at time of study conduct
according to guideline
other: "Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish acute toxicity test" (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5 , Kanpokihatsu No.110331009, March 31, 2011
Conducted according to guideline in effect at time of study conduct
GLP compliance:

Test material

Constituent 1
Reference substance name:
Cas Number:
Details on test material:
- Purity: 99.7944%

Sampling and analysis

Analytical monitoring:
Details on sampling:
1) Preparation of standard solutions
About 45 mL dimethylsulfoxide was collected in a 50-mL volumetric bottle, and the bottle with dimethylsulfoxide was treated as a tare at weighing of the test substance. The test substance was collected into a 10-mL glass centrifuge tube from cylinder, then added into the 50-mL volumetric bottle by decantation and weighed subtracting the tare weight. After the weighing, this solution was filled up to 50 mL with dimethylsulfoxide. The concentration of the test substance was calculated based on the measured weight, and this solution was diluted sequentially with dimethylsulfoxide to prepare standard solutions of 4 concentrations within the range of approximately 10-1000 mg/L. Dimethylsulfoxide was used as the standard solution at 0 mg/L.

Analysis of Test Solutions
Samples of the test solutions at the beginning and the end of the renewal period, i.e. when it is freshly prepared and immediately prior to renewal or at the end of exposure are referred to as one set. All sets of test solutions were analysed. The test solution of each test group was collected and analysed by gas chromatography/mass spectrometry (GC/MS). Samples were prepared by mixing 0.1 mL of test solution and 10.0 mL of ultra pure water.

Test solutions

Details on test solutions:
I. Preparation of stock solution
For the preparation of the stock solution, 5.8 L of dechlorinated water (cooled in the refrigerator prior to use) was used as dilution water.
(I) 17.4 g of the test substance was collected into a 50-mL Erlenmeyer flask from the cylinder. (A 50-mL Erlenmeyer flask, a stopper and tape for sealing the stopper were treated as a tare. The test substance was weighed subtracting the tare weight.
(2) The test substance was added into the dilution water by decantation while stirring with a magnetic stirrer, and then stirred for an additional 30 minutes.
(3) The resulting stock solution was allowed to rest at room temperature for 30 minutes and the supernatant was collected by decantation (about 2 L).
( 4) The collected solution was warmed up to the test temperature, and sonicated for 2 minutes prior to its use for the preparation of the test solutions.

2. Preparation of test solutions
(I) For the preparation of the test solutions for control, dilution water was prepared by stirring 5L of cooled dechlorinated water, allowing it to rest at room temperature and sonicating in the same manner as the stock solution.
(2) For the preparation of the test solutions for Concentrations 1-5, the stock solution was mixed with dechlorinated water (approx 24°C) in appropriate amounts for each concentration.

Test organisms

Test organisms (species):
Oryzias latipes
Details on test organisms:
- Age at study initiation: Less than a half year after hatching
- Feeding during test: no


Study design

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

Test conditions

Test temperature:
23.8 to 24.3 °C
6.7 to 7.4
Dissolved oxygen:
control ranged from 7.5 to 9.5. mg/L in test solutions ranged from 6.5 to 8.6. mg/L
Nominal and measured concentrations:
Nominal concentrations: 0, 20, 39, 77, 150 and 300 mg/L
Measured concentrations: 0, 1.33, 2.60, 4.91, 9.56 and 20.7 mg/L
Details on test conditions:
- Test vessel: 5-L glass vessel (cover: Teflon sheet), 5 L of test solution
- Renewal rate of test solution (frequency/flow rate): 24 hour renewal
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Photoperiod: 16-hour light/8-hour dark
- Light intensity: 1000 lux or less

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Test fish were observed at 3, 24, 48, 72 and 96 hours after the start of exposure. Any fish found dead were removed promptly to prevent deterioration of water quality.

- Range finding study results: Range finding nominal test concentrations were: 3.0, 30, 300 and 3000 mg/L. Percent mortality at 96 hours was 0, 0, 100 and 100%, respectively.

Results and discussion

Effect concentrations
96 h
Dose descriptor:
Effect conc.:
14.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% confidence limits: 9.56-20.7 mg/L, Binomial method. The results were calculated from the time-weighted mean of the measured concentrations.
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test solutions appeared colourless, and suspended substances, floating substances and precipitated substances were not observed in the test solutions during exposure.
Reported statistics and error estimates:
The LC50 was calculated using the probit method, moving average method and binomial method (TOXDAT3_02). 95% Confidence limits were calculated.

Any other information on results incl. tables

Sublethal observations / clinical signs:

96-hour LC0: 9.56 mg/L

96-hour LC100: 20.7 mg/L

In the control group, the mortality at 96-hour exposure was 0%.

In mean measured test solutions 1.33, 2.60, 4.91 , 9.56 and 20.7 mg/L, mortality at 96 hours was 0, 0, 0, 0 and 100%, respectively. Abnormal symptom (abnormal swimming) was observed in one animal at 72 hours in test solution 20.7 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
96-h LC50 = 14.1 mgL
Executive summary:

An acute, semi-static 96-hour toxicity test (with renewal on a 24 period) was conducted at concentrations of 0, 20, 39, 77, 150 and 300 mg/L. The mean measured concentrations used in LC50 calculations were 0, 1.33, 2.60, 4.91, 9.56 and 20.7 mg/L. Because of the volatile nature of the test item, the test was performed in 5 L glass vessel covered with Teflon sheets containing 5 L of test item solution. Control and test solutions were conducted with one replicate with 10 Oryzias latipes in each. In all the test groups, the temperature of the test solutions was in the range of 23.8 to 24.3 °C, the D.O. values were 6.5 mg/L or more, and the pH values were 6.7 to 7.4, fulfilling the test conditions. No control animals died.

Based on the mean measured concentrations, the 96-hour LC50 for was 14.1 mg/L.