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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
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 202 (Daphnia sp. Acute Immobilisation Test)
The study was 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
The study was 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:
Standard Curve:
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. The standard solutions were analysed by GC/MS

Test Solution:
Frequency: Two sets (One set consists of a new sample of test solution at the time of preparation and an old sample of the same test solution immediately prior to renewal or at the end of exposure.)
(i) At the time of preparation: Collected from the middle layer of the vessel for measuring the test conditions of each test group.
(ii) At the time immediately prior to renewal and at the end of exposure: Collected from the middle layer of one vessel of each test group.
The test solutions were analysed by Gas chromatography/mass spectrometry (GC/MS).

Test solutions

Details on test solutions:
1. Preparation of stock solution
For the preparation of the stock solution, 2.2 L of Elendt M4 medium (cooled in the refrigerator prior to use) was used as dilution water.

(1) 6.6 g of the test substance was collected into a 10-mL glass centrifuge tube from the cylinder. A 10-mL glass centrifuge tube, 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 1.5 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
(1) For the preparation of the test solutions for control, dilution water was prepared by stirring I.1 L of cooled Elendt M4 medium, 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 l-6, the stock solution was mixed with Elendt M4 medium (approx 20°C) in appropriate amounts for each concentration.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Strain: Daphnia magna
- Age at study initiation: < 24 hours
- Feeding during test: none

- Acclimation period: not reported

Study design

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

Test conditions

Test temperature:
20.1 - 20.3 °C
8.1 - 8.3
Dissolved oxygen:
7.5 - 9.5 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0, 100, 200, 390, 770, 1500 and 3000 mg/L
Measured concentrations: 0, 18.0, 25.0, 50.1, 98.8, 138 and 514 mg/L
Details on test conditions:
- Test vessel: 100-mL glass incubation bottle with ground-in stopper containing 95 mL of test solution
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- Adjustment of pH: no
- Photoperiod: 16-hour light / 8-hour dark
- Light intensity: 800 lux or less

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Frequency: 24-hour (before renewal) and 48-hour exposure.
Items: number immobile*1, other abnormal symptoms*2

* 1: Individuals that were unable to swim within 15 seconds after gentle agitation of the test vessel were considered immobile.
*2: Daphnids showing abnormal behaviour or appearance (such as discoloration) and floating daphnids on the surface of the water were recorded, if any.

- Range finding study:
Range finding nominal test concentrations were: 0, 3.0, 30, 300 and 3000 mg/L. Immobility at 48 hours was 0, 0, 0, 0 and 100%, respectively.

Results and discussion

Effect concentrations
48 h
Dose descriptor:
Effect conc.:
92.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: The results were calculated from the time-weighted mean of the measured concentrations. 95% confidence limits: 71.7-125 mg/L, Probit
Details on results:
- Behavioural abnormalities: Decline of activity was observed on a daphnid in 98.8 and 138 mg/L measured concentrations. A floating daphnid on the surface of the test solution in 514 mg/L measured was observed.
- Other biological observations: none
- Mortality of control: 0
- 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 EC50 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

48-hour EC0 = 25.0 mg/L

48-hour EC100 = 514 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).
48-hour EC50 = 92.9 mg/L
Executive summary:

An acute, semi-static 48-hour toxicity test (with renewal on a 24 period) was conducted at concentrations of 0, 100, 200, 390, 770, 1500 and 3000 mg/L. The mean measured concentrations used in EC50 calculations were 0, 18.0, 25.0, 50.1, 98.8, 138 and 514 mg/L. Because of the volatile nature of the test item, the test was performed in 100 mL glass stoppered vessels with 95 mL of test item solution. Control and test solutions were conducted in 4 replicates with 5 Daphnids in each. In all the test groups, the temperature of the test solutions was in the range of 20 ± 1 °C, the D.O. values were 7.5 mg/L or more, and the pH values were 8.1 to 8.3, fulfilling the test conditions. No control animals died.

Based on the mean measured concentrations, the 48-hour EC50 was 92.9 mg/L.