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Diss Factsheets

Administrative data

short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2007 - June 2007
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
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
adopted 29 December 1992
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
adopted 17 July 1992
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate
EC Number:
Cas Number:
Molecular formula:
ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate
Test material form:

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations:
Nominal: 0.1, 0.18, 0.32, 0.58 and 1.0 mg/L
Mean measured concentrations: 0.066, 0.112, 0.199, 0.375 and 0.627 mg/L
- Sampling method: The test solution was analysed for the active ingredient concentration along with the negative control and vehicle control at the beginning and the end of each day of treatment.

Test solutions

Details on test solutions:
- Method: Stock solution: A 100 mg sample of the test item was weighed into a 10 mL volumetric flask and brought to volume with the vehicle acetone (10000 mg/L). Individual test solutions were prepared directly in each test aquarium. Solutions were prepared by mixing calculated amounts of the stock solution into test medium (reconstituted water according to OECD 203) with a final volume of 40 litres.
- Controls: Negative control: Test medium; vehicle control: 4 mL of acetone mixed with 40 L test medium in a test aquarium

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: Zebra-fish
- Source: Marine Aquarium, Corporation Complex, Shivajinagar, Bangalore, India
- Length at study initiation: 2.8 ± 0.1 cm
- Weight at study initiation: 1.87 - 2.16 g

- Acclimation period: 7 days
- Acclimation conditions: Same as test conditions. The test medium in the aquaria was replaced at the rate of 10% per day on all working days
- Feeding frequency during acclimation: Once a day. Feeding was stopped 24 hours before the start of the treatment.
- Health during acclimation: No mortality observed

Study design

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

Test conditions

2.36 - 2.44 (m moles/L)
Test temperature:
22.1 to 22.6°C
7.70 to 8.04
Dissolved oxygen:
More than 83% of air saturation value
Not reported
The conductivity of the deionised water used in the preparation of the test medium was less than 5 μS/cm for both the range finding as well as the definitive test.
Details on test conditions:
- Test vessel: Aquarium
- Material, size, headspace, fill volume: Approximately 42 L capacity glass aquaria [46 cm (L) x 30 cm (W) x 31 cm (H)]
- Renewal rate of test solution: 24 h renewal
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: Max loading of 1.0 g fish/L

- Source/preparation of dilution water: Deionised water
- Ca/mg ratio: 4:1
- Intervals of water quality measurement: The temperature, pH and dissolved oxygen concentration of the negative control, vehicle control and the test solutions were checked once daily.

- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Fish were observed at 1, 2, 4, 24, 48, 72, and 96 hours after test initiation for visible abnormalities such as loss of equilibrium, changes in swimming and breathing patterns and mortality

- Range finding study
- Test concentrations: 0.01, 0.1, 1.0 mg/L
- Results used to determine the conditions for the definitive study:
A semi-static range finding test with 5 fish per concentration was conducted using a negative control, vehicle control and nominal concentrations of 0.01, 0.1 and 1.0 mg/L. Test solutions were renewed every 24 hours. At the end of 96 hours, 60% mortality was observed at the highest tested concentration of 1.0 mg/L. No mortality was observed in the negative, vehicle control and at test concentrations of 0.01 and 0.1 mg/L.
Reference substance (positive control):

Results and discussion

Effect concentrations
96 h
Dose descriptor:
Effect conc.:
0.51 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Determined by non linear extrapolation
Details on results:
- Behavioural abnormalities:
- Observations on body length and weight: Not reported
- Other biological observations: Signs of toxicity (sluggishness, , erratic swimming, loss of equilibrium, gasping, change i pigmentation) were observed at various degree in the two highest concentration groups.
- Mortality of control: No mortality
- Other adverse effects control: No effects observed in the control groups
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1: Definitive test, summary of toxic signs and mortality

GroupNominal concentration [m/L]Mean measured concentration [mg/L]Toxic sign (number of fish) at different observation time
1 hour2 hour4 hour24 hour48 hour72 hour96 hour
G1Negative control-N(10)N(10)N(10)N(10)N(10)N(10)N(10)
G2Vehicle control-N(10)N(10)N(10)N(10)N(10)N(10)N(10)
G60,580,375N(10)N(10)N(10)N(7), A(3)N(3), A(7)N(1), A(7), A&E(1), M(1)N(1), A&E(6), D(1), M(1)
G71,00,627N(10)N(10)N(10)N(1), A(9)A(5), A&E(2), M(3)A&B(2), A,C&E(4), M(1)B&E(1), D&E(2), M(3)

Toxic sign: N - Normal, A - Sluggishness, B - Erratic swimming, D - Gasping, E - Change in pigmentation, M - Mortality

Numbers in parentheses denote the number of fish

Applicant's summary and conclusion

Validity criteria fulfilled:
No mortality in controls at the end of the test. Average dissolved oxygen conc. of test medium was 92% of the air saturation value. Analysed conc. of test item 40-91% of nominal conc. Hence, toxicity results based on mean measured conc. of test item.
The LC50(96 h) for Danio Rerio of the test item was 0.51 mg/L based on the mean measured concentration.
Executive summary:

The acute toxicity (LC50, 96 h) of the test item to the Zebra-fish (Danio rerio) was investigated under semi-static condition according to OECD Guideline 203 and EEC, Guideline Part C.1.

The fish were exposed to a series of test concentrations of 0.1, 0.18, 0.32, 0.58 and 1.0 mg/L. A concurrent negative control (dilution water only) and a vehicle control (acetone) were also tested. The fish were exposed in a semi-static treatment method with the test solutions being renewed every 24 hours. One test chamber each was used for the test concentrations, dilution water and vehicle control with 10 fish in each chamber. At the start of the treatment, the average standard length of the fish was in the range of 2.8 ± 0.1 cm with an average body weight in the range of 1.87-2.16 g for the negative, vehicle control and treatment groups. HPLC analysis of the test solutions showed that there was a significant reduction in the measured concentration of the test item at the end of the test. Therefore, all results are reported based on the mean measured concentration.

Exposure of zebra-fish to nominal test item concentrations of 0.1, 0.18, 0.32, 0.58 and 1.0 mg/L (corresponding mean measured concentrations 0.066, 0.112, 0.199, 0.375 and 0.627 mg/L, respectively) resulted in 0, 0, 0, 20 and 70% mortality, respectively at the end of 96 hours. No mortality or sub-lethal effects were seen in the negative and vehicle control fish.

The LC50 of the test item at 96 hours was determined to 0.51 mg/L based on the mean measured concentration.