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Ecotoxicological information

Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 August to 25 August 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study undertaken to an internationally recognised guideline and at a GLP accredited laboratory.
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: 1.2, 10.0, 25.0, 50.0 and 100.0 mg a.i./l
- Sampling method: 50 ml samples were taken at 3, 6, 24, 48 and 96 hours
- Sample storage conditions before analysis: no data
Details on test solutions:
Preparation of Test Solution: A quantity of 5005 mg Ethyl 2-methylbutyrate was weighed separately and dissolved in 5 mL of acetone and diluted in 50 litres of water in a glass aquarium of 80 litres capacity to obtain the required test concentration of 100 mg a i/L. For vehicle control volumes of 5 mL acetone diluted m 50 litres of water in a glass aquarium of 80 litres capacity. The test solution was thoroughly mixed to achieve a uniform distribution of the test item within the aquarium. The test media in the aquarium was changed every 24h and replaced with freshly prepared (15 minutes prior to use) test item solution.

A photoperiod of 16h light and 8h dark was maintained with using a timer attached to the power supply
Test organisms (species):
Cyprinus carpio
Details on test organisms:
- Common name: Cyprinus carpio
- Source: Prem Fisheries Consultancy, Kapodra, Bh. Tapi Hotel, N.H. No. 8, Ankleshwar, DistBharuch, Gujarat, India. Cyprinus carpio were received at Jai Research Foundation on April 04, 2012 and maintained at Section - Ecotoxicology, JRF.
- Length at study initiation (length definition, mean, range and SD): between 2.0 and 4.0 cm
- Feeding during test: The fish were fed daily with ''TAIYO'' Floating type, supplied by Fish-N-Fun, Bilimora, Gujarat, India. Feed was withheld 24h before the exposure to Ethyl 2-methylbutyrate and throughout the exposure period.
- Feeding: None during test period

- Acclimation period: The fish were received into the experimental room and allowed to acclimatise for 8 days prior to testing in a glass aquarium containing water identical to that to be used in the study.
- Acclimation conditions (same as test or not): The water was maintained at a mean temperature of 21.6 ± 0.2°C (ranged between 21.3 and 21.9°C) with a mean dissolved oxygen content of 90.4 ± 1.3% (ranged between 88.6 and 93.0%) as air saturation value and total hardness of 248.0 mg/L as CaCO3.
- Health during acclimation (any mortality observed): No signs of physical and behavioural abnormalities or mortalities were observed during the acclimatisation period.
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
Post exposure observation period:
- Observations: Fish were observed at 3, 6, 24, 48, 72 and 96h of exposure. Recorded parameters included behavioural responses, clinical signs and mortality.
- Water quality parameters: Temperature, pH and dissolved oxygen content of the test media were measured daily before and after each renewal of the media with freshly prepared test solutions. Total hardness was anaylsed daily for each renewal of the diluent water during the study period.
Total hardness of test media: 240 to 248 mg/L as CaCO3.

Test temperature:
21.4 to 24.0°C

7.23 to 7.94

Dissolved oxygen:
76.4 to 93.9%
Nominal and measured concentrations:
Nominal: 100.0 mg/L
Measured: 97.17 mg/l
Details on test conditions:
- Test vessel: All glass aquaria; capacity 80 L
- Aeration: The test solutions were not aerated during the exposure
- Renewal rate of test solution (frequency/flow rate): Test solutions were changed every 24 h with freshly prepared solutions.
- No. of vessels per concentration (replicates): 10 fish per group
- No. of vessels per control (replicates): 10 fish
- No. of vessels per vehicle control (replicates): 10 fish

- Intervals of water quality measurement: Temperature, pH and dissolved oxygen content of the test media were measured daily before and after each renewal of the media with freshly prepared test solution. Total hardness was analysed daily for each renewal of the diluent water during the study period.

- Photoperiod: 16 h light and 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): 96h LC50

- Range finding study:
- Test concentrations: A preliminary range finding study was conducted with test concentrations of control [without Ethyl 2-methylbutyrate], vehicle control [with acetone], 1.2, 10.0, 25.0, 50.0, 100.0 mg Ethyl 2-methylbutyrate/L and 100 mg a.i/L for a period of 96h with ten fish in each group to determine the concentrations to be selected for the main study.
- Results used to determine the conditions for the definitive study: The percent mortalities observed were 0 in all the test concentrations.
Reference substance (positive control):
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Reported statistics and error estimates:
- Statistical Analysis: Since the study was conducted as a limit study and no mortality was recorded at the selected limit dose level of 100 mg a.i/L therefore the LC50 was not calculated.
Sublethal observations / clinical signs:

- Mortalities: There were no mortalities (over a period of 96h) observed at the test concentration of 100.0 mg a.i.

Ethyl 2-methylbutyrate/L as well as control [without Ethyl 2-methylbutyrate] and vehicle control [with acetone] (Table 1 of attached report).

- Observations: Fish exposed to control [without Ethyl 2-methylbutyrate], vehicle control [with acetone] and 100.0 mg a.i./L did not exhibit any treatment related clinical symptoms (Table 2 of attached report).

- Water Quality Parameters: Ethyl 2-methylbutyrate did not alter any of the selected water quality parameters (temperature, dissolved oxygen, total hardness and pH) of the test solution (Table 4 and Appendix 1 of attached report). Temperature and dissolved oxygen values of test media ranged between 21.4 to 24.0°C and 76.4 to 93.9% respectively. The pH values of test media ranged between 7.23 to 7.94. Total hardness of test media ranged between 240 to 248 mg/L as CaCO3. All the parameters were found to be within the guidelines limit.

- Analysis of Test Concentration: Test media samples were collected in glass bottles for each test concentration for determination of the stability and a.i. concentration of Ethyl 2-methylbutyrate in test media. During range finding study at the start of the test (0h) 97.00% (lowest concentration) and 97.61% (highest concentration) of the nominal concentrations were found. After 24h test duration 94.16% (lowest concentration) and 95.29% (highest concentration) of the nominal concentrations were found. During main study (0h) fish were exposed to 97.17% of nominal concentration. All reported results were related to nominal concentration of test item (Appendix 2 of attached report).

- Statistical Analysis: As no mortality was observed at the test concentration of 100 mg a.i./L. Probit analysis was not carried out.
Validity criteria fulfilled:
The acute fish toxicity (96h LC50) of Ethyl 2-methylbutyrate Common carp, Cyprinus carpio was found to be greater than 100 mg a.i./L.
Executive summary:

This study was performed to assess the acute toxicity (24, 48, 72 and 96h LC50) of Ethyl 2-methylbutyrate [Supplied by Toyo Gosei Co., Ltd., Japan] in Common carp, Cyprinus carpio. The method followed was as per the guidelines of OECD No. 203 (July 1992).

The study was conducted as a limit study in which one group of fish was exposed to a limit concentration of 100 mg a.i. Ethyl 2 methylbutyrate/L. A negative control (without Ethyl 2 methylbutyrate) and vehicle control (with acetone) were run concurrently. Each group comprising of 10 fish. Water quality parameters viz. temperature, pH, dissolved oxygen and total hardness were measured and recorded during the study and found to be within the guideline limits.

Fish exposed to 100.0 mg a.i./L as well as the control did not exhibit any clinical symptoms.


The percent mortalities observed during the course of the study were as follows:


Concentration of               Percentage mortality of fish    Cumulative frequency

Ethyl2-methylbutyrate/L        24h  48h  72h  96h             __(%) at 96h __                                     

Control                     0     0       0      0              0

Vehicle control               0     0       0      0               0

100 mg a.i/L                 0     0       0      0              0


Test media was analysed for Ethyl 2 -methylbutyrate stability and a.i. concentration to monitor the concentration of test solution at 0 and 24h during range finding (control, vehicle control, the lowest concentration and highest concentration) and at 0h during main study. The concentrations selected for range finding and the main study were found to be stable during exposure period within the guidelines limit.


The study was conducted as a limit test in which the 96h LC50 of Ethyl 2 methylbutyrate was found to be greater than 100.0mg a.i./L.

Description of key information

The LC50 is >100 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

The acute fish toxicity (96h LC50) of Ethyl 2-methylbutyrate Common carp, (Cyprinus carpio) was found to be greater than 100 mg a.i./L.