2,6-dimethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)cyclohex-2-en-1-ol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 25, 2012 to July 13, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD Guideline 203 with GLP certificate. This study is considered reliable with restrictions for the following reasons: - The substance is adequately identified, but some data on composition is missing. - The treatment solutions were prepared with a solvent (acetone), which is not the best method at the time being but in acceptable quantity (0,005 mL/L). - No information on the quantity of test substance included in the stock solution. - The test was performed under open conditions. - Measured concentrations in the daily new media were not reported in the study report and therefore the mean measured concentrations (which were based on nominal and old test solution concentrations) stated in the report cannot be verified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

July 1992

Deviations:

yes

Remarks:

-test item treatments not maintained within 80-120% of the nominal values. Calculations based on mean measured concentrations. -Mean measured concentrations in the new media not matching nominal values: initial concentrations taken as the nominal value

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate of Phytosafe signed on March 11, 2011.

Specific details on test material used for the study:

- Physical state : Light yellow translucent liquid
- Storage : The test substance was stored at room temperature protected from direct sun light.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Range-finding test : 0.01 - 0.1 - 1.0 - 10.0 and 100.0 mg/L ; Definitive test : 0.7 - 1.0 - 1.6 - 2.4 and 4.1 mg/L
- For the range-finding test : the test treatments were renewed each day without changing the mineral water. For the definitive test the test solutions were renewed each day and the test item concentrations were measured in the old and in the new media. Despite this, the test item treatments were not maintained within 80-120% of the nominal values during the test period (deviation of the study plan). The test concentrations were taken as mean of the initial concentration in the new media and in the final concentration in the old media throughout the 4-day test period.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The treatment solutions were prepared in acetone. In the light of previous validation (12-42-006-ES), it was anticipated that semi-static methodology would be required because of high instability of the test item. For the range-finding test : the test treatments were renewed each day without changing the mineral water. For the definitive test the test solutions were renewed each day and the test item concentrations were measured in the old and in the new media. Despite this, the test item treatments were not maintained within 80-120% of the nominal values during the test period (deviation of the study plan). The test concentrations were taken as mean of the initial concentration in the new media and in the final concentration in the old media throughout the 4-day test period. Because the test item was long to solubilise, the measured initial concentrations did not match the nominal values and were not retained. The test solutions were checked to match the required concentrations and the test item treatments were considered as totally set at each medium renewal. As a consequence the nominal value was used as the initial value for every successive day (deviation from the study plan).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium: 0.005 mL/L.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: The batch of fish was received on April 2012 and previously validated.
- Age at study initiation (mean and range, SD): not detailed
- Fish were maintained at 23 +/- 2°C, with a 16h/8h light/dark cycle, in the same reconstituted water as used for testing. The light was 200-260 lux.
- The fish were kept unfed kduring the exposure period.

Test type:

semi-static

Water media type:

other: Reconstituted water : ISO test water (ISO 6341) was freshly reconstituted by dilution of mineral stock solutions in pure water.

Limit test:

no

Total exposure duration:

96 h

Test temperature:

The temperature of the test solutions was maintained at 23 +/- 2°C.

pH:

from 7.8 to 8.2

Dissolved oxygen:

from 83.9 to 99.8%

Nominal and measured concentrations:

Concentrations : Range-finding test : 0.01 - 0.1 - 1.0 - 10.0 and 100.0 mg/L ; Definitive test : 1.0 - 1.5 - 2.2 - 3.3 - 5.0 mg/L.
The treatment solutions were prepared in acetone. In the light of previous validation (12-42-006-ES), it was anticipated that semi-static methodology would be required because of high instability of the test item. For the range-finding test : the test treatments were renewed each day without changing the mineral water. For the definitive test the test solutions were renewed each day and the test item concentrations were measured in the old and in the new media. Despite this, the test item treatments were not maintained within 80-120% of the nominal values during the test period (deviation of the study plan). The test concentrations were taken as mean of the initial concentration in the new media and in the final concentration in the old media throughout the 4-day test period. Because the test item was long to solubilise, the measured initial concentrations did not match the nominal values and were not retained. The test solutions were checked to match the required concentrations and the test item treatments were considered as totally set at each medium renewal. As a consequence the nominal value was used as the initial value for every successive day (deviation from the study plan).

Details on test conditions:

TEST SYSTEM
- Test vessel: range-finding test : 1,5 L capacity glass containers filled with 1 L of reconstituted water. One replicate unit for each the five test item treatments and for the controls. Seven fish per aquarium. Definitive test : 8,0 L capacity glass containers filled with 4L of reconstituted water. One replicate unit for each the five test item treatments and for the controls. Seven fish per aquarium.
- Type (delete if not applicable): open

- Renewal rate of test solution (frequency/flow rate): test solutions were renewed each day (range-finding test and definitive test).
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1

OTHER TEST CONDITIONS
- Photoperiod: 16h light/dark cycle
- Light intensity: 200-260 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test units were inspected for mortality 24h, 48h, 72h and 96h after the test initiation. Any abnormal behavior or appearance, if any, was reported.

TEST CONCENTRATIONS
- Range finding study : yes
- Test concentrations: 0.01 - 0.1 - 1.0 - 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: The viability was not affected for test item treatment up to and including 1.. mg/L. The final percentage of mortality was 85,7 % at 10.0 mg/L and 100 % at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Sensitivity to Potassium dichromate was assessed. The measured values for 96h-LC50 were within the expected interval of 50-250 mg/L.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: the nominal value was used as the initial value for every successive day (deviation from the study plan).

Details on results:

- Analytical check of the test item treatments: Due to high volatilisation of the test item from water, it was not possible to maintain the test item treatments between 80 and 120% of the nominal values. The treatments were renewed each day. Despite this, the results showed that the test item treatments decreased down to approximately 20-30% throughout the 24h standing period. The test item concentrations were thus anticipated to be the mean of the measured concentrations throughout the test period. The test item treatment solutions were checked not to deviate by more than 5% from the nominal value (data not shown). Despite this the measured concentrations in the daily new media did not match the nominal values (data not shown). It was concluded that the solubilisation was long to be achieved and that the measured concentrations did not reflect the actual values. As a consequence the measured initial concentrations were not retained, and the nominal values were taken as the test item concentrations for every daily new medium. Consequently the test item treatments were taken as 0.7, 1.0, 1.6, 2.4 and 4.1 mg/L instead of 1.0, 1.5, 2.2, 3.3 and 5.0 mg/L.

Sublethal observations / clinical signs:

Table 6.1.1/2: Definitive test - % recovery of the test item treatments over the test period

	Nominal treatment concentrations
	1,0 mg/L	1,5 mg/L	2,2 mg/L	3,3 mg/L	5,0 mg/L
Day 1, old media	0,31 mg/L 31,6%	0,55 mg/L 37,2%	0,88 mg/L 39,4%	1,29 mg/L 38,6%	2,09 mg/L 41,8%
Day 2, old media,	0,34 mg/L 33,9%	0,55 mg/L 36,8%	0,97 mg/L 43,5%	1,34 mg/L 40,1 %	2,34 mg/L 46,8 %
Day 3, old media,	0,44 mg/L 44,3%	0,60 mg/L 40,3 mg/L	1,00 mg/L 44,8%	1,73 mg/L 51,9%	(*)
End of test	0,53 mg/L 52,8%	0,70 mg/L 46,7%	0,99 mg/L 44,3%	1,83 mg/L 54,7%	(*)

Table 6.1.1/3: Definitive test - Observed mortality

	Mortality (%)
	24h	48h	72h	96h
Water control	0.0	0.0	0.0	0.0
Solvent control	0.0	0.0	0.0	0.0
0.7 mg/L 1.0 mg/L 1.6 mg/L 2.4 mg/L 4.1 mg/L	0.0 0.0 0.0 42.9 85.7	0.0 0.0 14.3 57.1 100	0.0 0.0 28.6 85.7 100	0.0 0.0 28.6 85.7 100

Validity criteria fulfilled:

no

Remarks:

The test item treatments were not maintained within 80-120% of the nominal values. The calculations were based on mean measured concentrations.

Conclusions:

Based on the results of this study, the 96h-LC50 value was determined at 1,9 mg/L, based on initial measured concentrations. Statistical analysis gave a 95%-confidence interval of 0.8 - 4,6 mg/L.

Executive summary:

The influence of the test substance on mortality of Danio rerio was investigated according to OECD Guideline 203 (Fish, Acute Toxicity Test) (July 1992) with GLP compliance statement. Danio rerio were exposed to the test item under open and semi-static conditions, for 96 h.

Based on the results of a range-finding test, a definitive test was performed, exposing Danio rerio to 5 different nominal concentrations : 1.0 - 1.5 - 2.2 - 3.3 and 5.0 mg/L. For the definitive test, the test solutions were renewed each day and the test item concentrations were measured in the old and in the new media.

As the initial test solution concentrations did not match the nominal concentrations, they rejected the initials data and kept the nominals. The results showed that the test item concentrations of old solutions decreased to approxiamately 20 -30% of the initials over each 24h standing period. The test item concentrations were thus calculated as the mean of the nominal and the measured old solution concentrations over the test period. Consequently the test item treatments were calculated as 0.7, 1.0, 1.6, 2.4 and 4.1 mg/L instead of 1.0, 1.5, 2.2, 3.3 and 5.0 mg/L.

Based on the results of this study, the 96h-LC50 value was determined at 1,9 mg/L, based on initial measured concentrations. Statistical analysis gave a 95%-confidence interval of 0.8 - 4,6 mg/L.

This study is considered reliable with restrictions for the following reasons:

- The substance is adequately identified, but some data on composition is missing.

- The treatment solutions were prepared with a solvent (acetone), which is not the best method at the time being but in acceptable quantity (0,005 mL/L).

- No information on the quantity of test substance included in the stock solution.

- The test was performed under open conditions.

- Measured concentrations in the daily new media were not reported in the study report and therefore the mean measured concentrations stated in the report cannot be verified.

Description of key information

OECD Guideline 203, GLP, key study, validity 2 :

96h-LC50 (Danio rerio) = 1,9 mg/L based on initial measured concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.9 mg/L

Additional information

The influence of the test substance on mortality of Danio rerio was investigated according to OECD Guideline 203 (Fish, Acute Toxicity Test) (July 1992) with GLP compliance statement. Danio rerio were exposed to the test item under open and semi-static conditions, for 96 h.

Based on the results of a range-finding test, a definitive test was performed, exposing Danio rerio to 5 different nominal concentrations : 1.0 - 1.5 - 2.2 - 3.3 and 5.0 mg/L. During the definitive test, the test solutions were renewed each day and the test item concentrations were measured in the new and old media. As the initial test solution concentrations did not match the nominal concentrations, they rejected the initials data and kept the nominals. The results showed that the test item concentrations of old solutions decreased to approxiamately 20 -30% of the initials over each 24h standing period. The test item concentrations were thus calculated as the mean of the nominal and the measured old solution concentrations over the test period. Consequently the test item treatments were calculated as 0.7, 1.0, 1.6, 2.4 and 4.1 mg/L instead of 1.0, 1.5, 2.2, 3.3 and 5.0 mg/L.

Based on the results of this study, the 96h-LC50 value was determined at 1,9 mg/L, based on initial measured concentrations. Statistical analysis gave a 95%-confidence interval of 0.8 - 4,6 mg/L.

This study is considered reliable with restrictions for the following reasons:

- The test substance is adequately identified, but some data on composition is missing.

- The treatment solutions were prepared with a solvent (acetone), which is not the best method at the time being but in acceptable quantity (0,005 mL/L).

- No information on the quantity of test substance included in the stock solution.

- The test was performed under open conditions.

- Measured concentrations in the daily new media were not reported in the study report and therefore the mean measured concentrations stated in the report cannot be verified.