2-methylcyclohexyl acetate

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 11th, 2012 to November 30th, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 4.6, 10, 22, 46 and 100 mg/L
- Sampling method: 3 mL form the approximate centre of the test vessels were taken at the start of the test and after 72 hours from the freshly prepared conditions and at the first renewal (t=24h) and the end of the test from the 24-hour old solutions.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a concentration of 100 mg/l applying approximately a 30-minute period of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.
- Controls:Test medium without test substance or other additives.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Length at study initiation (length definition, mean, range and SD): 2.7 ± 0.2 cm
- Weight at study initiation (mean and range, SD): 0.51 ± 0.09 g

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Pelleted fish food: Cyprico Crumble Excellent (300-500 um)
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): Mortality during the seven days prior to the start of the test was less than 5%.

FEEDING DURING TEST
- Frequency: No feeding from 24 hours prior to the test and during the total test period.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

180 mg CaCO3 per litre

Test temperature:

20.5-22.0 ºC

pH:

7.7 ± 0.3

Dissolved oxygen:

7.0-9.0 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0, 4.6, 10, 22, 46, 100 mg/L
Measured concentrations (average exposure 0-96 mg/L): 0, 3.3, 7.4, 15, 41*, 87*.
(*: Initial concentration. Nominal concentrations of 46 and 100 mg/L were not analysed since all fish were died at 24 hours).

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 litres, all-glass, containing 9.5 litres of test medium
- Type (delete if not applicable): Airtight closed
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): Daily renewal
- No. of organisms per vessel: 7 fish
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.38 g fish/litre

TEST MEDIUM / WATER PARAMETERS
- Test medium: Adjusted ISO medium
- Source/preparation of dilution water: RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Mortality and other effects were analysed at 2.5, 24, 48, 72 and 96 hour following the start of exposure. Fish were considered to be dead when no reaction was observed after touching the caudal peduncle and visible breathing movements were absent.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: Three fish per concentration were exposed to 0.1, 1.0, 10, 100 mg/L.
- Results used to determine the conditions for the definitive study: At 100 mg/L 100% mortality was observed within 2.5 hours. At 0.1 mg/L no mortality was observed. The expected LC50 was between 8.1 and 84 mg/l (based on initial measured concentrations).

Reference substance (positive control):

no

Remarks:

(Internal laboratory results of the reference test with pentachlorophenol were included)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 12-23 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

7.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Details on results: Within 2½ hours complete mortality was observed in the two highest concentrations of 41 and 87 mg/l. Four fish died in the intermediate group of 15 mg/l. No effects were observed at an average exposure concentrations of 3.3 and 7.4 mg/l, and neither in the control.
Analytical monitoring: The average exposure concentrations were calculated to correspond with 3.3, 7.4, 15, 41, 87 mg/l for the concentrations prepared at 4.6, 10, 22, 46 and 100 mg/l, respectively. Since measured concentrations deviated by more than 20% from nominal, results were based on the average exposure concentration.

Results with reference substance (positive control):

- Results with reference substance valid? Yes (historical data, pentachlorophenol)
- LC50: 96h-LC50 was 0.25 mg/l based on nominal concentrations, with a 95% confidence interval between 0.20 and 0.41 mg/l (January 2012).
- Other: The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/l based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.

Reported statistics and error estimates:

The 72 and 96-hour LC50 was determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations for an exposure period of 72 and 96 hours. The 24 and 48-hour LC50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.

Sublethal observations / clinical signs:

Incidence of mortality and total mortality during the range-finding test:

Average exposure concentration 2-methylcyclohexyl acetate (mg/l)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		2½h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
3.3	7	0	0	0	0	0	0
7.4	7	0	0	0	0	0	0
15	7	0	0	0	2	4	57
41	7	7	7	7	7	7	100
87	7	7	7	7	7	7	100

Average exposure concentration versus nominal concentration:

Nominal concentration (mg/l)	Concentration (mg/l)
	Measured at		Average exposure 0-24 h (mg/l)	Measured at		Average exposure 0-96 h (mg/l)
	t=0h	t=24h		t=72h	t=96h
0	n.d.	n.d.	0	n.d.	n.d.	0
4.6	4.19	2.74	3.4	4.40	2.32	3.3
10	9.04	7.60	8.3	8.69	4.90	7.4
22	19.5	14.0	17	15.6	11.8	15
46	41.4	n.d.	41*	n.d.	n.d.	41*
100	86.8	n.d.	87*	n.d.	n.d.	87*

n.d. not determined

* - initial concentration (not analysed since all fish died within 24h).

Effect parameters:

Parameter	Average exposure concentration (mg/l)	95%-confidence interval (mg/l)
NOEC	7.4
24h-LC50	26	17-41
48h-LC50	25	15-41
72h-LC50	17	14-29
96h-LC50	14	12-23

Validity criteria fulfilled:

yes

Remarks:

(No mortality in the control, test conditions maintained throughout the test, dissolved oxygen > 60% of air saturation, since measured concentrations deviated >20% results were based on the average exposure concentration)

Conclusions:

The 96h-LC50 of 2-methylcyclohexyl acetate in carps (basis for effect: mortality) was determined to be an average exposure concentration of 14 mg/L. The test item induced no lethal effects at or below an average exposure concentration of 7.4 mg/l (NOEC).

Executive summary:

A fish acute toxicity test was performed on 2 -methylcyclohexyl acetate in accordance with OECD Guideline 203. A final test was performed in a semi-static system airtight closed with daily renewals based on the results obtained in a preceding static range-finding test. Seven carp per test group were exposed to a control and solutions containing 4.6, 10, 22, 46 and 100 mg/l. The total test period was 96 hours. The average exposure concentrations were calculated to correspond with 3.3, 7.4, 15, 41, 87 mg/l for the concentrations prepared at 4.6, 10, 22, 46 and 100 mg/l, respectively. Since measured concentrations deviated by more than 20% from nominal, results were based on the average exposure concentration. 2-methylcyclohexyl acetate induced no lethal effects in carp at or below an average exposure concentration of 7.4 mg/l (NOEC). The 96h-LC50 was 14 mg/l based on average exposure concentrations (95% confidence interval between 12 and 23).

Description of key information

Key study: OECD Guideline 203. GLP study. The 96h-LC50 of 2-methylcyclohexyl acetate in carps (basis for effect: mortality) was determined to be an average exposure concentration of 14 mg/L. The NOEC was determined to be 7.4 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

14 mg/L

Additional information

Key study: A fish acute toxicity test was performed on 2 -methylcyclohexyl acetate in accordance with OECD Guideline 203. Seven carp per test group were exposed to a control and solutions containing 4.6, 10, 22, 46 and 100 mg/l in a semi-static airtight closed system. The total test period was 96 hours. The average exposure concentrations were calculated to correspond with 3.3, 7.4, 15, 41, 87 mg/l for the concentrations prepared at 4.6, 10, 22, 46 and 100 mg/l, respectively. Since measured concentrations deviated by more than 20% from nominal, results were based on the average exposure concentration. 2-methylcyclohexyl acetate induced no lethal effects in carp at or below an average exposure concentration of 7.4 mg/l (NOEC). The 96h-LC50 was 14 mg/l based on average exposure concentrations (95% confidence interval between 12 and 23).