2-methylcyclohexyl acetate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 11th, 2012 to November 28th, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 10, 18, 32, 56 and 100 mg/l
- Sampling method: 3 mL form the approximate centre of the test vessels were taken at the start (t=0) and at the end (t=48) of the test.
- 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 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea
- Strain/clone: Daphnia magna Straus
- Age at study initiation (mean and range, SD): <24 h
- Weight at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD):
- Method of breeding: In-house laboratory culture with a known history.
- Source: Start of each batch with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age of parental stock (mean and range, SD): Maximum age of the cultures: 4 weeks
- Feeding during test: no feeding

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L of CaCO3

Test temperature:

19.3 and 19.8 °C

pH:

7.9-8.1

Dissolved oxygen:

8.2-8.9 mg/L O2

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 10, 18, 32, 56, 100 mg/L
Initial measured concentrations: 0 (control), 7.5, 15, 25, 44, 85 mg/l
Measured concentrations at the end: 0 (control), 8, 14, 23, 42, 82.5 mg/L.
During the exposure period the measured concentration remained stable (95-110% of initial). Therefore, the effect parameters were expressed in terms of initially measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: ~60 ml, all-glass
- Type (delete if not applicable): completely filled and air-tight closed
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 5 per vessel

TEST MEDIUM / WATER PARAMETERS
- Test medium: Adjusted ISO medium
- Source/preparation of dilution water: Tap water purified by Reverse Osmosis.
- Culture medium different from test medium: Breeding medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Immobility (including mortality) was measured at 24 hours and at 48 hours of exposure.
Immobile are those animals not able to swim within 15 seconds after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: Twenty Daphnia per concentration (four replicated, 5 daphnids per vessel) were exposed to a blank control and a concentration of 100 mg/l. Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to 0.1, 1.0 and 10 mg/l.
- Results used to determine the conditions for the definitive study: Besides the highest concentration no effects were observed during the whole exposure period. In the highest concentration 95% of the daphnids became immobilised at the end of the test. Therefore, the expected EC50 was between 6.6 and 71 mg/l (measured concentration).

Reference substance (positive control):

no

Remarks:

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

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

34 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 30-40 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No significant immobilisation was observed in the control (5%) and the two lowest concentrations tested (0%). Note that the guideline allows a 10% effect in the control which is considered biologically not significant. The recorded immobility at the remaining concentrations ranged from 10 to 100% at the end of the test and increased with concentration.

Results with reference substance (positive control):

- Results with reference substance valid? Yes (historical data with potassium dichromate, November 2012)
- EC50/LC50: The 48h-EC50 was 0.38 mg/l with a 95% confidence interval between 0.34 and 0.45 mg/l.
- Other: The actual responses in this reference test with K2Cr2O7 were in the lower range of the expected historical responses at the different concentrations, i.e. a 48h-EC50 between 0.3 and 1.0 mg/l. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at WIL Research Europe.

Reported statistics and error estimates:

The EC50-value was calculated at 24 and 48 hours of exposure from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method.

Acute immobilisation of daphnids after 24 and 48 hours in the final test:

Initial concentration 2-methylcyclohexyl acetate (mg/l)	Vessel number	Number Daphnia Exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
control	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 1	5
7.5	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
15	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
25	A B C D	5 5 5 5	0 1 1 0	10	0 1 1 0	10
44	A B C D	5 5 5 5	4 2 3 4	65	4 4 3 5	80
85	A B C D	5 5 5 5	3 3 3 4	65	5 5 5 5	100

Effect parameters:

Parameter	Initial concentration 2-methylcyclohexyl acetate (mg/l)	95%-confidence interval (mg/l)
NOEC	15
24h-EC50	39	33-50
48h-EC50	34	30 -40

Validity criteria fulfilled:

yes

Remarks:

(in the control <10% were immobilised, oxygen concentration at the end was >3 mg/L)l

Conclusions:

The 48h-EC50 of 2-methylcyclohexyl acetate in Daphnia magna (basis for effect: mobility) was determined to be 34 mg/l based on the analytical confirmed initial concentrations. The test item did not induce acute immobilisation of Daphnia magna at an analytical confirmed initial concentration of 15 mg/l after 48 hours of exposure (NOEC).

Executive summary:

An acute toxicity study in Daphnia magna with 2 -methylcyclohexyl acetate was performed in accordance with OECD Guideline 202. A final test was performed based on the results obtained in a preceding combined limit/range-finding test. Twenty daphnia per group (5 per vessel, 4 vessels) were exposed to a control and solutions containing 10, 18, 32, 56 and 100 mg/l. The total test period was 48 hours. During the exposure period the measured concentration remained constant (95-110% of initial) and therefore, the exposure concentrations were based on initially measured concentrations. All the validity criteria were met. 2-methylcyclohexylacetate did not induce acute immobilisation of Daphnia magna at an analytical confirmed initial concentration of 15 mg/l (NOEC). The 48h-EC50 was 34 mg/l based on initially measured concentrations (95% confidence interval between 30 and 40 mg/l).

Description of key information

Key study: OECD Guideline 202. GLP study. The 48h-EC50 of 2-methylcyclohexyl acetate in Daphnia magna (basis for effect: mobility) was determined to be an analytical confirmed initial concentration of 34 mg/L. The NOEC was determined to be 15 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

34 mg/L

Additional information

Key study: An acute toxicity study in Daphnia magna with 2 -methylcyclohexyl acetate was performed in accordance with OECD Guideline 202. Twenty daphnia per group (5 per vessel, 4 vessels) were exposed to a control and solutions containing 10, 18, 32, 56 and 100 mg/l in an airtight closed static system. The total test period was 48 hours. During the exposure period the measured concentration remained constant (95-110% of initial) and therefore, the exposure concentrations were based on initially measured concentrations. All the validity criteria were met. 2-methylcyclohexylacetate did not induce acute immobilisation of Daphnia magna at an analytical confirmed initial concentration of 15 mg/l (NOEC). The 48h-EC50 was 34 mg/l based on initially measured concentrations (95% confidence interval between 30 and 40 mg/l).