Cyclohexanecarboxylic acid, 1-(2-ethylbutyl)-

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2010 – 06 January 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and EC guidelines and in compliance with GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

(2004)

Deviations:

yes

Remarks:

(Evaluation of effects was performed after 26 instead of 24 hours in the final test, but this has no impact on the final conclusion of the study.)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

(2008)

Deviations:

yes

Remarks:

(see OECD 202 deviation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Version / remarks:

(Third edition, 1996-04-01)

Deviations:

yes

Remarks:

(see OECD 202 deviation)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

yes

Remarks:

(see OECD 202 deviation)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-(2-ethylbutyl)cyclohexane-1-carboxylic acid

EC Number:

619-508-4

Cas Number:

381209-09-2

Molecular formula:

C13 H24 O2

IUPAC Name:

1-(2-ethylbutyl)cyclohexane-1-carboxylic acid

Details on test material:

- Name of test material (as cited in study report): CAT-Acid
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the range finding test, samples for analytical confirmation of actual exposure concentrations were taken at the start of the test and at the end of the test from the test vessels containing 10% and 100% of the WSF.
- Sampling volume: 2 ml from the approximate centre of the test vessels
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

During the final test singular samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
- Concentrations: at t=0 and t=48
- Sampling volume: 2 ml from the approximate centre of the test vessels (singular samples for regular analysis and singular reserve samples for possible re-analysis, stored in the freezer)
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of CAT-Acid tested was a clear orange liquid with big orange crystals and a purity of 99.7%.

The test substance was not completely soluble in test medium at the loading rate initially prepared. Note that CAT-Acid was heated up to 50ºC for 1-2 hours before weighing resulting in a yellow solution.

Preparation of test solutions started with a loading rate of 100 mg/l applying approximately 20 hours of stirring followed by a 2-2½ hour settlement period. This resulted in a clear and colourless solution containing a floating layer and precipitate. The middle fraction was subsequently collected using a tube filled with glass wool to remove any remaining larger undissolved particles. The resulting clear and colourless Water Soluble Fraction (WSF) was used as highest test concentration.

Lower test concentrations were prepared by serial dilution of the WSF prepared at a loading rate of 100 mg/l.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical
parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- Reason for selection: This system has been selected as an internationally accepted invertebrate species.
- Validity of batch: 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.
- Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no

Test conditions

Hardness:

ca. 180 mg/L (expressed as CaCO3 )

Test temperature:

19.8 - 20.9°C

pH:

7.6 - 7.8

Dissolved oxygen:

8.7 - 9.1

Nominal and measured concentrations:

nominal concentrations: WSF at 100 mg/L loading rate and dilutions thereof
measured concentrations (at the start of the final test): 5.2, 9.6, 16.4, 30.2 and 54.4 mg/l at test groups containing 10, 18, 32, 56 and 100% of the WSF, respectively. During the exposure period the measured concentrations remained constant. Consequently, all results were based on the initial measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass
- Fill volume: 80 ml
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2-4 (rf test), 4 (final test)
- No. of vessels per control (replicates): 4
- Biomass loading rate: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod daily
- Feeding: No feeding
- Introduction of daphnids: Within 20 minutes after preparation of the test solutions.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobility (including mortality): at 26 hours and at 48 hours

TEST CONCENTRATIONS
- Controls: Test medium without test substance or other additives
Combined limit/Range finding test:
- Test concentrations: 0.1%, 1%, 10% and 100% of the WSF at 100 mg/L loading rate (initially measured as 70 mg/L)
- Results used to determine the conditions for the definitive study: 10% and 100% of WSF (i.e. 7 and 70 mg/L)
Definitive study (final test):
- Test concentrations: 10%, 18%, 32%, 56% and 100% of WSF at 100 mg/L loading rate (initially measured as 5.2, 9.6, 16.4, 30.2 and 54.4 mg/L, respectively); During the exposure period the measured concentrations remained constant.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

29 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: immobility

Remarks on result:

other: (95% confidence interval between 25 and 36 mg/l)

Details on results:

Combined limit/range finding test:
All daphnids exposed to the WSF prepared at a loading rate of 100 mg/l were immobilised already after 24 hours of exposure. No immobility was observed in any of the dilutions of the WSF during the test period.
Samples taken from the WSF prepared at 100 mg/l and the ten-fold dilution of the WSF were analysed. The measured concentrations in the WSF and the ten-fold dilution were 70 and 7 mg/l, respectively and these concentrations remained stable during the 48-hour test period. Therefore, the expected EC50 was between 7 and 70 mg/L.

Final test:
see tables below

Results with reference substance (positive control):

The reference test was carried out to check the sensitivity of the test system as used at the test facility.
- Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l and to a control. Twenty daphnia were exposed per concentration.

- Acute immobilisation of Daphnia Magna observed in the reference study: 0% immobile at 0.32 mg K2Cr2O7/l (after 24 and 48 h), 40% immobile at 0.56 mg K2Cr2O7/l (after 48 hours), 95% immobile at 1.0 mg K2Cr2O7/l (already observed after 24 hours), 100% immobile at 1.8 mg K2Cr2O7/l (already observed after 24 hours)

The 24h-EC50 was 0.77 mg/l with a 95% confidence interval between 0.72 and 0.86 mg/l.
The 48h-EC50 was 0.57 mg/l with a 95% confidence interval between 0.52 and 0.66 mg/l.

The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was 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 the test facility.

Reported statistics and error estimates:

Calculation of EC50:
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 (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Any other information on results incl. tables

The test was accepeted for the following reasons:

1. In the control, no daphnids became immobilised.

2. The oxygen concentration at the end of the test was = 3 mg/l in control and test vessels.

Acute immobilisation of daphnids after 26 and 48 hours in the final test

Concentration CAT-Acid Initial (mg/l)	Vessel number	Number Daphnia exposed	Response at 26 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 0	0
5.2	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
9.6	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
16.4	A B C D	5 5 5 5	1 1 1 0	15	1 1 1 1	20
30.2	A B C D	5 5 5 5	2 1 1 2	30	2 1 1 2	30
54.4	A B C D	5 5 5 5	5 5 5 5	100	5 5 5 5	100

Effect parameters

Parameter	Concentration CAT-Acid (mg/l)	95%- confidence interval
NOEC	9.6
26h-EC50	30	25-36
48h-EC50	29	25-36

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the described study CAT-Acid did not induce acute immobilisation of Daphnia magna at an initial measured concentration of 9.6 mg/l after 48 hours of exposure (NOEC).
The 48h-EC50 was 29 mg/l based on initial measured exposure concentrations (95% confidence interval between 25 and 36 mg/l).