Cyclohexanecarboxylic acid, 1-(2-ethylbutyl)-

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2010 – 10 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 203 (Fish, Acute Toxicity Test)

Version / remarks:

(1992)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)

Version / remarks:

(Second edition, 1996-06-15)

Deviations:

no

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:

no

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, after 24 hours of exposure and at the end of the test from the test vessels containing 10% and 100% of the WSF.
- Sampling volume: 2 ml
- 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. The glass wool containing the undissolved residue was kept for possible analysis.
- Concentrations: at t=0 and t=96 all test concentrations and the control group; In addition at t=3 and t=72 h for those groups in which all fish had died.
- Sampling volume: 2 ml (singular samples for regular analysis and singular reserve samples for possible re-analysis)
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

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 20-22 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):

Cyprinus carpio

Details on test organisms:

Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length (1): Range-finding test: 3.2 ± 0.2 cm
Final test: 4.0 ± 0.2 cm
Mean weight(1): Range-finding test: 0.90 ± 0.16 g
Final test: 1.64 ± 0.34 g
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 54

(1) Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

no

Test conditions

Hardness:

180 mg CaCO3 per litre

Test temperature:

21.5 - 22.4°C

pH:

7.2 - 7.7

Dissolved oxygen:

5.8 - 9.4

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): 7.2, 12.8, 23.4, 40.8 and 76.2 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: 35 litres, all-glass, containing 20 litres of test solution.
- Aeration: The test media were aerated between 24 and 48 hours of exposure.
- No. of organisms per vessel: 7 per concentration
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.57 g fish/litre, i.e. 7 fish per 20 litres of test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod daily
- Introduction of fish: Within 11 minutes after preparation of the test media from a holding tank with comparable water quality parameters and
pH and temperature differences between test and holding tank media of less than 0.5 unit and 0.5°C.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- mortality: At 3¼, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 and every morning from day 1 to observe for any dead or severely distressed fish. Dead fish were removed when observed.

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 0.1%, 1%, 10% and 100% of 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
- Test concentrations: 10%, 18%, 32%, 56% and 100% of WSF at 100 mg/L loading rate (initially measured as 7.2, 12.8, 23.4, 40.8 and 76.2 mg/L, respectively); During the exposure period the measured concentrations remained constant.

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP)

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

20 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence interval between 18 and 27 mg/l)

Details on results:

Range finding test:
All fish exposed to the WSF prepared at 100 mg/l died within the 1st day of exposure, whereas no mortality or clinical effects were observed in any of the dilutions prepared from 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 96-hour test period. Therefore, the expected LC50 was between 7 and 70 mg/l.

Final test:
see tables below
Note that aeration was introduced after 24 hours of exposure, as the oxygen concentration tended to drop below the optimum level for testing with carp, i.e. below 5 mg/l.

Results with reference substance (positive control):

This reference test was carried out to check the sensitivity of the test system as used at the test facility.
- Concentrations: 0.10, 0.22 and 0.46 mg PCP/l in ISO-medium. (pentachlorophenol = PCP)
- Incidence of mortality observed in the reference study: 0% total mortality at 0.10 mg PCP/l (after 96 h), 100% total mortality at 0.22 and 0.46 mg PCP/l (already observed after 24 and 6.5 hours, respectively)
- 96h-LC50 for carp exposed to PCP: 0.15 mg/l (95 % confidence interval between 0.10 and 0.22 mg/l). This effect was already reached within 24 hours of exposure.

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

Any other information on results incl. tables

Sublethal observations / clinical signs:

The test was accepeted for the following reasons:

1. No mortality was observed in the control at the end of the test.

2. Test conditions were maintained constant throughout the test. (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C)

3. The dissolved oxygen concentration has been at least 60% of the air saturation value throughout the test (>5 mg/l at 22°C).

4. Since the measured concentrations deviated by more than 20% from nominal, but remained stable during the test period, results were based on the initial measured exposure concentration.

Mortality and other effects

Incidence of mortality and total mortality during the final test

 

Concentration CAT-Acid (mg/l)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		3¼h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
7.2	7	0	0	0	0	0	0
12.8	7	0	0	0	0	0	0
23.4	7	0	0	0	3	5	71
40.8	7	2	6	6	7	7	100
76.2	7	7	7	7	7	7	100

 

 

Clinical effects observed during the final test.

 

Concentration CAT-Acid (mg/l)	Time of recording (hours)	Specification of effects	Relative number
12.8	96	Swimming at the surface	3/7
		Swimming at the surface and loss of equilibrium	1/7
23.4	3¼	Swimming at the surface	6/7
	24	Swimming at the surface and discoloured	7/7
	48	Swimming at the surface and loss of righting reflex	7/7
	72	Swimming at the surface and loss of righting reflex	4/4
	96	Swimming at the surface and loss of equilibrium	2/2
40.8	3¼	Immobile and discoloured	3/5
		Loss of righting reflex and discoloured	1/5
		Discoloured	1/5
	24	Swimming at the surface and discoloured	1/1
	48	Swimming at the bottom	1/1

 

 

Effect parameters

 

Parameter	Concentration CAT-Acid (mg/l)	95%- confidence interval
24,48h-LC50	34	31-42
72h-LC50	23	20-31
96h-LC50	20	18-27

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the described test CAT-Acid induced no lethal effects in carp at or below an initial exposure concentration of 12.8 mg/l.
The 96h-LC50 was 20 mg/l based on initial exposure concentrations (95% confidence interval between 18 and 27 mg/l).