Cyclohexanecarboxylic acid, 1-(2-ethylbutyl)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2010 – 07 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 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2008, Amended by EC No. 761/2009, publication no. L220)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

(Second edition, 01 October 2004)

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 of the test, after 24 hours 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. The glass wool containing the undissolved residue was kept for possible analysis.
- Concentrations: all at t=0 h and t=72 h
- Sampling volume: 2 ml (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 with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start and at the end of the test period.

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 20-21 hours of stirring followed by a 2-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.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10^4 cells/ml.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21.5 - 22.6°C

pH:

7.6 - 8.1

Dissolved oxygen:

not determined

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): 0.59, 1.9, 6.3, 20.1 and 69.1 mg/l at test groups containing 1.0, 3.2, 10, 32 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, containing 50 ml of test solution
- Aeration: No
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density (mean of 6 values): 134.9 x 10^4 cells/mL (range finding test), 119.2 x 10^4 cells/mL (final test)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: 1 replicate of the highest concentration without algae.


GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium, according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (Milli-RO)

OTHER TEST CONDITIONS
- Sterile test conditions: not indicated
- Photoperiod and light intensity: no; Continuous illumination using TLD-lamps of the type "Cool-white" of 30 Watt, with a light intensity within the range of 86 to 98 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: t=0: microscope + counting chamber; t > 0: spectrophotometer at 720 nm with a cuvette (pathlength = 10 mm)
- Other: Appearance of the cells: At the end of the final test microscopic observations were performed on the test concentration closest to the EC50 to observe for any abnormal appearance of the algae

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 73 mg/L)
- Results used to determine the conditions for the definitive study: 10% and 100% of WSF (i.e. 7.3 and 73 mg/L)
Definitive study (final test):
- Test concentrations: 1.0%, 3.2%, 10%, 32% and 100% of WSF at 100 mg/L loading rate (initially measured as 0.59, 1.9, 6.3, 20.1 and 69.1 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

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: same NOEC for yield inhibition

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 69 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: is the concentration measured in a WSF prepared at a loading rate of 100 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% confidence interval between 5.7 and 12 mg/l)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Remarks on result:

other: (95% confidence interval between 8.1 and 22 mg/l)

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Remarks on result:

other: (95% confidence interval between 1.7 and 4.9 mg/l)

Details on results:

Combined limit/range finding test:
- The highest concentration tested , i.e. 100% of the WSF, reduced growth rate by 56.3% and inhibited yield by 94.4%. The second highest concentration tested , i.e. 10% of the WSF, reduced growth rate by 6.5% and inhibited yield by 27.3%.
- 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 73 and 7.3 mg/l, respectively and these concentrations remained stable during the 72-hour test period. Therefore, the expected EC50 for growth rate reduction approximated the concentration of 73 mg/l present in the highest test group, while the expected EC50 for yield inhibition was between 7.3 and 73 mg/l.

Final test:
see tables below

- Growth rates were in the range of the controls at the concentrations of 0.59 and 1.9 mg/l during the 72-hour test period, whereas the growth rate of algae exposed to 6.3 mg/l and higher were increasingly reduced. Statistically significant reduction of growth rate was found at concentrations of 6.3 mg/l and higher (Bonferroni t test, a = 0.05).

- No inhibition of yield was recorded up to 1.9 mg/l. Inhibition of yield increased with increasing concentration of CAT-Acid from 6.3 mg/l upwards resulting in 89% inhibition at 69.1 mg/l. Statistically significant inhibition of yield was found at test concentrations of 6.3 mg/l and higher (Bonferroni t test, a = 0.05).

- Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

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.
Algae (Pseudokirchneriella subcapitata, strain: NIVA CHL-1) were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a control. The initial cell density was 1.0 x 10^4 cells/mL.

- Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.32 mg/l and higher.

- The EC50 for growth rate reduction (ERC50: 0-72h) was 1.0 mg/l with a 95% confidence interval ranging from 0.66 to 1.4 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.

- The EC50 for yield inhibition (EYC50: 0-72h) was 0.37 mg/l with a 95% confidence interval ranging from 0.28 to 0.51 mg/l. The historical ranges of the 72h-EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

- For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman).
- Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
- Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding initial meaqsured concentrations of the test substance.
- No EC50-values could be calculated for growth (EC50 > maximum soluble concentration).

Any other information on results incl. tables

The test was accepted for the following reasons:

1. In the controls, cell density increased by an average factor of > 16 within two days.

2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.

3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

Mean cell densities (x 10⁴ cells/ml) during the final test

Concentration CAT-Acid (mg/l)	Exposure time (hours)
	0	24	48	72
control	1.0	5.7	27.3	119.2
0.59	1.0	5.9	26.9	114.7
1.9	1.0	6.5	27.4	120.7
6.3	1.0	5.1	20.2	86.7
20.1	1.0	4.9	13.9	40.3
69.1	1.0	3.5	7.9	13.7

Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Concentration CAT-Acid (mg/l)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
control	0.06635		118.22
0.59	0.06582	0.8	113.65	3.9
1.9	0.06656	-0.3	119.68	-1.2
6.3	0.06195	6.6	85.73	27.5
20.1	0.05128	22.7	39.25	66.8
69.1	0.03627	45.3	12.66	89.3

Percentage reduction of growth rate at different time intervals during the final test

Concentration CAT-Acid (mg/l)	Mean growth rate
	µ (0-24 h)	Reduction (%)	µ (24-48 h)	Reduction (%)	µ (48-72 h)	Reduction (%)
control	0.07215		0.06554		0.06135
0.59	0.07392	-2.4	0.06320	3.6	0.06035	1.6
1.9	0.07768	-7.7	0.06028	8.0	0.06174	-0.6
6.3	0.06806	5.7	0.05709	12.9	0.06069	1.1
20.1	0.06571	8.9	0.04399	32.9	0.04413	28.1
69.1	0.05074	29.7	0.03521	46.3	0.02287	62.7

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the described study with Pseudokirchneriella subcapitata, CAT-Acid reduced growth rate and inhibited the yield of this fresh water algae species significantly at an initially measured concentration of 6.3 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) exceeded an initial measured concentration of 69 mg/l, being the concentration measured in a WSF prepared at a loading rate of 100 mg/l.
The EC10 for growth rate reduction (ERC10: 0-72h) was 8.2 mg/l with a 95% confidence interval ranging from 5.7 to 12 mg/l (initial measured concentrations).

The EC50 for yield inhibition (EYC50: 0-72h) was 14 mg/l with a 95% confidence interval ranging from 8.1 to 22 mg/l (initial measured concentrations).
The EC10 for yield inhibition (EYC10: 0-72h) was 2.9 mg/l with a 95% confidence interval ranging from 1.7 to 4.9 mg/l (initial measured concentrations).

The NOEC for both growth rate reduction and yield inhibition was 1.9 mg/l based on initial measured concentrations.