cis-2-tert-butylcyclohexyl acetate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Nov 2008 to 28 Jan 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: ontrol (pooled replicates) and each test group (pooled replicates) at 0 and 72 hours
- Sample storage conditions before analysis: direct, given the volatile nature of the test material an additional control and test replicate was prepared at each test concentration and incubated alongside the test remaining unopened until the end of the test. A sample of each of these additional test replicates was taken for chemical analysis at 72 hours.

Vehicle:

no

Details on test solutions:

CULTURE MEDIUM
The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCI.The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/L of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system.

SATURATED SOLUTION PREPARATION
An amount of test material (1100 mg) was dispersed, in duplicate, in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for periods of 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 pm Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter)
• Filtration through a 0.2 pm Sartorius Sartopore filter (approximately 2 litres discarded in order to pre-condition the filter)

SOLVENT SPIKE PREPARATION
An amount of test material (50 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to a 50 mg/10 mL solvent stock solution. An aliquot (1000 pl) of this 50 mg/10 mL solvent stock solution was dispersed in 10 litres of culture medium with the aid of magnetic stirring at approximately 21°C for approximately 10 minutes to give the required test concentration of 0.50 mg/L prior to taking samples for chemical analysis after the following pre-treatments:
• Untreated
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 pm Gelman Acrocap filter (approximately 100 mL discarded in order to pre-condition the filter)
• Filtration through a 0.2 pm Gelman Acrocap filter (approximately 500 mL discarded in order to pre-condition the filter)

The remainder of the 0.50 mg/L test concentration was returned to the magnetic stirrer and stirred for a further 48 hours with samples being taken for analysis after both 24 and 48 hours stirring.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10E+04 - 10E+05 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No detail given in study report

Test temperature:

24 ± 1°C

pH:

8.1 at 0 hours
8.6 - 9.1 at 72 hours

Dissolved oxygen:

No detail given in study report

Salinity:

No detail given in study report

Nominal and measured concentrations:

- Nominal concentrations: 1.75, 3.5, 7.0, 14, 27 mg/L
- Measured concentrations: 0.39, 0.57, 0.81, 4.4, 26 mg/L

Details on test conditions:

TEST SYSTEM
Based on the results of the pre-study media preparation trial and range-finding test the following nominal test concentrations were assigned to the definitive test: 1.75, 3.5, 7.0, 14 and 27 mg/L. Chemical analysis of the test preparations at 0-Hours showed measured test concentrations of 1.4, 2.9, 6.0, 12 and 25 mg/L were obtained. The slight differences observed between the measured concentrations obtained from the pre-study media preparation trial and definitive test were considered to be due to slight differences in the stirring rate of the saturated solution. Due to the low aqueous solubility and high purity of the test material the test concentrations used in the definitive test were prepared by diluting (with culture medium) a saturated solution prepared from an initial test material dispersion at a concentration of 100 mg/L.
An amount of test material (1100 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 pm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution with a 0-Hour measured concentration of 25 mg/L. A series of dilutions was made from this saturated solution to give stock solutions of 12, 6.0, 2.9 and 1.4 mg/L. An aliquot (2 litres) of each of the stock solutions was separately inoculated with 9 mL algal suspension to give the 0-Hour measured test concentrations of 1.4, 2.9, 6.0, 12 and 25 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

EXPOSURE CONDITIONS
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each completely filled with test preparation were used for the control and three flasks each completely filled with test preparation were used for each treatment group. The control group was maintained under identical conditions but not exposed to the test material. Due to the volatile nature of the test material an additional replicate was prepared for the control and each treatment group at 0 hours and incubated alongside the test to provide samples for unopened vessel analysis at 72 hours.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 8.91 x 1 05 cells per mL. Inoculation of 2 litres of test medium with 9.0 mL of this algal suspension gave an initial nominal cell density of 4 x 103 cells per mL and had no significant dilution effect on the final test concentration. The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 3.0 - 5.9 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.57 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 1.4, 2.9 and 6.0 mg/L* test cultures were observed to be green dispersions. The 12 mg/L* test cultures were observed to be pale green dispersions whilst the 25 mg/L* test cultures were observed to be clear colourless solutions.

* 0-hour measured concentrations.

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures.

Results with reference substance (positive control):

The positive control was conducted between 26 May 2009 and 29 May 2009. Exposure of Desmodesmus subspicatus(CCAP 276/20) to the reference material gave the following results:
- ErC50 (0 - 72 h): 0.79 mg/L*
- ErC50 (0 - 72 h): 0.30 mg/L, 95% confidence limits 0.27 - 0.34 mg/L

* It was not possible to calculate 95% confidence limits as the data generated did not fit the models available for the calculation of confidence limits.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Where appropriate 95% confidence limits for the EC50 values were calculated, the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949) was used.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h ErC50 value is 4.2 mg/L and the 72-h NOErC is 0.57 mg/L in Desmodesmus subspicatus.

Executive summary:

The effect of the test substance on the growth of aquatic algae was determined in a GLP-compliant OECD Guideline 201 study. Green algae (Desmodesmus subspicatus) with an initial cell concentration of 4E+03 cells/mL) were exposed for 72 hours to nominal concentrations of 1.75, 3.5, 7.0, 14, and 27 mg/L test substance (3 replicates per concentration) and a control (6 replicates). Inhibition of algal growth rate and biomass (as yield) were monitored and all cultures were inspected microscopically at 72 hours. Samples of the test solutions were taken 0 and 72 hours and analysed by gas chromatography (GC). As the measured concentrations declined over the 72-hour testing period, the geometric mean of measured concentrations was used for calculating effect concentrations. The final concentration were all below the LoQ. Both growth rate and yield were significantly reduced compared to controls (p<0.05) at the three highest test levels. Based on these reductions the 72- h ErC50 and the 72-h NOErC were determined to be 4.2 and 0.57 mg/L, respectively. These effect concentrations are over-conservative values, because the LoQ is driving these toxicity values.

Description of key information

The effect of the test substance on the growth of aquatic algae was determined in a GLP-compliant OECD Guideline 201 study. Green algae (Desmodesmus subspicatus) with an initial cell concentration of 4E+03 cells/mL) were exposed for 72 hours to nominal concentrations of 1.75, 3.5, 7.0, 14, and 27 mg/L test substance (3 replicates per concentration) and a control (6 replicates). Inhibition of algal growth rate and biomass (as yield) were monitored and all cultures were inspected microscopically at 72 hours. Samples of the test solutions were taken 0 and 72 hours and analysed by gas chromatography (GC). As the measured concentrations declined over the 72-hour testing period, the geometric mean of measured concentrations was used for calculating effect concentrations. The final concentration were all below the LoQ. Both growth rate and yield were significantly reduced compared to controls (p<0.05) at the three highest test levels. Based on these reductions the 72- h ErC50 and the 72-h NOErC were determined to be 4.2 and 0.57 mg/L, respectively. These effect concentrations are over-conservative values, because the LoQ is driving these toxicity values.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.2 mg/L

EC10 or NOEC for freshwater algae:

0.57 mg/L