Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

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:

2 February 1996 to 22 February 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the solvent control and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.

Vehicle:

yes

Details on test solutions:

For the purpose of the definitive study the test material was prepared using a preliminary solution in ethanol.An amount of test material (800 mg) was dissolved in solvent and the volume adjusted to 25 ml to give an 800 mg/25 ml stock solution. Dilutions were made from this stock solution to produce 400, 200, 100 and 50 mg/25 ml solvent stock solutions.Aliquots (100 uL) of each stock solution were separately dispersed into 1 litre of algal suspension to give the test concentrations of 0.20, 0.40, 0.80, 1.6 and 3.2 mg/l.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria. Cultures were maintained in the laboratory by the replenishment of culture medium approximately once per week. The culture was maintained in the laboratory at a temperature of 21 +/- 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 +/-1° C

pH:

8.0 - 10.3

Salinity:

Not appliicable

Nominal and measured concentrations:

Definitive study
Based on the results of the range-finding study the following test concentrations were assigned to the definitive study 0.20, 0.40, 0.80, 1.6 and 3.2 mg/l.

Details on test conditions:

Range-finding study
The test concentrations to be used in the definitive study were determined by a preliminary range-finding study. The range-finding study was conducted by exposing Scenedesmus subspicatus cells to a series of nominal test concentrations of 0.020, 0.20, 2.0 and 20 mg/l for a period of 72 hours.The test material was known to be volatile and therefore the study was conducted in 250 ml glass conical flasks sealed with ground glass stoppers to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. The test material was dissolved in 1% v/v Tween 80-ethanol.
An amount of test material (2.0 g) was dissolved in solvent and the volume adjusted to 10 ml to give a 2.0 g/10 ml stock solution. Dilutions were made from this stock solution to produce 200 , 20 and 2.0 mg/10 ml solvent stock solutions. Aliquots (20 ul) of each stock solution were separately dispersed into 200 ml of algal suspension to give the test concentrations of 0.020, 0.20, 2.0 and 20 mg/l.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 uL/L of 1 % v/v Tween 80-ethanol.
At the start of the range-finding study a sample of each test and control culture was removed and the cell density estimated using a haemocytometer and light microscope. The flasks were then covered with aluminium foil and incubated (Callenkamp INR - 401-010W) at 24 +/- 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 hours.
After 72 hours the cell density of each flask was determined
Definitive study
Based on the results of the range-finding study the following test concentrations were assigned to the definitive study 0.20, 0.40, 0.80, 1.6 and 3.2 mg/l.
Exposure conditions
As in the range-finding study 250 ml glass conical flasks were used. Three flasks each containing 100 ml of solution were prepared for the control, solvent control and each treatment group.The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 ul of 1 % v/v Tween 80-ethanol.At initiation of the study the culture contained a nominal cell density of 10^4 cells per mL. The culture conditions gave an algal suspension in log phase growth characterised by an absorbance of 1.634 (at 665 nm). This suspension was diluted to an absorbance of 0.024 prior to use.The flasks were sealed with ground glass stoppers and incubated (Gallenkamp IN R-401 -010W) at 24 +/-1° C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 hours.Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined by direct counting with the aid of a haemocytometer. Absorbance values could not be used to monitor the growth of the test cultures due to the test material solutions absorbing light at the wavelength used to monitor algal growth (665 nm).

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.271 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Details on results:

Growth data
From the data , it is clear that both the growth (r) and the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material, over the 72 hour exposure period.

Accordingly the following results were determined from the data:
EbC50 (72 h): 0.90 mg/l
ErC50 (0 - 24 h): 1.2 mg/l

where EbC50, is the test concentration that reduced biomass by 50% and ErC50 is the test concentration that reduced specific growth rate by 50%.

Statistical analysis of the area under the growth curve data was carried out for the solvent control and all test concentrations using one way analysis of variance.
There were no statistically significant differences between the solvent control, 0.20 and 0.40 mg/l test concentrations (P >= 0.05), however all other test concentrations were significantly different (P <0.001) and, therefore the "No Observed Effect Concentration" (NOEC) is given as 0.40 mg/l.

The following data show that the cell concentration of the control cultures increased by a factor of 32 and the cell concentration of the solvent control cultures increased by a factor of 34 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours:

Mean cell density of control at 0 h: 1.23 x 10^4 cells/m
lMean cell density of control at 72 h: 3.89 x 10^5 cells/ml
Mean cell density of solvent control at 0 h : 1.23 x 10^4 cells/ml
Mean cell density of solvent control at 72 h: 4.16 x 10^5 cells/ml

Observations
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.The pH values of the control and test cultures were observed to increase from pH 8.0 at 0 hours to pH 8.5 - 10.3 at 72 hours. This effect is considered to be due to the large number of cells in the log phase of growth respiring oxygen and producing carbonates and bicarbonates as part of photosynthesis/ respiration which in solution give rise to alkaline conditions. The increase in pH observed over the 72-hour study period followed a concentration dependent pattern with the lower test material concentrations exhibiting a greater increase in pH. This effect is considered to be due to there being greater numbers of viable cells in the lower test concentrations and hence a greater production of carbonates and bicarbonates from photosynthesis/respiration.Temperature was maintained at 24 +/- 1 °C throughout the study.

Physico-chemical measurements
Temperature was maintained at 24 1 'C throughout the study.

Verification of test concentrations
Chemical analysis of the test solutions at 0 hours showed the measured test concentrations to be in excess of the required 80% of nominal.
Analysis of the solvent stock solutions used to prepare the test series at 0 hours showed the measured concentrations to be near nominal. At 72 hours a marked decline in measured test concentrations was observed with values of 53 - 70% of nominal.Since pre-study stability analysis shows the test material is stable over the study period, the decline in measured test concentrations is considered to be due to adsorption to glassware and/or algal cells, or the loss of test material from solution due to its volatile nature.
Adsorption was not a factor in the pre-study stability analysis since no algal cells were present and the glassware was rinsed with extraction solvent.The test material was known to be volatile and therefore the study was conducted in test vessels sealed with ground glass stoppers to prevent losses of the test material due to volatilization. Despite these precautions, a decline in the measured test concentrations was observed after 72 hours. This decline may be due to the requirement for the test vessels to be opened during the study to remove samples for the determination of the algal cell density.Volatilization of the test material was not a factor in the pre-study stability analysis since the bottles used were sealed. See details in the attached document.

In order to give a worst case analysis of the data, it was considered justifiable to base EC50 values on the 72-hour measured test concentrations.
The following resu Its are based on the 72-hour measured test concentrations:
EbC50 (72 h) : 0.60 mg/I
ErC50 (0 - 24 h) : 0.80 mg/I
"No Observed Effect Concentration" : 0.271 mg/l

Validity criteria fulfilled:

yes

Conclusions:

The effect of ET-344-SP on the growth of Scenedesmus subspicatus has been investigated over a 72-hour period and gave an EbC50 value of 0.90 mg/l and an ErC50 (0 - 24 h) value of 1.2 mg/l. The No Observed Effect Concentration at 72 hours was 0.40 mg/l.

Executive summary:

Methods

A study was performed to assess the effect of the test material, on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No. 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Procedure

Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at concentrations of 0.20, 0.40, 0.80, 1.6 and 3.2 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 +/-1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group.

Results

Exposure of Scenedesmus subspicatus to the test material gave an EbC50 (72 h) value of 0.90 mg/ l and an ErC50 (0 - 24 h) value of 1.2 mg/l. The No Observed Effect Concentration was found to be 0.40 mg/l.

Analysis of the test solutions at 0 and 72 hours showed a marked decline in test concentrations after 72 hours and so it was considered justifiable to base the results on the measured test concentrations also. The EbC50 (72 h) based on measured test concentrations was 0.60 mg/l and the ErC50 (0 - 24 h) was 0.80 mg/ l. The No Observed Effect Concentration was 0.271 mg/ l.

Description of key information

A study was performed to assess the effect of the test material, on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No. 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Exposure of Scenedesmus subspicatus to the test material gave an EbC50 (72 h) value of 0.90 mg/ l and an ErC50 (0 - 24 h) value of 1.2 mg/l. The No Observed Effect Concentration was found to be 0.40 mg/l.

Analysis of the test solutions at 0 and 72 hours showed a marked decline in test concentrations after 72 hours and so it was considered justifiable to base the results on the measured test concentrations also. The EbC50 (72 h) based on measured test concentrations was 0.60 mg/l and the ErC50 (0 - 24 h) was 0.80 mg/ l. The No Observed Effect Concentration was 0.271 mg/ l.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.8 mg/L

EC10 or NOEC for freshwater algae:

0.271 mg/L

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