1,2-Cyclohexanedicarboxylic acid, 1-[2-[(1-oxo-2-propen-1-yl)oxy]ethyl] ester

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:

The range-finding test was conducted between 1 September 2006 and 4 September 2006 and the definitive test between 8 September 2006 and 11 September 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 30th August 2005. Date of signature: 21/11/05

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Test concentrations in definitive test: 6.25, 12.5, 25, 50 and 100 mg/l.

- Sampling method: The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.

Samples were taken from the control and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
For the purpose of the definitive test, the test material was dissolved directly in culture medium.

An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 200 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 100, 50, 25 and 12.5 mg/I. An aliquot (250 ml) of each of the stock solutions
was separately mixed with algal suspension (250 ml) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/I.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

- Controls:
The control group was maintained under identical conditions but not exposed to the test material.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Method of cultivation: Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

ACCLIMATION
- Culturing media and conditions (same as test or not): The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Hardness:

Not stated.

Test temperature:

The flasks were incubated at 24 ± 1°C. The temperature within the incubator was recorded daily.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

Definitive Test:
Nominal Concentrations: 6.25, 12.5, 25, 50 and 100 mg/l

Details on test conditions:

TEST SYSTEM
As in the range-finding test 250 ml glass conical flasks were used. Three flasks each containing 100 ml of test preparation were used for the control and each treatment group.

The control group was maintained under identical conditions but not exposed to the test material.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.65 x10E6 cells per ml. This suspension was diluted to a cell density of 2.27 x 10E4 cells per ml prior to use. At initiation of the test the culture contained a nominal cell density of 10E4 cells per ml.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.

GROWTH MEDIUM
The culture medium is defined below:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

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 HCl. The prepared media was sterilised by 0.2 µm membrane filter.



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: particle counter
- Other: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS

- Range finding study
The test concentrations to be used in the definitive test were detennined by a preliminary range-finding test. The range-finding test was conducted by exposing Scenedesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.

After 72 hours the cell density of each flask was detennined using a Coulter® Multisizer Particle Counter.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 27 - 43 mg/l

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Observations on cultures:
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 at 6.25, 12.5, 25 and 50 mg/l, however no intact cells were observed to be present in the test cultures at 100 mg/I.

Observations on test material solubility:
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, 6.25 and 12.5 mg/l test cultures were observed to be pale green dispersions. The 25 mg/l test cultures were observed to be very pale green dispersions whilst the 50 and 100 mg/l test cultures were observed to be clear colourless solutions.

See results on range-finding and definitive tests below (any other information on results incl. tables section).

Results with reference substance (positive control):

No positive control.

Reported statistics and error estimates:

Statistical analysis of the area under the growth curve data was carried out for the control and all test concentrations using 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 (Dunnett 1955). There were no statistically significant differences between the control and 6.25 mg/l test concentration (P ≥0.05), however all other test concentrations were significantly different (P<0.05) and therefore the NOEC was 6.25 mg/l.

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Scenedesmus subspicatus to the test material during the range-finding test are given in Table 1 (see attched background material).

The results showed no significant effect on growth at the test concentrations of 0.10, 1.0 and 10 mg/l. However, growth was observed to be reduced at 100 mg/l.

Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l were selected for the definitive test.

Definitive Test

Growth data

From the data given in Tables 2 and 3 (see attached background information), 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.

The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figures 2 and 3 (see attached background material for figures).

Accordingly the following results were determined from the data:

EbC50 (72h): 18 mg/l; 95% confidence limits 17 - 20 mg/l

ErC50 (72h): 34 mg/l; 95% confidence limits 27 - 43 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%.

The following data show that the cell concentration of the control cultures increased by a factor of 53 after 72 hours. This increase was 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 hours: 1.01 x 10⁴ cells per ml

Mean cell density of control at 72 hours: 5.33 x 10⁵ cells per ml

Physico-chemical measurements

The pH values of each test and control flask are given in Table 2 (see attached background material). Temperature was maintained at 24 ± 1°C throughout the test.

The pH values of the control cultures (see Table 2) were observed to increase from pH 7.4 at 0 hours to pH 7.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

A concentration dependent decline in pH was observed at both 0 and 72 hours. This was considered to be due to an intrinsic property of the test material and as such no attempt was made to alter the pH.

Verification of test concentrations

Analysis of the test preparations at a and 72 hours (see Appendix 1 - attached background material) showed measured test concentrations to range from 86% to 99% of nominal and so it was considered justifiable to calculate the EC50 values in tenns of the nominal test concentrations only.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated over a 72-Hour period and gave an EbC50 (72 h) value of 18 mg/l; 95% confidence limits 17 - 20 mg/l and an ErC50 (0 - 72 h) value of 34 mg/l; 95% confidence limits 27 - 43 mg/l. The No Observed Effect Concentration at 72 hours was 6.25 mg/l.

Executive summary:

Introduction.

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).

Methods.

Following a preliminary range-finding test, Scenedesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 6.25, 12.5, 25, 50 and 100 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, using a Coulter Multisizer Particle Counter.

Results.

Exposure of Scenedesmus subspicatus to the test material gave an EbC50 (72 h) value of 18 mg/l; 95% confidence limits 17 - 20 mg/l and an ErC50 (0 - 72 h) value of 34 mg/l; 95% confidence limits 27 - 43 mg/l. The No Observed Effect Concentration was 6.25 mg/l.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 86% to 99% of nominal and so the results are based on nominal test concentrations only.

Description of key information

Based on the geometric mean measured test concentrations, the ErC50 (0 - 72 h) value was 34 mg/l with 95% confidence limits of 27 - 43 mg/l, the No Observed Effect Concentration at 72 hours was 6.25 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

34 mg/L

EC10 or NOEC for freshwater algae:

6.25 mg/L

Additional information

A study with Scenedesmus subspicatus exposed to concentrations of an analogue for 72 hours gave an ErC50 (0 - 72 h) value of 38 mg/l with 95% confidence limits 31 - 46 mg/l and an ErC10 of 2.1 mg/l. The No Observed Effect Concentration at 72 hours was 1.0 mg/l. Based on the geometric mean measured test concentrations, the ErC50 (0 - 72 h) value was 38 mg/l with 95% confidence limits of 31 - 47 mg/l, the ErC10 was estimated at 1.3 mg/l and the No Observed Effect Concentration at 72 hours was 0.53 mg/l. These results support the reliability of the key values for the target substance.