Reaction mass of bis(2-methylpropyl) (1R,2R,3R,6S)-3,6-dimethylcyclohexane-1,2-dicarboxylate and bis(2-methylpropyl) (1S,2S,3R,6S)-3,6-dimethylcyclohexane-1,2-dicarboxylate

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:

09 May 2012 - 23 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical Formula: C18H32O4
Molecular Weight: 312.4
CAS Number: 901311-65-7
Physical State: Colourless to pale yellow liquid
Batch Number: H1L002
Purity: 99.0%
Storage: Room temperature in the dark
Expiry Date: Re-test end of September 2013
Sample received: 26 September 2011

Analytical monitoring:

yes

Details on sampling:

The test concentrations of SIBE 138 were measured using gas chromatography with Mass Spectrometric detection (GC-MS). At the start of the definitive test, two samples (20 mL) were taken from the freshly-prepared control and test media. After 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. Samples were added to vials containing hexane (10 mL) to facilitate the extraction process.

Additional samples were also taken from the flask containing SIBE 138 at 0.100 and 100 mg/L but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.

On each occasion, one of the samples was analysed and the other was stored in a freezer in case further analysis was required.

Vehicle:

no

Details on test solutions:

The method of preparation used during the definitive test was based on the results of a range finding test and a formulation trial.

At 0.32 to 100 mg/L, the test media were individually prepared: an aliquot of the test substance was dispersed in sterile OECD medium in a volumetric flask or glass aspirator [nominally 207, 66, 20.7 or 6.6 μL in 2L; 5.2 μL in 5L and 3.3 μL in 10 L and corrected for specific gravity (0.965)]. Each preparation was stirred overnight in the dark and then left to stand for approximately 23 hours in the dark before an aliquot (1 L; WAF) was removed midvessel and used directly to provide Water Accommodated Fraction (WAF) with nominal loading rate of 100, 32.0, 10.0, 3.20, 1.00 or 0.320 mg/L.

At 0.1 mg/L, the volume of test substance required was too small to be accurately dispensed, so an aliquot (313 mL) of the WAF with a nominal loading rate of 0.320 mg/L was diluted with sterile OECD medium (1 L) to provide the test medium at 0.1 mg/L.
An aliquot (4.0 mL) of the secondary algal inoculum was added to a portion (500 mL) of the test medium at each concentration, to give an initial mean cell density of ca. 1x10^4 cells/mL. An aliquot (100 mL) of the appropriate inoculated test medium was added to each of the test vessels.

Test organisms (species):

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

Details on test organisms:

Axenic, uni-cellular, liquid slope cultures of algae were obtained.

The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C to provide an inoculum in the log phase of growth, characterised by a cell density of 1.25 x 10^6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

0 hours: 21.1 - 22.3°C
72 hours: 22.8 - 23.1°C

pH:

0 hours: 8.03 - 8.13
72 hours: 8.27 - 9.39

Nominal and measured concentrations:

Nominal loading rates: 0 (control), 0.1, 0.32, 1.00, 3.2, 10, 32, 100 mg/L
Measured concentrations, 0 hours: 0.0958, 0.303, 0.937, 2.11, 1.82, 4.04, 5.96 mg/L
Measured concentrations, 72 hours: 0.0795, 0.173, 0.617, 1.82, 1.23, 1.91, 2.44 mg/L

Measured concentrations of culture medium incubated under test conditions without algal cells, 72 hours (0.100 and 100 mg/L loading rates only): 0.0805, 2.52 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flask, 100 mL fill level. Flasks were loosely stoppered with a foam bung
- Initial cells density:
- Control end cells density: Approximately 1 x 10^4 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality:nominally 4440 to 8880 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

- Range finding study
The three range finding tests employed WAFs prepared at nominal loading rates from 1 to 100 mg/L. Although cell growth was variable between tests, overall the results indicated that algal growth was inhibited above 1 mg/L. The variability in algal growth due to the test substance being an UVCB and its low solubility, which made it difficult to formulate the test substance in aqueous media. However, the use of WAFs is the recommended way to formulate test substances with these properties (multi-component; UVCB).
Based on these results, the definitive test employed WAFs prepared at nominal loading rates of 0.100, 0.320, 1.00, 3.20, 10.0, 32.0 and 100 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: Growth rate and yield / biomass

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: Growth rate and yield / biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.81 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Growth rate and yield / biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.96 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Growth rate and yield / biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.26 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Details on results:

No microscopic abnormalities of the cells were detected as compared to the control group.

Less than 50% inhibition of the growth rate was seen at the highest loading level (16% inhibition was seen at the 100 mg/L test group).

Validity criteria:

For the test to be valid, the following criteria must be met:

- Cell concentration in control cultures should have increased by a factor of at least 16 within 72 hours. This corresponds to a specific growth rate of 0.92 day-1;

- The mean coefficient of variation for daily growth rates (Days 0-1, 1-2 and 2-3) in the control cultures must not exceed 35%;

- The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Validity criteria fulfilled:

yes

Conclusions:

The effect of SIBE 138 on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed over a 72 hour period.
Based on nominal loading rates, the 72-hour EC50 for growth rate and yield was >100 mg/L (as only 16% inhibition occurred at the highest test level) and 17.0 mg/L, respectively. The ‘no-observed effect loading rate’ for growth rate and yield was 1.00 mg/L and the lowest observed effect loading rate was 3.20 mg/L.
Based on mean measured concentrations of SIBE 138, the 72-hour EC50 for growth rate and yield was >3.81 mg/L and 2.26 mg/L, respectively. The ‘no-observed effect concentration’ for growth rate and yield was 0.760 mg/L and the lowest observed effect concentration was 1.96 mg/L.

Description of key information

E_rC₅₀, 72 Hours >100 mg/L nominal loading rate (>3.81 mg/L measured concentration (geometric mean))

E_yC₅₀, 72 Hours = 17.0 mg/L nominal loading rate (2.26 mg/L measured concentration (geometric mean))

No Observed Effect Loading Rate = 1.00 mg/L (0.760 mg/L measured concentration)

Lowest Observed Effect Loading Rate = 3.20 mg/L (1.96 mg/L measured concentration)