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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 2012 - 01 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: H1L002
- Expiration date of the lot/batch: Re-test end of September 2013
- Purity test date: 31 October 2011

Storage: Room temperature in the dark
Physical State: Colourless to pale yellow liquid
Chemical Formula: C18H32O4
Molecular Weight: 312.4
Purity: 99.0%

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 48 hours, the contents of the test vessels from each group were pooled and further samples were taken for analysis. Samples were added to vials containing hexane (10 mL) to facilitate the extraction process.

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.

At each concentration, an aliquot of the test substance was dispersed in Elendt M4 medium in volumetric flask (nominally 10.4 µL in 1 L, 9.4 µL in 2 L, or 10.7, 4.9 and 2.2 µL in 5 L).
The contents were stirred for approximately 24 hours in the dark and then left to stand for approximately 24 hours in the dark. An aliquot was then removed from each volumetric flask to provide a Water Accommodated Fraction (WAF) at each nominal loading rate of 0.427, 0.939, 2.07, 4.55 and 10.0 mg/L, which were used as the test media.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna (Straus) used in this study were cultured in-house and were obtained from a strain originating from the National Institute for Applied Chemical Research (IRCHA), France.
Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.5 to 0.8 litres of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20±2°C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. The culture medium was renewed three times each week.
Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial two days when a slightly lower ration was given. Culture conditions ensure that the stock animals reproduce by parthenogenesis.
The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test.

Test type:

static

Water media type:

other: Elendt M4 Medium

Limit test:

yes

Total exposure duration:

48 h

Hardness:

243 mg/L

Test temperature:

0h: 21.0 - 21.1 °C
48h: 19.8 - 20.0 °C

pH:

0h: 8.09 - 8.15
48h: 8.20 - 8.26

Dissolved oxygen:

0h: 95-99 %ASV
48h: 101-102 %ASV

Nominal and measured concentrations:

Nominal: 0 (control), 0.427, 0.939, 2.07, 4.55, 10.0 mg/L
Measured, 0 hours: 0, 1.20, 0.276, 0.638, 2.01, 2.84 mg/L
Measured, 48 hours: 0, 0.585, 0.101, 0.414, 0.730, 1.36 mg/L
Measured, Overall geometric mean: 0, 0.838, 0.167, 0.514, 1.21, 1.97 mg/L

It was noted that the 0.427 mg/L level had an anomalously high measured concentration (0h = 1.20 mg/L; 48h = 0.585 mg/L; mean = 0.838 mg/L), however it was considered not to have impacted the result as no immobilisation occured at measured concentrations up to 1.21 mg/L.

Details on test conditions:

TEST SYSTEM
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 20 mL medium per organism

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.54 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

4.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

6.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The measurements of water quality (temperature, pH, dissolved oxygen, total hardness and alkalinity) remained within acceptable limits during the study.
At all concentrations, the test media were colourless.

Reported statistics and error estimates:

Statistical analysis was performed using the SAFEStat LD50 application (version 1.3), SAS 9.1.3 (SAS Institute, 2002). Test results were expressed in terms of the nominal loading rate and mean measured concentrations. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant.

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal loading rates, the 48-hour EC50 of SIBE 138 for the immobilisation of Daphnia magna was 6.41 mg/L (95% confidence limits of 4.55 and 7.39 mg/L) and the ‘no observed effect concentration’ of SIBE 138 with Daphnia magna was 4.55 mg/L.

In terms of the mean measured concentrations, the 48-hour EC50 of SIBE 138 for the immobilisation of Daphnia magna was 1.50 mg/L (95% confidence limits of 1.21 and 1.63 mg/L) and the ‘no-observed effect concentration’ of SIBE 138 with Daphnia magna was 1.21 mg/L.

As the measured concentrations of SIBE 138 in the test media exceed its stated limit of aqueous solubility (0.046 mg/L), even though WAFs were employed, it is concluded that the test substance is not toxic to Daphnia magna at its limit of aqueous solubility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.5 mg/L