Ethyl 3,5,5-trimethylhexanoate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 September 2016 to 14 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 3,5,5-trimethylhexanoate

EC Number:

266-959-4

EC Name:

Ethyl 3,5,5-trimethylhexanoate

Cas Number:

67707-75-9

Molecular formula:

C11H22O2

IUPAC Name:

ethyl 3,5,5-trimethylhexanoate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

-Verification of test concentrations:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

-Test water:
Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined in Annex 4.
-Experimental preparation:
A nominal amount of test item (25 mg) was dispersed in 500 mL of test water (5 replicates) with the aid of orbital shaking at approximately 300 rpm for 24 hours. After 24 hours the shaking was stopped, the replicates pooled, and any undissolved test item removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 50, 25, 12.5 and 6.25% v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

-Test System and Supporting Information:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Annex 2) in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

250 mg/L as CaCO3

Test temperature:

21°C to 22°C at test initiation
20°C at test termination

pH:

7.6 at test initiation
7.8 to 8.1 at test termination

Dissolved oxygen:

7.7 to 7.9 at test initiation
7.4 to 8.3 at test termination

Nominal and measured concentrations:

-Nominal test concentration (% v/v saturated solution):
6.25, 12.5, 25, 50, 100 (%)
-0-hour measured test concentration:
0.699, 1.58, 3.22, 7.06, 14.7 (mg/L)
-48-hour measured test concentration:
0.610, 1.43, 2.93, 6.40, 14.3 (mg/L)

Details on test conditions:

-Exposure conditions:
As in the range-finding test 150 mL ground glass stoppered conical flasks completely filled with test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then sealed to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

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

The test preparations were not renewed during the exposure period.

-Test organism observations:
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Reference substance (positive control):

yes

Remarks:

A positive control (Envigo Study Number MS29MC) used potassium dichromate as the reference item. The positive control was conducted between 29 February 2016 and 02 March 2016.

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

-Immobilization data:
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. The relationship between percentage immobilization and concentration at 48 hours is given in Figure 1.

-Validation criteria:
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Results with reference substance (positive control):

A positive control (Envigo Study Number MS29MC) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:
EC50: 0.64 mg/L; 95% confidence limits Not possible to determine
No Observed Effect Concentration (NOEC) : 0.56 mg/L
Lowest Observed Effect Concentration (LOEC) : 1.0 mg/L

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

See attached Table 2 and Figure 1 for detailes on results

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item has been investigated and gave the following results based on the 0-Hour measured test concentrations:
EC50: 2.3 mg/L; 95% confidence limits 1.6-3.2 mg/L

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Information provided by the Sponsor indicated the test item was insoluble in water. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 17 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution for 48 hours at a temperature of 20 °C to 22 °C under static test conditions. The test item solutions were prepared by dispersing an excess (50 mg/L) of test item in test water with the aid of orbital shaking at approximately 300 rpm for 24 hours. After the shaking period any undissolved test item was removed by filtration (0.2 μm Gelman Acrocap filter, first approximate 100 mL discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Due to the volatile nature of the test item, testing was conducted in completely filled and sealed test vessels.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.70 to 15 mg/L. There was no significant change in the measured concentrations at 48 hours (87% to 97% of the 0-Hour measured concentrations) and so the results are based on 0-Hour measured test concentrations only. Exposure of Daphnia magna to the test item gave the following results based on the 0-Hour measured test concentrations:

EC50: 2.3 mg/L; 95% confidence limits 1.6-3.2 mg/L