Isobutyl phenylacetate

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:

The study was conducted between 17 November 2014 and 10 December 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl phenylacetate

EC Number:

203-007-9

EC Name:

Isobutyl phenylacetate

Cas Number:

102-13-6

Molecular formula:

C12H16O2

IUPAC Name:

isobutyl phenylacetate

Test material form:

liquid

Details on test material:

Identification: Isobutyl Phenyl Acetate
Chemical nature: 2-methylpropyl 2-phenylacetate
EC No.: 203-007-9
CAS No.: 102-13-6
Molecular weight: 192.2 g/mol
Appearance/physical description: clear, colourless liquid
Empirical formula: C12H16O2
Batch No: SC00010162
Purity: 100%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk fresh test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.

Duplicate samples were taken and stored frozen for further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in Appendix 6 of the attached study report.

Test solutions

Vehicle:

no

Details on test solutions:

Test Water
Reconstituted water (ISO medium) was used for both the range-finding and definitive tests.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

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 Appendix 2 of attached study report) in a temperature controlled room at approximately 20 '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

Nominal and measured concentrations:

Range-finding Test:
Nominal test concentrations of 0.10, 1.0, 10, and 100% v/v saturated solution.

Definitive Test:
Nominal test concentrations of 6.3, 12.5, 25, 50 and 100% v/v saturated solution.
Initial measured test concentrations of 5.1, 9.9, 21, 43, and 84 mg/L

Details on test conditions:

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10, and 100% v/v saturated solution.

A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 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 10, 1.0 and 0.10% v/v saturated solution.

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

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.

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

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.3, 12.5, 25, 50 and 100% v/v saturated solution.

Experimental Preparation
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours in the dark. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 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 6.3% v/v saturated solution.

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

Exposure Conditions
In the definitive test 100 mL conical flasks completely filled with test preparation and stoppered were used to minimize any losses due to volatility. 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 maintained in a temperature controlled room at approximately 21 °C in the dark. 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.

Evaluations
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 for approximately 15 seconds after gentle agitation.

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate and was conducted between 05 March 2014 and 07 March 2014.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

not determinable because of methodological limitations

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

9.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 of the attached report.

10 and 20% immobilization was observed in the 0.10 and 1.0% v/v saturated solution test concentrations respectively, however, this immobilization is considered not to be due to exposure to the test item as no immobilization was observed in the 10% v/v saturated solution test concentration. However, 100% immobilization was observed at 100% v/v saturated solution.

Based on this information test concentrations of 6.3, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.

Chemical analysis of the 10 and 100% v/v saturated solutions at 0 hours (see Appendix 6 in study report) showed measured test concentrations of 8.0 and 68 mg/L. There was significant decline in the measured concentrations at 48 hours to 5.9 and 59 mg/L indicating that the test item was not stable under test conditions. The loss of test item was considered to be due to test item volatility, therefore the definitive test was conducted in completely filled and stoppered test vessels.


Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours (see Appendix 6 in study report) showed measured test concentrations to range from 5.1 to 84 mg/L. Analysis of the test preparations at 48 hours showed 87 to 94% of the initial measured concentration was recovered, therefore the results are based on initial measured test concentrations only.


Immobilization Data
Analysis of the immobilization data by probit analysis using linear maximum-likelihood regression at 24 and 48 hours based on the initial measured test concentrations gave the following results:
The 24 h EC50 was 20 mg/L.
The 48 h EC50 was 14 mg/L.
The No Observed Effect Concentrations after 48 hours exposure was 9.9 mg/L. The Lowest Observed Effect Concentrations after 48 hours exposure was 21 mg/L.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41400711) 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 maximum-likelihood probit method (Finney, 1971) at 24 hours and by the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results:

The 24 h EC50 was 0.87 mg/L with 95 % confidence limits of .75 - 1.0 mg/L.
The 48 h EC50 was 0.71 mg/L with 95% confidence limits of 0.65 - 0.78 mg/L.
The NOEC was 0.56 mg/L and the NOEC was 1.0 mg/L.

The No Observed Effect Concentration is based upon 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.

Reported statistics and error estimates:

The EC50 values were calculated by probit analysis using linear maximum-likelihood regression. All results were calculated using the ToxRat Professional computer software package (ToxRat).
If there is no immobilization between 0% and 100% immobilization, then the concentrations resulting in 0% and 100% immobilization will be the 95% confidence limits.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 was 14 mg/L.
The No Observed Effect Concentrations after 48 hours exposure was 9.9 mg/L respectively. The Lowest Observed Effect Concentrations after 48 hours exposure was 21 mg/L respectively.

Executive summary:

Introduction

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.

 

Methods…….

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 6.3, 12.5, 25, 50 and 100% v/v saturated solution for 48 hours at a temperature of approximately 21°C in the dark under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

 

Results…….

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 5.1 to 84 mg/L. Analysis of the test preparations at 48 hours showed 87 to 94% of the initial measured concentration was recovered, therefore the results are based on initial measured test concentrations only.

Exposure of Daphnia magna to the test item gave the following results:

48 h EC50 was 14 mg/L. The NOEC was 9.9 mg/L and the LOEC was 21 mg/L.