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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Akzo Nobel Functional Chemicals b.v., batch: 100000005282
- Expiration date of the lot/batch: 31 December 2021
- Purity test date: 98.25%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and the test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 1.8 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.

The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures. Adult daphnids were maintained in 150 mL glass beakers containing 100 mL 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
21-22 °C
pH:
7.7
Dissolved oxygen:
8.5-9.9 mg O2/L
Reference substance (positive control):
yes
Remarks:
A positive control (Envigo Study Number XT01HS) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 2.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
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. There was no immobilization in 20 daphnids exposed to a test concentration of 2.4 mg/L for a period of 48 hours. No sub-lethal effects of exposure were observed throughout the test.

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 XT01HS) 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, however, throughout the definitive test the temperature range was recorded between 19 and
22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely
affected the results of the test. Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations.

The 48 hour EC50 was1.2 mg/L (95% Confidence Limits: 1.1-1.3 mg/L) and the NOEC was 0.56 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.

Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis
was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the Ethylene dibenzoate to the freshwater invertebrate Daphnia magna has been investigated and based on the 0-Hour measured test concentrations gave a 48-Hour EC50 value of greater than 2.4 mg/L. The No Observed Effect Concentration was 2.4 mg/L.

This study showed that there were no toxic effects at saturation.
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

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 1.8 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 a concentration of 100% v/v saturated solution for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Chemical analysis of the test preparations at 0 hours showed that a measured test concentration of 2.4 mg/L was obtained. There was no significant change in the measured concentration at 48 hours and so the results are based on 0-Hour measured test concentrations

only. Exposure of Daphnia magna to the test item gave EC50 values based on the 0-Hour measured test concentrations of greater than 2.4 mg/L. The No Observed Effect Concentration was 2.4 mg/L. This study showed that there were no toxic effects at saturation.

Description of key information

An OECD 202 study according to protocol under GLP is available.

Chemical analysis of the test preparations at 0 hours showed that a measured test concentration of 2.4 mg/L was obtained. There was no significant change in the measured concentration at 48 hours and so the results are based on 0-Hour measured test concentrations only.

Exposure of Daphnia magna to the test item gave EC50 values based on the 0-Hour measured test concentrations of greater than 2.4 mg/L. The No Observed Effect Concentration was 2.4 mg/L.

This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.4 mg/L

Additional information