Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

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:

Experimental start date 16 February 2021
Experimental completion date 19 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

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:

Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

EC Number:

243-497-1

EC Name:

Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

Cas Number:

20073-13-6

Molecular formula:

C13H20O3

IUPAC Name:

methyl 2-[(1S,2S)-3-oxo-2-[(2Z)-pent-2-en-1-yl]cyclopentyl]acetate

Specific details on test material used for the study:

Identification: Methyljsamonate
CAS Number: 39924-52-2
Batch: 0900175
Purity: 99.03%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 25 March 2023
Storage Conditions: Approximately 4 °C in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-finding Test
A sample of each test concentration was taken for immediate chemical analysis from the freshly prepared bulk test preparations at 0 hours and from the pooled replicates at 48 hours in order to determine the stability of the test item under test conditions. Two additional vessels for each test concentration were set up and ran alongside the study for duplicate samples at 48 hours. A duplicate set of samples was taken on each occasion and stored frozen for further analysis if required.

Definitive Test
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 48 hours.
Samples were taken from the control and each of the test groups from the bulk test
preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis.
All samples were taken for immediate analysis. Duplicate sets of samples were taken at 0
and 48 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
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 Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019). Therefore, a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

Preliminary Media Preparation Trial
1. Saturated Solution Preparation
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 L of Elendt M4 media with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Gelman Acrocap filter (approximately 100 mL discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Gelman Acrocap filter (approximately 500 mL discarded in order to pre-condition the filter)

Based on this information the test item was prepared using a prolonged stir method of reparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours and as a precautionary measure, 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 nominal test concentration of approximately 96 mg/L.

Range-finding Test
A nominal amount of test item (1100 mg) was dispersed in 11 L of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (the first approximate 500 mL used to pre-condition the filter was discarded), to give a 100mg/L test solution. A series of dilutions were made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10 mg/L test solutions.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (1100 mg) was dispersed in 11 L of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (the first approximate 500 mL used to pre-condition the filter was
discarded), to give a 100 mg/L test solution. A series of dilutions were made from this saturated solution to give further test concentrations of 56, 32, 18 and 10 mg/L test solutions.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

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 vessels containing 100 mL Elendt M4 medium 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 Raphidocelis subcapitata) and GEMMA Micro 300 fish 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:

other: Reconstituted water (Elendt M4 medium)

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

Temperature was maintained at 19 °C to 20 °C

pH:

7.5 - 7.7 at 0 hours. 7.5 - 7.8 at 48 hours.
There were no treatment related differences for pH

Dissolved oxygen:

9.0 - 9.1 mg O2/L at 0 hours. 9.0 - 9.1 mg O2/L at 48 hours.
There were no treatment related differences for oxygen

Nominal and measured concentrations:

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

Based on the results of the range-finding test the following test concentrations were assigned
to the definitive test: 10, 18, 32, 56 and 100 mg/L.
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 10 to 109 mg/L (99% to 109% of nominal). There was no significant change in the measured concentrations at 48 hours and so the results are based on nominal test concentrations.

Details on test conditions:

Range-finding Test
In the range-finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. 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 immediate chemical analysis from the freshly prepared bulk test preparations at 0 hours and from the pooled replicates at 48 hours in order to determine the stability of the test item under test conditions. Two additional vessels for each test concentration were set up and ran alongside the study for duplicate samples at 48 hours. A duplicate set of samples was taken on each occasion and stored frozen for further analysis if required.

Definitive Test
As in the range-finding test 150 mL glass vessels containing approximately 100 mL of test preparation were used. At the start of the test five 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 covered 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 and 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 48-Hour exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Immobilization Data
The No Observed Effect Concentration (NOEC) after 24 hours was determined to be greater than 100 mg/L and at 48 hours exposure was 18 mg/L. The Lowest Observed Effect Concentration (LOEC) after 24 hours exposure could not be determined and at 48 hours exposure was 32 mg/L.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in 18, 32, 56 and 100 mg/L test concentrations.
This response was reduced mobility

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.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear
colorless solutions.

Results with reference substance (positive control):

A positive control (Covance study number 8461001) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The positive control was conducted between 05 January 2021 and 29 January 2021.
Analysis of the immobilization data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package. Accordingly the following results were obtained:

24 hours
EC50: 0.86 mg/L (0.21 - 3.5 95% confidence limit)
NOEC: 0.32 mg/L
LOEC: 0.56 mg/L

48 hours
EC50: 0.50 mg/L (0.43 - 0.58 95% confidence limit)
NOEC: 0.32 mg/L
LOEC: 0.56 mg/L

The NOEC 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*.

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 nominal test concentrations:

48 hours
EC50: 35 mg/L (30 - 42 95% confidence limit)
NOEC: 18 mg/L
LOEC: 32 mg/L

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 96 mg/L was obtained from a prolonged stir method of preparation indicating that the test item is readily available in the test media.
Following a preliminary range-finding test and initial experiments, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg/L saturated solution for 48 hours at a temperature of 19 °C to 20 °C under static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (the first approximate 500 mL
used to pre-condition the filter was discarded), to give a 100 mg/L solution of the test item. This 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.

Results
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 10 to 109 mg/L (99% to 109% of nominal). There was no significant change in the measured concentrations at 48 hours and so the results are based on the nominal test concentrations only.
Exposure of Daphnia magna to the test item gave the following results based on the nominal test concentrations:
48 hours
EC50: 35 mg/L (30 - 42, 95% confidence limit)
NOEC: 18 mg/L
LOEC: 32 mg/L