Ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-ene-1-carboxylate

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:

07 July to 31 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

rel-(1R,6S)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Molecular formula:

C15H24O3

IUPAC Name:

rel-(1R,6S)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Constituent 2

Reference substance name:

rel-(1R,6R)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Molecular formula:

C15H24O3

IUPAC Name:

rel-(1R,6R)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as stated in the report): Calyxol
- Batch No. VE00412958
- Expiration date of the lot/batch: 15.11.2017

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. The nominal volume per sample was 20 mL. In order to avoid potential adsorption of test item to pipette inner walls, the sample volumes were weighed directly into the sample bottles on a balance. The volume of each sample was recorded.
After sampling and before shipment, all samples were stored in amber glass bottles in the dark at a temperature of ≤ -18 °C. A record was kept for each sample. Samples of untreated test medium were provided to the analytical laboratory in order to determine the recovery of the analytical method with the specific test medium. Samples were transferred to the test site for chemical analysis under the required storage conditions.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution (S1) of the test item was prepared by dissolving 0.0152 g of the test item in 500 mL of test medium, resulting in a nominal concentration of 30 mg/L. This stock solution was stirred for 3-h at ambient temperature in the dark. Subsequent inspection of the solution showed neither undissolved matter (no oily droplets on the surface) nor any Tyndall effect. The stock solution was used immediately to prepare the desired test item concentrations. The stock solution was used, undiluted, as the highest test concentration. The diluted test solutions were stirred for another 10- 20 min.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: The test system used in this study was Daphnia magna STRAUS (clone M10), a water flea, cultured at ECT Oekotoxikologie GmbH since December 22, 2011.
Origin: The organisms were originally supplied by KU Leuven, Belgium
Age of exposed daphnids at start: < 24 hours
Food: Algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
Feeding frequency: 4 times per week

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

14.0 °dH

Test temperature:

21.0°C

pH:

7.9

Dissolved oxygen:

8.2 mg/L

Conductivity:

661 μS/cm

Nominal and measured concentrations:

Nominal concentration: 1.28, 2.82, 6.20, 13.6 and 30.0 mg test item/L.
Measured concentrations (test period: 0 day) : 1.01, 2.61, 5.58, 13.2 and 29.1 mg test item/L.
Measured concentrations (test period: 2 days) : 0.99, 2.53, 5.89, 12.6 and 27.4 mg test item/L.

Details on test conditions:

Target temperature: 18 - 22 °C
Volume of test solution per test vessel: 50-60 mL
Number of daphnids per test vessel: 5
Photoperiod: 16 h light / 8 h dark
Aeration: none
Feeding during exposure: none
Test units: 60mL glass beakers
Number of replicates per concentration: 4
Number of replicates in the controls: 4
Data evaluation: Evaluation of immobility. Weibull analysis, Fisher exact test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

12.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Any other information on results incl. tables

Since the mean of the measured concentrations was between 80 and 120% of nominal, and since the biologically relevant concentrations (6.20, 13.6 and 30 mg/L) remained within 90% or more of the nominal concentrations, the biological endpoints are expressed based on nominal concentrations.

No significant interferences from the test medium were detected at the retention time corresponding to the analyte in any of the control specimens.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled as required by the test guideline

Conclusions:

A clear concentration-response relationship was observed. The EC50(48h) of Calyxol has been determined to be 12.43 mg/L according to these test conditions.

Executive summary:

Acute Toxicity Study in Daphnia magna with Calyxol was determined according to OECD guideline No. 202.

The batch of Calyxol tested was a colorless to pale yellow liquid. A stock solution (S1) of the test item was prepared by dissolving 0.0152 g of the test item in 500 mL of test medium, resulting in a nominal concentration of 30 mg/L. This stock solution was stirred for 3-h at ambient temperature in the dark. Subsequent inspection of the solution showed neither undissolved matter (no oily droplets on the surface) nor any Tyndall effect. The stock solution was used immediately to prepare the desired test item concentrations.

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to the nominal test concentrations: controle, 1.28, 2.82, 6.20, 13.6 and 30.0 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test ( Day 0 and Day 2).

During the exposure period, the mean of the measured concentrations was between 80 and 120% of nominal, and since the biologically relevant concentrations (6.20, 13.6 and 30 mg/L) remained within 90% or more of the nominal concentrations, the biological endpoints are expressed based on nominal concentrations.

No significant interferences from the test medium were detected at the retention time corresponding to the analyte in any of the control specimens.

The study met the acceptability criteria prescribed by the study plan and was considered valid. The 48h-EC50 was 12.43 mg/L based on nominal concentrations.