2-ethoxyethyl ethyl carbonic acid ester

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 June 2018 to 10 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The chemistry analysis from the initial experiment (originally planned as a definitive test) did not show that the nominal test material concentrations were obtained and maintained from 0 hours to 48 hours. Also there were some doubts over the integrity of the original sample of test material due to white inclusions found in another departments’ sample; therefore, a second sample was sent at the Sponsors’ request and this was used in the definitive test.
The results of the initial experiment were not used for reporting purposes.

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100 mg/L 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. A set of duplicate samples was taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

- Range Finding Test
A nominal amount of test material (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

- Definitive Test
A nominal amount of test material (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration. The solution was then stirred for 1 hour to ensure that the test material was fully dissolved.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: in-house laboratory cultures
- Age of parental stock: < 24 hours
- Feeding during test: no

CULTURE CONDITIONS
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 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 (Desmodesmus subspicatus) and GM 300 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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20 - 21 °C

pH:

7.9 - 8.1

Dissolved oxygen:

8.5 - 9.1 mg O2/L

Nominal and measured concentrations:

Range Finding test: 0.10, 1.0, 10 and 100 mg/L (nominal)
Definitive test: 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type (delete if not applicable): closed (test vessels were covered to reduce evaporation)
- Fill volume: 100 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M7 medium) was used for both the range-finding and definitive tests.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light / 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 857 to 897 Lux
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.

EFFECT PARAMETERS MEASURED
Any immobilisation 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 immobilised if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

> Range Finding Test
Immobilisation was, at most 5 %, in the control and at the test concentrations of 0.1 and 10 mg/L; however 50 % immobilisation was observed at 1.0 which was considered to be an anomalous result as less immobilisation was observed at the higher concentrations of 10 and 100 mg/L.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 45 % to 104 % of nominal concentrations. There was a significant decline in the measured concentrations at 48 hours for the test concentrations at 0.10, 1.0 and 10 mg/L indicating that the test material was not stable under test conditions at these concentrations; however at 100 mg/L 82 % of nominal was measured at 48 hours.
Based on the results of the range finding test, a single test concentration of four replicates, at 100 mg/L.

> Initial Test
An initial test was conducted on the first sample (Expiry date 17 May 2019) but the results of chemical analysis did not show that the nominal test material concentrations were obtained or maintained from 0 hours to 48 hours. The measured concentration increased over time which may indicate that it was not fully in solution at 0 hours. The definitive test method was adjusted to include a stirring period to ensure that the test material was fully dissolved in the test preparations before use. There were also some doubts over the integrity of the original samples of test material (white inclusions were seen in another departments sample) therefore at the Sponsors request a new sample of test material was supplied for the definitive test.

> Definitive Test
- Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near the nominal concentration (103 and 106 % of nominal) and so the results are based on the nominal test concentration.
- Immobilisation Data
There was no immobilisation in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 48 hours. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.
- Sub-Lethal Effects
At the 100 mg/L test concentration some daphnids were trapped at the surface of the media or covered in debris.
- Validation Criteria
The test was considered to be valid given that no more than 10% of the control daphnids showed immobilisation 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 Material Solubility
At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.

Results with reference substance (positive control):

A positive control (study number LK67NP) 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 immobilisation data was carried out using the Trimmed Spearman-Karber method at 24 hours and the Binomial Distribution 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 a 48 hour EC value of 0.75 mg/L. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

An estimate of the EC50 values was given by inspection of the immobilisation data.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test material gave a 48-hour EC50 value in excess of 100 mg/L.

Executive summary:

The toxicity of the test material to the aquatic invertebrate, Daphnia magna, was investigated in a study which was conducted in accordance with the standardised guideline OECD 202 and EU Method C.2, under GLP conditions.

Following a preliminary range-finding and initial test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a nominal concentration of 100 mg/L for 48 hours at a temperature of 20 - 21 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be between 103 and 106 % of the nominal value and so the results are based on the nominal test concentration.

Exposure of Daphnia magna to the test material gave a 48-hour EC50 value in excess of 100 mg/L.

Description of key information

Exposure of Daphnia magna to the test material gave a 48-hour EC50 value in excess of 100 mg/L.

Additional information

The toxicity of the test material to the aquatic invertebrate, Daphnia magna, was investigated in a study which was conducted in accordance with the standardised guideline OECD 202 and EU Method C.2, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Following a preliminary range-finding and initial test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at a nominal concentration of 100 mg/L for 48 hours at a temperature of 20 - 21 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be between 103 and 106 % of the nominal value and so the results are based on the nominal test concentration.

Exposure of Daphnia magna to the test material gave a 48-hour EC50 value in excess of 100 mg/L.