Ethyl 2-ethylhexanoate

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:

From : 17 June 2016 to 02 August 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

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:

Ethyl 2-ethylhexanoate

EC Number:

221-043-3

EC Name:

Ethyl 2-ethylhexanoate

Cas Number:

2983-37-1

Molecular formula:

C10H20O2

IUPAC Name:

ethyl 2-ethylhexanoate

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h and t=48 h
Volume 3.0 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer (≤ -15°C) until analysis.

At the end of the exposure period, the replicates were not pooled at each concentration before sampling. Additionally, reserve samples of 3.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤ -15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of IROTYL tested was a colourless liquid with a purity of 100.0% (by GLC) and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with a loading rate of 100 mg/L applying 3 days of slow magnetic stirring followed by a settlement period of approximately 2 hours. Settlement resulted in a clear and colourless solution with a floating layer of undissolved test item. The fraction containing the Saturated Solution (SS) was collected and used as highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

20-21°C

pH:

7.7-7.8

Dissolved oxygen:

7.9-8.8

Nominal and measured concentrations:

Nominal loading rate: 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L
Measured concentration (0h): 1.59, 4.19, 8.28, 16.7, 48.1 mg/L
Measured concentration (48h): 1.86, 4.65, 8.45, 18.8, 43.5 mg/L
Average exposure: 1.7, 4.4, 8.4, 18, 46 mg/L

Details on test conditions:

-Combined limit/range-finding test
The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a SS solution prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to 0.10, 1.0 and 10% of the SS in the combined range-finding test.
• Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

-Final test
Test concentrations: 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L.
Controls: Test medium without test item or other additives.
Test duration: 48 hours
Test type: Static
Test vessels: 50 mL, all-glass, airtight closed
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 50 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 48 minutes after preparation of the test solutions.

-Measurements and recordings
Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

-Acceptability of the test
1. In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

-Combined limit/range-finding test
No immobility was observed in dilutions containing 0.10 to 10% of the SS during the test period. Daphnids exposed to the undiluted SS (100% SS) were all immobile within 24 hours. Samples taken from 10% SS were analysed. The measured concentration decreased from 3.8 mg/L at the start to 2.8 mg/L after 48 hours. Therefore, the expected EC50 was between concentrations of 3.3 and 33 mg/L.

-Final test
The responses recorded in this test allowed for reliable determination of an EC50. The responses recorded were in agreement with what was expected based on the results of the combined limit/range-finding test.

Results with reference substance (positive control):

-48-hour Acute Toxicity Study in Daphnia magna with Potassium Dichromate (K2Cr2O7) (Project 514941).
Start: 18 July 2016
End: 20 July 2016

-The study procedures described in this report were based on the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004 and the ISO International Standard 6341.
-The reference test was carried out to check the sensitivity of the test system as used by Charles River Den Bosch.
-Daphnids were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a control. Twenty daphnids were exposed per concentration.
-The reference item, potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K44879664) was obtained from Merck, Darmstadt, Germany.

-The actual responses in this reference test with K2Cr2O7 were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was within the expected range of 0.30 to 1.0 mg/L. Hence, the sensitivity of the tested batch of Daphnia magna was comparable to the sensitivity of batches previously tested at Charles River Den Bosch.
-The 48h-EC50 was 0.39 mg/L with a 95% confidence interval ranging from 0.33 to 0.44 mg/L. The historical ranges for the 48h-EC50 lie between 0.28 and 0.90 mg/L. The observed 48h-EC50 corresponds with this range.

-The study plan, raw data and report from this study are kept in the Charles River Den Bosch archives. The test described above was performed under GLP with a QA-check.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 was 6.3 mg/L based on average exposure concentrations (95% confidence interval between 5.7 and 7.1 mg/L).

Executive summary:

Acute Toxicity Study inDaphnia magnawith IROTYL.

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of theCommissionRegulation (EC) No440/2008,Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000.

The batch of IROTYL tested was a colourless liquid with a purity of 100.0% (by GLC) and not completely soluble in test medium at the loading rate initially prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying 3 days of slow magnetic stirring followed by a settlement period of approximately 2 hours. Settlement resulted in a clear and colourless solution with a floating layer of undissolved test item. The fraction containing the Saturated Solution (SS) was collected and used as highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to IROTYL test groups representing 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 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.

Analysis of the samples taken at the start and the end of the final test showed that measured concentrations remained relatively stable during the 48-hour test period. The range tested based on average measured concentrations was 1.7, 4.4, 8.4, 18 and 46 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The 48h-EC₅₀was 6.3 mg/L based on average exposure concentrations (95% confidence interval between 5.7 and 7.1 mg/L).