2-isopropyl-N,2,3-trimethylbutyramide

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:

April - December 2017

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)

Version / remarks:

April 2004

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: WS-23
CAS Number: 51115-67-4
Physical state/Appearance: White solid
Batch: 80100005
Purity: 99.80%
Expiry Date: 13 October 2018
Storage Conditions: Room temperature in the dark
Intended use/Application: Aroma chemical

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and 100 mg/L test group from the freshly prepared bulk test preparation at 0 hours and from the old/expired 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.

Details on test solutions:

The test concentration selected for the definitive test was determined by a preliminary range-finding test. In the range-finding test D. magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L. A nominal amount of test item (50 mg) was dissolved in Elendt M7, with the aid of ultra-sonication for 15 minutes 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 test solution was inverted several times to ensure adequate mixing and homogeneity.

Based on the results of the range-finding test a limit test was conducted at a concentration of 100 mg/L. A nominal amount of test item (100 mg) was dissolved in Elendt M7, with the aid of ultra-sonication for 5 minutes and the volume adjusted to 1 litre to give the 100 mg/L test concentration. The prepared test solution was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by analytical determination at 0 and 48 hours which was carried out on 19 June 2017.

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 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 (not first brood progeny) 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:

other: Reconstituted Water – Elendt M7 Medium

Remarks:

Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 +/- 25 mg/L (as CaCO3). Actual values of 236 and 260 mg/L (as CaCO3) were achieved for the range-finding and definitive tests, respectively.

Test temperature:

21°C

pH:

7.9 +/- 0.3

Dissolved oxygen:

Dissolved oxygen concentrations was maintained between 8.6-9.1 mg O2/L.

Nominal and measured concentrations:

The nominal concentrations were analytically confirmed. The analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal, therefore the results were based on nominal concentrations only.

Details on test conditions:

Range finding study
Two replicate vessels were prepared for each test item concentration and control treatment, each containing five daphnids. The vessels were 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 (with 20 minute dawn and dusk periods) for a period of 48 hours. Each 150 mL test 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 (i.e. untreated Elendt M7 media).

Definitive test
At the start of the test five daphnids were placed in each test and control test item concentration and control treatment. The test vessels (150 mL glass beakers containing approximately 100 mL of test solution) were then covered to reduce evaporation and kept 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 (i.e.-untreated Elendt M7 media). The test preparations were not renewed during the exposure period.

Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. Daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Remarks:

immobilization

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Remarks:

immobilization

Details on results:

Range-finding Test
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1. No immobilization was observed at test concentrations of 0.10, 1.0, 10 and 100 mg/L. Based on this information, a single test concentration of four replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a limit test to confirm that at the maximum test concentration given in the OECD Test Guidelines, no
immobilization or adverse reactions to exposure were observed.

Analytical verification of the 100 mg/L test solution at 0 hours showed a measured test concentration of 105% of nominal. There was no significant change in the measured concentration at 48 hours (103% of nominal) indicating that the test item was stable under test conditions.

Definitive test
Cumulative immobilization data and other observations from the exposure of D. magna to the test item during the definitive test are given in Table 2. There was no significant immobilization in 20 daphnids exposed to a test concentration of100 mg/L for a period of 48 hours. Exposure of D. magna to the test item gave an EC50 value of >100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L. A sub-lethal effect of exposure was observed in one daphnid in the 100 mg/L test concentration at 24 hours. This response was trapping at the surface.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal (96% and 98% of nominal, respectively) and so the results are based on nominal test concentrations. 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 (actual value was 8.6 mg/L) in the control and test vessels. Temperature was maintained at 21-°C to 22-°C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 461 to 552 Lux. 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):

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Table 1. Cumulative Immobilization Data and Observations in the Range-finding Test.

Nominal Concentration (mg/l)		Cumulative Immobilized Daphnia (Initial Population: 5/replicate) @ 24 hours		Observations @ 24 hours		Cumulative Immobilized Daphnia (Initial Population: 5/replicate) @ 48 hours		Observations @ 48 hours
		R1	R2	R1	R2	R1	R2	R1	R2
Control		0	0	5N	5N	0	0	5N	5N
0.10		0	0	5N	5N	0	0	5N	5N
1.0		0	0	5N	5N	0	0	5N	5N
10		0	0	5N	5N	0	0	5N	5N
100		0	0	5N	5N	0	0	5N	5N

R1 - R2 = Replicates 1 and 2. N = No sub-lethal effects observed

Table 2. Cumulative Immobilization Data and Observations in the Definitive Test

Nominal concentration (mg/l)

24 hours

Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5N

5N

5N

5N

100

0

0

0

0

0

0

5N

5N

5N

1T 4N

Nominal concentration (mg/l)

48 hours

Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5N

5N

5N

5N

100

0

0

0

1

1

5

5N

5N

5N

4N

R1–R4 = Replicates 1 to 4. N = No sub-lethal effects observed. T = Trapping at the surface.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of WS-23 to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour Effect Concentration for 50% of the population (EC50) value of >100 mg/L.  The No Observed Effect Concentration (NOEC) was 100 mg/L.

Executive summary:

The short term toxicity of the test item to Daphnia magna was determined following the methods described in the OECD 202 guidelines. Chemical analysis was carried out at 0 and 48 hours for the test solution. The chemical analysis showed that the measured concentration was near the nominal. The nominal concentration has therefore been used for the results. The Effect Concentration for 50% of the population (EC50) for the substance is therefore >100 mg/L after 48 hours. The No Observed Effect Concentration (NOEC) was 100 mg/L.

Description of key information

The short term toxicity of the test item to Daphnia magnawas determined following the methods described in the OECD 202 guidelines. Chemical analysis was carried out at 0 and 48 hours for the test solution. The chemical analysis showed that the measured concentration was near the nominal. The nominal concentration has therefore been used for the results. The Effect Concentration for 50% of the population (EC50) for the substance is therefore >100 mg/L after 48 hours. The No Observed Effect Concentration (NOEC) was 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L