Isopropyl acetoacetate

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:

2020-06-11 to 2020-08-27

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:

Adopted April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

June 6, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test and at day 1. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the aged test media of all test concentrations and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the test beakers of each treatment. All samples remained undiluted until analysis.

- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light, until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the only test item concentration of nominal 100 mg/L was prepared by dissolving 51.7 and 49.0 mg test item into 517 and 490 mL test water by intense stirring for 25 and 20 minutes. The test media was prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.

- Controls: In the control, test water was used without addition of the test item.

- Evidence of undissolved material: There were no remarkable observations

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna (Straus), clone 5
- Stage and instar at study initiation: from 1.5 to 16.25 hours old
- Breeding conditions: The daphnids were bred in the laboratory under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with freshly grown green algae (Desmodesmus subspicatus).
Source: ibacon's in-house laboratory culture

ACCLIMATION
- Acclimation period: Was not necessary, since the test was performed in the same medium used for the culture

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

20.3 to 20.4 °C in the freshly prepared media;
20.2 to 20.3°C in the aged test media

pH:

7.9 to 8.0 in the freshly prepared media;
8.0 in the aged test media

Dissolved oxygen:

8.6 to 8.8 mg/L in the freshly prepared media;
8.7 to 8.8 mg/L in the aged test media

Nominal and measured concentrations:

Nominal conc.: 100 mg/L; Measured concentration: 96% of nominal measured concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers of 100 mL volume containing approximately 60 mL of test medium.
- Type: closed (The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions).
- Volume of solution: ca. 60 mL
- Renewal of test solution: test medium renewal was done on Day 1
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (Elendt "M4"):
- Culture medium different from test medium: no, the same

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light: 8 h dark
- Light intensity: 260 to 440 lux

EFFECT PARAMETERS MEASURED: The number of immobile daphnids were determined by visual observation and recorded after 24 and 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 100 mg test item/L and a control.
- 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

Duration:

48 h

Dose descriptor:

EC10

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

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and the only test item concentration of 100 mg test item/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 48-h EC50: 0.986 mg/L

Table 1: Summary of Biological Results

Nominal Concentration [mg test item/L]	% of immobilised daphnids after
	24 hours	48 hours
Control	0	0
100	0	0
EC50 [mg test item/L]:	> 100	> 100
95 % CI [mg test item/L]:	n.d.	n.d.
EC20 [mg test item/L]:	> 100	> 100
95 % CI [mg test item/L]:	n.d.	n.d.
EC10 [mg test item/L]:	> 100	> 100
95 % CI [mg test item/L]:	n.d.	n.d.
NOEC [mg test item/L]:	≥ 100	≥ 100
LOEC [mg test item/L]:	> 100	> 100

 

Values refer to nominal test concentrations

CI: Confidence interval

n.d.: not determinable

NOEC and LOEC were determined directly from the raw data.

 

Table 2: Summary of Analytical Results

Sample Description (mg test item/L)	% of nominal^1	RSD (%)
Control	n.a.	n.a.
100	96	1

 

¹ mean value of all measured samples per treatment group

RSD: relative standard deviation per treatment group

n: number of analysed samples

n.a.: not applicable

 

Table 3: Influence of the test item on the Mobility of Daphnia magna and Sublethal Effects

 

Nominal concentration (mg test item/L)	No. of Daphnia tested	No. of immobilised Daphnia after		% of immobilised Daphnia after		Observations
		24 h	48 h	24 h	48 h	24 h	48 h
Control	5	0	0	0	0	0	0
	5	0	0			0	0
	5	0	0			0	0
	5	0	0			0	0
100	5	0	0	0	0	0	0
	5	0	0			0	0
	5	0	0			0	0
	5	0	0			0	0

Abbreviations for observations:

0: No abnormalities

 

Table 4: Results of the Analytical Determination of the Test Item in the Test Samples

Sample Description			Concentration found		Concentration calculated [mg test item/L]	Concentration nominal [mg test item/L]	% of nominal^1
	Sampling Age
[mg test item/L]	day	[h]	[mg test item/L]	D.F.
Control	0	0	<LOD	1	n.a	0.000	n.a
Control	0	0	<LOD	1	n.a	0.000	n.a
Control	1	24	<LOD	1	n.a	0.000	n.a
Control	1	24	<LOD	1	n.a	0.000	n.a
Control	1	0	<LOD	1	n.a	0.000	n.a
Control	1	0	<LOD	1	n.a	0.000	n.a
Control	2	24	<LOD	1	n.a	0.000	n.a
Control	2	24	<LOD	1	n.a	0.000	n.a
100	0	0	95.062	1	95.062	100.000	95
100	0	0	95.841	1	95.841	100.000	96
100	1	24	95.286	1	95.286	100.000	95
100	1	24	94.553	1	94.553	100.000	95
100	1	0	97.621	1	97.621	100.000	98
100	1	0	97.330	1	97.330	100.000	97
100	2	24	96.879	1	96.879	100.000	97
100	2	24	95.585	1	95.585	100.000	96
						mean value (n=8):	96
						RSD (n=8):	1

 

¹ The tabulated results represent rounded results calculated on the exact raw data LOD: Limit of Detection = 0.3 mg test item/L

n.a.: not applicable

RSD: Relative Standard Deviation

D.F.: Dilution factor

Validity criteria fulfilled:

yes

Conclusions:

In the 48-h acute toxicity of the test item on Daphnia magna according to OECD T 202, no immobilisation was observed in the control and in the 100 mg/L concentration group. In both groups no abnormal behaviour or appearance of test animals was noted. The 48-h NOEC was determined to be > 100 mg/L. The 48-h LOEC and the EC50 values were determined to be > 100 mg/L.

Executive summary:

The influence of the test item on the mobility of Daphnia magna was determined in a 48-h “Acute Immobilisation Test” according to OECD TG 202 and under GLP conditions. For this purpose, young daphnids (< 24 hours old) were exposed in a semi-static test for 48 hours to the only concentration of nominal 100 mg/L under defined conditions. The recorded effects were the mobility of the daphnids after 24 and 48 hours. This limit-test was performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the daphnids up to this concentration. The test method and the test species Daphnia magna are recommended by the test guidelines. This study encompassed two treatment groups (one test item concentration at nominal 100 mg/L, and one control) each containing 20 individuals. The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Environmental conditions were recorded. All measured values remained within acceptable ranges. No immobilisation or any abnormal behaviour was observed in the control and the 100 mg/L concentration group after 48 hours of exposure. All validity criteria were within acceptable limits and therefore, the study was considered valid. The quantification of the test item in the test samples was performed using high performance liquid chromatography with HPLC-UV detection. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium. The analytically measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the nominal concentration. The 48-h NOEC was determined to be > 100 mg/L. The 48-h LOEC and the EC50 values were determined to be > 100 mg/L.

Description of key information

In the 48-h acute toxicity of the test item on Daphnia magna according to OECD T 202, no immobilisation was observed in the control and in the 100 mg/L concentration group. In both groups no abnormal behaviour or appearance of test animals was noted. The 48-h NOEC was determined to be > 100 mg/L. The 48-h LOEC and the EC₅₀ values were determined to be > 100 mg/L (reference 6.1.3-1).

Additional information

The influence of the test item on the mobility of Daphnia magna was determined in a 48-h “Acute Immobilisation Test” according to OECD TG 202 and under GLP conditions. For this purpose, young daphnids (< 24 hours old) were exposed in a semi-static test for 48 hours to the only concentration of nominal 100 mg/L under defined conditions. The recorded effects were the mobility of the daphnids after 24 and 48 hours. This limit-test was performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the daphnids up to this concentration. The test method and the test species Daphnia magna are recommended by the test guidelines. This study encompassed two treatment groups (one test item concentration at nominal 100 mg/L, and one control) each containing 20 individuals. The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Environmental conditions were recorded. All measured values remained within acceptable ranges. No immobilisation or any abnormal behaviour was observed in the control and the 100 mg/L concentration group after 48 hours of exposure. All validity criteria were within acceptable limits and therefore, the study was considered valid. The quantification of the test item in the test samples was performed using high performance liquid chromatography with HPLC-UV detection. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium. The analytically measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the nominal concentration. The 48-h NOEC was determined to be > 100 mg/L. The 48-h LOEC and the EC₅₀ values were determined to be > 100 mg/L.