Potassium isobutyrate

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:

03 December - 05 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

yes

Remarks:

M4-medium (husbandry) was switched to Dilution water (test). See justification below.

Principles of method if other than guideline:

Deviations from the Guideline
Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to cause any detrimental effects for test daphnia, therefore no acclimation period of 48 h was performed. This is a deviation from OECD Guideline No. 202 and EU-Method C.2, which can be considered uncritical.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium isobutyrate

EC Number:

243-077-8

EC Name:

Potassium isobutyrate

Cas Number:

19455-20-0

Molecular formula:

C4H8O2.K

IUPAC Name:

potassium 2-methylpropanoate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / 181101001
- Expiration date of the lot/batch: 31 Oct 2020
- Purity test date: 01 Nov 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel at room temperature (16.2 – 23.7 °C) away from humidity and under inert gas.
- Solubility and stability of the test substance in the solvent/vehicle:
Stability: H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: > 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was solid, because of its hygroscopic properties, and therefore, it was dried overnight at 145 °C.
A test solution containing 101.6 mg/L test item in dilution water was prepared for the test.
- Final preparation of a solid: as above.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : In solution

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Sample preparation: Before measurement the taken samples were diluted (10 fold) with dilution water, to stay in the calibrated range.

On each sampling day, validity of calibration was controlled by measuring QC samples (10 mg/L).
At the beginning of the study a new calibration was performed. The measured QC sam-ples were not taken into account. The mean QC value at the end of the experiment (107.7 %, mean of 108.7 %, 105.9 %, 108.0 %, 107.7 %, 107.4 % and 108.3 %) was higher than ± 3 % and therefore, taken into account.

Test solutions

Vehicle:

yes

Details on test solutions:

A test solution containing 101.6 mg/L test item in dilution water was prepared for the test.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: STRAUS
- Age at study initiation (mean and range, SD): 0-24 hours
- Method of breeding: Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical
- Source: Umweltbundesamt Berlin
- Feeding during test : No
- Food type: green algae (Desmodesmus subspicatus)
- Frequency: twice a week

ACCLIMATION
- Acclimation period: None

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

24 h

Post exposure observation period:

No

Test conditions

Hardness:

Not reported

Test temperature:

18.1-20.5

Nominal and measured concentrations:

100 mg/L (nominal concentration)
The concentrations to be tested are based on the result of a non-GLP pre-test.

Details on test conditions:

TEST SYSTEM
- Test vessel: flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass beakers, nominal volume 50 mL, tall shape
- Volume of solution: 50 mL
- Aeration: test system was not aerated. Dilution water was aerated after preparation.
- Renewal rate of test solution (frequency/flow rate): N/A
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 vessels, each containing 20 ± 5 mL test solution and 5 daphnia
- No. of vessels per vehicle control (replicates): 4 vessels, each containing 20 ± 5 mL test solution and 5 daphnia


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water (Daphnia medium) with the following specification was used (stated in SOP 11800201)

Parameter Concentration in mg/L
CaCl2*2H2O 293.80
MgSO4*7H2O 123.30
NaHCO3 64.80
KCl 5.80

Deviations from the nominal weighted loads were less than 5 %. Exact values are record-ed in the raw data. After preparation, the dilution water was aerated and the pH was measured. The pH was 7.7.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours using neon tubes
- Light intensity: not recorded


VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study (201910R201).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Results with reference substance (positive control):

The 24h-EC50 value was determined as 2.07 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as speci-fied in the OECD guideline.

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®).

Any other information on results incl. tables

Findings

Immobility

In the blank control, none of the daphnia was immobilised (see table below).

Table 4. Immobility

Nominal Concentration in mg/L	Immobility 24 hours					Immobility 48 hours
	absolute				in %	absolute				in %
Blank control	0	0	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0	0	0

 

pH and O₂

The pH values and the concentration of dissolved oxygen in the test media and the blank control are given in the following table:

Table 5.pH and O₂-values

Nominal Concentration in mg/L	pH		O2-Concentration in mg/L
	0 h	48 h	0 h	48 h
Blank control	7.6	7.6	8.2	9.1
100	7.7	7.6	8.1	9.1

 

Analytical Determinations

At the beginning and at the end of the test, the content of the test item in the test solutions was determined usingHPLC-UV-determination. The concentration determined at the start of the test was 102 % of the nominal concentration. At the end of the test the determined concentration was 113 % of the nominal concentration. Therefore, the determination of the biological results was based on the nominal concentration (see Guidance Doc. No.23 (§176)).

At the beginning of the test, a new calibration was performed.

Detailed data are given in the following tables:

Table 6.Measured Concentrations at 0 h

Nominal Concentration Test Item	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean (including accuracy of 102.2 %)
mg/L	mAU*min	mg/L	%	--	mg/L	mg/L
Blank control	n. a.	--	--	--	--	--
100	0.2910	10.11	--	10	101.10	101.72
	0.3082	10.68	--	10	106.82

Table 7.Measured Concentrations after 48 h

Nominal Concentration Test Item	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean (including accuracy of 102.2 %)
mg/L	mAU*min	mg/L	%	--	mg/L	mg/L
10 (QC sample)	0.1528	5.43 *	108.7	--	5.00	--
	0.1488	5.30 *	105.9	--	5.00
	0.1518	5.40 *	108.0	--	5.00
	0.1514	5.39 *	107.7	--	5.00
Blank control	n. a.	--	107.7	--	--	--
100	0.3637	12.52	107.7	10	116.26	112.95
	0.3583	12.34	107.7	10	114.61
10 (QC sample)	0.1510	5.37 *	107.4	--	5.00	--
	0.1523	5.42 *	108.3	--	5.00

* Because of a sample preparation error, the real concentration of the QC sample was 5 mg/L.

 

Table 8.% of Nominal Concentration

Nominal Concentration Test Item	% of Nominal Concentration
mg/L	t = 0 h	t = 48 h
Blank control	--	--
100	102	113

Biological Results

The biological results are presented in the following table:

Table 9.     Biological Results Test Item

Parameter	Value
24h EC50	> 100 mg/L
48h EC50	> 100 mg/L
48h NOEC	≥100 mg/L
48h LOEC	> 100 mg/L

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

One valid experiment was performed.
The study was performed as a limit test with the nominal concentration of 100 mg/L. For the test concentration and the blank control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 23.5 and 48 hours, the immobilised daphnia were counted.
None of the animals was immobilised in the blank control and the test concentration.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.
At the beginning and at the end of the test, the content of the test item in the test solutions was determined using HPLC-UV-determination. The concentration determined at the start of the test was 102 % of the nominal concentration. At the end of the test the determined concentration was 113 % of the nominal concentration. Therefore, the determination of the biological results was based on the nominal concentration (see Guidance Doc. No.23 (§176)).
No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met.
The result of the test is considered valid.

Executive summary:

Findings and Results:

 

One valid experiment was performed.

The study was performed as a limit test with the nominal concentration of 100 mg/L. For the test concentration and the blank control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.

None of the animals was immobilised in the blank control and the test concentration.

Potassium dichromate K₂Cr₂O₇(CAS No. 7778-50-9) was used as positive controlin a current reference study to assure that the test conditions are reliable.

At the beginning and at the end of the test, the content of the test item in the test solutions was determined usingHPLC-UV-determination. The concentration determined at the start of the test was 102 % of the nominal concentration. At the end of the test the determined concentration was 113 % of the nominal concentration. Therefore, the determination of the biological results was based on the nominal concentration (see Guidance Doc. No.23 (§176)).

 

The following results were determined for the test itemPotassium isobutyrate (species:Daphnia magna).

48h-NOEC≥100 mg/L
48h-LOEC> 100mg/L
24h-EC₅₀> 100 mg/L
48h-EC₅₀> 100 mg/L