Potassium isobutyrate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Dec 2019 - 17 Jan 2020

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 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

See comments in 'principles of method if other than guideline'

Principles of method if other than guideline:

Deviations from the Guideline
The following deviations were observed:
• The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.
• The pre-culture was prepared 5 days before the start of experiment 1. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
• The pre-culture was prepared 5 days before the start of experiment 2. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
• The temperature in the experiment 1 was in the range 22.8 – 24.7 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and expo-nential growth was observed in the blank control this was stated as uncritical.
• The temperature in the experiment 2 was in the range 21.6 – 25.6 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and expo-nential growth was observed in the blank control this was stated as uncritical.
• The temperature during pre-culture incubation for experiment 1 was in the range 23.0 – 27.0 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
• The temperature during pre-culture incubation for experiment 2 was in the range 22.5 – 24.5 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
The deviations were assessed and signed by the study director on 27. Jan. 2020.

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 number 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: Room Temperature (20 ± 5 °C); Keep away from humidity; keep under inert gas
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Stability in solvents H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility in solvents 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:
Because of its hygroscopic properties, the test item was dried over night at 140 – 150 °C.
A stock solution containing 101.6 mg/L (first experiment) resp. 100.0 mg/L (second exper-iment) test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. Lower test concentrations were prepared by dilution of the stock solution in algal medium

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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sample Preparation
Before measurement the taken samples were diluted, if necessary, to stay in the calibrated range.
Samples with nominal concentration 100 mg/L were diluted tenfold, samples with nominal concentration 32 mg/L were diluted twofold with test medium.

Miscellaneous
On each sampling day, validity of calibration was controlled by measuring QC samples (1 and 10 mg/L). If the deviation was higher than ± 3 % and lower than or equal ± 15 %, the sensitivity correction factor from the QC samples was taken into account
First experiment:
At the beginning of the study a new calibration was performed and the measured QC samples were not taken into account. The mean recovery of the QC samples at the end of the experiment (109.2 % and 105.5 %) were higher than ± 3 % and therefore, taken into account.
Second experiment:
In the second experiment the mean recovery of the QC sample was taken into account at the beginning (92.8 % and 95.5 %), after 24 hours (93.2 %) and at the end of the test (96.5 %).

Test solutions

Vehicle:

yes

Details on test solutions:

Chemicals
All solutions were sterilized before use.
Composition of the Solutions:
Stock Solution I
NH4Cl 1500 mg
MgCl2*6H2O 1200 mg
CaCl2*2H2O 1800 mg
MgSO4*7H2O 1500 mg
KH2PO4 160 mg
H2O deionised ad 1000 mL

Stock Solution II
FeCl3*6H2O 64 mg
Na2EDTA*2H2O 100 mg
H2O deionised ad 1000 mL



Stock Solution III
H3BO3 185 mg
MnCl2*4H2O 415 mg
ZnCl2 3 mg
CoCl2*6H2O 1.5 mg
CuCl2*2H2O 0.01 mg
Na2MoO4*2H2O 7 mg
H2O deionised ad 1000 mL

Stock Solution IV
NaHCO3 50 g
H2O deionised ad 1000 mL

Algal medium
Stock solution I 10.0 mL
Stock solution II 1.0 mL
Stock solution III 1.0 mL
Stock solution IV 1.0 mL
H2O deionised ad 1000 mL

Stock solutions I and III and the deionised water were sterilized using an autoclave. Stock solutions II and IV were sterilized using filtration. The algal medium corresponds to the OECD TG 201 medium.

Deviations from the nominal weighted loads were less than 5 %.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: SAG Strain number: 86.81
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Test temperature:

Experiment 1: 22.8 – 24.7 °C
Experiment 2: 21.6 – 25.6 °C

pH:

Experiment 1
Nominal concentration in mg/L 0 h 72 h
Blank control 7.7 7.7
0.32 7.7 7.5
1 7.7 7.7
3.2 7.7 7.8
10 7.7 7.6
32 7.7 7.6
100 7.7 7.6

Experiment 2
Nominal Concentration in mg/L 0 h 72 h
Blank control 7.3 7.6
0.32 7.4 7.6
1 7.4 7.5
3.2 7.4 7.8
10 7.4 7.7
32 7.4 7.6
100 7.4 7.6

Nominal and measured concentrations:

0.32, 1, 3.2, 10, 32, 100 mg/L

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 97.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Significant inhibition of algal growth was observed at all concentrations. However, a clear dose-response relationship was observed.
The content of the test item in the test solutions was determined using HPLC. In the first experiment the content of the test item was determined at the beginning and at the end of the test. In the second experiment the content of the test item was measured daily.
At the lowest concentration 0.32 mg/L no test item was detectable. In the 5 higher concen-trations, at the beginning of the test, the correlation between measured concentration and nominal concentration was very good. At the following measurements a decrease of the measured concentrations could be observed, especially in the lower concentrations. At the end of the test, test item could only be detected in the 2 highest concentrations 32 mg/L and 100 mg/L.

Results with reference substance (positive control):

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were de-termined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Any other information on results incl. tables

Findings (Experiment 1)

Not reported since the study was repeated.

Findings (Experiment 2)

Cell Numbers

The cell numbers were determined with an electronic particle counter, the details are given inAnnex 3: Detailed Data (Experiment 2). The means and standard deviations of the cell numbers of the blank control and the treatments are presented in the following table:

Cell Number/mL

NominalConcentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2060	12917	89713	861843
	SD	0	772	8317	45228
0.32	Mean	2060	13793	124927	730667
	SD	0	1324	8248	21510
1	Mean	2060	13447	117673	702513
	SD	0	1629	5767	28652
3.2	Mean	2060	17327	116287	656253
	SD	0	2329	5946	20956
10	Mean	2060	14347	124607	562493
	SD	0	1202	5645	25633
32	Mean	2060	13060	111113	438627
	SD	0	897	4380	26660
100	Mean	2060	11140	91367	298907
	SD	0	924	7007	27506

SD = Standard Deviation

Light Intensity

The light intensity is checked monthly and recorded in a control chart. The lighting corresponded to the specified range of 4440 - 8880 lux.

 

Microscopical Observations

In the following table, the appearance of the algae at the end of the test is stated:

Microscopical Observations

Nominal Concentrationin mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
0.32	Yes
1	Yes
3.2	Yes
10	Yes
32	Yes
100	Yes

 

Measured Concentrations at 0 h

Nominal Concentration Test Item	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean
mg/L	mAU*min	mg/L	%	--	mg/L	mg/L
QC 1 mg/L *	0.0282	0.95	94.6	--	1.00	--
	0.0280	0.94	93.9	--	1.00
Blank control	n. a.	--	92.8	0.93	--	--
	n. a.	--	92.8	0.93	--	--
0.32	n. a.	--	92.8	0.93	--	--
	n. a.	--	92.8	0.93	--
1	0.0305	1.03	92.8	0.93	1.03	1.02
	0.0304	1.02	92.8	0.93	1.02
QC 1 mg/L *	0.0275	0.92	92.1	--	1.00	--
	0.0271	0.91	90.7	--	1.00
QC 10 mg/L	0.2764	9.54	95.4	--	10.00	--
	0.2767	9.55	95.5	--	10.00
3.2	0.0976	3.35	92.8	0.93	3.35	3.32
	0.0962	3.30	92.8	0.93	3.30
10	0.2764	9.98	95.5	--	10.45	10.64
	0.2767	10.34	95.5	--	10.83
32	0.2891	16.44	95.5	1.86	31.96	31.96
	0.2996	16.44	95.5	1.86	31.96
100	0.4758	10.85	95.5	9.29	105.47	105.55
	0.4757	10.87	95.5	9.29	105.64
QC 10 mg/L	0.3143	9.57	95.7	--	10.00	--
	0.3148	9.55	95.5	--	10.00

n.a. = not analysable

* seeDeviations

 

Measured Concentrations at 24 h

Nominal Concentration Test Item	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean
mg/L	mAU*min	mg/L	%	--	mg/L	mg/L
QC 1 mg/L *	0.0274	0.92	91.8	--	1.00	--
	0.0282	0.95	94.6	--	1.00
Blank control	n. a.	--	93.2	0.93	--	--
	n. a.	--	93.2	0.93	--
0.32	n. a.	--	93.2	0.93	--	--
	n. a.	--	93.2	0.93	--
1	0.0293	0.98	93.2	0.93	1.03	0.93
	0.0261	0.87	93.2	0.93	1.02
3.2	0.0931	3.19	93.2	0.93	3.35	3.14
	0.0905	3.10	93.2	0.93	3.30
QC 1 mg/L *	0.0280	0.94	93.9	--	1.00	--
	0.0276	0.92	92.5	--	1.00
QC 10 mg/L	0.2919	10.07	100.7	--	10.00	--
	0.2914	10.06	100.6	--	10.00
10	0.2919	10.59	--	0.93	10.45	9.83
	0.2914	10.58	--	0.93	10.83
32	0.3068	16.72	--	1.86	31.96	30.86
	0.3064	16.52	--	1.86	31.96
100	0.4838	10.30	--	9.29	105.47	95.67
	0.478	10.31	--	9.29	105.64
10	0.2983	10.05	100.5	--	10.00	--
	0.2988	10.05	100.5	--	10.00

n.a. = not analysable

* * seeDeviations

 

Measured Concentrations at 48 h

Nominal Concentration Test Item	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean
mg/L	mAU*min	mg/L	%	--	mg/L	mg/L
QC 1 mg/L *	0.031	1.04	104.2	--	1.00	--
	0.0319	1.07	107.4	--	1.00
Blank control	n. a.	--	--	0.93	--	--
	n. a.	--	--	0.93	--
0.32	n. a.	--	--	0.93	--	--
	n. a.	--	--	0.93	--
1	0.0206	0.68	--	0.93	0.63	0.65
	0.0215	0.71	--	0.93	0.66
3.2	0.0878	3.01	--	0.93	2.79	2.80
	0.0880	3.02	--	0.93	2.80
QC 1 mg/L *	0.0271	0.91	90.7	--	1.00	--
	0.0322	1.08	108.4	--	1.00
QC 10 mg/L	0.2967	10.24	102.4	--	10.00	--
	0.2941	10.15	101.5	--	10.00
10	0.3073	10.61	--	0.93	9.85	9.70
	0.2981	10.29	--	0.93	9.55
32	0.4709	16.27	--	1.86	30.21	30.26
	0.4723	16.32	--	1.86	30.30
100	0.2996	10.34	--	9.29	96.01	96.17
	0.3006	10.37	--	9.29	96.33
QC 10 mg/L	0.2965	10.23	102.3	--	10.00	--
	0.2961	10.22	102.2	--	10.00

n.a. = not analysable

* seeDeviations

 

Measured Concentrations at 72 h

Nominal Concentration Test Item	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean
mg/L	mAU*min	mg/L	%	--	mg/L	mg/L
QC 1 mg/L *	0.0306	1.03	102.9	--	1.00	--
	0.0287	0.96	96.2	--	1.00
Blank control	n. a.	--	--	0.93	--	--
	n. a.	--	--	0.93	--
0.32	n. a.	-- **	--	0.93	-- *	-- **
	n. a.	-- **	--	0.93	-- *
1	n. a.	-- **	--	0.93	-- *	-- **
	n. a.	-- **	--	0.93	-- *
3.2	n. a.	-- **	--	0.93	-- *	-- **
	n. a.	-- **	--	0.93	-- *
QC 1 mg/L *	0.0299	1.00	100.4	--	1.00	--
	0.0289	0.97	96.9	--	1.00
QC 10 mg/L	0.2795	9.64	96.4	--	10.00	--
	0.2773	9.57	95.7	--	10.00
10	n. a.	-- **	96.5	0.93	-- *	-- **
	n. a.	-- **	96.5	0.93	-- *
32	0.0690	2.36	96.5	1.86	2.83	4.75
	0.0754	2.58	96.5	1.86	3.70
100	0.2795	9.65	96.5	9.29	93.18	93.05
	0.2811	9.70	96.5	9.29	94.90
QC 10 mg/L	0.2792	9.63	96.3	--	10.00	--
	0.2829	9.76	97.6	--	10.00

n.a. = not analysable

* seeDeviations

* lower than the LOQ (0.5 mg/L)

 

% of Nominal Concentration

Nominal Concentration Test Item	% of Nominal Concentration				Geometric Mean
mg/L	t = 0 h	t = 24 h	t = 48 h	t = 72 h	mg/L
0.32	* < LOQ	* < LOQ	* < LOQ	* < LOQ	--
1	102 %	93 %	65 %	* < LOQ	**0.63
3.2	104 %	98 %	87 %	* < LOQ	**1.64
10	106 %	98 %	97 %	* < LOQ	**3.99
32	100 %	96 %	95 %	4.75	19.40
100	106 %	96 %	96 %	93.05	97.50

* Measured concentrationlower than the LOQ (0.5 mg/L).

** ½ LOQ 0.25 mg/L was used for the measuring point 72 hours

Growth Rate, Yield

From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Growth Rate µ, Yield

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72 h) [day^-1]	Yield (0-72 h) [Cell Concentration/mL]
Blank control	Mean	2.01	859783
	SD	0.02	45228
0.32	Mean	1.96	728607
	SD	0.01	21510
1	Mean	1.94	700453
	SD	0.01	28652
3.2	Mean	1.92	654193
	SD	0.01	20956
10	Mean	1.87	560433
	SD	0.02	25633
32	Mean	1.79	436567
	SD	0.02	26660
100	Mean	1.66	296847
	SD	0.03	27506

SD = Standard Deviation

Inhibition

The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

Inhibition Values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72 h)	Yield (0-72 h)
Blank control	0	0
0.32	2.72	15.26
1	3.38	18.53
3.2	4.50	23.91
10	7.06	34.82
32	11.19	49.22
100	17.58	65.47

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibition of algal growth was observed at all concentrations. However, a clear dose-response relationship was observed.

Biological Results of the Test Item
Parameter Value 95 % confidence interval
NOEC (Growth Rate) 72 h < 0.63 mg/L --
NOEC (Yield) 72 h < 0.63 mg/L --
LOEC (Growth Rate) 72 h ≤ 0.63 mg/L --
LOEC (Yield) 72 h ≤ 0.63 mg/L --
72h ErC10 13.52 mg/L 11.13 – 16.16 mg/L
72h EyC10 0.15 mg/L 0.07 – 0.34 mg/L
72h ErC50 > 97.5 mg/L --
72h EyC50 20.71 mg/L 7.34 – 58.95 mg/L

Executive summary:

Findings and Results:

 

Two valid experiments were performed.

In the first experiment the measured values at the end of the test are not usable, because of an error during the analytical measurement or sample preparation. Therefore, the experiment was repeated. Because the test item is not stable at low concentrations, in the second experiment analytical measurements were performed every 24 hours. The biological results were based on the result of the second experiment only.

 

The study was performed using 6 concentrations ranging from 0.32 to 100 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus)was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield[1]were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at all concentrations. However, a clear dose-response relationship was observed.

 

The content of the test item in the test solutions was determined using HPLC. In the first experiment the content of the test item was determined at the beginning and at the end of the test. In the second experiment the content of the test item was measured daily.

At the lowest concentration 0.32 mg/L no test item was detectable. In the 5 higher concentrations, at the beginning of the test, the correlation between measured concentration and nominal concentration was very good. At the following measurements a decrease of the measured concentrations could be observed, especially in the lower concentrations. At the end of the test, test item could only be detected in the 2 highest concentrations 32 mg/L and 100 mg/L.

The measured concentrations within the exposure period were not within the range of ± 20 % of the nominal values. Therefore, the determination of the results was based on the geometric mean of the measured concentrations (see § 39 OECD 201). The lowest concentration 0.32 mg/L was excluded from evaluation because no test item was detectable at any measurement.

For the treatments 1 mg/L, 3.2 mg/L and 10 mg/L the half of the limit of quantification was used for calculation of the geometric mean because no test item was detectable at the end of the test.

[1]Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period. Calculation see under 8.2 Growth and growth inhibition are quantified from measurements of the algal biomass as a function of time. Algal biomass is defined as the dry weight per volume, e.g. mg algae/L test solution. However, dry weight is difficult to measure and therefore surrogate parameters are used. Of these surrogates, cell counts are most often used.