Potassium propionate

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:

2021-03-02 to 2021-04-xx

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

07 Decmber 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium propionate

EC Number:

206-323-5

EC Name:

Potassium propionate

Cas Number:

327-62-8

Molecular formula:

C3H6O2.K

IUPAC Name:

potassium propionate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0,12.5, 25, 50, 100, 200 mg/L
- Sampling method: After 0 h, 24 h, 48 h and 72 h exposure, the additional vessels of NC, A, B, C, D and E were sampled: 2 samples of 1 mL per treatment group.
- Sample storage conditions before analysis: The samples were filled directly into 2 mL glass vials and stored in the freezer (≤ -18 °C). Of each sampled treatment, one of the analytical samples from 0 h, 24 h, 48 h and 72 h was sent to the analytical laboratory at the test site biochemA GmbH for chemical analysis using an insulated box with thermal packs. The remaining samples were stored as retain samples in the freezer (≤ 18 °C) until finalisation of the study.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: Test material was dissolved in OECD TG 201 medium which served as vehicle control
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): The stock solution was prepared by adding 222 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) to 1000 mL test medium and shaking rigorously.
The stock solution was used as highest test item concentration in the test. The further test item concentrations were prepared by diluting the stock solution with test medium according to the following scheme (spacing factor of 2).
- Test concentration separation factor: 2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Raphidocelis subcapitata
- Source (laboratory, culture collection): originates from the Culture Collection of Algae at the University of Goettingen.
- Age of inoculum (at test initiation): Before the start of the test, 1.640 mL of the algae stock suspension was diluted with 48.360 mL test medium to obtain a concentration of 5 E+04 cells/mL. This pre-culture was incubated for 4 d at 22.6 – 23.0 °C and 80.3 µE m-² s-1 ± 6.2 % continuous lighting.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21-24°C

pH:

The pH was 7.6 – 7.8 in the control and 7.6 – 7.9 in the test item treatment

Nominal and measured concentrations:

nominal: 0, 12.5, 25, 50, 100, 200 mg/L,
chemical analysis revealed no difference between the nominal and the measured concentrations above 20% thus, all effect level are reported in relation to the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: glass, 100 mL volume, fill-volume: 50 mL
- Aeration: no
- Initial cells density: 7 E+04 cells/mL
- Control end cells density: 3.05 E+06 cells/mL
- No. of vessels per concentration (replicates): triplicate
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: The test vessels were continuously illuminated by LEDs (warm white) with a light intensity of 80.3 µE m-² s-1 ± 6.2 %.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: The algal biomass was monitored by measuring the chlorophyll fluorescence after 0 h, 24 h, 48 h and 72 h. From each test vessel, 200 µL test solution was transferred into a 96-well microplate and the fluorescence measured with the fluorescence microplate reader at an excitation wavelength of 465 nm and an emission wavelength of 670 nm.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: A preliminary range finding test without GLP was performed before the start of this GLP-study. Nominal concentrations of 4, 20 and 100 mg/L test item were tested and resulted in inhibition of -2.0 %, -0.8 % and 5.4 % for growth rate and -9.2 %, -3.0 % and 22.8 % for yield (72 h), respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Reported statistics and error estimates:

Chlorophyll fluorescence values are correlated with measured cell concentrations. The slope of the curve gives the conversion factor. By means of the conversion factor, the surrogate parameter chlorophyll fluorescence is converted into cell concentration.

Yield is defined as biomass (cell concentration) at the end of the test (72 h) minus biomass (cell concentration) at the start of the test (0 h). The mean yield is calculated for each test item concentration and the control. The inhibition of the yield is calculated according to the following formula:
%Iy = Yc-YT/Yc * 100
%Iy: Percent inhibition of yield
Yc: Mean for yield in the control
YT: Yield for the treatment replicate

The specific growth rates are calculated for each test concentration and the control according to the formula:
µi-j = lnXj - ln Xi/tj - ti (day-1)
µi-j: Average specific growth rate from time i to j
Xi: Biomass at time i
Xj: Biomass at time j

The coefficient of variation of the replicates (RSD, relative standard deviation) is calculated according to the formula:

RSD = Standard Deviation of the Mean / Mean*100

The percentage inhibition of the growth rate in relation to the control is calculated for each test vessel according to the following formula:
%Ir = µc -µT/µc * 100
%Ir: Percent inhibition in average specific growth rate
µc: Mean for average specific growth rate (µ) in the control
µT: Average specific growth rate for the treatment replicate

With the statistical software ToxRat Professional 3.3.0 (ToxRat Solutions GmbH, Alsdorf, Germany), ECx and LOEC/NOEC for yield and growth rate were determined.

ECx values were determined with nonlinear regressions.
LOEC/NOEC was determined with Shapiro-Wilk´s Test on Normal Distribution, Levene´s Test on Variance Homogenity, Trend analysis by Contrasts and Williams Multiple Sequential t-test Procedure.

Any other information on results incl. tables

Table 1: Inhibition of yield after 24 h, 48 h and 72 h exposure

	Nominal test item concentration [mg/L]		Inhibition of yield [%]
			24 h		48 h		72 h
NC		--		--		--
12.5		-11.4		10.3		21.9
25		-40.7		-23.3		-20.0
50		-1.7		6.0		22.7
100		-2.0		14.1		19.3
200		-8.0		3.8		31.3

Table 2: Inhibition of growth rate after 24 h, 48 h and 72 h exposure

	Nominal test item concentration [mg/L]		Inhibition of growth rate [%]
			24 h		48 h		72 h
NC		--		--		--
12.5		-7.2		4.0		6.4
25		-23.5		-7.8		-4.6
50		1.2		3.0		7.2
100		0.6		5.7		5.6
200		-4.7		1.4		11.3

Table 3: Effect concentrations for yield and growth rate at the end of the test

(Lowest/No Observed) Effect Concentration	Nominal test item concentration [mg/L]
95 % Confidence Limits (CL)	Yield (72 h)	Growth rate (72 h)
EC50	> 200	> 200
CL	n.d.	n.d.
EC20	112.4	> 200
CL	n.d.	n.d.
EC10	53.8	184.3
CL	n.d.	n.d.
LOEC	200	200
NOEC	100	100

n.d.: not determined due to mathematical reasons or inappropriate data

 

 

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the results reported in the present study the 72h EC50 value for yield and growth rate of Raphidocelis subcapitata exposed to 0, 12.5, 25, 50, 100 and 200 mg/L dipotassium propionate is > 200 mg/L for both parameters. The respective NOEC was determined to be 100 mg/L for both parameters.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Raphidocelis subcapitata were exposed to dipotassium propionate at nominal concentrations of {0, 12.5, 25, 50, 100, and 200 mg a.i./L under static conditions in accordance with OECD TG 201 (March 2006). The NOEC and EC50 values based on yield and growth rate were 100 and 200 mg a.i./L, respectively.  The % growth inhibition in the treated algal culture as compared to the control ranged from 5.6 to 11.3 %.

 

There were no compound related phytotoxic effects.

This toxicity study is classified as acceptable and satisfies the guideline requirements for a Toxicity to aquatic algae and cyanobacteria study.

 

Results Synopsis

Test Organism: green alga (Raphidocelis subcapitata)

Test Type: Static

 

72 hr EC50: > 200.mg a.i./L}                95% C.I.: n.d.

72 hr NOEC: 100 mg a.i./L

 

Endpoint(s) Effected: yield and growth rate