Sodium pyruvate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2018-04-10 to 2018-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 23rd March 2006, corrected 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

01 March 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sample preparation: Three parallel samples were taken from each test concentration and six samples from the control solutions on each day of the analytical occasions. Formulation samples were diluted with ultrapure water before the analysis or directly injected.
- Sample storage conditions before analysis: No storage

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions used in the test was prepared by mechanical dispersion without using any solubilising agents. A stock solution was first prepared by dissolving an amount of 0.08 g test item in 800 mL dilution water (OECD Medium) to obtain the nominal concentration of 100 mg/L. The test solutions of subsequent lower test concentrations were prepared by appropriate diluting of this stock solution. After the formulation procedure algal cells were immediately introduced into test solution. Untreated control ran parallel in the test.
- Controls: Untreated control, toxic reference control (with the reference substance potassium dichromate )

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum: 4 days

ACCLIMATION
- Acclimation period: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.4 – 22.8 °C

pH:

8.09 – 9.51

Nominal and measured concentrations:

Nominal concentration: 2.6, 6.4, 16.0, 40.0 and 100.0 mg/L
Calculated geometric mean measured concentrations: 0.47, 0.70, 1.18, 1.90 and 3.02 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, fill volume: Glass, ~250 mL volume, 100 mL test medium
- Aeration: No
- Initial cells density: N = 1 × 10^4 cells/mL
- Control end cells density: N = 79.33× 10^4 cells/mL (mean value)
- No. of vessels per concentration: 3
- No. of vessels per untreated control: 6

GROWTH MEDIUM
- Standard medium used: Yes, OECD Medium

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water. Separate stock solutions were prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE Direct-Q 5 water purification system). Then, the growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 7971 lux, fluorescent lamps (400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and, therefore, provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At 24, 48 and 72 h manual cell counting using microscope with counting chamber

RANGE FINDING TEST
- Test concentrations: 0.1 , 1, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes

STATISTICAL ANALYSIS
Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 24 h and 48 h, and at the end of the test (72 hours after the start of the test) using Excel for Windows software. Percentage inhibition of growth rate (µ) and yield (y) were calculated using Excel for Windows software. The ECx values and their confidence limits for growth rate and yield were calculated using Probit analysis by SPSS software. For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using analysis of variance (ANOVA) and Dunnett’s test (α = 0.05; 2-sided) by SPSS software.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 2.11 - 2.78

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 3.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 2.71 - 3.83

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.02 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 3.81 - 8.10

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% conf. limits: 1.50 - 1.87

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% conf. limits: 1.84 - 2.17

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% conf. limits: 2.58 - 3.04

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Unusual cell shape: Some swollen cells were noticed in one replicate of the 1.18 mg/L concentration (measured). Thin cells were observed in the concentrations of 1.18, 1.90 and 3.02 mg/L (measured) together with the normal cells during the test.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- 72h ErC50: 1.08 mg/L (95 % confidence limits: 0.79 – 1.51 mg/L)
- 72h EyC50: 0.61 mg/L (95 % confidence limits: 00.49 – 0.76 mg/L)

Reported statistics and error estimates:

Statistical procedure for ErCx (growth rate) and for EyCx (yield) estimation for Growth rates: Probit analysis, Pearson Goodness-of-Fit Test.
Statistical procedure for NOEC estimation: Dunnett´s test

Table 1: Algal cell number (× 104 cell/mL) during the test period of 72 hours and Yield

Algal cell number (× 104 cell/mL) during the test period of 72 hours and YieldConcentration			Number of Cells (× 10^4/mL)				Yield
Nominal [mg/L]	measured [mg/L]		0 h	24 h	48 h	72 h
Control	Not detected		1	4	27	71	70
			1	4	26	103	102
			1	4	21	62	61
			1	4	25	83	82
			1	4	23	102	101
			1	3	21	55	54
		Mean	1.00	3.83	23.83	79.33	78.33
		SD	0.00	0.41	2.56	20.25	20.25
2.6	0.47		1	4	27	90	89
			1	2	23	79	78
			1	4	29	71	70
		Mean	1.00	3.33	26.33	80.00	79.00
		SD	0.00	1.15	3.06	9.54	9.54
6.4	0.70		1	5	25	103	102
			1	4	23	82	81
			1	3	15	90	89
		Mean	1.00	4.00	21.00	91.67	90.67
		SD	0.00	1.00	5.29	10.60	10.60
16.0	1.18		1	3	25	98	97
			1	4	12	85	84
			1	3	21	78	77
		Mean	1.00	3.33	19.33	87.00	86.00
		SD	0.00	0.58	6.66	10.15	10.15
40.0	1.90		1	4	17	48	47
			1	4	24	80	79
			1	3	19	64	63
		Mean	1.00	3.67	20.00	64.00	63.00
		SD	0.00	0.58	3.61	16.00	16.00
100.0	3.02		1	2	13	36	35
			1	4	13	40	39
			1	3	15	29	28
		Mean	1.00	3.00	13.67	35.00	34.00
		SD	0.00	1.00	1.15	5.57	5.57

Table 2: The specific Growth Rates during the test period of 72 hours

Concentration			Growth Rates (µ)
Nominal [mg/L]	measured [mg/L]		0 – 24 h	0 – 48 h	0 – 72 h
Control	Not detected		0.0578	0.0687	0.0592
			0.0578	0.0679	0.0644
			0.0578	0.0634	0.0573
			0.0578	0.0671	0.0614
			0.0578	0.0653	0.0642
			0.0458	0.0634	0.0557
		Mean	0.0558	0.0660	0.0604
		SD	0.0049	0.0023	0.0036
2.6	0.47		0.0578	0.0687	0.0625
			0.0289	0.0653	0.0607
			0.0578	0.0702	0.0592
		Mean	0.0481	0.0680	0.0608
		SD	0.0167	0.0025	0.0016
6.4	0.70		0.0671	0.0671	0.0644
			0.0578	0.0653	0.0612
			0.0458	0.0564	0.0625
		Mean	0.0569	0.0629	0.0627
		SD	0.0107	0.0057	0.0016
16.0	1.18		0.0458	0.0671	0.0637
			0.0578	0.0518	0.0617
			0.0458	0.0634	0.0605
		Mean	0.0498	0.0608	0.0620
		SD	0.0069	0.0080	0.0016
40.0	1.90		0.0578	0.0590	0.0538
			0.0578	0.0662	0.0609
			0.0458	0.0613	0.0578
		Mean	0.0538	0.0622	0.0575
		SD	0.0069	0.0037	0.0036
100.0	3.02		0.0289	0.0534	0.0498
			0.0578	0.0534	0.0512
			0.0458	0.0564	0.0468
		Mean	0.0441	0.0544	0.0493
		SD	0.0145	0.0017	0.0023

Table 3: Growth Rates (µ) and percentage inhibition of µ during the test period

Concentration		Growth Rates (µ)and % Inhibition ofµ
Nominal [mg/L]	measured [mg/L]	0–24 h		0–48 h		0–72 h
		µ	%	µ	%	µ	%
Control	Not detected	0.0558	–	0.0660	–	0.0604	–
2.6	0.47	0.0481	13.7	0.0680	-3.2	0.0608	-0.7
6.4	0.70	0.0569	-2.0	0.0629	4.6	0.0627	-3.9
16.0	1.18	0.0498	10.7	0.0608	7.9	0.0620	-2.7
40.0	1.90	0.0538	3.6	0.0622	5.7	0.0575	4.8
100.0	3.02	0.0441	20.8	0.0544	17.5	0.0493	18.4

Table 4: Yield (y) and percentage inhibition of y during the test period       

Concentration		Yield y and % Inhibition of y
Nominal [mg/L]	measured [mg/L]	0–72 h
		y	%
Control	Not detected	78.33	–
2.6	0.47	79.00	-0.9
6.4	0.70	90.67	-15.7
16.0	1.18	86.00	-9.8
40.0	1.90	63.00	19.6
100.0	3.02	34.00	56.6

Table 5: Sodium pyruvate concentration measured on 10 April 2018 (at the start of the test)       

Nominal concentration (mg/L)	Mean of the measured concentrations (mg/L)	Measured concentration in % of the nominal
2.6	2.45	94
6.4	6.10	95
16	14.9	93
40	37.1	93
100	92.3	92

Table 6: Sodium pyruvate concentration measured on 11 April 2018

Nominal concentration (mg/L)	Mean of the measured concentrations (mg/L)	Measured concentration in % of the nominal
2.6	2.01	77
6.4	3.98	62
16	12.8	80
40	35.0	87
100	90.7	91

Table 7: Sodium pyruvate concentration measured on 12 April 2018

Nominal concentration (mg/L)	Mean of the measured concentrations (mg/L)	Measured concentration in % of the nominal
2.6	0	-
6.4	0	-
16	0	-
40	0	-
100	below the quantitation limit	-

On 13 April 2018 (at the end of the test) concentrations of all the samples were zero. The test item was not detected in the control samples.

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata was determined according to the principles of OECD 201, Council Regulation (EC) No. 761/2009 C.3 and EPA OPPTS 850.5400. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 3.02 mg/L and 2.78 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.45 mg/L and 1.71 mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 1.90 mg/L. In conclusion, the test item had significant toxic effects on the growth of Raphidocelis subcapitata.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) was determined according to the principles of OECD 201, Council Regulation (EC) No. 761/2009 C.3 and EPA OPPTS 850.5400. The study was conducted in compliance with GLP criteria and under static conditions. The definitive test was carried out at nominal concentrations of 2.6, 6.4, 16.0, 40.0 and 100.0 mg/L because of the results of the pretest. The corresponsing measured concentrations were 0.47, 0.70, 1.18, 1.90 and 3.02 mg/L. In this study, the test item was found to inhibit the growth of the freshwater green alga Raphidocelis subcapitata after 72 hours with the following effect values (based on geometric means of measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were >3.02 mg/L (95 % conf. limits: 3.81 - 8.10) and 2.78 mg/L (95 % conf. limits: 2.58 - 3.04), respectively. The EC-10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.45 mg/L (95 % conf. limits: 2.11 – 2.78) and 1.71 (95% conf. limits: 1.50 - 1.87), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 1.90 mg/L. In conclusion, the test item had significant toxic effects on the growth of Raphidocelis subcapitata. All validity criteria were met, therefore, the study is considered as valid.

Description of key information

The EC50-values of the substance for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours are > 3.02 mg/L and 2.78 mg/L (geometric mean measured), respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.45 mg/L and 1.71 mg/L (geometric mean measured), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 1.90 mg/L (geometric mean measured).

Key value for chemical safety assessment

EC50 for freshwater algae:

3.02 mg/L

EC10 or NOEC for freshwater algae:

2.45 mg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) was determined according to the principles of OECD 201, Council Regulation (EC) No. 761/2009 C.3 and EPA OPPTS 850.5400. The study was conducted in compliance with GLP criteria and under static conditions. The definitive test was carried out at nominal concentrations of 2.6, 6.4, 16.0, 40.0 and 100.0 mg/L because of the results of the pretest. The corresponsing measured concentrations were 0.47, 0.70, 1.18, 1.90 and 3.02 mg/L. In this study, the test item was found to inhibit the growth of the freshwater green alga Raphidocelis subcapitata after 72 hours with the following effect values (based on geometric means of measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were >3.02 mg/L (95 % conf. limits: 3.81 - 8.10) and 2.78 mg/L (95 % conf. limits: 2.58 - 3.04), respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.45 mg/L (95 % conf. limits: 2.11 – 2.78) and 1.71 (95 % conf. limits: 1.50 - 1.87), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 1.90 mg/L. In conclusion, the test item had significant toxic effects on the growth of Raphidocelis subcapitata. All validity criteria were met, therefore, the study is considered as valid (reference 6.1.5-1).