Sodium hydrogen L-aspartate

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:

2017-08-15 to 2017-08-18

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:

- Concentrations: six replicate samples (5 mL per replicate) were taken from the test solution (100 mg/L) and from the control at the start and at the end of the test
- Sampling method: HPLC method with UV detection
- Sample storage conditions before analysis: No storage

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test solution used in the test was prepared by mechanical dispersion without using of any solubilising agent.

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

Post exposure observation period:

None

Test temperature:

The temperature in the flask was in the range of 22.9 – 23.6 °C and within the climate chamber between 22.2 and 24.0 °C.

pH:

8.01 – 8.94

Nominal and measured concentrations:

Nominal concentration: 6.25, 12.5, 25, 50 and 100 mg/L
Calculated geometric mean measured concentrations: 1.77, 2.48, 3.52, 4.99 and 7.11 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 × 10E4 cells/mL
- Control end cells density: 93.50 × 10E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 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
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 7990 lux, fluorescent lamps (400-700 nm)

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

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 6.25, 12.5, 25, 50, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

STATISTICAL ANALYSIS
Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using Independent Samples T-Test (2-tailed; α = 0.05) by SPSS software.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 5.95 – 7.67

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 8.04 – 12.13

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.47 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated value for anhydrous substance

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 13.79 – 30.25

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.15 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated value for anhydrous substance

Key result

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:

LOEC

Effect conc.:

4.99 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.:

3.66 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% conf. limits: 2.77 – 4.34

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.88 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% conf. limits: 4.08 – 5.88

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% conf. limits: 6.77 – 13.23

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:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.99 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 the 2.48 mg/L concentration (measured). Swollen and thin cells were observed in the concentrations of 3.52, 4.99 and 7.11 mg/L 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
- ErC50 = 0.99 mg/L; EyC50 = 0.59 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 (× 10⁴cell/mL)during the test period of 72 hours and Yield

Concentration [mg/L]			Number of Cells (× 10^4/mL)				Yield
nominal	measured		0 h	24 h	48 h	72 h
Untreated control	Not detected		1	4	19	93	92
			1	4	19	87	86
			1	4	24	104	103
			1	5	26	97	96
			1	4	23	96	95
			1	3	16	84	83
		Mean	1.00	4.00	21.17	93.50	92.50
		SD	0.00	0.63	3.76	7.23	7.23
6.25	1.77		1	4	15	87	86
			1	5	21	92	91
			1	4	19	91	90
		Mean	1.00	4.33	18.33	90.00	89.00
		SD	0.00	0.58	3.06	2.65	2.65
12.5	2.48		1	5	24	97	96
			1	4	20	90	89
			1	5	23	95	94
		Mean	1.00	4.67	22.33	94.00	93.00
		SD	0.00	0.58	2.08	3.61	3.61
25	3.52		1	5	18	88	87
			1	4	23	87	86
			1	5	16	93	92
		Mean	1.00	4.67	19.00	89.33	88.33
		SD	0.00	0.58	3.61	3.21	3.21
50	4.99		1	3	23	85	84
			1	4	18	62	61
			1	3	19	78	77
		Mean	1.00	3.33	20.00	75.00	74.00
		SD	0.00	0.58	2.65	11.79	11.79
100	7.11		1	4	21	52	51
			1	4	24	56	55
			1	4	25	51	50
		Mean	1.00	4.00	23.33	53.00	52.00
		SD	0.00	0.00	2.08	2.65	2.65

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

Concentration [mg/L]			Growth Rates (µ)
nominal	measured		0 – 24 h	0 – 48 h	0 – 72 h
Untreated control	Not detected		0.0578	0.0613	0.0630
			0.0578	0.0613	0.0620
			0.0578	0.0662	0.0645
			0.0671	0.0679	0.0635
			0.0578	0.0653	0.0634
			0.0458	0.0578	0.0615
		Mean	0.0573	0.0633	0.0630
		SD	0.0068	0.0038	0.0011
6.25	1.77		0.0578	0.0564	0.0620
			0.0671	0.0634	0.0628
			0.0578	0.0613	0.0627
		Mean	0.0609	0.0604	0.0625
		SD	0.0054	0.0036	0.0004
12.5	2.48		0.0671	0.0662	0.0635
			0.0578	0.0624	0.0625
			0.0671	0.0653	0.0632
		Mean	0.0640	0.0646	0.0631
		SD	0.0054	0.0020	0.0005
25	3.52		0.0671	0.0602	0.0622
			0.0578	0.0653	0.0620
			0.0671	0.0578	0.0630
		Mean	0.0640	0.0611	0.0624
		SD	0.0054	0.0039	0.0005
50	4.99		0.0458	0.0653	0.0617
			0.0578	0.0602	0.0573
			0.0458	0.0613	0.0605
		Mean	0.0498	0.0623	0.0598
		SD	0.0069	0.0027	0.0023
100	7.11		0.0578	0.0634	0.0549
			0.0578	0.0662	0.0559
			0.0578	0.0671	0.0546
		Mean	0.0578	0.0656	0.0551
		SD	0.0000	0.0019	0.0007

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

Concentration [mg/L]		Growth Rates (µ)and % Inhibition ofµ
nominal	measured	0–24 h		0–48 h		0–72 h
		µ	%	µ	%	µ	%
Untreated control	Not detected	0.0573	–	0.0633	–	0.0630	–
6.25	1.77	0.0609	-6.2	0.0604	4.6	0.0625	0.8
12.5	2.48	0.0640	-11.6	0.0646	-2.1	0.0631	-0.2
25	3.52	0.0640	-11.6	0.0611	3.5	0.0624	1.0
50	4.99	0.0498	13.2	0.0623	1.6	0.0598	5.0
100	7.11	0.0578	-0.8	0.0656	-3.6	0.0551	12.5

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

Concentration [mg/L]		Yield y and % Inhibition of y
nominal	measured	0–72 h
		y	%
Untreated control	Not detected	92.50	–
6.25	1.77	89.00	3.8
12.5	2.48	93.00	-0.5
25	3.52	88.33	4.5
50	4.99	74.00	20.0
100	7.11	52.00	43.8

Table 5: The section-by-section specific growth rates in the control cultures

Section-by-Section Growth Rate
Expressed in hours	0-24 h	24-48 h	48-72 h
	0.0573	0.0693	0.0624
Expressed in days	0-1 day	1-2 day	2-3 day
	1.3755	1.6633	1.4966

Table 6: Morphological changes of algal cells

Concentration[mg/L]		Symptoms
nominal	measured	24 h	48 h	72 h
Untreated control	Not detected	normal cells	normal cells	normal cells
6.25	1.77	normal cells	normal cells	normal cells
12.5	2.48	normal cells	normal cells some swollen cells	normal cells some swollen cells
25	3.52	normal cells	normal cells swollen cells	normal cells
				swollen cells
				thin cells
50	4.99	normal cells	normal cells	normal cells
			swollen cells	swollen cells
				thin cells
100	7.11	normal cells	normal cells	normal cells
			swollen cells	swollen cells
				thin cells

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to the unicellular freshwater green alga 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 EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 18.39 mg/L and 8.44 mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 3.52 mg/L, respectively. The 72-h EC50 and 72-h NOEC (growth rate) for the anhydrous substance was calculated to be EC50 = 16.47 mg/L and NOEC = 3.15 mg/L. In conclusion, L-aspartic acid sodium salt monohydrate had significant toxic effects on the growth of Raphidocelis subcapitata.

Executive summary:

The toxicity of the hydrated test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata (formerly 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 study was conducted in compliance with GLP criteria and under static conditions. The definitive test was carried out at concentrations of 6.25, 12.5, 25, 50, 100 mg/L because of the results of the pretest. The corresponsing measured concentrations were 1.77, 2.48, 3.52, 4.99 and 7.11 mg/L. In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella 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 18.39 mg/L (95% conf. limits: 13.79 – 30.25) and 8.44. (95% conf. limits: 6.77 – 13.23 ), respectively. The EC-10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 6.62 mg/L (95% conf. limits: 5.95 – 7.67) and 3.66 (95% conf. limits: 2.77 – 4.34), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 3.52 mg/L. The 72-h EC50 and 72-h NOEC (growth rate) for the anhydrous substance was calculated to be EC50 = 16.47 mg/L and  NOEC = 3.15 mg/L. In conclusion, L-aspartic acid sodium salt monohydrate had significant toxic effects on the growth of Pseudokirchneriella subcapitata. All validity criteria were met, therefore, the study is considered as valid.

Description of key information

In a growth inhibition test with freshwater algae, conducted according to OECD TG 201, EU Method C.3 and OPPTS 850.5400, the EC50-values (hydrated substance) for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 18.39 mg/L and 8.44 mg/L, respectively (reference 6.1.5 -1). The NOEC-value (hydrated substance) for both inhibition of growth rate and yield after 72 hours was 3.52 mg/L, respectively. The 72-h EC50 and 72-h NOEC (growth rate) for the anhydrous substance was calculated to be EC50 = 16.47 mg/L and  NOEC = 3.15 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

16.47 mg/L

EC10 or NOEC for freshwater algae:

3.15 mg/L

Additional information

The toxicity of the hydrated test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata (formerlyRaphidocelis subcapitata) was determined according to the principles of OECD 201, EU Method C.3 and EPA OPPTS 850.5400 (reference 6.1.5 -1). The study was conducted in compliance with GLP criteria and under static conditions. The definitive test was carried out with at concentrations of 6.25, 12.5, 25, 50, 100 mg/L based on the results of a preliminary study. Biological results are based on the geometric mean of corresponding measured concentrations of 1.77, 2.48, 3.52, 4.99 and 7.11 mg/L. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 18.39 mg/L (95% conf. limits: 13.79 – 30.25) and 8.44. (95% conf. limits: 6.77 – 13.23 ), respectively. The EC-10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 6.62 mg/L (95% conf. limits: 5.95 – 7.67) and 3.66 (95% conf. limits: 2.77 – 4.34), respectively. The NOEC-value for both inhibition of growth rate and yield after 72 hours was 3.52 mg/L. The 72-h EC50 and 72-h NOEC (growth rate) for the anhydrous substance was calculated to be EC50 = 16.47 mg/L and  NOEC = 3.15 mg/L. All validity criteria were met, therefore, the study is considered as valid.