L-Aspartic acid, N,N'-1,2-ethanediylbis-, sodium salt (1:3)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 01 to 04 June, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

not specified

Details on sampling:

- Concentrations: 0, 0.032, 0.065, 0.125, 0.25, 0.50 and 1.0 mg/L
- Sampling method: no data
- Sample storage conditions before analysis: no data

Vehicle:

not specified

Details on test solutions:

no data

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: CCAP 211/11B
- Source (laboratory, culture collection): LISEC laboratory culture
- Age of inoculum (at test initiation): no data
- Method of cultivation: no data

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

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

no data

Hardness:

24 mg CaCO3/L

Test temperature:

25.7 +/- 1.8oC (refer to Table 2)

pH:

7.98 - 8.01 (refer to Table 2)

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

nominal concentrations of 0, 0.032, 0.065, 0.125, 0.25, 0.50 and 1.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: borosilicate glass Erlenmeyers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 150 mL containing 100 mL of test solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- Renewal rate of test solution (frequency/flow rate): no data
- Initial cells density: 8.2 x 10 6 cell/mL (after 4 days of incubation)
- Control end cells density: 8.2 x 10 6 cell/mL (after 4 days of incubation)
- No. of organisms per vessel: 1.2 x 10 4cells/mL of Chlorella vulgaris
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 0


GROWTH MEDIUM
- Standard medium used: yes (refer to Table 1)
- Detailed composition if non-standard medium was used: no



OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 72-h light period
- Light intensity and quality: 5558 +/-669 lux continuous uniform illumination provided by Philips TL-33 lamps
- Salinity (for marine algae): no data


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
- Chlorophyll measurement: no data
- Other: no data


TEST CONCENTRATIONS
- Spacing factor for test concentrations: a geometric series at a concentration ratio of 2
- Justification for using less concentrations than requested by guideline: no data
- Range finding study: yes
- Test concentrations: no data
- Results used to determine the conditions for the definitive study: no data

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 1.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: no data
- Colour differences: no data
- Flocculation: no data
- Adherence to test vessels: no data
- Aggregation of algal cells: no data
- Other:
- Any stimulation of growth found in any treatment: no data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: no data

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

a t-test was used to check if the average of the absorbances at different test concentrations was significantly different (95% confidence limit) from that of the control.

Table 3: Measured absorbances of the test solutions.

		Measured absorbance at time t (h)
		0 h	24 h	48 h	72 h
Control	A	1.000	3.000	27.000	160.000
	B	2.000	3.000	5.000	9.000
	C	2.000	5.000	34.000	234.000
	D	3.000	3.000	36.000	230.000
	E	2.000	5.000	36.000	221.000
	F	2.000	15.000	23.000	180.000
Average		2.000	6.200	31.200	205.000
Standard deviation		0.707	5.020	5.891	33.061
0.032 mg/L	A	1.000	3.000	32.000	192.000
	B	1.000	3.000	26.000	160.000
	C	2.000	2.000	31.000	174.000
Average		1.333	2.667	29.667	175.333
Standard deviation		0.577	0.577	3.215	16.042
0.65 mg/L	A	3.000	2.000	26.000	142.000
	B	3.000	5.000	31.000	213.000
	C	2.000	2.000	28.000	202.000
Average		2.667	3.000	28.333	185.667
Standard deviation		0.577	1.732	2.517	38.214
0.125 mg/L	A	1.000	4.000	30.000	199.000
	B	2.000	3.000	25.000	133.000
	C	2.000	3.000	24.000	158.000
Average		1.667	3.333	26.333	163.333
Standard deviation		0.577	0.577	3.215	33.322
0.25 mg/L	A	2.000	3.000	19.000	100.000
	B	1.000	3.000	20.000	114.000
	C	2.000	1.000	16.000	106.000
Average		1.667	2.333	18.333	106.667
Standard deviation		0.577	1.155	2.082	7.024
0.5 mg/L	A	2.000	2.000	12.000	63.000
	B	3.000	3.000	15.000	65.000
	C	2.000	3.000	17.000	61.000
Average		2.333	2.667	14.667	63.000
Standard deviation		0.577	0.577	2.517	2.000
1 mg/L	A	1.000	2.000	12.000	25.000
	B	2.000	3.000	15.000	38.000
	C	2.000	3.000	14.000	32.000
Average		1.667	2.667	13.667	31.667
Standard deviation		0.577	0.577	1.528	6.506

Validity criteria fulfilled:

yes

Conclusions:

In a GLP study conducted according to OECD Guideline 201, the 72-h NOEC, EC50 (0-72 h) for growth and EC50 (24-72h) for growth rate for trisodium EDDS were 0.125, 0.29 and 1.57 mg/L to the green alga (Chlorella vulgaris) in freshwater.

Executive summary:

In a GLP study conducted according to OECD Guideline 201, the effect of trisodium EDDS on the growth of a unicellular green algal species (Chlorella vulgaris) was assessed under semi-static conditions in freshwater. The test was carried out with an initial concentration of 1.2 x 10⁴ cells/mL of C. vulgaris in the test cultures (in triplicate), for an exposure duration of 72 h. The nominal concentrations tested were 0, 0.032, 0.065, 0.125, 0.25, 0.50 and 1.0 mg/L for the determination of the concentration that caused 50% reduction (EC50; using the measured absorbances of the algal suspensions) in either growth or growth rate relative to the control culture.

Under the conditions of this study, the 72-h NOEC, EC50 (0-72 h) for growth and EC50 (24-72 h) for growth rate of C. vulgaris were 0.125, 0.29 and 1.57 mg/L, respectively, following exposure to trisodium EDDS in freshwater.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

medium contained altered trace metals and EDTA

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
no data

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.37, 0.75, 1.2, 2.5, 3.7, 6.1 and 12.3 mg/L
- Alga growth inhibition test: one sample was taken from each flask after 0, 21.5, 48, 72.5, 92.5 and 143 h, and the number of particles per mL in the samples was analysed
- Photostability test: at the start, and after 1, 3, 5 and 7 days of incubation, single 50 mL samples were taken from one flask with and one test flask without algae.
- Sample storage conditions before analysis: to each sample 1.35 mL of 37% formaldehyde was added for preservation. The samples were stored in a refrigerator.

Vehicle:

no

Details on test solutions:

no data

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: SAG 211/11b
- Source (laboratory, culture collection): collection of Algal Cultures, Institute for Plant Physiology, University of Gottingen, Nicolausberger Weg 18, D-3400 Gottingen, Germany.
- Age of inoculum (at test initiation): no data
- Method of cultivation: no data

ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

143 h

Remarks on exposure duration:

66 h for range-finding test

Post exposure observation period:

no data

Hardness:

24 mg CaCO3/L

Test temperature:

23 +/- 1oC

pH:

8.3-8.4

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

0, 0.37, 0.75, 1.2, 2.5, 3.7, 6.1 and 12.3 mg/L (nominal in final growth inhibition test; refer to Table 2 for concentrations in the other tests)

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 200 mL (no details on headspace or fill volume).
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- Renewal rate of test solution (frequency/flow rate): no data
- Initial cells density: 1 x 10(6) cells/mL
- Control end cells density: 0.95 x 10(4) cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 0


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: (refer to Table 2 for details)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: no data
- Total organic carbon: no data
- Particulate matter: no data
- Metals: refer to table 1
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 60-120 micromol/s m2, fluorescent lamps
- Salinity (for marine algae): not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no data


TEST CONCENTRATIONS
- Spacing factor for test concentrations: no data
- Justification for using less concentrations than requested by guideline: no data
- Range finding study
- Test concentrations: 0, 0.33, 1.0 and 3.0 mg/L
- Results used to determine the conditions for the definitive study: no relation

Reference substance (positive control):

not specified

Duration:

72.5 h

Dose descriptor:

EC50

Effect conc.:

ca. 9.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: biomass-independent

Duration:

72.5 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72.5 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72.5 h

Dose descriptor:

EC50

Effect conc.:

ca. 1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72.5 h

Dose descriptor:

EC90

Effect conc.:

ca. 2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

92.5 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

92.5 h

Dose descriptor:

EC50

Effect conc.:

ca. 2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

92.5 h

Dose descriptor:

EC90

Effect conc.:

ca. 3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

143 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

143 h

Dose descriptor:

EC50

Effect conc.:

ca. 4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

143 h

Dose descriptor:

EC90

Effect conc.:

ca. 13.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72.5 h

Dose descriptor:

EC10

Effect conc.:

> 12.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72.5 h

Dose descriptor:

EC50

Effect conc.:

> 12.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72.5 h

Dose descriptor:

EC90

Effect conc.:

> 12.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

92.5 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

92.5 h

Dose descriptor:

EC50

Effect conc.:

ca. 2.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

92.5 h

Dose descriptor:

EC90

Effect conc.:

ca. 4.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

143 h

Dose descriptor:

EC10

Effect conc.:

ca. 2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

143 h

Dose descriptor:

EC50

Effect conc.:

ca. 4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

143 h

Dose descriptor:

EC90

Effect conc.:

ca. 7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): in the presence of trisodium EDDS increased cell sizes and coagulation of cells were observed, whereas cultures containing different trace metals and EDTA concentrations revealed no abnormalities.
- Any stimulation of growth found in any treatment: there was an an increase in the mean particle size during the first 21.5 hours of incubation
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not observed
- Effect concentrations exceeding solubility of substance in test medium: no data

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

EC values with respect to the inoculum viability followed by logistic growth (EeC values) were calculated by means of parametric model developed by Kooijman et al. (1983) and by weighted least square fittings of logistic function to the total particle volumes at each test concentration. The area under the growth curves was calculated from the cell numbers and from the total cell volume values by the method given in OECD Guideline 201.

Table 3. Individual growth parameters calculated from the results of the final growth inhibition test.

Concentration of trisodium EDDS (mg/L)	Calculated inoculum		Calculated yield		Calculated growth rate
	10^6µm^3/ml	% of control	10^6µm^3/ml	% of control	(h)	% of control
0 ^1)	0.09	¿	189.6	¿	0.087	¿
0	0.095	100	147.5	100	0.082	100
0.37	0.011	12	112.3	76	0.121	147
0.75	0.019	20	155.2	105	0.105	128
1.2	0.068	72	166.9	113	0.091	111
2.5	0.01	11	157.4	107	0.083	101
3.7	0.021	22	^2)	¿	0.063	76
6.1	0.064	68	^2)	¿	0.051	62
12.3	0.197	208	^2)	¿	0.036	43

¹⁾Standard OECD test medium.

²⁾Exponential growth; no yield.

Validity criteria fulfilled:

yes

Conclusions:

In a GLP study conducted according to a modified OECD Guideline 201, the 72.5-h EC50 of trisodium EDDS with respect to growth rate of the freshwater algae, Chlorella vulagaris, was determined to be approximately 9.5 mg/L and the 72.5-h NOEC 1.2 mg/L.

Executive summary:

The growth inhibition of trisodium EDDS to the freshwater green alga, Chlorella vulgaris, under semi-static conditions in relation to the medium concentration of the trace metals cobalt (Co), copper (Cu) and zinc (Zn), and in relation to the presence of the chelating agent, ethylenediamine tetra acetic acid (EDTA), was assessed.

 

Three concentrations of trisodium EDDS (0, 0.33, 1.0 and 3.0 mg/L) were used in a range-finding test based on OECD Guideline 201 with the standard OECD medium, or the standard medium containing higher concentrations of trace metals (476 times Co, 16393 [medium 2a] and 178690 [medium 2b] times Cu, and 350 times Zn) and higher EDTA concentrations (100 times). The algal growth rate in medium 2b (with the highest Cu concentrations) was considerably lower than in the standard test medium. In the presence of trisodium EDDS, the algal growth rate was significantly reduced at all tested concentrations in medium 2a (with the lowest Cu concentrations) but only significantly reduced at 3.0 mg/L in medium 2b.

 

Based on the results of the range-finding test, a final growth inhibition test according to a modified OECD Guideline 201 and in compliance with GLP was performed at test concentrations 0, 0.37, 0.75, 1.2, 2.5, 3.7, 6.1 and 12.3 mg/L in either the standard OECD medium (additional control) or a different mediium (Medium 2M) containing trace metals Co, Cu and Zn at about 100, 850 and 145 times higher, respectively, than in the standard medium. One sample was taken from each flask after 0, 21.5, 48, 72.5, 92.5 and 143 h, and the number of particles counted. The increased trace metal concentrations in Medium 2M resulted in a lower algal growth rate than compared with the standard OECD test medium.

 

In the presence of trisodium EDDS it was observed that addition of up to 1.2 mg/L to Medium 2M resulted in a similar or higher growth rate than in the controls (considered the NOEC in this study), but reduced growth was seen at 2.5 mg/L and above (particularly after 72.5 h), and the EC50 of approximately 9.5 mg/L was derived. The EC50 with respect to inoculum viability was 1.8 mg/L, and the corresponding EC10 and EC90 values were 1.0 and 3.1 mg/L, respectively. The effect pattern of trisodium EDDS on algal growth can be best explained by trace metal toxicity and deficiency, due to complexation by the test substance.

Description of key information

In a GLP study conducted according to OECD Guideline 201, the 72-h NOEC, EC50 (0-72 h) for growth and EC50 (24-72 h) for growth rate for trisodium EDDS were 0.125, 0.29 and 1.57 mg/L for the green alga (Chlorella vulgaris) in freshwater (Neven and Henderix, 1990). For algae, the findings from this test are not considered as measures of direct toxicity, but an effect of overchelation of the essential metals in the growth media and resulting nutrient depletion (Schowanek et al. 1996).

In several, subsequent modified OECD Guideline 201 studies to GLP, the influence of varying medium concentrations of trace metals (cobalt, copper and zinc) with the use of natural river water, and in addition increasing levels of water hardness and use of media different from the OECD standard, were assessed in an attempt to understand their influence on the growth inhibition of trisodium EDDS. With increasing concentrations of trace metals, algal growth was increasingly less inhibited by EDDS acid at 1 mg/L (Hanstveit and Oldersma, 1993). The use of river water or increasing water hardness did not significantly influence the algae growth inhibition effects (Hanstveit and Oldersma, 1994a,b). In the presence of trisodium EDDS it was observed that addition of up to 1.2 mg/L to Medium 2M resulted in a similar or higher growth rate than in the controls (considered the NOEC in this study), and a EC50 of approximately 9.5 mg/L was derived. The effect pattern of trisodium EDDS and EDDS acid on algal growth can be best explained by trace metal toxicity and deficiency, due to complexation by the test substance (Hanstveit et al. 1994).

In a reliable study, [S,S]-EDDS acid, at concentrations of up to 100 mg/L, caused only minimal inhibition of photosynthesis (up to 14% after 8 min incubation) to the freshwater green alga, Selenastrum capricornutum. The EC50 was therefore greater than 100 mg/L (Schowanek et al. 1996).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.29 mg/L

EC10 or NOEC for freshwater algae:

0.125 mg/L

Additional information

Standard growth inhibition study (with trisodium EDDS):

In a GLP study conducted according to OECD Guideline 201, the effect of trisodium EDDS on the growth of a unicellular freshwater green algal species, Chlorella vulgaris, was assessed under semi static conditions. The test was carried out with an initial concentration of 1.2 x 10⁴ cells/mL of C. vulgaris in the test cultures (in triplicate), for an exposure duration of 72 h. The nominal concentrations tested were 0, 0.032, 0.065, 0.125, 0.25, 0.50 and 1.0 mg/L for the determination of the concentration that caused 50% reduction (EC50; using the measured absorbances of the algal suspensions) in either growth or growth rate relative to the control culture. Under the conditions of this study, the 72-h NOEC, EC50 (0-72 h) for growth and EC50 (24-72 h) for growth rate of C. vulgaris were 0.125, 0.29 and 1.57 mg/L, respectively, following exposure to trisodium EDDS in freshwater (Neven and Henderix, 1990).

For algae, the findings from a standard OECD test are not considered as measures of direct toxicity, but an effect of overchelation of the essential metals in the growth media and resulting nutrient depletion (Schowanek et al. 1996). If algal laboratory data are extrapolated to the field in this way an unrealistically low PNEC value would be obtained (Jaworska et al. 1999). A number of more recent studies have been designed in an attempt to better understand the intrinsic toxic effects of trisodium EDDS on algae.

 

Effect of metal-enriched algal medium (with EDDS acid):

In a GLP study conducted according to a modified OECD Guideline 201 method, the relationship between the medium concentration of trace metals cobalt (Co), copper (Cu) and zinc (Zn) and the growth inhibition of EDDS acid to C. vulgaris, were determined. Addition of EDDS acid at 1 mg/L (only tested concentration) to OECD medium strongly inhibitted algal growth. However, with increasing concentrations of trace metals, algal growth was increasingly less inhibited by EDDS acid (Hanstveit and Oldersma, 1993).

 

River water spiked with OECD nutrients (with EDDS acid):

In a GLP study conducted according to a modified OECD Guideline 201 method, the addition of EDDS acid at a concentration of 1 mg/L (the only tested concentration) to natural river water medium for 96 h reduced the growth rate of C. vulgaris by 35% compared to controls (Hanstveit and Oldersma, 1994a).

 

Effect of water hardness (with trisodium EDDS):

In a GLP study conducted according to a modified OECD Guideline 201 method, addition of 1 mg/L of trisodium EDDS inhibited the growth rate of C. vulgaris by an average of 27% over 163.5 h, compared to controls. The lower growth rate, however, resulted in the same biomass yield as in the control but after a longer incubation period. It was observed that the water hardness level did not affect the inhibition of algal growth by trisodium EDDS (Hanstveit and Oldersma, 1994b).

 

Effect of metal-enriched algal medium (with trisodium EDDS):

In a GLP study conducted according to a modified OECD Guideline 201 method, the growth inhibition of trisodium EDDS to C. vulgaris, under semi-static conditions, in relation to the medium concentration of the trace metals Co, Cu and Zn was assessed. Trisodium EDDS concentrations of 0, 0.37, 0.75, 1.2, 2.5, 3.7, 6.1 and 12.3 mg/L were added to either the standard OECD medium (additional control) or a different media (Medium 2M) containing the trace metals Co, Cu and Zn at about 100, 850 and 145 times higher, respectively, than in the standard medium. One sample was taken from each flask after 0, 21.5, 48, 72.5, 92.5 and 143 h, and the number of cells and cell volumes assessed. The increased trace metal concentrations in Medium 2M resulted in a lower algal growth rate than compared with the standard OECD test medium. In the presence of trisodium EDDS it was observed that addition of up to 1.2 mg/L to Medium 2M resulted in a similar or higher growth rate than in the controls (considered the NOEC in this study), but reduced growth was seen at 2.5 mg/L and above (particularly after 72.5 h), and a EC50 of approximately 9.5 mg/L was derived. The EC50 with respect to inoculum viability was 1.8 mg/L. The effect pattern of trisodium EDDS on algal growth can be best explained by trace metal toxicity and deficiency, due to complexation by the test substance (Hanstveit et al. 1994).

 

Effect on inhibition of photosynthesis (with EDDS acid):

In a good-quality study, [S,S]-EDDS acid was tested for its ability to inhibit photosynthesis in the freshwater green alga, Selenastrum capricornutum. This method was used because the test substance is a chelating agent capable of binding metals essential for growth that are present in culture media; an effect that could confound the interpretation of a standard growth test. The algal cells were incubated in pH 7.5 buffer with levels of EDDS acid at up to 100 mg/L under a light source for 30 min to equilibrate before the addition of radiolabelled sodium bicarbonate solution as a source of CO2 for photosynthesis. Duplicate chambers were prepared for each test concentration. Samples were removed at 2, 4, 8 and 20 min and an equal volume of 2N hydrochloric acid was added to volatilise the remaining non-fixed carbon. The cells were then dried and the carbon uptake determined by liquid scintillation counting. At 100 mg/L, the test substance caused a minimal reduction in ¹⁴CO₂ fixation rate of about 10% after 4 min and 14% after 8 min; the fixation rate was linear up to about 8 min, after which the supply of ¹⁴CO₂ became limiting. All other concentrations produced no reduction, or actually stimulated photosynthesis. The positive control (copper) induced a 50% inhibition of photosynthesis at 0.1 mg/L. The EC50 for photosynthesis was greater than 100 mg/L when [S,S]-EDDS acid was incubated with S. capricornutum in an algal short-term photosynthesis inhibition test (Schowanek et al. 1996).

 

This test assesses the interaction of the test substance with the photosynthesis process, an endpoint integrating various cellular processes, and a possible alternative endpoint to growth (Peterson and Nyholm, 1993). The short timescale of the test allows the algae to survive on stored nutrients, thus eliminating any effects due to nutrient availability. The test has been shown to be less sensitive than, but to correlate well overall with, longer growth tests (Versteeg, 1990).

 

[Information on EDDS free acid is considered relevant to use for understanding the potential effects of trisodium EDDS on algal growth, and is acceptable for using as read-across information.]

 

No data on marine algae or cyanobacteria are currently available.

 

References (not included elsewhere in IUCLID dossier - need to move to reference list in CSR)

Jaworska JS et al. (1999). Environmental risk assessment for trisodium [S,S]-ethylene diamine disuccinate, a biodegradable chelator used in detergent applications. Chemosphere 38, 3597-3625.

Peterson HG and Nyholm N (1993). Algal Bioassays for Metal Toxicity Identification. Water Pollution Research, Journal of Canada 28, 129-153.

Versteeg DJ (1990). Comparison of short and long-term toxicity test results for the green alga, Selenastrum capricornutum. In: "Plants for Toxicity Assessment", ASTM STP 1091. Wang W, JW Gorsuch and WR Lower (eds). American Society for Testing and Materials, Philadelphia pp. 40-48.