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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

not specified

Principles of method if other than guideline:

Although only limited reporting in the EU Risk Assessment Report (RAR) for EDTA, this study was conducted according to OECD guidelines and was considered by the EU experts as the most relevant to determine the PNEC for microorganisms.

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES (OF CaNa2 EDTA)
- Melting point: no data
- Boiling point: 614.2oC
- Vapour pressure: 1.15E-16 mmHg at 25oC
- Water solubility (under test conditions): no data
- Henry's law constant: 1.47E-25 atm*m3/mole; 25 °C
- log Pow: -10.42
- pKa: no data
- Stability in water: no data
- Stability in light: no data
- pH dependance on stability: no data

OTHER PROPERTIES:
no data

Analytical monitoring:

not specified

Details on sampling:

- Concentrations: no data
- Sampling method: no data
- Sample storage conditions before analysis: no data

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: no data
- Eluate: no data
- Differential loading: no data
- Controls: no data
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no data
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): no data
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: no applicable
- Method of cultivation: not applicable
- Preparation of inoculum for exposure: no data
- Pretreatment: no data
- Initial biomass concentration: no data but test carried out according to OECD 209 which stipulates 1.5 g/L of suspended solids in the final test and control solutions

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Post exposure observation period:

no data

Hardness:

no data.

Test temperature:

no data. Test conducted according to OECD 209 which stipulates a temperature of 20 ± 2oC

pH:

no data. Test conducted according to OECD 209 which stipulates a pH of 7.5 ± 0.5

Dissolved oxygen:

no data. Test conducted according to OECD 209 which stipulates a dissolved oxygen concentration of 60-70%

Salinity:

no data

Nominal and measured concentrations:

125, 250 and 500 mg/L (nominal)

Details on test conditions:

According ot the EU RAR, "Na2H2EDTA was used in concentrations of 125, 250, and 500 mg/l (referred as H4EDTA). Equimolar amounts of CaCl2 were added, thus CaEDTA was formed in the stock solutions."

Reference substance (positive control):

not specified

Duration:

30 min

Dose descriptor:

EC10

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: not applicable

Duration:

30 min

Dose descriptor:

NOEC

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: not applicable

Details on results:

No significant inhibition of respiration rate was seen after 30 minutes at any test concentration.

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

no data

Validity criteria fulfilled:

yes

Conclusions:

In a guideline study, calcium disodium EDTA did not significantly affect the respiration rate of activated sludge microorganisms when tested at up to 500 mg/L and the oxygen consuption was measured after 30 min.

Executive summary:

In a study conducted according to OECD Guideline 209, calcium disodium EDTA was studied for its effect on the respiration rate of microorganisms present in a sample of activated sludge taken from a domestic sewage plant. Disodium EDTA was used at concentrations of 125, 250 and 500 mg/L "(referred as H₄EDTA)" and equimolar amounts of calcium chloride were added, thus calcium EDTA was formed in the stock solutions. No significant inhibition of the respiration rate was detected after 30 minutes at any tested concentration. The EU Risk Assessment Report (2004) on EDTA concluded that both the EC10 and NOEC were greater than 500 mg/L. This study was considered by the EU experts to be the most relevant for the determination of the PNEC for microorganisms.

[Due to the structural similarity of the two substances, data on calcium disodium EDTA is relevant to use for understanding the effects of trisodium EDDS on the inhibition of microbial respiration rate, and is acceptable for using as read-across information].

Reference 2

Endpoint:

toxicity to microorganisms, other

Remarks:

Inhibition of the metabolic conversion of chemical energy into bioluminescence in the bacterium Vibrio fischeri

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline available

Principles of method if other than guideline:

The assay measures the ability of substances to inhibit the metabolic conversion of chemical energy into bioluminescence in the bacterium Vibrio fischeri; light emission is measured photometrically. Full details of method not given; this assay is more appropriate as a screen assay according to the investigators.

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 316.39
- Boiling point: 564.65
- Vapour pressure: 3.22E-013 mm Hg,25 deg C
- Water solubility (under test conditions): no data
- Henry's law constant: 1.07E-026 atm-m3/mole
- log Pow: -5.44
- pKa: no data
- Stability in water: no data
- Stability in light: no data
- pH dependance on stability: no data

Analytical monitoring:

not specified

Details on sampling:

no data

Vehicle:

not specified

Details on test solutions:

no data

Test organisms (species):

other: Vibrio fischeri

Details on inoculum:

- Laboratory culture: no; obtained as a freeze-dried culture
- Method of cultivation: none required
- Preparation of inoculum for exposure: bacteria reconstituted in 1 mL purified distilled water
- Pretreatment: none
- Initial biomass concentration: 10(8) cells/mL

Test type:

static

Water media type:

brackish water

Limit test:

no

Total exposure duration:

22 h

Post exposure observation period:

None; light emission measured at the end of the incubation period

Hardness:

no data

Test temperature:

27oC

pH:

7.5

Dissolved oxygen:

no data

Salinity:

2%

Nominal and measured concentrations:

no data on the 5 concentrations tested, but includes 0.125 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: cuvettes
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: no data
- Aeration: no
- No. of organisms per vessel: 10(6)/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): no data
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultrapure distilled water
- Culture medium different from test medium: no culture medium was used
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: yes, if outside the range of pH 6-8

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : light emission

TEST CONCENTRATIONS
- Spacing factor for test concentrations: serial dilution
- Justification for using less concentrations than requested by guideline: no guideline available
- Range finding study: no data
- Test concentrations: no data
- Results used to determine the conditions for the definitive study: no data

Reference substance (positive control):

yes

Remarks:

copper sulphate

Duration:

22 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.15 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: inhibition of metabolism

Remarks on result:

other: 0.06-0.2 (95% CL)

Duration:

22 h

Dose descriptor:

other: EC20

Effect conc.:

ca. 0.17 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: inhition of metabolism

Remarks on result:

other: 0.1-0.24 (95% CL)

Duration:

22 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.23 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: inhibition of metabolism

Remarks on result:

other: 0.19-0.26 (95% CL)

Duration:

22 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.125 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: inhibition of metabolism

Remarks on result:

other: CL not applicable

Details on results:

- 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):

- Results with reference substance valid? yes
- Relevant effect levels: NOEC 0.125 mg/L

Reported statistics and error estimates:

no data

Validity criteria fulfilled:

yes

Conclusions:

A 22-h NOEC and EC50 of 0.125 and 0.23 mg/L, respectively, were calculated for EDDS in the Microtox test (which measures the ability of a substance in inhibit the metabolic conversion of chemical energy to bioluminescence) in the marine bacterium Vibrio fischeri.

Executive summary:

EDDS was tested for its potential toxicity (decrease in the ability of the bacteria to produce luminescence) in the bacterium Vibrio fischeri.

Using a Microtox chronic test kit, which supplied all the reagents and the freeze-dried bacterium, the reconstituted bacterial cells were incubated for 22-h at 27oC with five unspecified concentrations of the test substance in a saline culture medium; each test was prepared in quadruplicate. Inhibition of luminescence (which is a combined function of cell division, metabolism, growth and luciferase induction) was measured photometrically. Full details of method not given.

In conclusion, the 22-h EC10, EC20, EC50 and NOEC were 0.15, 0.17, 0.23 and 0.125 mg/L

This assay is more appropriate as a screen assay according to the investigators. The bacterium used requires a saline environment, and therefore the results are only of limited relevance for assessing the potential toxicity to micoro-organisms residing in sewage treatment plants. [Due to the similarity in stucture to EDDS, trisodium EDDS is likely to exhibit a similar level of toxicity to Vibrio fischeri.]

Description of key information

No activated sludge respiration tests were identified for trisodium EDDS. However, in a test for biodegradability in sludge, 85% of trisodium EDDS was degraded after 28 days, demonstrating that the test substance did not significantly inhibit the activity of the sludge microflora (Lisec, 1993).

A 22-h NOEC and EC50 of 0.125 and 0.23 mg/L, respectively, were calculated for EDDS in the Microtox test (which measures the ability of a substance to inhibit the metabolic conversion of chemical energy to bioluminescence) in the marine bacterium Vibrio fischeri (Radix et al. 1999). [The bacterium used requires a saline environment, and therefore the results are only of limited relevance for assessing the potential toxicity to microorganisms residing in sewage treatment plants.

However, in a test conducted according to OECD Guideline 209, no significant inhibition of the respiration rate was detected in an activated domestic sewage sludge treated with disodium EDTA at up to 500 mg/L ("referred as H4EDTA") after 30 minutes. Equimolar amounts of CaCl2 were added, thus calcium EDTA was formed in the stock solutions. Both the EC10 and NOEC values were concluded to be above 500 mg/L (van Ginkel and Stroo, 2000). The EU RAR (2004a,b) considers the EC10 of 500 mg/L determined in this study as the most relevant to use for deriving a PNEC.

Key value for chemical safety assessment

EC50 for microorganisms:

500 mg/L

EC10 or NOEC for microorganisms:

500 mg/L

Additional information

No activated sludge respiration tests were identified for trisodium EDDS. However, in a test for biodegradability in sludge, 85% of trisodium EDDS was degraded after 28 days, demonstrating that the test substance did not significantly inhibit the activity of the sludge microflora (Lisec, 1993). However, relevant data on related compounds were identified in the literature.

In an agar inhibition test, with the fungi Aspergillus niger and Pycnoporus sanguineus and the bacterium Staphylococcus aureus, no growth inhibition was noted with 100 mM EDDS complexes with Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ or Pb²⁺. EDDS complexed with Hg²⁺at 10 mM slightly inhibited the growth of A. niger and S. aureus, but the zone of inhibition was equal to that of the free metal ion alone. Similarly, comparable zones of inhibition were seen with Cd²⁺-EDDS and Cd²⁺ when tested with P. sanguineus at 100 mM, although this chelate was slightly more toxic to A. niger than the free metal ion (presumably by facilitating the delivery of the toxic metal into the fungus). These results demonstrate that metal chelates of EDDS do not add to the toxicity of the free metal ions (Costa et al. 2008).

EDDS acid was tested for its potential toxicity (decrease in the ability of the bacteria to produce luminescence) in the bacterium Vibrio fischeri. Using a Microtox chronic test kit, which supplied all the reagents and the freeze-dried bacterium, the reconstituted bacterial cells were incubated for 22 h at 27oC with five unspecified concentrations of the test substance in a saline culture medium; each test was prepared in quadruplicate. Inhibition of luminescence (which is a combined function of cell division, metabolism, growth and luciferase induction) was measured photometrically. Full details of method not given. The 22-h EC10, EC20, EC50 and NOEC were 0.15, 0.17, 0.23 and 0.125 mg/L (Radix et al. 1999). [The bacterium used requires a saline environment, and therefore the results are only of limited relevance for assessing the potential toxicity to microorganisms residing in sewage treatment plants].

In a test conducted according to OECD Guideline 209, no significant inhibition of the respiration rate was detected in an activated domestic sewage sludge. Disodium EDTA was tested for 30 minutes in concentrations up to 500 mg/L ("referred as H₄EDTA"), however equimolar amounts of CaCl2 were added, thus CaEDTA was formed in the stock solutions. Thus, both the EC10 and NOEC values were concluded to be above 500 mg/L (van Ginkel and Stroo, 2000). The EU RAR (2004a,b) considers the EC10 of 500 mg/L determined in this study as the most relevant to use for deriving a PNEC.

In a bacterial oxygen consumption test conducted according to the method of Robra with disodium EDTA, the 30-minute EC10 was 55 mg/L (i.e. 48 mg/L EDTA) (BASF, 1990). A test on the growth inhibition of Pseudomonas putida with tetrasodium EDTA gave a 16-h toxic effect concentration (EC3) of 105 mg/L (i.e. 81 mg/L EDTA) (Bringmann and Kühn, 1976). [According to the citing source (EU, 2004a,b) no data on the test conditions were available for either test.]

The inhibition of cell multiplication with different protozoa was assessed in several studies, but apparently under identical experimental conditions. Stock and preliminary cultures of the test protozoa were fed with viable bacteria, whereas the test cultures were fed with inactivated bacteria. Tetrasodium EDTA was used as the test substance. The test medium (pH 6.9) contained 290 mg/L Ca(NO₃)₂.4 H2O and 70 mg/L Mg(NO₃)₂.6 H2O, therefore the EDTA was completely complexed with Ca and Mg. At the end of the test period, the cells were counted, and the toxic effect concentration (i.e. EC5) was determined as EDTA equivalents. The toxic effect concentrations were: 13 mg/L (20 h), 510 mg/L (48 h) and 28 mg/L (72 h) for Uronema parduczi, Chilomonas paramaecium and Entosiphon sulcatum, respectively (Bringmann, 1978; Bringmann et al. 1980; Bringmann and Kühn, 1980). The citing source (EU, 2004a,b) considered these studies were not well documented and felt that nutrient deficiency could not be excluded as the cause of these results.

[Due to their structural similarity, data on EDTA (and its simple salts) are considered relevant to use for understanding the effects of trisodium EDDS on the inhibition of microbial respiration rate, and is acceptable for using as read-across information].

References (for which no ESR has been created; need to move to reference list in CSR)

BASF (1990a). Sauerstoffkonsumptionstest nach Robra, Titriplex III (cited in EU, 2004a,b).

Bringmann G (1978). Bestimmung der biologischen Schadwirkung wassergefährdender Stoffe gegen Protozoen. Z. Wasser Abwasser Forsch. 11, 210-15 (cited in EU, 2004a,b).

Bringmann G and Kühn R (1976). Vergleichende Befunde der Schadwirkung wassergefährdender Stoffe gegen Bakterien (Pseudomonas putida) und Blaualgen (Microcystis aeruginosa). Gwf-wasser/abwasser 117, 410-413 (cited in EU, 2004a,b).

Bringmann G and Kühn R (1980). Bestimmung der biologischen Schadwirkung wassergefährdender Stoffe gegen Protozoen, II. Bakterienfressende Cilliaten. Z. Wasser Abwasser Forsch. Nr. 1, 26-31 (cited in EU, 2004a,b).

Bringmann G, Kühn R and Winter A (1980). Bestimmung der biologischen Schadwirkung wassergefährdender Stoffe gegen Protozoen. III. Saprozoische Flagellaten. Z. Wasser Abwasser Forsch. 13, 170-173 (cited in EU, 2004a,b).

Costa NSJ et al. (2008). Antimicrobial activity of ethylenediaminedisuccinate metal complexes. Short cummunication. Chemistry and Diversity 5, 2156 -2159.

EU (2004a). European Union Risk Assessment Report (RAR); edetic acid (EDTA). Vol. 49. European Chemicals Bureau (ECB). Final report available at http://ecb.jrc.ec.europa.eu/DOCUMENTS/Existing-Chemicals/RISK_ASSESSMENT/REPORT/edtareport061.pdf.

EU (2004b). European Union Risk Assessment Report (RAR); tetrasodium ethylenediaminetetraacetate (Na4EDTA). Vol. 51. European Chemicals Bureau (ECB). Final report available at http://ecb.jrc.ec.europa.eu/DOCUMENTS/Existing-Chemicals/RISK_ASSESSMENT/REPORT/na4edtareport062.pdf.

Lisec (1993) Adsorption/Desorption of E-4591.01 to Activated Sludge with 14C Analysis. Study No. WG-03- 002, LISEC Genk, Belgium (Unpublished report submitted by Procter and Gamble Manufacturing Pty Ltd) (cited in NICNAS, 2003).

NICNAS (2003). Full public report on trisodium ethylene diamine disuccinate. STD/1044. Available at http://www.nicnas.gov.au/publications/CAR/new/std/stdFULLR/std1000FR/std1044FR.pdf.

van Ginkel and Stroo (2000). Toxicity of EDTA to Activated Sludge. Final Research Report August 9, 2000 (cited in EU, 2004a,b).