Diammonium dihydrogen ethylenediaminetetraacetate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP screening study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent nitrification.

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301 D) were introduced; a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river/lake water instead of an effluent/extract/mixture was used as inoculum.

GLP compliance:

no

Remarks:

Non-GLP preliminary screening study performed in a GLP lab under identical conditions as the two GLP studies with EDTA-CaNa2 and EDTA-Na2H2

Specific details on test material used for the study:

- Chemical name: Ethylenediaminetetraacetic acid, disodium salt
- purity: 89.9% (water 9.6%)
- pH of a 1% w/v solution: 4.6
- Batch/lot No.: FC-C 9715
- Appearance: white powder
- Expiry date: May 2021

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

river/lake water

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The river water spiked with mineral salts of the nutrient medium was used undiluted.
Lake water was sampled from Lake Veluwe, a bordering lake in the Netherlands, between the provinces of Flevoland on the west side and Gelderland on the east side near the city of Hardewijk. This water was also aerated for 7 days and particles were removed by sedimentation after 1 day while moderately aerating. The lake water was also used undiluted.

Duration of test (contact time):

84 d

Initial conc.:

5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test was performed according to Test Guidelines (OECD 1992). The lake and river water were spiked per liter with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
The test substances were dosed using aqueous stock solutions. The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 bottles containing only river water or lake water used as controls. In addition series of 3 bottles with the respective test items and river water or lake water were filled. The final concentration of the test items (actives) in the bottles was 3.0 (EDTA-NH4)2H2 and 5.0 mg/L (others). All test items dissolved well.
Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium.
Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

Reference substance:

not required

Test performance:

Test conditions
The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of waters was 8.0 (river) and 8.8 (lake) at the start of the tests. The pH of the river water ranged from 7.8 to 8.0 at the end of the tests. The pH at the end of the tests measured in lake water was 8.8±0.1. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption.

Key result

Parameter:

% degradation (O2 consumption)

Value:

78

Sampling time:

56 d

Remarks on result:

other:

Remarks:

river water

Key result

Parameter:

% degradation (O2 consumption)

Value:

74

Sampling time:

42 d

Remarks on result:

other:

Remarks:

lake water

Details on results:

Table: EDTA complexes and their ThOD used to calculate the biodegradation percentages.
Substance ThODNO3 (g O2/g test item)
EDTA-Na2H2 1.19

Toxicity
The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. No inhibition of the endogenous respiration of the inoculum was detected.

Results with reference substance:

For the non-GLP screening test no reference substance was included.

Percentages biodegradation of EDTA complexes in Closed Bottle tests inoculated with lake water and river water.

Substance	Inoculum	Biodegradation percentage at day
		7	14	21	28	42	56	84
EDTA-Na2H2	River water	2	0	0	0	55	78	81
	Lake water	1	10	13	52	74

Validity criteria fulfilled:

not specified

Interpretation of results:

other: Non-persistency was demonstrated for EDTA-Na2H2

Conclusions:

Non-GLP screening tests performed on GLP lab according to guideline OECD 301D indicated that EDTA-Na2H2 is not readily biodegradable but also not persistent in the environment.

Executive summary:

In order to assess the biotic degradation of EDTA-Na2H2, non-GLP ready biodegradability tests were performed which allows the evaluation of the biodegradability under aerobic conditions. The ready biodegradability was determined in the Closed Bottle tests performed according to slightly modified OECD, EU and ISO Test Guidelines, and performed on a lab which works in for final studies in compliance with the OECD principles of Good Laboratory Practice. In river water biodegradation percentages in excess of 60 was found with EDTA-Na2H2. Thus the EDTA complex is considered to be not persistent (according to REACH criteria). The biodegradation rate in lake water is faster compared to the biodegradation in river water, which is most likely due to difference in pH.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2018 - September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

non-GLP screening study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent nitrification.

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301 D) were introduced; a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river water instead of an effluent/extract/mixture was used as inoculum.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Chemical name: Ethylenediaminetetraacetic acid, disodium salt
- purity: 89.9% (water 9.6%)
- pH of a 1% w/v solution: 4.6
- Batch/lot No.: FC-C 9715
- Appearance: white powder
- Expiry date: May 2021

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

river water

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands (13-07-2018). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.

Duration of test (contact time):

60

Initial conc.:

5 mg/L

Based on:

test mat.

Initial conc.:

4.5 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test (OECD TG 301 D) was performed according to the study plan. The study plan was develo¬ped from ISO Test Guidelines (1994). Use was made of 10 bottles con-taining only river water, 6 bottles con¬taining river water and sodium acetate, 10 bottles containing river water with test substance. The concentrations of the test substance, and sodium ace¬tate in the bottles were 8.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bot¬tles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.
One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992). The oxygen concentration was measured at day 42 and 60.

Reference substance:

acetic acid, sodium salt

Test performance:

Test conditions
The pH of the media was 7.8 at the start of the test. The pH of the medium at day 28 was 7.8 (test and control). The temperature ranged from 22.5 to 22.9°C which is within the prescribed temperature range of 22 to 24°C.

Key result

Parameter:

% degradation (O2 consumption)

Value:

80

Sampling time:

60 d

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Details on results:

Theoretical oxygen demand (ThOD)
The ThODs of Ethylenediaminetetraacetic acid, disodium salt and water are 1.19 g/g (89.9%), and 0.0 g/g (9.6%), respectively. It is assumed that the ThODs of the unknown constituents (0.5%) are equal to the ThOD of Ethylenediaminetetraacetic acid, disodium salt. The ThOD of the test item calculated is 1.08 g/g. The ThOD of sodium acetate is 0.78 g/g

Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected (Table I). Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Validity of the test
The validity of the test is demonstrated by an endogenous respiration of 1.2 mg/L at day 28 (Table I). Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 82 (Table II and Figure). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

The Closed Bottle test results
Ethylenediaminetetraacetic acid, disodium salt was biodegraded by 2% at day 28 and should therefore not be classified as readily biodegradable (Figure and Table II). In the prolonged Closed Bottle test, the test item was biodegraded by 80% at day 60 (enhanced biodegradability testing). The test item should therefore be classified as not persistent.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 82.

Table I Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)	Oxygen concentration (mg/L)
	Oc	Ot	Oa
0	8.7	8.7	8.7
	8.7	8.7	8.7
Mean (M)	8.7	8.7	8.7
7	8.0	8.0	4.1
	8.1	8.1	4.2
Mean (M)	8.1	8.1	4.2
14	7.6	7.6	3.1
	7.6	7.5	3.4
Mean (M)	7.6	7.6	3.3
21	7.5	7.5
	7.6	7.6
Mean (M)	7.6	7.6
28	7.5	7.5
	7.4	7.3
Mean (M)	7.5	7.4
42	7.1	4.7
	7.2	5.0
Mean (M)	7.2	4.9
60	6.8	2.4
	7.0	2.7
Mean (M)	6.9	2.6

O_c         River water with nutrients.

O_t         River water with nutrients, and test substance (5.0 mg/L). 

O_a         River water with nutrients and sodium acetate (6.7 mg/L).

 

Table II Oxygen consumption (mg/L) and the percentages biodegradation of the test substance (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	0.0	3.9	0	75
14	0.0	4.4	0	85
21	0.0		0
28	0.1		2
42	2.3		43
60	4.3		80

 

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, fulfilling specific criteria

Conclusions:

Valid test performed according to guideline OECD 301D with minor acceptable deviations applying GLP conditions.

Executive summary:

In order to assess the biotic degradation of Ethylenediaminetetraacetic acid, disodium salt, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The test item did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Ethylenediaminetetraacetic acid, disodium salt was biodegraded by 2% at day 28 and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test, the test item was biodegraded by 80% at day 60 (enhanced biodegradability testing). The test item should therefore be classified as not persistent.

The test is valid as shown by an endogenous respiration of 1.2 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 82% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP screening study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent nitrification.

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301 D) were introduced; a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river/lake water instead of an effluent/extract/mixture was used as inoculum.

GLP compliance:

no

Remarks:

Non-GLP preliminary screening study performed in a GLP lab under identical conditions as the two GLP studies with EDTA-CaNa2 and EDTA-Na2H2

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

river/lake water

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The river water spiked with mineral salts of the nutrient medium was used undiluted.
Lake water was sampled from Lake Veluwe, a bordering lake in the Netherlands, between the provinces of Flevoland on the west side and Gelderland on the east side near the city of Hardewijk. This water was also aerated for 7 days and particles were removed by sedimentation after 1 day while moderately aerating. The lake water was also used undiluted.

Duration of test (contact time):

84 d

Initial conc.:

5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test was performed according to Test Guidelines (OECD 1992). The lake and river water were spiked per liter with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
The test substances were dosed using aqueous stock solutions. The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 bottles containing only river water or lake water used as controls. In addition series of 3 bottles with the respective test items and river water or lake water were filled. The final concentration of the test items (actives) in the bottles was 3.0 (EDTA-NH4)2H2 and 5.0 mg/L (others). All test items dissolved well.
Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium.
Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

Reference substance:

not required

Test performance:

Test conditions
The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of waters was 8.0 (river) and 8.8 (lake) at the start of the tests. The pH of the river water ranged from 7.8 to 8.0 at the end of the tests. The pH at the end of the tests measured in lake water was 8.8±0.1. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption.

Key result

Parameter:

% degradation (O2 consumption)

Value:

77

Sampling time:

56 d

Remarks on result:

other:

Remarks:

river water

Key result

Parameter:

% degradation (O2 consumption)

Value:

66

Sampling time:

28 d

Remarks on result:

other:

Remarks:

lake water

Details on results:

Table: EDTA complexes and their ThOD used to calculate the biodegradation percentages.
Substance ThODNO3 (g O2/g test item)
EDTA-MgNa2 1.12

Toxicity
The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. No inhibition of the endogenous respiration of the inoculum was detected.

Results with reference substance:

For the non-GLP screening test no reference substance was included.

Percentages biodegradation of EDTA complexes in Closed Bottle tests inoculated with lake water and river water.

Substance	Inoculum	Biodegradation percentage at day
		7	14	21	28	42	56	84
EDTA-MgNa2	River water	0	0	3	22	55	77	80
	Lake water	2	14	15	66	78

Validity criteria fulfilled:

not specified

Interpretation of results:

other: Non-persistency was demonstrated for EDTA-MgNa2

Conclusions:

Non-GLP screening tests performed on GLP lab according to guideline OECD 301D indicated that EDTA-MgNa2 is not readily biodegradable but also not persistent in the environment.

Executive summary:

In order to assess the biotic degradation of EDTA-MgNa2, non-GLP ready biodegradability tests were performed which allows the evaluation of the biodegradability under aerobic conditions. The ready biodegradability was determined in the Closed Bottle tests performed according to slightly modified OECD, EU and ISO Test Guidelines, and performed on a lab which works in for final studies in compliance with the OECD principles of Good Laboratory Practice. In river water biodegradation percentages in excess of 70 was found with EDTA-MgNa2. Thus the EDTA complex is considered to be not persistent (according to REACH criteria). The biodegradation rate in lake water is faster compared to the biodegradation in river water, which is most likely due to difference in pH.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP screening study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent nitrification.

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301 D) were introduced; a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river/lake water instead of an effluent/extract/mixture was used as inoculum.

GLP compliance:

no

Remarks:

Non-GLP preliminary screening study performed in a GLP lab under identical conditions as the two GLP studies with EDTA-CaNa2 and EDTA-Na2H2

Specific details on test material used for the study:

- purity: 87.1% (water 11.5%)
- pH of a 1% w/v solution: 6.8
- Batch/lot No.: FC-C 10332
- Appearance: white powder
- Expiry date: June 2021

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

river/lake water

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The river water spiked with mineral salts of the nutrient medium was used undiluted.
Lake water was sampled from Lake Veluwe, a bordering lake in the Netherlands, between the provinces of Flevoland on the west side and Gelderland on the east side near the city of Hardewijk. This water was also aerated for 7 days and particles were removed by sedimentation after 1 day while moderately aerating. The lake water was also used undiluted.

Duration of test (contact time):

84 d

Initial conc.:

5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test was performed according to Test Guidelines (OECD 1992). The lake and river water were spiked per liter with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
The test substances were dosed using aqueous stock solutions. The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 bottles containing only river water or lake water used as controls. In addition series of 3 bottles with the respective test items and river water or lake water were filled. The final concentration of the test items (actives) in the bottles was 3.0 (EDTA-NH4)2H2 and 5.0 mg/L (others). All test items dissolved well.
Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium.
Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

Reference substance:

not required

Test performance:

Test conditions
The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of waters was 8.0 (river) and 8.8 (lake) at the start of the tests. The pH of the river water ranged from 7.8 to 8.0 at the end of the tests. The pH at the end of the tests measured in lake water was 8.8±0.1. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption.

Key result

Parameter:

% degradation (O2 consumption)

Value:

69

Sampling time:

56 d

Remarks on result:

other:

Remarks:

river water

Key result

Parameter:

% degradation (O2 consumption)

Value:

79

Sampling time:

42 d

Remarks on result:

other:

Remarks:

lake water

Details on results:

Table: EDTA complexes and their ThOD used to calculate the biodegradation percentages.
Substance ThODNO3 (g O2/g test item)
EDTA-CaNa2 1.07

Toxicity
The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. No inhibition of the endogenous respiration of the inoculum was detected.

Results with reference substance:

For the non-GLP screening test no reference substance was included.

Percentages biodegradation of EDTA complexes in Closed Bottle tests inoculated with lake water and river water.

Substance	Inoculum	Biodegradation percentage at day
		7	14	21	28	42	56	84
EDTA-CaNa2	River water	0	3	3	11	40	69	76
	Lake water	3	11	17	53	79

Validity criteria fulfilled:

not specified

Interpretation of results:

other: Non-persistency was demonstrated for EDTA-CaNa2

Conclusions:

Non-GLP screening tests performed on GLP lab according to guideline OECD 301D indicated that EDTA-CaNa2 is not readily biodegradable but also not persistent in the environment.

Executive summary:

In order to assess the biotic degradation of EDTA-MgNa2, non-GLP ready biodegradability tests were performed which allows the evaluation of the biodegradability under aerobic conditions. The ready biodegradability was determined in the Closed Bottle tests performed according to slightly modified OECD, EU and ISO Test Guidelines, and performed on a lab which works in for final studies in compliance with the OECD principles of Good Laboratory Practice. In river water biodegradation percentages in excess of 60 was found with EDTA-CaNa2. Thus the EDTA complex is considered to be not persistent (according to REACH criteria). The biodegradation rate in lake water is faster compared to the biodegradation in river water, which is most likely due to difference in pH.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2018 - September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

non-GLP screening study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent nitrification.

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301 D) were introduced; a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river water instead of an effluent/extract/mixture was used as inoculum.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- purity: active 87.1% (water 11.5%)
- pH of a 1% w/v solution: 6.8
- Batch/lot No.: FC-C 10332
- Appearance: white powder
- Expiry date: June 2021

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

river water

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands (13-07-2018). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.

Duration of test (contact time):

60 d

Initial conc.:

6 mg/L

Based on:

test mat.

Initial conc.:

5.2 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test (OECD TG 301 D) was performed according to the study plan. The study plan was develo¬ped from ISO Test Guidelines (1994). Use was made of 10 bottles con-taining only river water, 6 bottles con¬taining river water and sodium acetate, 10 bottles containing river water with test substance. The concentrations of the test substance, and sodium ace¬tate in the bottles were 8.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bot¬tles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.
One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992). The oxygen concentration was measured at day 42 and 60.

Reference substance:

acetic acid, sodium salt

Test performance:

Test conditions
The pH of the media was 7.8 at the start of the test. The pH of the medium at day 28 was 7.8 (test and control). The temperature ranged from 22.5 to 22.9°C which is within the prescribed temperature range of 22 to 24°C.

Key result

Parameter:

% degradation (O2 consumption)

Value:

84

Sampling time:

60 d

Parameter:

% degradation (O2 consumption)

Value:

23

Sampling time:

28 d

Details on results:

Theoretical oxygen demand (ThOD)
The ThODs of Ethylenediamine-tetraacetic acid, calcium-disodium complex and water are 1.07 g/g (87.1%), and 0.0 g/g (11.5%), respectively. It is assumed that the ThODs of the unknown constituents (1.4%) are equal to the ThOD of Ethylenediamine-tetraacetic acid, calcium-disodium complex. The ThODNO3 of the test item calculated is 0.95 g/g. The ThOD of sodium acetate is 0.78 g/g

Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected (Table I). Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Test conditions
The pH of the media was 7.8 at the start of the test. The pH of the medium at day 28 was 7.8 (test and control). The temperature ranged from 22.5 to 22.9°C which is within the prescribed temperature range of 22 to 24°C.

Validity of the test
The validity of the test is demonstrated by an endogenous respiration of 1.2 mg/L at day 28 (Table I). Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 82 (Table II and Figure). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability
Ethylenediamine-tetraacetic acid, calcium-disodium complex was biodegraded by 23% at day 28 and should therefore not be classified as readily biodegradable (Figure and Table II). In the prolonged Closed Bottle test, the test item was biodegraded by 84% at day 60 (enhanced biodegradability testing). The test item should therefore be classified as not persistent.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 82.

Table I Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)	Oxygen concentration (mg/L)
	Oc	Ot	Oa
0	8.7	8.7	8.7
	8.7	8.7	8.7
Mean (M)	8.7	8.7	8.7
7	8.0	8.0	4.1
	8.1	8.0	4.2
Mean (M)	8.1	8.0	4.2
14	7.6	7.6	3.1
	7.6	7.6	3.4
Mean (M)	7.6	7.6	3.3
21	7.5	7.3
	7.6	7.5
Mean (M)	7.6	7.4
28	7.5	6.3
	7.4	6.0
Mean (M)	7.5	6.2
42	7.1	3.9
	7.2	4.0
Mean (M)	7.2	4.0
60	6.8	1.9
	7.0	2.2
Mean (M)	6.9	2.1

O_c         River water with nutrients.

O_t         River water with nutrients, and test substance (6.0 mg/L). 

O_a         River water with nutrients and sodium acetate (6.7 mg/L).

 

Table II Oxygen consumption (mg/L) and the percentages biodegradation of the test substance (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	0.1	3.9	2	75
14	0.0	4.4	0	85
21	0.2		4
28	1.3		23
42	3.2		56
60	4.8		84

 

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, fulfilling specific criteria

Conclusions:

Valid test performed according to guideline OECD 301D with minor acceptable deviations applying GLP conditions.

Executive summary:

In order to assess the biotic degradation of Ethylenediamine-tetraacetic acid, calcium-disodium complex, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The test item did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Ethylenediamine-tetraacetic acid, calcium-disodium complex was biodegraded by 23% at day 28 and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test, the test item was biodegraded by 84% at day 60 (enhanced biodegradability testing). The test item should therefore be classified as not persistent.

The test is valid as shown by an endogenous respiration of 1.2 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 82% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP screening studies

Justification for type of information:

Category justification document is attached in chapter 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

% degradation (O2 consumption)

Value:

78

Sampling time:

56 d

Remarks on result:

other: river water

Remarks:

EDTA-Na2H2 (139-33-3); screening study

Key result

Parameter:

% degradation (O2 consumption)

Value:

80

Sampling time:

60 d

Remarks on result:

other: river water

Remarks:

EDTA-Na2H2 (139-33-3); GLP study

Key result

Parameter:

% degradation (O2 consumption)

Value:

74

Sampling time:

42 d

Remarks on result:

other: lake water

Remarks:

EDTA-Na2H2 (139-33-3); screening study

Key result

Parameter:

% degradation (O2 consumption)

Value:

77

Sampling time:

56 d

Remarks on result:

other: river water

Remarks:

EDTA-MgNa2 (14402-88-1); screening study

Key result

Parameter:

% degradation (O2 consumption)

Value:

66

Sampling time:

28 d

Remarks on result:

other: lake water

Remarks:

EDTA-MgNa2 (14402-88-1); screening study

Key result

Parameter:

% degradation (O2 consumption)

Value:

69

Sampling time:

56 d

Remarks on result:

other: river water

Remarks:

EDTA-CaNa2 (62-33-9); screening study

Key result

Parameter:

% degradation (O2 consumption)

Value:

84

Sampling time:

60 d

Remarks on result:

other: river water

Remarks:

EDTA-CaNa2 (62-33-9); GLP study

Key result

Parameter:

% degradation (O2 consumption)

Value:

79

Sampling time:

42 d

Remarks on result:

other: lake water

Remarks:

EDTA-CaNa2 (62-33-9); screening study

Details on results:

Table: EDTA complexes and their ThOD used to calculate the biodegradation percentages.
Substance ThODNO3 (g O2/g test item)
EDTA-Na2H2 1.19
EDTA-CaNa2 1.07
EDTA-MgNa2 1.12

Toxicity
The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption.

The Closed Bottle test results
The ThODs used to calculate the biodegradation percentages are given above. In river water biodegradation percentages in excess of 60 were found with EDTA-Na2H2 and EDTA-MgNa2 after 56 days. EDTA complexed with NH4 Ca, Na, and Mg are considered to be not persistent (REACH). The biodegradation found in lake water is faster compared to the biodegradation in river water which is most likely due to difference in pH.

The results broadly confirm that only EDTA-metal complexes with a stability constants of <10^15 are directly accessible to microorganisms (van Ginkel 2017).

Validity criteria fulfilled:

not specified

Interpretation of results:

other: Non-persistency was demonstrated for EDTA-metal complexes with stability constant <10^15

Reference 7

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP screening study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent nitrification.

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301 D) were introduced; a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river/lake water instead of an effluent/extract/mixture was used as inoculum.

GLP compliance:

no

Remarks:

Non-GLP preliminary screening study performed in a GLP lab under identical conditions as the two GLP studies with EDTA-CaNa2 and EDTA-Na2H2

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

river/lake water

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The river water spiked with mineral salts of the nutrient medium was used undiluted.
Lake water was sampled from Lake Veluwe, a bordering lake in the Netherlands, between the provinces of Flevoland on the west side and Gelderland on the east side near the city of Hardewijk. This water was also aerated for 7 days and particles were removed by sedimentation after 1 day while moderately aerating. The lake water was also used undiluted.

Duration of test (contact time):

84 d

Initial conc.:

3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test was performed according to Test Guidelines (OECD 1992). The lake and river water were spiked per liter with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
The test substances were dosed using aqueous stock solutions. The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 bottles containing only river water or lake water used as controls. In addition series of 3 bottles with the respective test items and river water or lake water were filled. The final concentration of the test items (actives) in the bottles was 3.0 mg/L.
Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium.
Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

Reference substance:

not required

Test performance:

Test conditions
The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of waters was 8.0 (river) and 8.8 (lake) at the start of the tests. The pH of the river water ranged from 7.8 to 8.0 at the end of the tests. The pH at the end of the tests measured in lake water was 8.8±0.1. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption.

Key result

Parameter:

% degradation (O2 consumption)

Value:

56

Sampling time:

56 d

Remarks on result:

other:

Remarks:

river water

Key result

Parameter:

% degradation (O2 consumption)

Value:

83

Sampling time:

28 d

Remarks on result:

other:

Remarks:

lake water

Details on results:

Table: EDTA complexes and their ThOD used to calculate the biodegradation percentages.
Substance ThODNO3 (g O2/g test item)
EDTA-MgNa2 1.62

Toxicity
The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. No inhibition of the endogenous respiration of the inoculum was detected.

Results with reference substance:

For the non-GLP screening test no reference substance was included.

Percentages biodegradation of EDTA complexes in Closed Bottle tests inoculated with lake water and river water.

 

Substance	Inoculum	Biodegradation percentage at day
		7	14	21	28	42	56	84
EDTA-NH4)2H2	River water	9	29	44	45	48	56	65
	Lake water	19	44	51	83	85

Neg  = negative %

Validity criteria fulfilled:

not specified

Interpretation of results:

other: Non-persistency was demonstrated for EDTA-(NH4)2H2

Conclusions:

Non-GLP screening tests performed on GLP lab according to guideline OECD 301D indicated that EDTA-(NH4)2H2 is not readily biodegradable but also not persistent in the environment.

Executive summary:

In order to assess the biotic degradation of EDTA-(NH4)H2, non-GLP ready biodegradability tests were performed which allows the evaluation of the biodegradability under aerobic conditions. The ready biodegradability was determined in the Closed Bottle tests performed according to slightly modified OECD, EU and ISO Test Guidelines, and performed on a lab which works in for final studies in compliance with the OECD principles of Good Laboratory Practice. In river water biodegradation percentages in excess of 60 was found with EDTA-(NH4)2H2. Thus the EDTA complex is considered to be not persistent (according to REACH criteria). The biodegradation rate in lake water is faster compared to the biodegradation in river water, which is most likely due to difference in pH.

Description of key information

Diammonium dihydrogen ethylenediaminetetraacetate (CAS 20824-56-0) is not readily biodegradable according to OECD criteria. In standard OECD 301D ready biodegradability tests with natural river and lake water less than 60% biodegradation was observed for the substance and other subcategory 1 members after 28d incubation time. In these same tests however > 60% biodegradation was observed when prolonged to 60 days indicating that Diammonium dihydrogen ethylenediaminetetraacetate (CAS 20824-56-0) and other subcategory 1a members, having stability constants < 10E13, are inherently and ultimately biodegradable in a ready biodegradation test under enhanced conditions and can thus be classified as "not persistent".

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Type of water:

freshwater

Additional information

Numerous degradation tests are available for EDTA and its metal complexes listed in subcategory 1a. For justification ofthe read-across approach between subcategory 1a members within the category see the category justification document in Chapter 13. Results from an OECD guideline test indicate that Diammonium dihydrogen ethylenediamintetraacetate is not readily biodegradable. Several intinsic/extrinsic factors are known to influence the biodegradation behaviour:

 

pH

It could be shown that a change of the pH-value has significant influence on the biodegradability of EDTA and its complexes. In a SCAS test (OECD 302 A) biodegradation of EDTA could be observed at pH 8-9, but not significantly at pH 6.5 (Van Ginkel et al. 1997). Similar results obtained in a DOC removal test according to the principles of the OECD guideline 301 using natural surface water from the river Rhine as inoculum. After 60 days up to 100 % EDTA was degraded at pH 8.5 but less than 10 % at pH 6.5 (BASF 2000). These slightly alkaline conditions are realistic in environmental surface water compartments.

 

Adaptation

An enhanced biodegradability of EDTA could be shown after long adaption. In guideline tests according to OECD 301 EDTA was moderately biodegradable and well eliminable from water using adapted inoculum (BASF 2001, 2002). The adaptation potential of EDTA degradation shows also an industrial wastewater treatment plant from a Finnish paper mill. Using activated sludge from this plant EDTA was biodegraded about > 80 % CO2 evolution and about 99 % DOC removal in a laboratory study according to OECD 301B (Kaluza et al. 1998). This study represents a low-level pre-adaption test system and can be regarded as an enhanced biodegradation screening test (Guidance for Implementation of REACH, Chapter R.7b, 2008).

 

Influences of the stability constant

As a chelating agent, EDTA forms complexes with cationic ions. Fundamental EDTA exists naturally as a mixture of chelate complexes. The biodegradability differs between the acid resp. their salts and on the other side the metal complexes. Investigations show, that EDTA complexes with a thermodynamic stability constant below 10E13, like Ca, Mg and Mn, were degraded. On contrast heavy metal EDTA complexes with stability constants above 10E13, such as Cu and Zn, were not significantly degraded (Klüner et al. 1998; Van Ginkel, 1999, 2018, 2019; Nörtemann 2003).

 

Degradation pathway

Several investigations revealed that it is possible to enrich cultures of EDTA-utilizing microorganisms. Different bacteria strains were isolated which can mineralize EDTA completely (Nörtemann 1992; Van Ginkel 1999). The degradation pathway of EDTA was described from Klüner et al. (1998) and summarised in the EU Risk Assessment (2004). The first intermediate described is ethylenediaminetriacetate (ED3A). ED3A can react spontaneously to ketopiperazinediacetate (KPDA) by intramolecular cyclisation (Ternes et al. 1996). KPDA itself is biodegradable which could be shown by Van Ginkel & Stroo (1999).

 

Recently performed enhanced ready biodegradation tests with several members of subcategory 1 show that in standard OECD 301D tests using natural river and lake water as inoculum, EDTA complexes with a stability constant lower than 10E13 like EDTA-NH42H2, EDTA-Na4, EDTA-CaNa2, EDTA-MgNa2 etc. biodegrades to an extent of less than 60% after 28 days confirming that these substances should be not classified as readily biodegradable. However, in these tests > 60% biodegradation was observed after 60 days indicating that these complexes, having stability constants < 10E13, are not persistent. This stability constant threshold of 10E13 will be dependent on the concentration balance between the starting complex and free metal ions (alkali and alkaline earth metals). The lower the starting concentration of the EDTA-metal complex the higher this stability constant threshold for biodegradation. Based on this result Diammonium dihydrogen ethylenediaminetetraacetate is assessed to be inherently biodegradable fulfilling specific criteria and thus not persistent in the environment.