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Biodegradation in water: screening tests

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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.

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 d
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

 

Oc         River water with nutrients.

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

Oa         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.

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.

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.:
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.

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.

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

 

Oc         River water with nutrients.

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

Oa         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.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
GLP compliance:
no
Specific details on test material used for the study:
Batch No.: 405876/1 43300
Oxygen conditions:
aerobic
Inoculum or test system:
other: fresh water from the river Rhine
Details on inoculum:
Mixture from fresh Rhinewater and adapted Rhinewater from Scas-Test.
Duration of test (contact time):
44 d
Initial conc.:
20 mg/L
Based on:
DOC
Initial conc.:
72 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Parameter:
% degradation (CO2 evolution)
Value:
> 30 - < 40
Sampling time:
28 d
Key result
Parameter:
% degradation (DOC removal)
Value:
> 90 - < 100
Sampling time:
28 d
Details on results:
Test duration (days): 44
Duration of the adaptation phase (days): 16
Duration of the degradation phase (days): 28

Biodegradation degree (CO2/ThCO2) after 28 days: 30-40 % (14 %, 27 %, 55 %)
Elimination degree (DOC) after 28 days: 90-100 % (76 %, 100 %, 101 %)

The deviation of the degradation degree of the test substance in the plateau phase (> 20 %) was justified in the high pH value from > 9.0 and the difference in the test-vessels. So CO2 was solved as (CO3)2- and not stripped and measured in the absorption-vessels.

The test substance is in this test moderately or partially biodegradable.
But the test substance is in this test well eliminable from water.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
GLP compliance:
no
Specific details on test material used for the study:
- Batch No.: 405876/1 43300
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Remarks:
river rhine water
Duration of test (contact time):
72 d
Initial conc.:
40 mg/L
Based on:
DOC
Initial conc.:
140 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
The test substance was added directly to the inoculum without addition of mineral test medium.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (DOC removal)
Value:
> 90 - < 100
Sampling time:
72 d
Parameter:
% degradation (DOC removal)
Value:
> 0 - < 10
Sampling time:
28 d
Details on results:
Test duration (days) : 72

pH: 7 - 7.5
Degradation degree of the test substance at the end of the test (% DOC): < 10

pH: 8
Duration of adaptation phase (days): 28 (mean)
Duration of the biodegradation phase (days): 20
Degradation degree of the test substance at the end of the 10-day window (% DOC): 0
Degradation degree of the test substance at the end of the test (% DOC): 80 - 90

pH: 8.5
Duration of adaptation phase (days): 28 (mean)
Duration of the biodegradation phase (days): 30
Degradation degree of the test substance at the end of the 10-day window (% DOC): 0
Degradation degree of the test substance at the end of the test (% DOC): 90 - 100


Biodegradation degree (DOC removal) after 28 days: < l0%
Biodegradation degree (DOC removal) after 72 days: 90 -100%


Inhibition control (sodium benzoate):
Inhibition control (DOC removal) after 14 days (pH 7.0): 40 - 50%
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
GLP compliance:
no
Specific details on test material used for the study:
Batch No.: 405876/1 43300
Oxygen conditions:
aerobic
Inoculum or test system:
other: fresh water from the river Rhine
Details on inoculum:
Rhine-water from the gauging station in Bad Honeff. Concentration of dry substance was not measured.
Duration of test (contact time):
70 d
Initial conc.:
20 mg/L
Based on:
DOC
Initial conc.:
104 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (DOC removal)
Value:
> 0 - < 10
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
80 - 90
Sampling time:
59 d
Details on results:
Test duration (days): 70
Duration of the adaptation phase (days): 50
Duration of the degradation phase (days): 10

Degradation of the test substance at the end of the 10-day window (% DOC): 0

Degradation degree of the test substance after 59 days at the end of test 1 (% DOC): 80-90
Degradation degree of the test substance after 70 days at the end of test 2 (% DOC): 80-90

Biodegradation degree (DOC removal) after 28 days: 0-10 %
Biodegradation degree (DOC removal) after 59 days: 80-90 %

Kinetic of test substance (in %):
= 0 after 28 day(s)
= 4 after 49 day(s)
= 86 after 57 day(s)
Kinetic of control substance (in %):
= 90 ... 100 after 14 day(s)
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: PN-88/C-05561
Deviations:
not specified
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
The activated sludge is a biocoenosis which, owing to the diversity of micoorganisms contained in it, is able to decompose organic compounds.
Duration of test (contact time):
20 d
Initial conc.:
4 other: g/dm3
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The tested compound in the amount of about 4 g/dm3 dissolved in a synthetic mineral substrate inoculated with standard activated sludge was placed in three 3 dm3 flasks arranged in a row on magnetic stirrers and connected by an oxygen feeding tube. Besides the tested compound, 100 ml of 0.1% glucose (an agent aiding the biodegradation of some organic compounds, being a source of easily decomposed carbon and energy for microorganisms), was added. The third flask was a reference flask.
Reference substance:
not specified
Preliminary study:
no data available
Test performance:
no data availble
Key result
Parameter:
% degradation (O2 consumption)
Value:
54.9
Sampling time:
20 d
Details on results:
For EDTA the degree of biodegradation (the compound content reduction in the sample) was found to be about 55% at a concentration of 3.3 g/dm3 after 20 days of biodegradation. Based on the results EDTA can be assigned as biodegradable, but not readily biodegradable.
COD decreased proportionally to the decay of the compound in the sample – by 40% for the permanganate method both with and without glucose and by more than 50% for the dichromate method.
Results with reference substance:
no data available
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Principles of method if other than guideline:
Method: The carbon dioxide evolution test was conducted according to Sturm (1973). Sturm RN (1973). J. Amer. Oil Chem. Soc. 50: 159-167
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: sewage treatment plant effluent, adapted
Duration of test (contact time):
42 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter:
% degradation (CO2 evolution)
Value:
10
Sampling time:
28 d
Details on results:
CO2 evolution: 10 %
DOC removal: 22 %
Interpretation of results:
other: Under the conditions of the test, the test substance was poorly biodegradable.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Principles of method if other than guideline:
The closed bottle test was conducted according to Fischer et al. (1974). Fischer WK, Gerike P, Schmid R (1974). Z. Wasser Abwasser Forsch. 7: 99-118
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic (adaptation not specified)
Details on inoculum:
Inoculum: 1 drop of effluent/L
Duration of test (contact time):
30 d
Initial conc.:
1 mg/L
Based on:
DOC
Parameter:
% degradation (O2 consumption)
Value:
0 - 10
Sampling time:
30 d
Interpretation of results:
under test conditions no biodegradation observed
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Principles of method if other than guideline:
Method: The OECD Screening test was conducted according to a method described by  the Umweltbundesamt (1978) and the OECD (1976).
Umweltbundesamt (1978). OECD Chemicals Testing Programm. Expert Group C  "Persistence", Draft Working Papers, 31 Mar 1978 OECD Environment Directorate (1976). Proposed Method for the  Determination of the Biodegradability of Surfactants used in Synthetic  Detergents,
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic (adaptation not specified)
Duration of test (contact time):
19 d
Initial conc.:
3 - 20 mg/L
Based on:
DOC
Parameter:
% degradation (DOC removal)
Value:
10
Sampling time:
19 d
Interpretation of results:
other: Under the conditions of the test, the test substance was poorly biodegradable.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Method: Zahn-Wellens test conducted according to the method described by Zahn and Wellens (1974). Zahn R, Wellens H (1974). Chemiker Z. 98: 228-232
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial (adaptation not specified)
Duration of test (contact time):
14 d
Initial conc.:
400 mg/L
Based on:
DOC
Parameter:
% degradation (DOC removal)
Value:
30 - 40
Sampling time:
14 d
Interpretation of results:
other: Under the conditions of the test, the test substance was partly eliminated from water.
Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication
Principles of method if other than guideline:
other
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: soil-water-system (close to environmental conditions)

Haberer & Ternes (1996) studied the metabolisation of EDTA in a soil-water-system under aerobic conditions over a 100-day period. Metabolites formed under the conditions of a soil passage (close to environmental conditions) are ethylendiamine triacetate (ED3A) and ketopiperacindiacetate (KPDA). Ethylendiaminetriacetate spontaneously polymerised to the ketopiperacine derivative. The authors assume, that the open and the cyclic form exist simultaneously in the soil solution. The metabolites could be identified by GC/MS-, GC/MS/MS-spectra and different NMR-technics.

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:
other: Acceptable, well documented publication
Qualifier:
according to guideline
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Principles of method if other than guideline:
Biodegradability tests were performed using a combined CO2/DOC method that allows the simultaneous determination of CO2 formation and of DOC removal according to the updated ISO 9439, 1998.
Additional a monitoring study conducted at a industrial wastewater treatment plant dedicated to treat the effluent from a paper mill. The monitoring measurements on the influent and the treated effluent showed an EDTA elimination of about 80 % by test substance specific analysis.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial, adapted
Details on inoculum:
Activated sludge taken from a Finnish industrial wastewater treatment plant.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Monitoring study (waste water treatment plant): 50 separate samples of influent and treated effluent were taken daily over a period of two weeks.
Parameter:
% degradation (CO2 evolution)
Value:
80 - 90
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
90 - 100
Sampling time:
28 d
Details on results:
In the present publication, it was shown that EDTA is ultimately biodegraded under practical industrial wastewater treatment conditions, involving a Finnish plant dedicated to treat the effluent from a paper mill. Monitoring measurements on the influent and the treated effluent showed an EDTA elimination of about 80 % by test substance specific analysis. The mean EDTA concentration in the influent was 23.8 mg/L and in the corresponding effluent 5.8 mg/L. The biodegradability of EDTA was verified in the laboratory with activated sludge from the treatment plant. Using a combined CO2/DOC method the total mineralization of EDTA was indicated by > 80 % CO2 formation and >= 99 % DOC removal
Interpretation of results:
other: ultimately biodegradable
Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Metabolism study with isolated bacteria strain BNC1.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: isolated bacteria strain BNC1
Details on results:
The first catabolic enzyme appears to be an EDTA monooxygenase since it requires O2, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded via N,N'-ethylenediaminediacetate. Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues of EDTA.

The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts of strain BNCI was investigated. Metal-EDTA chelates with thermodynamic stability constants below 10E12 were readily mineralized by whole cells. With the exception of ZnEDTA, chelates with stability constants greater than 10E12 were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded after pretreatment by addition of calcium and magnesium salts in the pH range 9-11.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication
Principles of method if other than guideline:
Metabolism study with isolated bacteria strain BNC1.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source test material: BASF AG , Name Trilon BS
Oxygen conditions:
aerobic
Inoculum or test system:
other: isolated bacteria strain BNC1

The first catabolic enzyme appears to be an EDTA monooxygenase since it requires O2, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded via N,N'-ethylenediaminediacetate. Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues of EDTA.

The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts of strain BNCI was investigated. Metal-EDTA chelates with thermodynamic stability constants below 1E+12 were readily mineralized by whole cells. With the exception of ZnEDTA, chelates with stability constants greater than 10E12 were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded after pretreatment by addition of calcium and magnesium salts in the pH range 9-11.

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:
other: Acceptable, well documented publication
Principles of method if other than guideline:
Method: Cells of the mixed culture were employed in a fluidized bed reactor with sand as support material.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial, adapted
Details on inoculum:
EDTA-degrading bacterial mixed culture from industrial sewage receiving EDTA-containing wastewater effluents and two isolated bacteria strains (BNC1 and BNC2) were used as inoculum, obtained from industrial sewage receiving EDTA-containing waste water effluents. A special enrichment procedure was applied.
Initial conc.:
292 mg/L
Based on:
test mat.
Parameter:
% degradation (test mat. analysis)
Value:
ca. 90 - ca. 100
Sampling time:
10 d

The bacterial mixed culture which was obtained from sewage by a special  enrichment procedure, utilized EDTA as the sole source of carbon and  nitrogen for growth. The chemical speciation did not influence the  biodegradability of EDTA.

A gram-negative isolate from the mixed population (BNC1) also metabolized  EDTA in monoculture. Growth of the pure culture was slow compared to the  original community and always accompanied by the accumulation of  unidentified metabolites. In contrast, a two species culture of strain BNC1 and a gram-positive isolate (BNC2) grew resembling the original mixed community without noticeable accumulation of metabolites.

Interpretation of results:
other: The test compound was biodegradable after acclimation under the conditions of this study.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Duration of test (contact time):
100 d
Initial conc.:
65 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Parameter:
% degradation (DOC removal)
Value:
90 - 100
Sampling time:
28 d

It could be shown in three different experiments that a change of the  pH-value can result in a biodegradation of EDTA.
 
In a SCAS facility which had been inoculated with activated sludge from a  municipal wastewater treatment plant, a removal by biodegradation of EDTA  (65 mg/L) added to domestic wastewater could be observed at pH of 8.0 to  9.0. With a pH of 6.5 no biodegradation could be obtained. The removal at  pH 8.5 started 3 weeks after inoculation with a result up to 100 %  degradation after 28 days. The SCAS unit ran with sludge retention times  > 12 days. A maximum EDTA removal rate of 0.2 kg/m³/day was achieved.

In order to confirm the biodegradation of EDTA, closed bottle tests were  carried out with sludge originating from the SCAS unit. The tests were  conducted at a pH range from 8.0 to 8.5. The lag period was only few  days. The time to reach a BOD/ThOD ratio of 0.6 was 3 weeks after  initiation of detectable biodegradation. EDTA was not biodegraded at pH  7.0.

The removal of EDTA was investigated in a full-scale activated sludge  plant operated at pH values between 7.5 and 8.5 with dairy wastewater  containing ca. 30 mg/L EDTA. The dairy wastewater was treated at a  hydraulic residence time of one day and a sludge retention time of 20  days. The analysis of influent, effluent and sludge concentration resulted in approximately 90 % removal. At a pH of 6.7 no biodegradation took place.

Interpretation of results:
other: ultimately biodegradable
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:
other: Reliable Data taken from EU Risk Assessment
Principles of method if other than guideline:
Method: Environmental samples from a river, a ditch and a lake were examined in the closed bottle test.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: Environmental samples from a river, a ditch and a lake were examined in the closed bottle test.
Key result
Parameter:
% degradation (DOC removal)
Value:
>= 53 - <= 72
Sampling time:
28 d
Remarks on result:
other: pH8

Recent data suggest that under alkaline conditions EDTA can be degraded.

Environmental samples from a river, a ditch and a lake were examined in  the closed bottle test for their potential to degrade CaNa2EDTA in a concentration of 8.0 mg/L at pH 6.5 and 8.0 over a period of few weeks. The results show for all environmental samples that at pH 6.5 no or little biodegradation (2-12 %) occurred within the first 28 days. After 49 days a biodegradation between 60 and  83 % was obtained. At pH 8, rates of 53, 62 and 72 % were obtained after 28 days and 75-89 % after 35 days (Van Ginkel, 1999).

Different investigations show, that it is possible to obtain enrichment  cultures of EDTA-utilising microorganisms. So far three strains of  bacteria have been isolated breaking down EDTA completely. One of them could be identified as Agrobacterium sp. Counter-ions exert effects on  the biodegradation of chelating agents. The different results show, that metal-EDTA complexes with a thermodynamic stability constant below 1E+12,  like Ca, Mg and Mn, were degraded. Chelates with stability constants above 1E+12, such as Cu and Fe, were not degraded (Van Ginkel, 1999).

Results obtained at a pH of 8 could be relevant because the pH value of  lake and river water ranges from 7.7 to 8.5. However, in surface waters  EDTA is preferably complexed with heavy metal ions. Regarding the  degradation tests cited above, no biological degradation is expected. Ca-EDTA can only occur in the environment where strong point sources release this species into a river with a low flow.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
yes
Remarks:
Water from the river Rhine was used as inoculum
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: fresh water from the river Rhine
Details on inoculum:
Water from the river Rhine was used as inoculum which had been taken upstream from the spot where the effluent of waste water treatment plant was discharged into the river.
Duration of test (contact time):
60 d
Initial conc.:
20 mg/L
Based on:
DOC
Initial conc.:
62 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
The pH of the test medium was adjusted to 6.5 at test start and during the course of the study controlled and if necessary readjusted.
Reference substance:
aniline
Key result
Parameter:
% degradation (DOC removal)
Value:
> 0 - < 10
Sampling time:
60 d
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
yes
Remarks:
Water from the river Rhine was used as inoculum
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: fresh water from the river Rhine
Details on inoculum:
Water from the river Rhine was used as inoculum which had been taken upstream from the spot where the effluent of waste water treatment plant was discharged into the river.
Duration of test (contact time):
60 d
Initial conc.:
20 mg/L
Based on:
DOC
Initial conc.:
62 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
The pH of the test medium was adjusted to 8.5 at test start and during the course of the study controlled and if necessary readjusted.
Reference substance:
aniline
Key result
Parameter:
% degradation (DOC removal)
Value:
> 70 - < 80
Sampling time:
60 d
Details on results:
Kinetic of test substance (in %):
= 0 after 28 day(s)
= 5 after 32 day(s)
= 22 after 35 day(s)
= 52 after 46 day(s)
= 73 after 60 day(s)

The substance was determined to be biodegradable after adaptation.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
yes
Remarks:
Water from the river Rhine was used as inoculum
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: fresh water from the river Rhine
Details on inoculum:
Water from the river Rhine was used as inoculum which had been taken downstream from the spot where the effluent of waste water treatment plant was discharged into the river.
Duration of test (contact time):
60 d
Initial conc.:
20 mg/L
Based on:
DOC
Initial conc.:
62 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
The pH of the test medium was adjusted to 6.5 at test start and during the course of the study controlled and if necessary readjusted.
Reference substance:
aniline
Key result
Parameter:
% degradation (DOC removal)
Value:
> 0 - < 10
Sampling time:
60 d
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
yes
Remarks:
Water from the river Rhine was used as inoculum
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: fresh water from the river Rhine
Details on inoculum:
Water from the river Rhine was used as inoculum which had been taken downstream from the spot where the effluent of waste water treatment plant was discharged into the river.
Duration of test (contact time):
60 d
Initial conc.:
20 mg/L
Based on:
DOC
Initial conc.:
62 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
The pH of the test medium was adjusted to 8.5 at test start and during the course of the study controlled and if necessary readjusted.
Reference substance:
aniline
Key result
Parameter:
% degradation (DOC removal)
Value:
90
Sampling time:
60 d
Details on results:
Kinetic of test substance (in %):
= 0 after 28 day(s)
= 16 after 40 day(s)
= 57 after 49 day(s)
= 76 after 54 day(s)
= 89 after 60 day(s)
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
The inoculum was activated sludge, adapted to the test substance, taken from a previous biodegradation study (Project No. 99/0668/21/2).
Duration of test (contact time):
28 d
Initial conc.:
61.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Reference substance:
aniline
Key result
Parameter:
% degradation (CO2 evolution)
Value:
> 0 - < 10
Sampling time:
28 d
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
The inoculum was activated sludge adapted to the test substance taken from a previous biodegradation study (Project No. 99/0668/21/4).
Duration of test (contact time):
28 d
Initial conc.:
61.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Reference substance:
aniline
Key result
Parameter:
% degradation (CO2 evolution)
Value:
> 0 - < 10
Sampling time:
28 d
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial (adaptation not specified)
Details on inoculum:
The activated sludge inoculum used in this study originated from an industrial wastewater treatment plant (BASF AG). The concentration of dry substance was 1 g/L.
Duration of test (contact time):
28 d
Initial conc.:
400 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Key result
Parameter:
% degradation (DOC removal)
Value:
> 0 - < 10
Sampling time:
28 d
Details on results:
Kinetic of test substance (in %):
= 0 after 3 hour(s)
= 0 after 7 day(s)
= 5 after 11 day(s)
= 8 after 28 day(s)
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
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
Key result
Parameter:
% degradation (O2 consumption)
Value:
56
Sampling time:
56 d
Remarks on result:
other: river water
Remarks:
EDTA-(NH4)2H2 (20824-56-0); screening study
Key result
Parameter:
% degradation (O2 consumption)
Value:
83
Sampling time:
28 d
Remarks on result:
other: lake water
Remarks:
EDTA-(NH4)2H2 (20824-56-0); 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
EDTA-(NH4)2H2 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.

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, EDTA-MgNa2, and EDTA-CaNa2 after 56 days. EDTA-(NH4)2H2 was degraded by 56% at day 56 and 65% at day 84. The immediate degradation of EDTA-(NH4)2H2 is attributed to the oxidation of the ammonium ions. 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

Description of key information

Edetic acid (CAS 60-00-4) 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 subcategory 1 members after 28d incubation time (Ginkel 2018). In these same tests however > 60% biodegradation was observed when prolonged to 60 days indicating that edetic acid (CAS 60-00-4) 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". This is supported by various "older" ready and inherent biodegradation tests available for edetic acid. however, the "newly performed" enhanced ready biodegradation tests with subcategory 1 members were basically used to cover the endpoint, since they are considered to be of highest quality within the abundant data set.

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 OECD guideline tests indicate that EDTA 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. 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-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 results edetic acid is assessed to be inherently biodegradable fulfilling specific criteria and thus not persistent in the environment.