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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
June 2018 - September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
As a chelating agent, EDTA forms complexes with cationic ions. Fundamental EDTA exists naturally as a mixture of chelate complexes. Between these EDTA complexes there is clearly a common functional group, common precursor/breakdonw producs and common mechanism of action. The biodegradability however 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 Fe, were not significantly degraded [Klüner & al. 1998, Van Ginkel, 1999 and Nörtemann, 2003]. In addition a degradation of Zn-EDTA was observed by Satroutdinov, 2003.

Degradation pathway
Several investigations revealed that it is possible to enrich cultures of EDTA-utilizing microorganisms. Different bacteria strains were isolated which can mineralised EDTA completely [Nörtemann, 1992 and Van Ginkel, 1999]. The degradation pathway of EDTA was described from Klüner & 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).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The only difference between the different EDTA-metal complexes is the metal complexed. Because the stability constants between these complexes differ the fate of these complexes is directly linked to this degree of binding. This binding degree is expressed in the stability constant.

3. ANALOGUE APPROACH JUSTIFICATION
EDTA is not readily biodegradable according to OECD criteria. It was shown that with natural river water as inoculum, in standard OECD 301D tests, EDTA complexes with a stability constant lower than 10E13 like EDTA-Na4, EDTA-CaNa2, EDTA-MgNa2 etc. less than 60% biodegradation was observed after 28 days indicating that these substances should be not classified as readily biodegradable but in these tests > 60% biodegradation was observed after 60 days in the prolonged (enhanced) tests indicating that these compexes, 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.
EDTA-Fe(OH)K2 has a stability constant higher than 10E13 under test conditions and is therefore considered justified to consider EDTA-Fe(OH)K2 be persistent according to the REACH requirements.
Reason / purpose:
read-across source
Qualifier:
according to
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. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Remarks:
river water instead of an effluent/extract/mixture was used as inoculum.
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.
Remarks:
89.9% purity
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:
other: Not included in the screening test
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:
2
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
80
Sampling time:
60 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.

Description of key information

EDTA was not found to be readily biodegradable according to OECD criteria. In standard OECD 301D ready biodegradability tests with natural river water as inoculum it was shown that EDTA complexes with a stability constant lower than 10E14 like EDTA-Na4, EDTA-CaNa2, EDTA-MgNa2 etc. less than 60% biodegradation was observed after 28 days indicating that these substances should indeed not be classified as readily biodegradable. In these same tests however > 60% biodegradation was observed when these tests are prolonged to day 60 (Ginkel, 2018) indicating that these complexes, having stability constants < 10E14, are ultimately biodegradable and should be classified as "not persistent".

Complexes with a stability constant >= 10E14 like EDTA-ZnX complexes (where X stands for K2, Na2 or (NH4)2) should be considered "Completely and inherently biodegradable". The dissociation rates are however considered too low to allow classification as not persistent.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria
Type of water:
freshwater

Additional information

EDTA is not found to be readily biodegradable according to OECD criteria. In standard OECD 301D ready biodegradability tests with natural river water as inoculum it was shown that EDTA complexes with a stability constant lower than 10E14 like EDTA-Na4, EDTA-CaNa2, EDTA-MgNa2 etc. less than 60% biodegradation was observed after 28 days indicating that these substances should indeed not be classified as readily biodegradable. In these same tests however > 60% biodegradation was observed when these tests are prolonged to day 60 (Ginkel, 2018) indicating that these complexes, having stability constants < 10E14, are ultimately biodegradable and should be classified as "not persistent".

EDTA-CaNa2 has a stability constant < 10E14 and is therefore under the conditions applied considered to be inherently biodegrable fulfilling specific criteria.

Complexes with a stability constant >= 10E14 like EDTA-CuX complexes (where X stands for K2, Na2 or (NH4)2) should be considered "Completely and inherently biodegradable". The dissociation rates are however considered too low to allow classification as not persistent.

 

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 10E14, like Ca, Mg and Mn, were degraded. On contrast heavy metal EDTA complexes with stability constants above 10E14, such as Cu and Fe, were not significantly degraded [Klüner & al. 1998, Van Ginkel, 1999 and Nörtemann, 2003]. In addition a degradation of Zn-EDTA was observed by Satroutdinov, 2003.

 

Degradation pathway

Several investigations revealed that it is possible to enrich cultures of EDTA-utilizing microorganisms. Different bacteria strains were isolated which can mineralised EDTA completely [Nörtemann, 1992 and Van Ginkel, 1999]. The degradation pathway of EDTA was described from Klüner & 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).

 

Conclusion

EDTA is not readily biodegradable according to OECD criteria. It was shown that with natural river water as inoculum, in standard OECD 301D tests, EDTA complexes with a stability constant lower than 10E14 like EDTA-Na4, EDTA-CaNa2, EDTA-MgNa2 etc. less than 60% biodegradation was observed after 28 days indicating that these substances should be not classified as readily biodegradable but in these tests > 60% biodegradation was observed after 60 days in the prolonged (enhanced) tests indicating that these complexes, having stability constants < 10E14, are not persistent. This stability constant threshold of 10E14 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.