Registration Dossier

Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
March - April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: Ammonium chloride was omitted from the medium more details see principles other than guideline
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
Version / remarks:
1994
Principles of method if other than guideline:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: 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).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical name 2,2’(octadecylimino) bisethanol
CAS no. 10213-78-2
Batch no. 20190290
Composition (Annex 2)
Organic content 100.0 % m/m
Water 0.00 % m/m
Expiry date 13-Oct-2022
Appearance White solids
Storage Store in closed container in a cool, dry, ventilated area
Chemical stability Stable under normal conditions
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Secondary activated sludge (15-Mar-2021) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter was dried for 1.5 hour at 104.8 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.2 g/L.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992).

The Colony forming units (CFU) of the preconditioned and diluted inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned and diluted inoculum as used in the closed bottles (2 mg/L dry weight) was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar.
Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.6 – 22.8 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 1.10^6 CFU/L.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
2 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stocks
Deionized water used in the Closed Bottle test contained per liter of water 8.50 mg KH2PO4, 21.75 mg K2HPO4, 33.41 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.51 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L. The test substance was administered using a stock solution of 1.0 g/L in dichloromethane.

Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum with nutrients, 10 bottles to which dichloromethane was first added and subsequently evaporated containing only inoculum with nutrients, 10 bottles containing test substance and inoculum with nutrients, and 10 bottles containing sodium acetate and inoculum with nutrients.
Accurate administration of the poorly water-soluble test substance at a concentration of 2 mg/L was accomplished by dissolving it in dichloromethane (Nyholm and Seiero, 1989). The test substance in dichloromethane was directly added to bottles. Before the start of the test, the solvent in the test and control bottles was allowed to evaporate by placing the bottles on a roller bank in a ventilated hood for 3 hours to obtain an even distribution of the test substance on the walls of the bottles. Next the bottles were taken from the roller bank and placed in the ventilated hood for another ~72 hours in order to ensure complete volatilization of the dichloromethane from the bottles. The concentration of sodium acetate in the bottles was 6.7 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 zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles 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.
Reference substance:
acetic acid, sodium salt
Preliminary study:
76% on day 28 based on ThOD-NO3
Test performance:
As expected readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NO3
Value:
70
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test results
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NH3
Value:
74
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test result
Details on results:
RESULTS AND DISCUSSION
Theoretical oxygen demand (ThOD)
The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.87 and 3.05 g oxygen/g active ingredient, respectively. These ThODs were calculated with an average molecular formula of the active ingredients of the 2,2’(octadecylimino) bisethanol (annex 2). The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

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 for the test substance was 6.9 and for the control, the dichloromethane control and reference substance were 6.8 at the start of the test. The pH of the medium in the reference bottles measured at day 14 was 6.7. The pH of the medium at day 28 was 6.7, 6.5 and 6.8 for the control, the test substance and the dichloromethane control, respectively. 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 0.80 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 87 (Table II and Figure 1). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability
2,2’(Octadecylimino) bisethanol was biodegraded by 74% (based on ThODNH3) at day 28 in the Closed Bottle test (Table II). Assuming complete nitrification, and calculating the biodegradation based on the ThODNO3 the test substance was biodegraded by 70% in the Closed Bottle test at day 28 (Table II, Figure 1). Over 60% biodegradation was achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion is herewith fulfilled however should not be applied because 2,2’(octadecylimino) bisethanol, is a poorly water-soluble surfactant of which the hydrophobic and hydrophilic moiety are most likely degraded sequentially by at least two microorganisms. The biodegradation of the test substance is therefore an addition of different biodegradation curves. Hence, the time window is an inappropriate pass/fail criterion for fatty amine surfactants (Richterich and Steber, 2001). 2,2’(Octadecylimino) bisethanol is classified as readily biodegradable based on the ≥60% biodegradation reached at day 28.
Results with reference substance:
87% biodegradation on day 14

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

Time (days)

Oxygen concentration (mg/L)

 

Oc

Oa

Ocd

Ot

0

8.9

8.9

8.9

8.9

 

8.9

8.9

8.9

8.9

Mean (M)

8.90

8.90

8.90

8.90

7

8.5

4.4

8.5

7.8

 

8.5

4.4

8.5

7.8

Mean (M)

8.50

4.40

8.50

7.80

14

8.4

3.8

8.3

4.8

 

8.4

3.9

8.3

4.7

Mean (M)

8.40

3.85

8.30

4.75

21

8.2

 

8.2

4.3

 

8.2

 

8.2

4.0

Mean (M)

8.20

 

8.20

4.15

28

8.1

 

8.1

3.7

 

8.1

 

8.1

4.0

Mean (M)

8.10

 

8.10

3.85

Oc      Mineral nutrient solution with only inoculum.

Ocd    Mineral nutrient solution with inoculum, before filling the bottles, dichloromethane (0.6 mL) was added to these bottles and subsequently allowed to evaporate.

Ot      Mineral nutrient solution with inoculum, test substance (2.0 mg/L test substance = 2.0 mg/L active ingredient) administered using a stock solution in dichloromethane, before filling the bottles the dichloromethane was allowed to evaporate

Oa       Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L).

 

 

Table II Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of sodium acetate and the test substance in the Closed Bottle test. Biodegradation of the test substance is calculated both without nitrification (BOD/ThODNH3) and with nitrification (BOD/ThODNO3).

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

ThODNH3

ThODNO3

0

0.00

0.00

0

0

0

7

0.70

4.10

12

11

78

14

3.55

4.55

62

58

87

21

4.05

 

71

66

 

28

4.25

 

74

70

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test is valid as shown by an endogenous respiration of 0.80 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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.
2,2’(Octadecylimino) bisethanol was biodegraded by 70% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion is herewith fulfilled however should not be applied because 2,2’(octadecylimino) bisethanol, is a poorly water-soluble surfactant of which the hydrophobic and hydrophilic moiety are most likely degraded sequentially by at least two microorganisms. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion.
Executive summary:

To assess the biotic degradation of 2,2’(octadecylimino) bisethanol, 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 (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles and incubated in the dark at 22.5 to 22.9 °C for 28 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, 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. Biodegradation percentages were calculated assuming a complete oxidation (nitrification) of the test substance organic nitrogen. The theoretical oxygen demand including the nitrification was used to correct for the oxygen consumption by the nitrification. 2,2’(Octadecylimino) bisethanol was biodegraded by 70% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion is herewith fulfilled however should not be applied because 2,2’(octadecylimino) bisethanol, is a poorly water-soluble surfactant of which the hydrophobic and hydrophilic moiety are most likely degraded sequentially by at least two microorganisms. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion. 2,2’(Octadecylimino) bisethanol is classified as readily biodegradable based on the ≥60% biodegradation reached at day 28.

The test is valid as shown by an endogenous respiration of 0.80 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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:
supporting study
Study period:
March - April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: Ammonium chloride was omitted from the medium more details see principles other than guideline
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
Version / remarks:
1994
Principles of method if other than guideline:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: 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).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical name 2,2’-(C16-18 (even numbered) alkyl imino) diethanol
CAS no. 1218787-30-4
Batch no. 20192048
Composition (Annex 2)
Organic content 99.93 % m/m
Water 0.07 % m/m
Expiry date 25-Oct-2022
Appearance White solids
Storage Store in closed container in a cool, dry, ventilated area
Chemical stability Stable under normal conditions
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Secondary activated sludge (15-Mar-2021) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter was dried for 1.5 hour at 104.8 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.2 g/L.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992).

The Colony forming units (CFU) of the preconditioned and diluted inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned and diluted inoculum as used in the closed bottles (2 mg/L dry weight) was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar.
Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.6 – 22.8 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 1.10^6 CFU/L.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
1.999 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stocks
Deionized water used in the Closed Bottle test contained per liter of water 8.50 mg KH2PO4, 21.75 mg K2HPO4, 33.41 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.51 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L. The test substance was administered using a stock solution of 1.0 g/L in dichloromethane.

Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum with nutrients, 10 bottles to which dichloromethane was first added and subsequently evaporated containing only inoculum with nutrients, 10 bottles containing test substance and inoculum with nutrients, and 10 bottles containing sodium acetate and inoculum with nutrients.
Accurate administration of the poorly water-soluble test substance at a concentration of 2 mg/L was accomplished by dissolving it in dichloromethane (Nyholm and Seiero, 1989). The test substance in dichloromethane was directly added to bottles. Before the start of the test, the solvent in the test and control bottles was allowed to evaporate by placing the bottles on a roller bank in a ventilated hood for 3 hours to obtain an even distribution of the test substance on the walls of the bottles. Next the bottles were taken from the roller bank and placed in the ventilated hood for another ~72 hours in order to ensure complete volatilization of the dichloromethane from the bottles. The concentration of sodium acetate in the bottles was 6.7 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 zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles 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.
Reference substance:
acetic acid, sodium salt
Preliminary study:
67% on day 28 based on ThOD-NO3
Test performance:
As expected readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NO3
Value:
61
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test results
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NH3
Value:
65
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test result
Details on results:
RESULTS AND DISCUSSION
Theoretical oxygen demand (ThOD)
The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.85 and 3.04 g oxygen/g active ingredient, respectively. These ThODs were calculated with an average molecular formula of the active ingredients of the 2,2’-(C16-18 (even numbered) alkyl imino) diethanol (annex 2). The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

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 for the test substance was 6.9 and for the control, the dichloromethane control and reference substance were 6.8 at the start of the test. The pH of the medium in the reference bottles measured at day 14 was 6.7. The pH of the medium at day 28 was 6.7, 6.6 and 6.8 for the control, the test substance and the dichloromethane control, respectively. 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 0.80 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 87 (Table II and Figure 1). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability
2,2’-(C16-18 (even numbered) alkyl imino) diethanol was biodegrade d by 65% (based on ThODNH3) at day 28 in the Closed Bottle test (Table II). Assuming complete nitrification, and calculating the biodegradation based on the ThODNO3 the test substance was biodegraded by 61% in the Closed Bottle test at day 28 (Table II, Figure 1). Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because 2,2’-(C16-18 (even numbered) alkyl imino) diethanol is a mixture of different constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the mixture of constituents suggests that the test substance is not readily biodegradable. The time window is therefore an inappropriate pass/fail criterion for mixtures of chemicals (Richterich and Steber, 2001). 2,2’-(C16-18 (even numbered) alkyl imino) diethanol is classified as readily biodegradable based on the ≥60% biodegradation reached at day 28.
Results with reference substance:
87% biodegradation on day 14

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

Time (days)

Oxygen concentration (mg/L)

 

Oc

Oa

Ocd

Ot

0

8.9

8.9

8.9

8.9

 

8.9

8.9

8.9

8.9

Mean (M)

8.90

8.90

8.90

8.90

7

8.5

4.4

8.5

8.4

 

8.5

4.4

8.5

8.4

Mean (M)

8.50

4.40

8.50

8.40

14

8.4

3.8

8.3

6.0

 

8.4

3.9

8.3

5.9

Mean (M)

8.40

3.85

8.30

5.95

21

8.2

 

8.2

4.8

 

8.2

 

8.2

4.9

Mean (M)

8.20

 

8.20

4.85

28

8.1

 

8.1

4.4

 

8.1

 

8.1

4.4

Mean (M)

8.10

 

8.10

4.40

Oc      Mineral nutrient solution with only inoculum.

Ocd    Mineral nutrient solution with inoculum, before filling the bottles, dichloromethane (0.6 mL) was added to these bottles and subsequently allowed to evaporate.

Ot      Mineral nutrient solution with inoculum, test substance (2.0 mg/L test substance = 1.9986 mg/L active ingredient) administered using a stock solution in dichloromethane, before filling the bottles the dichloromethane was allowed to evaporate

Oa       Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L).

 

 

Table II Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of sodium acetate and the test substance in the Closed Bottle test. Biodegradation of the test substance is calculated both without nitrification (BOD/ThODNH3) and with nitrification (BOD/ThODNO3).

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

ThODNH3

ThODNO3

0

0.00

0.00

0

0

0

7

0.10

4.10

2

2

78

14

2.35

4.55

41

39

87

21

3.35

 

59

55

 

28

3.70

 

65

61

 

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test is valid as shown by an endogenous respiration of 0.80 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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.
2,2’-(C16-18 (even numbered) alkyl imino) diethanol was biodegraded by 61% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because 2,2’-(C16-18 (even numbered) alkyl imino) diethanol is a mixture of constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion.
Executive summary:

To assess the biotic degradation of 2,2’-(C16-18 (even numbered) alkyl imino) diethanol, 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 (2 mg/L) was exposed to activated sludge which was spiked to a mineral nutrient solution, dosed in closed bottles and incubated in the dark at 22.5 to 22.9 °C for 28 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, 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. Biodegradation percentages were calculated assuming a complete oxidation (nitrification) of the test substance organic nitrogen. The theoretical oxygen demand including the nitrification was used to correct for the oxygen consumption by the nitrification.

2,2’-(C16-18 (even numbered) alkyl imino) diethanol was biodegraded by 61% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because 2,2’-(C16-18 (even numbered) alkyl imino) diethanol is a mixture of constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion. 2,2’-(C16-18 (even numbered) alkyl imino) diethanol is classified as readily biodegradable based on the ≥ 60% biodegradation reached at day 28.

The test is valid as shown by an endogenous respiration of 0.80 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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:
supporting study
Study period:
March - April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: Ammonium chloride was omitted from the medium more details see principles other than guideline
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
Version / remarks:
1994
Principles of method if other than guideline:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: 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).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical name 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol
CAS no. 1218787-32-6
Batch no. 20200030
Composition (Annex 2)
Organic content 99.96 % m/m
Water 0.04 % m/m
Expiry date 25-Jan-2023
Appearance light yellow liquid
Storage Store in closed container in a cool, dry, ventilated area
Chemical stability Stable under normal conditions
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Secondary activated sludge (15-Mar-2021) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter was dried for 1.5 hour at 104.8 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.2 g/L.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992).

The Colony forming units (CFU) of the preconditioned and diluted inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned and diluted inoculum as used in the closed bottles (2 mg/L dry weight) was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar.
Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.6 – 22.8 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 1.10^6 CFU/L.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
1.999 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stocks
Deionized water used in the Closed Bottle test contained per liter of water 8.50 mg KH2PO4, 21.75 mg K2HPO4, 33.41 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.51 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L. The test substance was administered using a stock solution of 1.0 g/L in dichloromethane.

Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum with nutrients, 10 bottles to which dichloromethane was first added and subsequently evaporated containing only inoculum with nutrients, 10 bottles containing test substance and inoculum with nutrients, and 10 bottles containing sodium acetate and inoculum with nutrients.
Accurate administration of the poorly water-soluble test substance at a concentration of 2 mg/L was accomplished by dissolving it in dichloromethane (Nyholm and Seiero, 1989). The test substance in dichloromethane was directly added to bottles. Before the start of the test, the solvent in the test and control bottles was allowed to evaporate by placing the bottles on a roller bank in a ventilated hood for 3 hours to obtain an even distribution of the test substance on the walls of the bottles. Next the bottles were taken from the roller bank and placed in the ventilated hood for another ~72 hours in order to ensure complete volatilization of the dichloromethane from the bottles. The concentration of sodium acetate in the bottles was 6.7 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 zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles 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.

Reference substance:
acetic acid, sodium salt
Preliminary study:
68% on day 28 based on ThOD-NO3
Test performance:
As expected readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NO3
Value:
60
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test results
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NH3
Value:
63
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test result
Details on results:
RESULTS AND DISCUSSION
Theoretical oxygen demand (ThOD)
The ThOD-NH3 and ThOD-NO3 of the test substance used to calculate the biodegradation percentages is 2.84 and 3.02 g oxygen/g active ingredient, respectively. These ThODs were calculated with an average molecular formula of the active ingredients of the 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol (annex 2). The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

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 for the test substance was 6.9 and for the control, the dichloromethane control and reference substance were 6.8 at the start of the test. The pH of the medium in the reference bottles measured at day 14 was 6.7. The pH of the medium at day 28 was 6.7, 6.6 and 6.8 for the control, the test substance and the dichloromethane control, respectively. 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 0.80 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 87 (Table II and Figure 1). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability
2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol was biodegrade d by 63% (based on ThODNH3) at day 28 in the Closed Bottle test (Table II). Assuming complete nitrification, and calculating the biodegradation based on the ThODNO3 the test substance was biodegraded by 60% in the Closed Bottle test at day 28 (Table II, Figure 1). Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol is a mixture of different constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the mixture of constituents suggests that the test substance is not readily biodegradable. The time window is therefore an inappropriate pass/fail criterion for mixtures of chemicals (Richterich and Steber, 2001). 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol is classified as readily biodegradable based on the 60% biodegradation reached at day 28.
Results with reference substance:
87% biodegradation on day 14

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

Time (days)

Oxygen concentration (mg/L)

 

Oc

Oa

Ocd

Ot

0

8.9

8.9

8.9

8.9

 

8.9

8.9

8.9

8.9

Mean (M)

8.90

8.90

8.90

8.90

7

8.5

4.4

8.5

8.4

 

8.5

4.4

8.5

8.4

Mean (M)

8.50

4.40

8.50

8.40

14

8.4

3.8

8.3

7.0

 

8.4

3.9

8.3

6.5

Mean (M)

8.40

3.85

8.30

6.75

21

8.2

 

8.2

5.3

 

8.2

 

8.2

5.7

Mean (M)

8.20

 

8.20

5.50

28

8.1

 

8.1

4.4

 

8.1

 

8.1

4.6

Mean (M)

8.10

 

8.10

4.50

Oc      Mineral nutrient solution with only inoculum.

Ocd    Mineral nutrient solution with inoculum, before filling the bottles, dichloromethane (0.6 mL) was added to these bottles and subsequently allowed to evaporate.

Ot      Mineral nutrient solution with inoculum, test substance (2.0 mg/L test substance = 1.9992 mg/L active ingredient) administered using a stock solution in dichloromethane, before filling the bottles the dichloromethane was allowed to evaporate

Oa       Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L).

 

 

Table IIOxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of sodium acetate and the test substance in the Closed Bottle test. Biodegradation of the test substance is calculated both without nitrification (BOD/ThODNH3) and with nitrification (BOD/ThODNO3).

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

ThODNH3

ThODNO3

0

0.00

0.00

0

0

0

7

0.10

4.10

2

2

78

14

1.55

4.55

27

26

87

21

2.70

 

48

45

 

28

3.60

 

63

60

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test is valid as shown by an endogenous respiration of 0.80 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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.
2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol was biodegraded by 60% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol, is a mixture of constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion.
Executive summary:

To assess the biotic degradation of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol, 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 (2 mg/L) was exposed to activated sludge which was spiked to a mineral nutrient solution, dosed in closed bottles and incubated in the dark at 22.5 to 22.9 °C for 28 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, 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.

Biodegradation percentages were calculated assuming a complete oxidation (nitrification) of the test substance organic nitrogen. The theoretical oxygen demand including the nitrification was used to correct for the oxygen consumption by the nitrification. 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol was biodegraded by 60% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol, is a mixture of constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion. 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol is classified as readily biodegradable based on the 60% biodegradation reached at day 28.

The test is valid as shown by an endogenous respiration of 0.80 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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
Study period:
March - April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: Ammonium chloride was omitted from the medium more details see principles other than guideline
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
Version / remarks:
1994
Principles of method if other than guideline:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: 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).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical name 2,2’-(Octade-9-enylimino)bisethanol
CAS no. 25307-17-9
Batch no. 1950627
Composition (Annex 2)
Organic content 99.97 % m/m
Water 0.03 % m/m
Expiry date 6-Mar-2023
Appearance yellow liquid
Storage Store in closed container in a cool, dry, ventilated area
Chemical stability Stable under normal conditions
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Secondary activated sludge (15-Mar-2021) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter was dried for 1.5 hour at 104.8 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.2 g/L.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992).

The Colony forming units (CFU) of the preconditioned and diluted inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned and diluted inoculum as used in the closed bottles (2 mg/L dry weight) was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar.
Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.6 – 22.8 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 1.10^6 CFU/L.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
1.999 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stocks
Deionized water used in the Closed Bottle test contained per liter of water 8.50 mg KH2PO4, 21.75 mg K2HPO4, 33.41 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.51 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L. The test substance was administered using a stock solution of 1.0 g/L in dichloromethane.

Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum with nutrients, 10 bottles to which dichloromethane was first added and subsequently evaporated containing only inoculum with nutrients, 10 bottles containing test substance and inoculum with nutrients, and 10 bottles containing sodium acetate and inoculum with nutrients.
Accurate administration of the poorly water-soluble test substance at a concentration of 2 mg/L was accomplished by dissolving it in dichloromethane (Nyholm and Seiero, 1989). The test substance in dichloromethane was directly added to bottles. Before the start of the test, the solvent in the test and control bottles was allowed to evaporate by placing the bottles on a roller bank in a ventilated hood for 3 hours to obtain an even distribution of the test substance on the walls of the bottles. Next the bottles were taken from the roller bank and placed in the ventilated hood for another ~72 hours in order to ensure complete volatilization of the dichloromethane from the bottles. The concentration of sodium acetate in the bottles was 6.7 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 zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles 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.
Reference substance:
acetic acid, sodium salt
Test performance:
As expected readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NO3
Value:
61
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test results
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NH3
Value:
65
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test result
Details on results:
RESULTS AND DISCUSSION
Theoretical oxygen demand (ThOD)
The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.84 and 3.02 g oxygen/g active ingredient, respectively. These ThODs were calculated with an average molecular formula of the active ingredients of the 2,2’-(octade-9-enylimino)bisethanol (annex 2). The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

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 for the test substance was 6.9 and for the control, the dichloromethane control and reference substance were 6.8 at the start of the test. The pH of the medium in the reference bottles measured at day 14 was 6.7. The pH of the medium at day 28 was 6.7, 6.6 and 6.8 for the control, the test substance and the dichloromethane control, respectively. 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 0.80 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 87 (Table II and Figure 1). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability
2,2’-(Octade-9-enylimino)bisethanol was biodegrade d by 65% (based on ThODNH3) at day 28 in the Closed Bottle test (Table II). Assuming complete nitrification, and calculating the biodegradation based on the ThODNO3 the test substance was biodegraded by 61% in the Closed Bottle test at day 28 (Table II, Figure 1). Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because 2,2’-(octade-9-enylimino)bisethanol is a mixture of different constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the mixture of constituents suggests that the test substance is not readily biodegradable. The time window is therefore an inappropriate pass/fail criterion for mixtures of chemicals (Richterich and Steber, 2001). 2,2’-(Octade-9-enylimino)bisethanol is classified as readily biodegradable based on the ≥60% biodegradation reached at day 28.
Results with reference substance:
87% biodegradation on day 14

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

Time (days)

Oxygen concentration (mg/L)

 

Oc

Oa

Ocd

Ot

0

8.9

8.9

8.9

8.9

 

8.9

8.9

8.9

8.9

Mean (M)

8.90

8.90

8.90

8.90

7

8.5

4.4

8.5

8.4

 

8.5

4.4

8.5

8.3

Mean (M)

8.50

4.40

8.50

8.35

14

8.4

3.8

8.3

5.8

 

8.4

3.9

8.3

5.9

Mean (M)

8.40

3.85

8.30

5.85

21

8.2

 

8.2

5.1

 

8.2

 

8.2

5.0

Mean (M)

8.20

 

8.20

5.05

28

8.1

 

8.1

4.3

 

8.1

 

8.1

4.5

Mean (M)

8.10

 

8.10

4.40

Oc      Mineral nutrient solution with only inoculum.

Ocd    Mineral nutrient solution with inoculum, before filling the bottles, dichloromethane (0.6 mL) was added to these bottles and subsequently allowed to evaporate.

Ot      Mineral nutrient solution with inoculum, test substance (2.0 mg/L test substance = 1.9994 mg/L active ingredient) administered using a stock solution in dichloromethane, before filling the bottles the dichloromethane was allowed to evaporate

Oa       Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L).

 

 

Table II Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of sodium acetate and the test substance in the Closed Bottle test. Biodegradation of the test substance is calculated both without nitrification (BOD/ThODNH3) and with nitrification (BOD/ThODNO3).

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

ThODNH3

ThODNO3

0

0.00

0.00

0

0

0

7

0.15

4.10

3

2

78

14

2.45

4.55

43

41

87

21

3.15

 

55

52

 

28

3.70

 

65

61

 

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test is valid as shown by an endogenous respiration of 0.80 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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.
Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because 2,2’-(octade-9-enylimino)bisethanol, is a mixture of constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion.
Executive summary:

To assess the biotic degradation of 2,2’-(octade-9-enylimino)bisethanol, 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 (2 mg/L) was exposed to activated sludge which was spiked to a mineral nutrient solution, dosed in closed bottles and incubated in the dark at 22.5 to 22.9 °C for 28 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, 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.

Biodegradation percentages were calculated assuming a complete oxidation (nitrification) of the test substance organic nitrogen. The theoretical oxygen demand including the nitrification was used to correct for the oxygen consumption by the nitrification. 2,2’-(Octade-9-enylimino)bisethanol was biodegraded by 61% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because 2,2’-(octade-9-enylimino)bisethanol, is a mixture of constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion. 2,2’-(Octade-9-enylimino)bisethanol is classified as readily biodegradable based on the ≥60% biodegradation reached at day 28.

The test is valid as shown by an endogenous respiration of 0.80 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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:
supporting study
Study period:
March - April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: Ammonium chloride was omitted from the medium more details see principles other than guideline
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
Version / remarks:
1994
Principles of method if other than guideline:
One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: 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).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical name Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs
CAS no. 71786-60-2
EC no. 276-014-8
Batch no. 20201158
Composition (Annex 2)
Organic content 99.9 % m/m
Water 0.1 % m/m
Expiry date 10-Feb-2023
Appearance Light yellow liquid
Storage Store in closed container in a cool, dry, ventilated area
Chemical stability Stable under normal conditions
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Secondary activated sludge (22-Mar-2021) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter was dried for 1.5 hour at 103.4 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.2 g/L.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992).

The Colony forming units (CFU) of the preconditioned and diluted inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned and diluted inoculum as used in the closed bottles (2 mg/L dry weight) was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar.
Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.6 – 22.8 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 1.10^6 CFU/L.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
1.998 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stocks
Deionized water used in the Closed Bottle test contained per liter of water 8.50 mg KH2PO4, 21.75 mg K2HPO4, 33.41 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.51 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L. The test and reference substance were added to the bottles using aqueous stock solutions of 1 g/L. A clear aqueous stock solution of the test substance was obtained by acidifying the stock solution to pH 5.0 using a 2 M HCl solution.

Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum with nutrients, 10 bottles containing test substance and inoculum with nutrients, and 6 bottles containing sodium acetate and inoculum with nutrients. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L 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 bottles 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.
Reference substance:
acetic acid, sodium salt
Preliminary study:
63% biodegradation on day 28 based on ThOD-NO3 (2.95 mg O2/mg)
Test performance:
As expected readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NO3
Value:
60
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test results
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Based on ThOD-NH3
Value:
65
Sampling time:
28 d
Remarks on result:
other: Readily biodegradable based on measured test result
Details on results:
RESULTS AND DISCUSSION
Theoretical oxygen demand (ThOD)
The ThOD-NH3 and ThOD-NO3 of the test substance used to calculate the biodegradation percentages is 2.73 and 2.95 g oxygen/g active ingredient, respectively. These ThODs were calculated with an average molecular formula of the active ingredients of the ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs (annex 2). The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

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 for at the start of the test was 7.1 for the test substance, the control, and the reference substance. The pH of the medium in the reference bottles measured at day 14 was 6.8. The pH of the medium at day 28 was 7.0 and 6.8 for the control and test substance, respectively. 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 1.10 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 90 (Table II and Figure 1). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability
Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs was biodegraded by 65% (based on ThODNH3) at day 28 in the Closed Bottle test (Table II). Assuming complete nitrification and calculating the biodegradation with the ThOD-NO3 to correct for the oxygen consumption by the nitrification resulted in 60% biodegradation at day 28 (Table II, Figure 1). Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs is a mixture of different constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the mixture of constituents suggests that the test substance is not readily biodegradable. The time window is therefore an inappropriate pass/fail criterion for mixtures of chemicals (Richterich and Steber, 2001). Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs is classified as readily biodegradable based on the 60% biodegradation reached at day 28.
Results with reference substance:
90% biodegradation on day 14

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

Time (days)

Oxygen concentration (mg/L)

 

Oc

Oa

Ot

 

0

8.9

8.9

8.9

 

 

8.9

8.9

8.9

 

Mean (M)

8.90

8.90

8.90

 

7

8.2

4.1

6.6

 

 

8.2

4.1

7.2

 

Mean (M)

8.20

4.10

6.90

 

14

8.1

3.4

5.8

 

 

8.1

3.4

5.8

 

Mean (M)

8.10

3.40

5.80

 

21

7.9

 

4.7

 

 

7.9

 

4.6

 

Mean (M)

7.90

 

4.65

 

28

7.8

 

4.2

 

 

7.8

 

4.3

 

Mean (M)

7.80

 

4.25

 

Oc      Mineral nutrient solution with only inoculum.

Ot      Mineral nutrient solution with inoculum, test substance (2.0 mg/L test substance = 1.998 mg/L active ingredient)

Oa       Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L).

 

 

Table II Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of sodium acetate and the test substance in the Closed Bottle test. Biodegradation of the test substance is calculated both without nitrification (BOD/ThODNH3) and with nitrification (BOD/ThODNO3).

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

ThODNH3

ThODNO3

0

0.00

0.00

0

0

0

7

1.30

4.10

24

22

78

14

2.30

4.70

42

39

90

21

3.25

 

60

55

 

28

3.55

 

65

60

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test is valid as shown by an endogenous respiration of 1.10 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 90% 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.
Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs was biodegraded by 60% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs, is a mixture of constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion.
Executive summary:

To assess the biotic degradation of ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs, 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 (2 mg/L) was exposed to activated sludge which was spiked to a mineral nutrient solution, dosed in closed bottles and incubated in the dark at 22.5 to 22.9 °C for 28 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, 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. Biodegradation percentages were calculated assuming a complete oxidation (nitrification) of the test substance organic nitrogen. The theoretical oxygen demand including the nitrification was used to correct for the oxygen consumption by the nitrification.

Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs was biodegraded by 60% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs, is a mixture of constituents. The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should not be used as a pass/fail criterion. Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derivs is classified as readily biodegradable based on the 60% biodegradation reached at day 28.

The test is valid as shown by an endogenous respiration of 1.10 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 90% 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

Primary fatty amine ethoxylates are poorly soluble in water and at test concentrations required for ready biodegradability testing according to OECD TG 301, toxic to the micro-organisms in the inoculum. Sorption of the substance to silica gel or the wall of the test vessel reduces the exposure concentration and facilitates the slow release of the test substance (Timmer 2019, 2020). Under these conditions, the primary fatty amine ethoxylates are observed to be readily biodegradable in a closed bottle test. This has been demonstrated with 5 different primary fatty amine ethoxylates.


Timmer, N., Gore, D., Sanders, D., Gouin, T and Droge, S.T.J., (2019) Toxicity mitigation and bioaccessibility of the cationic surfactant cetyltrimethylammonium bromide in a sorbent-modified biodegradation study. Chemosphere 222: 461 - 468


Timmer, N., Gore, D., Sanders, D., Gouin, T and Droge, S.T.J. (2020). Application of 7 different clay types in sorbent-modified biodegradation studies with cationic biocides. Chemosphere, 245: 125643

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

For primary fatty amines ethoxylates with chain lengths of C12 to C18 (saturated and unsaturated) many ready biodegradability test results 60% at day 28 are available. Biodegradation in excess of 60% was not achieved in a few tests. The low biodegradability percentages obtained in these tests should be attributed to the biocidal properties of primary fatty amine ethoxylates and should consequently be ignored.

 

A primary fatty amine ethoxylate is a chemical consisting of a hydrophilic group linked to a hydrophobic moiety. Biodegradation of both moieties of surfactants requires the concerted action of at least two micro-organisms as a single organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). In ready biodegradability tests, the two moieties of this fatty amine derivative are therefore degraded sequentially.The degradation curve will therefore be the sum of two growth curves.The biodegradation of the two moieties may be fully in line with the time-day window criterion when judged as separate chemicals. The time window criterion was developed on the assumption that a compound is degraded according to the “standard” growth curve in ready biodegradability tests. The time-window should therefore be ignored as a pass fail criterion for these surfactants.

Chemically primary fatty amine ethoxylates have an alkyl chain linked directly to a nitrogen atom of diethanolamine through a covalent bond. The alkyl group may be derived from different sources like dodecyl, coco, tallow, or oleyl. Biodegradation of surfactants refers to the reduction in complexity of the chemical through metabolic activity of micro-organisms utilizing the substance as carbon and energy source. If a surfactant is to serve as a carbon and energy source for aerobic micro-organisms then it has to be converted into a form that can enter the central metabolism of micro-organisms. Normally this involves converting the surfactant into one, or more, low molecular weight intermediates of the tricarboxylic acid (TCA) cycle or compounds that feed into it. These conversions are described in biodegradation pathways for cationic surfactants(van Ginkel, 2007). Although micro-organisms capable of degrading surfactants are immensely diverse, the central metabolism (b-oxidation and TCA cycle) is remarkably similar. Kluyver and Donker (1926) first described this similarity known as the unity of biochemistry. This unity is the key to justification of the use of read-across of biodegradability test results.

Most surfactant-degrading consortia interact commensalistically through production and release of the hydrophilic part of the molecule by alkyl chain degrading bacteria. To understand the metabolic basis of degradation by microorganisms, the pathway of octadecylbis(2-hydroxyethyl)amines has been studied (van Ginkel and Kroon, 1993). Starting from an enrichment culture a bacterium capable of degrading octadecylbis(2-hydroxyethyl)amine was isolated. Alkylbis(2-hydroxyethyl)amineswith tallow, octadecenyl and coco alkyl chains, alkanals, and fatty acids can also serve as a carbon and energy source. Simultaneous adaptation studies showed that acetate, octadecanal and octadecanoate are respired by octadecylbis(2-hydroxyethyl)amine-grown cells indicating that the long alkyl chain is utilized for microbial growth. Dehydrogenase activity present in cell-free extract of octadecylbis(2-hydroxyethyl)amine-grown cells catalysed the liberation of the alkyl chain. The biodegradation of octadecylbis(2-hydroxyethyl)amine is therefore initiated by cleavage of the Calkyl-N bond. Another dehydrogenase, producing fatty acids from alkanals was also detected in cell-free extracts of octadecylbis(2-hydroxyethyl)amine-grown cells. The fatty acids are degraded through the b-oxidation cycle. In each cycle, the alkyl chain is progressively shortened by two carbons yielding one molecule of acetyl-CoA. The acetyl-CoA generated in b-oxidation enters the TCA cycle, where it is further oxidised to carbon dioxide and water. A single micro-organism can degrade both saturated and unsaturated chains with varying chain lengths. The alkyl chains are therefore completely degraded by micro-organisms with comparable potential. Diethanolamine is stoichiometrically produced by the pure culture of microorganisms growing with octadecylbis(2-hydroxyethyl)amine as sole source of carbon. Diethanolamine is readily biodegradable (van Ginkel and Kroon, 1993; Davis and Carpenter, 1997). Moreover, Williams and Callely (1982) isolated a bacterium capable of converting diethanolamine into biomass, carbon dioxide, water and ammonia. A consortium of microorganisms degrading the alkyl chain of alkylbis(2-hydroxyethyl)amines and diethanolamine are therefore capable of complete (ultimate) degradation of alkylbis(2-hydroxyethyl)amines.

Based on the broad substrate specificity of micro-organisms degrading fatty amine derivatives with respect to the alkyl chain length it is unlikely that the biodegradability of these surfactants differs significantly with varying alkyl chain lengths. Biocidal effects explain negative results obtained in ready biodegradability tests.

The adequate ready biodegradability test results obtained and the scientific evidence that consortia of diethanolamine and alkyl-utilizing micro-organisms through a joint biodegradation pathway degrade all primary fatty amine ethoxylates lead to the conclusion that all primary fatty amine ethoxylates (alkylbis(2-hydroxyethyl)amines)are readily biodegradable.