N-{3-[C12-14-(even numbered)-alkyloxy]propyl}propane-1,3-diamine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

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

Deviations:

no

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

Deviations:

no

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)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge (10-Jan-2022) 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 101.8 °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 3.6 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 22.7 – 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 1.3 .10^6 CFU/L.

Duration of test (contact time):

60 d

Initial conc.:

2 mg/L

Based on:

test mat.

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, 27.50 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification that is not related to the biodegradation of the test substance.
The test substance was added to the bottles using an aqueous stock emulsion of 1 g/L. A homogenous emulsion of the test substance was prepared by stirring the test substance in demineralized water and adjusting the pH to 8.0 using a 2M HCl solution. Humic acid was added from a 1 g/L stock solution as sorbent for detoxification of the test substance. The reference substance sodium acetate was added to the bottles using an aqueous stock solution of 1.0 g/L. Next the bottles were filled with nutrient medium with inoculum and closed.

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 with test substance and inoculum; 10 bottles only containing inoculum; 10 bottles with humic acid, test substance and inoculum; 10 bottles containing humic acid and inoculum; and 6 bottles with sodium acetate and inoculum.
The concentrations of the test substance, humic acid, and sodium acetate in the bottles were 2.0 mg/L, 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. 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 at day 42 and 60 using the bottles of day 28 and 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).

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

3

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

12

Sampling time:

60 d

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

2

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

10

Sampling time:

60 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

41

Sampling time:

60 d

Remarks on result:

other: activated slude + humic acid

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

9

Sampling time:

28 d

Remarks on result:

other: activated slude + humic acid

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

46

Sampling time:

60 d

Remarks on result:

other: activated slude + humic acid

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

11

Sampling time:

28 d

Remarks on result:

other: Activated sludge + humic acid

Theoretical oxygen demand (ThOD)

The average molecular formula of the analyzed constituents (constituents analyzed up to 99.6 % w/w according to CoA Annex II) of the test substance was used as representative for all organics (99.8 % w/w) present in the test substance (based on 0.2 % w/w water content in the test substance). The ThOD_NH3 and ThOD_NO3 of the test substance calculated from this molecular formula and used to calculate the biodegradation percentages is 2.81 and 3.20 g oxygen/g test substance, respectively. 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 is optional in the OECD guideline and was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition can be detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of the test substance (higher oxygen concentration in bottles with test substance compared to the concentration in the control bottles). A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7 when the test substance was dosed without humic acid (Table II). The test substance dosed together with the humic acid, added for detoxification of the test substance, did not show an inhibition of the endogenous respiration. The "high" initial test substance concentration (2 mg/L) may therefore have hampered the biodegradation in the test without addition of humic acid however no inhibition of the biodegradation is expected in the test supplemented with humic acid (Table II).

 

Test conditions

At the start of the test the pH of the media in the duplicate control, reference substance, control with humic acid, test substance and test substance with humic acid bottles was 6.9. The pH of the medium in the duplicate reference bottles measured at day 14 was 7.0. The pH of the medium in the duplicate bottles at day 60 was 7.1 for the control, the control with humic, the test substance, and test substance with humic acid bottles. The temperature ranged from 22.7 to 22.9 °C which is within the prescribed temperature range of 20 to 24°C.

 

Validity of the test

The validity of the test is demonstrated by an endogenous respiration of 0.90 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 84% (Table II and Figure 1 and 2). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

 

Biodegradability

C12-C14 alkyletherdiamine tested without addition of humic acid was biodegraded by 3% and 12% (based on ThOD_NH3) at day 28 and 60 in the Closed Bottle test, respectively (Table II, Figure 1 and 2). The biodegradation of the test substance in the absence of humic acid starts ~day 60 whereas in the test with humic acid addition the biodegradation starts ~day 28 (Table II). The slower start of the biodegradation also suggests that the biodegradation in the test without humic acid addition was hampered by the initial high test substance concentration (2 mg/L). C12-C14 alkyletherdiamine dosed with humic acid for detoxification was biodegraded by 11% (based on ThOD_NH3) at day 28 in the Closed Bottle test (Table II, Figure 1). The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 46% at day 60 (Table II, Figure 1). Assuming complete nitrification, and calculating the biodegradation based on the ThOD_NO3 the test substance was biodegraded by 41% in the Closed Bottle test at day 60 (Table II, Figure 2). Biodegradation percentages between 20% and 60% do not allow a classification as not persistent.

However, the biodegradation curve did not level off at day 60 suggesting that the biodegradation of C12-C14 alkyletherdiamine continues (Figure 1 and 2). In addition, the lack of complete biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

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

Time (days)	Oxygen concentration (mg/L)
	Mc	Mt	Ma	Mch	Mth
0	9.0	9.0	9.0	9.0	9.0
	9.0	9.0	9.0	9.0	9.0
Mean (M)	9.00	9.00	9.00	9.00	9.00
7	8.6	8.7	4.4	8.5	8.5
	8.6	8.7	4.3	8.5	8.5
Mean (M)	8.60	8.70	4.35	8.50	8.50
14	8.4	8.2	4.0	8.3	8.0
	8.4	8.5	4.0	8.3	8.1
Mean (M)	8.40	8.35	4.00	8.30	8.05
21	8.2	8.1		8.1	7.9
	8.2	8.1		8.1	7.9
Mean (M)	8.20	8.10		8.10	7.90
28	8.1	8.0		8.0	7.7
	8.1	7.9		8.0	7.1
Mean (M)	8.10	7.95		8.00	7.40
42	7.9	7.6		7.8	5.8
	7.9	7.8		7.8	7.3
Mean (M)	7.90	7.70		7.80	6.55
60	7.8	7.2		7.7	4.9
	7.8	7.1		7.8	5.4
Mean (M)	7.80	7.15		7.75	5.15

Mc                        Mineral nutrient solution with only inoculum.

M_t               Mineral nutrient solution with inoculum and test substance (2.0 mg/L)

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

M_ch             Mineral nutrient solution with inoculum and humic acid (2 mg/L)

M_th      Mineral nutrient solution with inoculum, test substance (2.0 mg/L) and humic acid (2.0 mg/L)

 

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

Biodegradation of the test substance with and without addition of humic acid is calculated both without a correction for the nitrification (BOD/ThOD_NH3) and with a nitrification correction (BOD/ThOD_NO3).

Time (days)	Oxygen consumption (mg/L)			Biodegradation (%)
	Test substance	Test substance + humic acid	Acetate	Test substance		Test substance + humic acid		Acetate
				ThODNH3	ThODNO3	ThODNH3	ThODNO3
0	0.00	0.00	0.00	0	0	0	0	0
7	-0.10	0.00	4.25	-2	-2	0	0	81
14	0.05	0.25	4.40	1	1	4	4	84
21	0.10	0.20		2	2	4	3
28	0.15	0.60		3	2	11	9
42	0.20	1.25		4	3	22	20
60	0.65	2.60		12	10	46	41

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Remarks:

(11% after 28 days); but (41 % after 60 days- prolonged exposure): further degradation expected because a plateau has not been reached.

Conclusions:

Under the study conditions, the test substance was determined to be inherently biodegradable with 41% biodegradation (based on ThODNO3) after 60 days.

Executive summary:

To assess the biotic degradation of C12-C14 alkyletherdiamine 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 substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles supplemented with 2 mg/L humic acid for detoxification of the test item, and incubated in the dark at 22.7 to 22.9 °C for 60 days.

The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, the test substance did not cause an inhibition of the biodegradation when tested in the presence of humic acid. C12-C14 alkyletherdiamine dosed with humic acid for detoxification was biodegraded by 11% (based on ThODNH3) at day 28 in the Closed Bottle test. The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 46% at day 60. Assuming complete nitrification, and calculating the biodegradation based on the ThODNO3 the test substance was biodegraded by 41% in the Closed Bottle test at day 60. Biodegradation percentages between 20% and 60% do not allow a classification as not persistent. However, the biodegradation curve did not level off at day 60 suggesting that the biodegradation of C12-C14 alkyletherdiamine continues. In addition, the lack of complete biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

The test is valid as shown by an endogenous respiration of 0.90 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 84% 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

Etherdiamine C12-14 is a cationic surfactant, which was found to be toxic to the activated sludge bacteria used for the inoculum. To mitigate this toxicity 2 mg/L humic acid found to be necessary. 

In the ready test, the test substance is not found to be readily biodegradable as only 9% (based on ThODNO3) biodegradation was obeserved after 28 days. After 60 days 41% (based on ThODNO3) biodegradation was observed. Within a second screening test according to OECD 301D the test was prolonged to 112 days and after 112 days 60% (ased on ThODNO3, 2 mg/L humic acid) biodegradation was observed indicating that the substance is ultimately biodegradable and based on these results not considered to be persistent in the aquatic compartment. 

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater

Additional information

Supporting study: A preliminary non-GLP supporting study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the test substance, etherdiamine C12-14 (99.8% active, EC 701-437-6), according to OECD Guideline 301D. Due to the well-known toxicity of the this etherdiamine, the test substance was evaluated using detoxification methods through the addition of humic acid at one concentration. Activated sludge or river water was used as inoculum in the Closed Bottle test. In addition, a sorbent free test group without any deviations from the guideline was included as a ‘negative control’ and to demonstrate the toxicity of the test substance and to demonstrate the positive detoxifying effects of the sorbents. Ammonium chloride was omitted from the medium to prevent nitrification for all groups except the sorbent free group. The tests were performed in triplicates using 0.3 L BOD bottles with glass stoppers. The test substance was dosed to the bottles using an aqueous stock emulsion of 1 g/L. A homogenous emulsion of the test substance was prepared by stirring the test substance in demineralized water and adjusting the pH to 8.0 using a 2M HCl solution. Humic acid was added from a 1 g/L stock solution as sorbent for detoxification of the test substance. The tests were performed in 0.3 L BOD bottles with glass stoppers. Two series of tests were performed. In the first series activated sludge and river water were used as inoculum and no humic acid was added to the bottles for detoxification. In the second series only activated sludge was used as inoculum and test with ant without the addition of humic acid were started. In both series use was made of 3 control bottles containing only respective inoculum and 3 test bottles with test substance and the respective inoculum. Humic acid was dosed both to the control and test bottle when added for detoxification of the test substance.
The concentrations of the test substance and (if applicable) humic acid in the bottles were 2.0 mg/L. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium. Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of the media was 7.1 (activated sludge) and 8.0 (river water) at the start of the test. The pH was 7.1±0.3 (activated sludge) and 8.0±0.2 (river water) at day 112. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected in all tests. The "high" initial test substance concentration may therefore have hampered the biodegradation in the tests even when humic acid was dosed for detoxification.

The ThODNH3 and ThODNO3 of the test substance calculated from the average molecular formula and used to calculate the biodegradation percentages was 2.81 and 3.20 g oxygen/g test substance, respectively.
Biodegradation of <60% were found within four weeks for both inocula and for the tests with and without addition of humic acid. The test substance is therefore considered to be not readily biodegradable. Biodegradation percentages of <60% were still found in all tests at day 56 demonstrating that C12-C14 alkyletherdiamine cannot be classified as not persistent. Biodegradation percentages ≥60, demonstrating the slow mineralization of the test substance, were however achieved for C12-C14 alkyletherdiamine after day 56 in the tests with activated sludge as inoculum and with and without the addition of humic acid for detoxification. 
Biodegradation of C12-C14 alkyletherdiamine starts in all tests after a long lag-phase which ranges from 42-84 days. The start of the biodegradation in the ready biodegradation test is possible hampered by some toxicity of the initial high test substance concentration. Detoxification of the initial high test substance concentration by addition of humic acid seems partly successful as the biodegradation in the tests with humic acid addition starts a bit earlier compared to the test without. Variation in the activated sludge inoculum might however have a larger effect on the duration of the lag time in the ready biodegradation test. For the main test the use of activated sludge as inoculum and test with and withou the addition of humic acid for detoxification for the main study was considered (Geerts, 2022).  

 

Key study:

The study was performed with the structural analogue etherdiamine C12-14 (99.8% active, EC 701-437-6) within read-across. The test substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles supplemented with 2 mg/L humic acid for detoxification of the test item, and incubated in the dark at 22.7 to 22.9 °C for 60 days.

The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, the test substance did not cause an inhibition of the biodegradation when tested in the presence of humic acid. C12-C14 alkyletherdiamine dosed with humic acid for detoxification was biodegraded by 11% (based on ThODNH3) at day 28 in the Closed Bottle test. The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 46% at day 60. Assuming complete nitrification, and calculating the biodegradation based on the ThODNO3 the test substance was biodegraded by 41% in the Closed Bottle test at day 60. Biodegradation percentages between 20% and 60% do not allow a classification as not persistent. However, the biodegradation curve did not level off at day 60 suggesting that the biodegradation of C12-C14 alkyletherdiamine continues. In addition, the lack of complete biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

The test is valid as shown by an endogenous respiration of 0.90 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 84% 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.

 

Etherdiamine C12-14 acetate and Etherdiamine C12-14 are etheramine surfactants characterized by a hydrophobic moiety i.e. tridecyl and a structurally related hydrophilic moiety of dipropylenetriamine linked together with an ether bond. Biodegradation of surfactants refers to the reduction in complexity of the chemical through metabolic activity of microorganisms.If a surfactant is to serve as a carbon and energy source for aerobic microorganisms then it has to be converted into a form that can enter the central metabolism of microorganisms. 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. Many of these conversions are described in pathways for 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 (1920) first described this similarity known as the unity of biochemistry. This unity is the key to justification of the use of read-across.

In general, micro-organisms produce enyzmes, which can initiate degradation of surfactants at three locations i.e. both far-ends and central cleavage (van Ginkel, 1996). For ether diamines the cleavage at the ether bond, the ώ-oxidation of the far-end of the alkyl chain, and removal of propylamine from ether diamines are theoretically three important steps to degrade this type of surfactant.

 

Etherdiamine C12-14 (ace.) is therefore expected to be not ready biodegradable. Indeed, etherdiamine C12-14 was biodegraded 9% (based on ThODNO3) at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test (enhanced biodegradability testing) a biodegradation percentage of 41% (based on ThODNO3) was achieved at day 60. This result could only be achieved by using humic acid with a concentration of 2 mg/L(Geerts et al, 2022).

Within a second screening test (supporting study) with etherdiamine C12-14 (CAS 68187-46-2) according to OECD 301D the test was prolonged to 112 days and after 112 days 60% biodegradation (based on ThODNO3, with 2 mg/L humic acid) was observed indicating that the substance is ultimately biodegradable and based on these results should be classified as NOT persistent because of the prolonged Closed Bottle test (enhanced test) result and the biodegradability of structurally related substances.