Quaternary ammonium compounds, benzyl-C16-18-alkyldimethyl, chlorides

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Remarks:

(preliminary screening study)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Preliminary test

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

preliminary test

Deviations:

yes

Remarks:

ommission of ammonium from test medium

GLP compliance:

no

Remarks:

Preliminary non-GLP study; main study has been planned to be conducted under GLP conditions.

Oxygen conditions:

aerobic

Inoculum or test system:

other: activated sludge, domestic, non-adapted and river water

Details on inoculum:

(a) Activated sludge wasobtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to 2.0 mg DW/in the biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992).
(b) River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The river water spiked with mineral salts of the nutrient medium was used undiluted.

The Colony forming units (CFU) of the preconditioned river water inoculum and diluted preconditioned activated sludge 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 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 – 22.9°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 bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively.

Duration of test (contact time):

ca. 42 d

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test procedures of Closed Bottle test
The Closed Bottle test was performed according to Test Guidelines (OECD 1992). The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. On request of the sponsor to perform these “negative control” tests without deviations from the guideline 0.5 mg/L ammonium chloride was included in the sorbent free tests. Test substance (solvent free) and humic acid were dosed using an aqueous stock solution of 1 g/L in water. Isopropanol was dosed from a 0.1 g/L stock solution in demiwater. The tests were performed in 0.3 L BOD bottles with glass stoppers. In the tests without sorbent use was made of 3 bottles with the test substance and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 µg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance). In the sorbent modified tests use was made of 3 bottles containing the test substance, the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 µg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance), and silica gel or humic acid. The tests without sorbent were used to demonstrate the toxic effects of the test substance to the inoculum in the Closed Bottle test and to illustrate the positive detoxifying effects of the sorbents. Silicagel and humic acid concentrations in the bottles (test and control) were 1 and 2 g /bottle and 1 and 2 mg acid/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 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).

Analyses
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using a EUTECH instruments pH meter. The temperature was measured and recorded with a thermo couple connected to a data logger. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a pre-weighed 12 um cellulose nitrate filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. The dry weight was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

12

Sampling time:

28 d

Remarks on result:

other: activated sludge as inoculum and without sorbent

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

59

Sampling time:

28 d

Remarks on result:

other: 66% after 42 d; (using activated sludge as inoculum and 2 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

64

Sampling time:

28 d

Remarks on result:

other: 68% after 42 d; (using activated sludge as inoculum and 1 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

58

Sampling time:

28 d

Remarks on result:

other: 62% after 42 d; (using activated sludge as inoculum and 2 mg/L humic acid / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

56

Sampling time:

28 d

Remarks on result:

other: 62% after 42 d; (using activated sludge as inoculum and 1 mg/L humic acid / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

-1

Sampling time:

28 d

Remarks on result:

other: river water as inoculum and without sorbent

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

54

Sampling time:

28 d

Remarks on result:

other: 57% after 42 d; (using river water as inoculum and 2 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

55

Sampling time:

28 d

Remarks on result:

other: 63% after 42 d; (using river water as inoculum and 1 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

56

Sampling time:

28 d

Remarks on result:

other: 65% after 42 d; (using river water as inoculum and 2 mg/L humic acid / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

52

Sampling time:

28 d

Remarks on result:

other: 63% after 42 d; (using river water as inoculum and 1 mg/L humic acid / bottle for detoxification)

Results

Test conditions

The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period and by differences of less than 20% for the triplicate values at day 28. Furthermore, the endogenous respiration at day 28 was <1.5 mg/L in the tests were ammonium chloride was omitted from the medium. In the tests with ammonium chloride in the medium (the “negative control” test) the endogenous respiration was 1.5 and 1.6 mg/L for the activated sludge and river water inoculum, respectively. The pH of the media was 7.4 (activated sludge) and 8.2 (river water) at the start of the test. The pH was 7.2±0.1 (activated sludge) and 8.0±0.1 (river water) at day 42. 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. Hampering of the biodegradation by inhibition of the endogenous respiration of the inoculum was clearly detected until day 21-28 in the sorbent free ready biodegradation tests. Silica gel and humic acid were added as sorbent for detoxification of the test substance. Detoxification by the sorbents in the closed bottle tests was successful except for the 1 mg/L humic acid sorbent which still showed an inhibition of the endogenous respiration at day 7. No inhibition of the biodegradation due to the “high” initial test substance concentration is therefore expected in the presence of the sorbents: silica gel (1 and 2 g/bottle) and humic acid (2 mg/L).

The Closed Bottle test results

The ThOD_NH3 and ThOD_NO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.89 g/g and 3.05 g/g, respectively. In OECD 301 tests growth-linked biodegradation takes place. This means that carbon and nitrogen is built into microorganisms (new biomass). Calculating the biodegradation by using the ThOD_NO3 assumes that all the organic nitrogen present in a test substance is transiently degraded to ammonium nitrogen and subsequently oxidized to nitrate. The C:N ratio of test substance (solvent free) is ~26:1. In the sorbent modified tests the organic nitrogen of the test substance is the only nitrogen present for growth due to the omission of ammonium nitrogen from the nutrient medium. With a C:N ratio of about 7:1 for growth of biomass (not exact) this would mean that most likely all the organic nitrogen present in the test substance will be built into new biomass. No oxidation of test substance nitrogen to nitrate is therefore expected in the CBT. The use of ThOD_NH3 can however only be justified if the low nitrate/nitrite concentrations in the CBT can be demonstrated by analysis. However, analysis is probably not accurate enough to demonstrate this small possible increase in nitrate/nitrite concentrations (maximum theoretical amount of NO₃formed from organic nitrogen present in the test substance is 0.3 mg/L and the “background” NO₃concentration in CBT blank with activated sludge and river water is ~0.7 mg/L and ~10 mg/L, respectively). The biodegradation of test substance (solvent free) is for this reason calculated with the “worst-case approach” assuming a complete oxidation of the organic nitrogen to nitrate (using the ThOD_NO3). The biodegradation percentages at day 28 using activated sludge as inoculum were slightly higher compared to results achieved with river water. More than 60% biodegradation was still achieved within 28 days using activated sludge as inoculum. Test substance (solvent free) should therefore be classified as readily biodegradable. For the final GLP test it is recommended to use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the test substance.

Table I Percentages biodegradation of test substance (CAS 68607-20-5) solvent free in Closed Bottle tests inoculated with activated sludge and river water. Biodegradation percentages were calculated using the ThOD_NH3.

Inoculum	Sorbent	Biodegradation percentage at day
		7	14	21	28	42
Activated sludge	No sorbent*	-10	-15	-9	13	-
	2 g silica gel /bottle	2	33	47	62	69
	1 g silica gel / bottle	1	32	52	68	72
	2 mg/L humic acid	5	33	43	61	65
	1 mg/L humic acid	-4	38	53	59	65
River water	No sorbent*	-13	-20	-16	-1	-
	2 g silica gel /bottle	3	40	55	57	60
	1 g silica gel / bottle	1	39	51	58	67
	2 mg/L humic acid	2	32	44	59	69
	1 mg/L humic acid	-6	36	47	55	66

* NH₄Cl (0.5 mg/L) was included in the nutrient medium as prescribed in the OECD 301D guideline.

Table II Percentages biodegradation of test substance (CAS 68607-20-5) solvent free in Closed Bottle tests inoculated with activated sludge and river water. Biodegradation percentages were calculated using the ThOD_NO3.

Inoculum	Sorbent	Biodegradation percentage at day
		7	14	21	28	42
Activated sludge	No sorbent*	-10	-14	-8	12	-
	2 g silica gel /bottle	2	32	45	59	66
	1 g silica gel / bottle	1	31	49	64	68
	2 mg/L humic acid	5	31	41	58	62
	1 mg/L humic acid	-4	36	50	56	62
River water	No sorbent*	-12	-19	-15	-1	-
	2 g silica gel /bottle	3	38	52	54	57
	1 g silica gel / bottle	1	37	48	55	63
	2 mg/L humic acid	2	31	41	56	65
	1 mg/L humic acid	-6	35	45	52	63

* NH₄Cl (0.5 mg/L) was included in the nutrient medium as prescribed in the OECD 301D guideline.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the test substance was determined to be readily biodegradable following the use of sorbent material which reduces the toxicity of the test substance to the inoculum.

Executive summary:

A preliminary non-GLP study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the test substance, C16-18 ADBAC (95.2% active), according to the OECD Guideline 301D. Due to the well-known toxicity of the quaternary substances, the test substance was evaluated using detoxification methods through the addition of the sorbents such as silica gel and humic acid at two different concentrations and two different inocula (activated sludge, river water).In addition, a sorbent free test group without any deviations from the guideline was also 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 inoculum concentration in the bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively. The tests were performed in triplicates using 0.3 L BOD bottles with glass stoppers. In the tests ‘without sorbent’ use was made of 3 bottles with the test substance (at 2 mg/L) and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 μg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the test substance (at 2 mg/L), the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 μg/L isopropanol, and silica gel or humic acid. Silicagel and humic acid concentrations in the bottles (test and control) were 1 and 2 g /bottle and 1 and 2 mg acid/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 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. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The theoretical oxygen demand (ThOD) of test substance was calculated from its molecular formula and molecular weight. The BOD (mg/mg) of the test substance was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle.

The ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.89 g/g and 3.05 g/g, respectively. The biodegradation percentages at Day 28 using activated sludge as inoculum were slightly higher compared to results achieved with river water. Using the conservative ThODNO3 to calculate the biodegradation of test substance still >60% biodegradation was achieved within 28 days using activated sludge as inoculum and 1 g silica gel / bottle for detoxification. 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 7.4 and 7.2±0.1 (activated sludge) and 8.2 and 8.0±0.1 (river water) at the start and end of Day 42 of the test respectively. 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 and this was clearly detected until day 21-28 in the sorbent free ready biodegradation tests. Silica gel and humic acid were added as sorbent for detoxification of the test substance. Detoxification by the sorbents in the closed bottle tests was successful except for the 1 mg/L humic acid sorbent which still showed an inhibition of the endogenous respiration at day 7. No inhibition of the biodegradation due to the “high” initial test substance concentration is therefore expected in the presence of the sorbents: silica gel (1 and 2 g/bottle) and humic acid (2 mg/L). Under the study conditions, the test substance was determined to be readily biodegradable and the use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the test substance was considered further for the main study (Geerts, 2020).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 Feb 2020 to 16 Mar 2020

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)

Deviations:

yes

Remarks:

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:

other: activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge (10-02-2020) 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 103.9 °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 22.7 – 22.9 °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 1E+6 CFU/L.

Duration of test (contact time):

ca. 28 d

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Reference substances and chemicals
The silica gel Davisil grade 636, pore size 60A, 35-60 mesh particle size (MKCH4201) was purchased from Sigma-Aldrich. Isopropanol analytical grade (lot 1913436) was obtained from Fisher Chemicals. All other chemicals used were of reagent grade quality.

Deionized water
Deionized water containing <1.0 mg/L of organic carbon was prepared in a water purification system.

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 8.51 mg KH2PO4, 21.75 mg K2HPO4, 33.42 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. The test substance, sodium acetate and isopropanol were added to the bottles using aqueous stock solution of 1 g/L, 1 g/L and 0.1 g/L, respectively. Silica gel was added as sorbent in the test bottles for detoxification of the test substance at a concentration of 1 g silica gel / bottle. 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 containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. The concentration isopropanol added to the control bottles with silica gel was 35 µg/L which is comparable to the isopropanol content present in the test bottles. 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.

Analyses
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using an Eutech pH meter. The temperature was measured and recorded with a sensor connected to a data logger.

Reference substance:

benzoic acid, sodium salt

Remarks:

Purity: 99.9%, Batch/lot number: BCBP8197V, Appearance: white powder

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

61

Sampling time:

28 d

Remarks on result:

other: using activated sludge as inoculum and 1 g silica gel / bottle for detoxification

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

65

Sampling time:

28 d

Remarks on result:

other: using activated sludge as inoculum and 1 g silica gel / bottle for detoxification

Results

Theoretical oxygen demand (ThOD)

The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.89 and 3.05 g oxygen/g active ingredient, respectively. These ThODs were calculated with an average molecular formula of the active ingredient (of the test substance) using an average alkyl chain length. The average alkyl chain length is calculated from the alkyl chain distribution. 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 in the presence of silica gel sorbent was detected at day 7. Therefore, it is expected that the biodegradation in the first week of the test was hampered due to the "high" initial test substance concentration.

Test conditions

The pH of the media for the control, control with silica gel, reference and test substance was 7.3 at the start of the test. The pH of the medium in the reference bottles measured at day 14 was 7.2. The pH of the medium at day 28 was 7.0 for the test and 7.1 for the control and control with silica gel. The temperature ranged from 22.7 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. 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 75. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability

Solvent free test substance was biodegraded by 65% (based on ThODNH3) at day 28 in the Closed Bottle test. 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. Solvent free test substance is classified as readily biodegradable based on the >60% biodegradation reached at day 28.

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

Time (days)	Oxygen concentration (mg/L)
	Oc	Oa	Ocs	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	4.5	8.3	8.6
	8.5	4.6	8.4	8.5
Mean (M)	8.50	4.55	8.35	8.55
14	8.0	4.2	8.0	7.1
	8.1	4.1	8.0	7.0
Mean (M)	8.05	4.15	8.00	7.05
21	8.0		8.0	5.6
	8.0		8.0	5.8
Mean (M)	8.00		8.00	5.70
28	7.8		7.8	4.3
	7.8		7.8	4.2
Mean (M)	7.80		7.80	4.25

O_c Mineral nutrient solution with only inoculum.

O_cs Mineral nutrient solution with inoculum, silica gel and isopropanol

O_t Mineral nutrient solution with inoculum, test substance (2.0 mg/L test substance = 1.90 mg/L active ingredient) and silica gel

O_a 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/ThOD_NH3) and with nitrification (BOD/ThOD_NO3) 

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance		Acetate
			ThODNH3	ThODNO3
0	0.00	0.00	0	0	0
7	-0.20	3.95	-4	-3	76
14	0.95	3.90	17	16	75
21	2.30		42	40
28	3.55		65	61

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the test substance was determined to be readily biodegradable with 61% biodegradation after 28 days.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, C16-18 ADBAC (95.2% active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the domestic wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. The measured dry weight of the inoculum was 3.2 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles. The inoculum concentration in the BOD bottles determined by colony count was 1E+6 CFU/L. 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 1 g silica gel/bottle as sorbent for detoxification of the test substance, and incubated in the dark at 22.7 to 22.9°C for 28 days. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. The concentration isopropanol added to the control bottles with silica gel was 35 µg/L which is comparable to the isopropanol content present in the test bottles. Each of the prepared solutions was dispensed into the respective group of biochemical oxygen demand (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. Endogenous respiration, theoretical oxygen demand (ThOD), biochemical oxygen demand (BOD) and biodegradation were calculated. The degradation of the test substance was assessed by the measurement of oxygen consumption. The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.89 and 3.05 g oxygen/g active ingredient, respectively. The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate. 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. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected. The validity of the test is demonstrated by an endogenous respiration of 1.10 mg/L at Day 28. 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 75%. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period. Solvent free test substance was biodegraded by 65% (based on ThODNH3) at Day 28 in the Closed Bottle test. 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. Under the study conditions, the test substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020).

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

acceptable deviations

Principles of method if other than guideline:

Two minor deviations from the guidelines of the Closed Bottle test were introduced; a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), b) humic acid (4.0 mg/L) was added to reduce the toxicity of the test substance in the test.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands (04-02-2010). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation.

Although the specific inoculumn concentration was not determined in the study, this is not expected to impact the validity of the study due to the below reason:

The concentration of the incolumn can be justified to be small, based on the fact that the test substance fulfills the validity criteria ≤ 1.5 mg/L oxygen consumption in the control group at Day 28 in the Closed Bottle test. This criteria fulfilment indicates low endogenous respiration, which is due to presence of small amount of inoculum. Further, the river water was used undiluted and was not pre-exposed to the test substance after sampling, which is another critical pre-requisite for ready biodegradation tests with regard to the inoculum.

Duration of test (contact time):

ca. 28 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 stock solutions
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Sodium acetate and
the test substance were added to the bottles using stock solution and suspension of 1.0 g/L.

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan was develo¬ped from ISO Test Guidelines (1994). Use was made of 10 bottles containing only river water (inoculum and medium), 10 bottles containing river water and humic acids (4 mg/L), 10 bottles containing
river water, the test substance and humic acid, and 6 bottles containing sodium acetate and river water The concentrations of the test substance
(active) and sodium acetate in the bottles were 2.0 (1.6) 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.

Calculation of the results

Calculation of endogenous respiration
The endogenous respiration (oxygen depletion in the control) was calculated as follows;
Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28)
Mc is the mean oxygen level in the control bottle inoculated with river water.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of the test substance, and sodium acetate were calculated from their molecular formulae and molecular weights
The calculated theoretical oxygen demand of the test substance is 2.5 mg/mg. The theoretical oxygen demand of sodium acetate is 0.8 mg/mg.
Calculation of the biochemical oxygen demand (BOD)
Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference
compound bottles were calculated as follows:
Oxygen consumptionn (mg/L) by test substance = Mch - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or ch = the mean oxygen level in the control bottles inoculated with river water, n days after the start of the test (c) or the control
bottles with humic acid (ch).
Mt or a = the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a),
and inoculated with river water, n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test substanceand sodium acetate was calculated by dividing the oxygen consumption by the
concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

64

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

60

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Details on results:

Theoretical oxygen demand (ThOD)
The calculated theoretical oxygen demand of the test substance is 2.5 mg/mg. The theoretical oxygen demand of sodium acetate is 0.8 mg/mg.

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 substance to microorganisms degrading acetate is not considered to be relevant. Humic acid was added to the bottles with the test substance, because this substance may be toxic to the competent bacteria. Inhibition of the endogenous respiration of the inoculum by the test substance in the presence of humic acid was not detected. Inhibition is usually detected prior to the onset of the biodegradation of the test substance through suppression of the endogenous respiration (lower oxygen consumption in the presence of a test substance as compared to the control). The biodegradation of the test substance was already initiated at day 7. Therefore, no inhibition of the biodegradation due to the presence of the test substance is expected.

Test conditions
The pH of the media was 8.0 at the start of the test. The pH of the medium at day 28 was 7.9 (control and test), 8.0 (control with humic acid).
Temperatures were within the prescribed temperature range of 22 to 24°C.

Validity of the test
The validity of the test is demonstrated by the following findings:
• Endogenous respiration of 1.4 mg/L at day 28 (see below Table I) indicating viability of microorganuisms
• Differences between the replicate values at day 28 less than 20%
• 87 % biodegradation of the reference compound, sodium acetate, at day 14
• Oxygen concentrations >0.5 mg/L in all bottles during the test period.

Results with reference substance:

The reference substance showed 85% degradation by Day 7 (see below table II)

Details on results:

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

Time (days)	Oxygen concentration (mg/L)
	Och	Ot	Oc	Oa
0	8.8	8.8	8.8	8.8
	8.8	8.8	8.8	8.8
Mean (M)	8.8	8.8	8.8	8.8
7	7.7	6.3	7.9	3.3
	7.9	6.1	7.8	3.2
Mean (M)	7.8	6.2	7.9	3.3
14	7.4	5.6	7.5	2.7
	7.4	5.7	7.4	2.8
Mean (M)	7.4	5.7	7.5	2.8
21	7.3	4.9	7.4
	7.3	4.8	7.4
Mean (M)	7.3	4.9	7.4
28	7.4	4.2	7.3
	7.4	4.1	7.4
Mean (M)	7.4	4.2	7.4

O_ch     Mineral nutrient solution without test material but with inoculum and humic acid.

O_t       Mineral nutrient solution with test material (2.0 mg/L), humic acid, and inoculum.

O_c       Mineral nutrient solution with only inoculum.

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

 

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

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	1.6	4.6	33	85
14	1.7	4.7	38	87
21	2.4		48
28	3.2		64

Influence of 2 -propanol presence in the test sample:

With regard to the influence of 2-propanol on biodegradation, the Theoretical Oxygen Demand (ThOD) equation currently used in the study report considers all the constituents by multiplying their respective weight percentages with their respective ThODs.Therefore, the overall percentage biodegradation calculation for the test substance does take into account possible oxygen consumption due to the biodegradation of 2-propanol, therefore not overestimating the biodegradability of the active. Please refer to the below section for the ThOD and percentage biodegradation calculations Table as well as for the justification for using ThOD_NH3equation.

The active substance is marketed with co-solvents because this improves handling. Easy handling allows a more accurate administration of the test substance.  

The possible influence due to presence of alcohol on the biodegradability of the quaternary ammonium compound is considered insignificant because 2-propanol and the quaternary ammonium compound are degraded by different sets of micro-organismsfrom the test substance. The inoculum used in the biodegradation tests are derived from the environment, which contain numerous microbial species degrading complex mixtures of chemicals. Most of these species are specialists only capable of degrading a limited number of organic substances. It is therefore very unlikely that ethanol and alkylbenzyldimethylammonium salts will be degraded by the same species.   

Influence of omission of ammonia from the test medium:

Ammonium chloride is omitted from the test medium to prevent oxygen consumption by nitrifying bacteria. The reason for this omission is to lower the endogenous oxygen consumption in the BOD bottles, thereby increasing the accuracy of the biodegradation assessment. This is reflected in the validity criterion of less than 1.5 mg/L of oxygen consumption in the inoculum control bottles at Day 28. Omission of ammonium should not hamper the biodegradation of organic compounds in the Closed Bottle Test. The biodegradation of the reference substance (sodium acetate) demonstrates that nitrogen is not limiting growth and that the nitrogen introduced with the inoculum is sufficient to fulfill the nitrogen requirement of the microorganisms.

Nitrification of the nitrogen present in the test substance itself could occur. This could be a reason for using the ThOD_NO3instead of the ThOD_NH3 to assess biodegradation. In the present case, using the ThOD_NO3of 2.66 g/g would result in a biodegradation percentage of 60.1, allowing classification of the substance as readily biodegradable.

	Molecular formula	MW	ThODNH3 (g/g)	ThODNO3(g/g)	Weight (%)
C16-18 ADBAC (chosen n=C17)	C26H48NCl	410.13	2.89	3.04	0.78
2-propanol	C3H8O	60.1	2.40	2.40	0.12
Water	H2O		0	0	0.08
The ThODNH3of the test substance is (0.78 x 2.89) + (0.12 x 2.40) + (0.08 x 0.00) =					2.54
The ThODNO3of the test substance is (0.78 x 3.04) + (0.12 x 2.40) + (0.08 x 0.00) =					2.66

 

Day	O2 consumption	BOD	ThODNH3	% biodegradation	ThODNO3	% biodegradation
7	1.6	0.8	2.54	31.5	2.66	30.1
14	1.7	0.85		33.5		31.9
21	2.4	1.2		47.3		45.1
28	3.2	1.6		63.0		60.1

Test conc: 2 mg/L

However, the use of ThOD_NO3is not obligatory for all nitrogen-containing test substances. The choice of the ThOD used to estimate biodegradation should not be based on possible formation of nitrite or nitrate. Tests of the OECD 301 series were developed to assess the biodegradability and mineralization of organic substances. Nitrogen-containing substances are biodegraded in ready biodegradability tests by heterotrophic micro-organisms capable of utilizing these substances as carbon and energy source. This usually results in the formation of biomass (growth), water, carbon dioxide and ammonium (mineralization). The ammonium formed may subsequently be oxidized by nitrifying bacteria. These nitrifying bacteria utilizing ammonium as energy source and carbon dioxide as carbon source (autotrophic growth) are not involved in the biodegradation of nitrogen-containing substances. Biodegradation percentages calculated with the ThOD_NH3therefore do represent the biodegradability and mineralization of most nitrogen-containing substances. The formation of nitrite and nitrate during the degradation of organic substances is rare and only occurs when organic nitrogen is for example present in the form of a nitro group. Organic nitrogen is always liberated by microorganisms as ammonium when nitrogen is present as primary amine (amino group), secondary amine group, tertiary amine or quaternary ammonium group.

The test substance, C16 -18 ADBAC has a quaternary ammonium group. Benzyldimethylamine was found as first metabolite of alkylbenzyldimethylammonium salts degradation byAeromonas hydrophila, activated sludge and aPseudomonas spp. community (Patrauchan and Oriel, 2003; van Ginkel 2004; Tezelet al.,2012). Alkyl-N fission is therefore the most probable strategy to gain access to the carbon of the alkyl chain. The next step in the biodegradation of these quaternary ammonium compounds was the successive removal of the two methyl groups. Benzylamine formed, in turn, is converted into benzaldehyde and ammonium (Patrauchan and Oriel, 2003). In contrast to the alkylbenzyldimethylammonium salts biodegradation pathway reported by Patrauchan and Oriel, (2003), neither benzylmethylamine nor benzylamine were identified as metabolites by two enrichment cultures. Exposure of activated sludge to decylbenzyldimethyl­ammonium chloride probably selects for three microorganisms that utilize the alkyl chain, the aromatic moiety, and dimethylamine (van Ginkel, 2004). Kinetic assays demonstrated that benzylmethylamine and benzylamine were not intermediates of alkylbenzyldimethylammonium salts transformation by the enrichedPseudomonasspp. community (Tezelet al., 2012). Thus, benzyldimethylamine is thought to be transformed to dimethylamine and benzoic acid via debenzylation. Dimethylamine is degraded by the successive removal of both methyl groups resulting in the formation of ammonium (Large, 1971). Both the pure and mixed culture studies showed that the degradation of the alkyl chain of alkylbenzyldimethylammonium chloride results in the formation of water, carbon dioxide and ammonium (Please refer to the figure in the attachment).

Alkylbenzyldimethylammonium salts are biodegraded by microorganisms first utilizing the alkyl chain. Subsequently, the methyl and benzyl groups are removed.  In conclusion, estimation of biodegradation based on the ThOD_NH3is thought to be a more appropriate choice for the test substance.

References

• Van Ginkel CG (2007). Ultimate biodegradation of ingredients of cleaning agents. In: Handbook of Cleaning Agents/Decontamination of Surfaces, Eds. I Johansson and P Somasundaran, Elsevier Amsterdam, The Netherlands. Volume 2: 655-694.
• Large P(1971). The oxidative cleavage of alkyl nitrogen bonds in microorganisms. Xenobiotica 1:457-467.
• Patrauchan MA and Oriel PJ (2003). Degradation of benzyldimethylalkylammonium chloride byAeromonas hydrophihlasp K. J. Appl.Microbiol. 94:266-272.
• TezelU,TandukarM, MartinezRJ,SobeckyPA, andPavlostathisSG(2012).Aerobic biotransformation ofn-tetradecylbenzyldimethylammonium chloride by an enrichedPseudomonasspp.Community.Environ. Sci. Technol.46(16):8714–8722.

 

 

Validity criteria fulfilled:

yes

Remarks:

Endogenous respiration of 1.4 mg/L at day 28. Differences between the replicate values at Day 28 less than 20%. 87% biodegradation of the reference compound, sodium acetate, at Day 14. Oxygen concentrations >0.5 mg/L in all bottles during the test.

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the test substance was determiend to be readily .

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, C16-18 ADBAC (78% active), in water according to OECD Guideline 301, in compliance with GLP.The test was performed with river water receiving domestic waste water as incolumn into 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 10 bottles containing river water and humic acids (4 mg/L), 10 bottles containing river water with the test substance and humic acid 6 bottles containing river water and sodium acetate. The concentrations of the test substance and sodium acetate in the bottles were 2 mg/L (1.6 mg a.i./L) and6.7 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. The test was found to be valid as shown by an endogenous respiration of 1.4 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 day. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability was determined to be 64% and 60% within 28 days using ThODNH3 and ThODNO3 equations respectively. Therefore the substance can be considered readily biodegradable in water. Furthermore, the test substance did not cause a reduction in the endogenous respiration under the chosen test conditions; as such, hence was considered to be non-inhibitory to the inoculum. The 10-day window pass criterion is not applicable for UVCB surfactant substances. Under the study conditions, the test substance can be considered to be readily biodegradable (van Ginkel, 2010).

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

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

Adequacy of study:

supporting study

Study period:

From October 4, 2005 to November 02, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Qualifier:

according to guideline

Guideline:

EU Method C.6 (Degradation: Chemical Oxygen Demand)

Version / remarks:

Degradation-biotic degradation: Closed Bottle test

Deviations:

yes

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)

Deviations:

yes

Principles of method if other than guideline:

The test was modified to permit prolonged measurements (van Ginkel and Stroo 1992).

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, The Netherlands. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. A minor deviation of the test procedures described in the guidelines was introduced: instead of an effluent/extract/mixture, activated sludge was used as an inoculum. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to a concentration of 2 mg DW/L in the BOD bottles (van Ginkel and Stroo 1992).

Duration of test (contact time):

ca. 28 d

Initial conc.:

1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The oxygen concentration was measured with a special funnel which enabled testing without sacrificing bottles. This funnel exactly fitted into the BOD bottle. Subsequently, the oxygen electrode was inserted into 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 flew back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo, 1992). The oxygen concentrations were measured in quadruplicate bottles instead of the prescribed duplicate bottle to improve accuracy. Use was therefore made of 4 bottles containing only inoculum, 4 bottles containing test substance and inoculum, and 4 bottles containing sodium acetate and inoculum. The concentrations of the test substance and sodium acetate in the bottles were 1.0 and 6.7 mg/L, respectively. The inoculum was diluted to 2 mg DW/L in the closed bottles. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.

Reference substance:

acetic acid, sodium salt

Remarks:

concentration in the bottles: 6.7 mg/L

Preliminary study:

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 substance to microorganisms degrading acetate is not relevant. A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test substance is expected. This toxicity was the reason for testing at an initial test substance concentration of 1.0 mg/L.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

77

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

73

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Details on results:

The calculated theoretical oxygen demand of the test substance was 2.9 mg/mg. This theoretical oxygen demand is calculated by assuming formation of ammonium chloride.
The pH of the media was 7.0 at the start of the test. The pH of the medium at Day 28 was 6.8. Temperatures ranged from 19 to 21°C.

Key result

Parameter:

ThOD

Value:

ca. 2.9 other: mg O2/mg

Remarks on result:

other: (NH3)

Key result

Parameter:

ThOD

Value:

ca. 3.06 mg O2/g test mat.

Remarks on result:

other: (NO3)

Results with reference substance:

The ThOD of sodium acetate was 0.8 mg/mg.
The biodegradation percentage at Day 14 was 66%.

Validity of the test:

The validity of the test is demonstrated by an endogenous respiration of 1.1 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference substance, sodium acetate, at day 14 was 66. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period. Please refer to the tables appended under 'attached background materials'.

Lower test concentrations than the guideline:

The test substance was tested at 1 mg/L, due to toxicity of the substance on the inoculum which was demonstrated in the other biodegradation studies conducted with the test substance in the concentration range of 2-4 mg/L

Omission of ammonium from the test medium:

Ammonium chloride is omitted from the test medium to prevent oxygen consumption by nitrifying bacteria. The reason for this omission is to lower the endogenous oxygen consumption in the BOD bottles, thereby increasing the accuracy of the biodegradation assessment. This is reflected in the validity criterion of less than 1.5 mg/L of oxygen consumption in the control bottles at Day 28. Omission of ammonium is not considered to hamper the biodegradation of organic compounds in the Closed Bottle Test. The biodegradation of the reference substance (sodium acetate) demonstrates that nitrogen is not limiting growth and that the nitrogen introduced with the inoculum is sufficient to fulfill the nitrogen requirement of the microorganisms.

Further, due to the presence of nitrogen in the test substance, there is small likelihood of occurence of nitrification, although its probability in case of quaternary ammonium substances was found to be low (see further explanation below).

Nitrification corrections:

Therefore, the biodegradation assessment based on theroretical oxygen demand (ThOD_NO3) with nitrification has been additionally evaluated and found to be 72.8%, allowing classification of the substance as readily biodegradable. See below for calculation details:

	Molecular formula	MW	ThODNH3 (g/g)	ThODNO3(g/g)	Weight (%)
C18 TMAC	C21H44NCl	348.06	2.90	3.08	0.995
The ThODNH3of the test substance is =					2.88
The ThODNO3of the test substance is =					3.06

 

 

Day	O2 consumption	BOD	ThODNH3	% biodegradation	ThODNO2	% biodegradation
7	0	0	2.88	0.0	3.06	0.0
14	0.5	0.5		17.4		16.3
21	1.5	1.5		52.1		48.9
28.00	2.23	2.23		77.4		72.8
Test conc:	1	mg/L

However, in general the use of ThOD_NO3is not obligatory for all nitrogen-containing test substances. The choice of the ThOD used to estimate biodegradation should not be based on possible formation of nitrite or nitrate. Tests of the OECD 301 series were developed to assess the biodegradability and mineralization of organic substances. Nitrogen-containing substances are biodegraded in ready biodegradability tests by heterotrophic micro-organisms capable of utilizing these substances as carbon and energy source. This usually results in the formation of biomass (growth), water, carbon dioxide and ammonium (mineralization). The ammonium formed may subsequently be oxidized by nitrifying bacteria. These nitrifying bacteria utilizing ammonium as energy source and carbon dioxide as carbon source (autotrophic growth) are not involved in the biodegradation of nitrogen-containing substances. Biodegradation percentages calculated with the ThOD_NH3therefore do represent the biodegradability and mineralization of most nitrogen-containing substances. The formation of nitrite and nitrate during the degradation of organic substances is rare and only occurs when organic nitrogen is for example present in the form of a nitro group. Organic nitrogen is always liberated by microorganisms as ammonium when nitrogen is present as primary amine (amino group), secondary amine group, tertiary amine or quaternary ammonium group.

C16-18 and C18-unsatd. TMAC has a quaternary ammonium group. To understand the metabolic basis of degradation by microorganisms, the pathway of alkyltrimethylammonium salts has been studied with a pure culture. Bacteria identified asPseudomonas spcapable of degrading alkyltrimethylammonium salts were isolated from activated sludge (van Ginkelet al.,1992; Takenakaet al.,2007). Alkyltrimethylammonium salts with octadecyl, hexadecyl, tetradecyl, dodecyl, decyl, octyl, hexyl and coco alkyl chains supported growth of the isolates, showing the broad substrate specificity with respect to the alkyl chain length. Alkanals, and fatty acids can also serve as a carbon and energy source (van Ginkelet al.,1992; Takenakaet al.,2007). In simultaneous adaptation studies,¹H nuclear magnetic resonance spectrometry (¹H-NMR) and GC-MS showed that acetate, alkanals and alkanoates are the main intermediates of alkyltrimethylammmonium salt degradation, indicating that the long alkyl chain is utilized for microbial growth (van Ginkelet al.,1992; Nishiyama and Nishihara, 2002; Takenakaet al.,2007). Trimethylamine is stoichiometrically produced by pure cultures of microorganisms growing with the alkyl chain of alkyltrimethylammonium chloride as the sole source of carbon. The cleavage of the C-alkyl-N bond of alkyltrimethylammonium salts resulting in the formation of trimethylamine is initiated by a mono-oxygenase (van Ginkelet al.,1992). Additional evidence of the cleavage of the C-alkyl-N bond as the initial degradation step of alkyltrimethylammonium salts was presented by Nishiyamaet al.(1995) and Takenakaet al.(2007).

Dehydrogenase activity present in cell-free extract of hexadecyltrimethylammonium chloride-grown cells catalysed the oxidation of alkanal to fatty acids. The route of the fatty acid degradation is by β-oxidation. Trimethylamine, a naturally occurring compound is readily biodegradable (Pitter and Chudoba 1990). Complete degradation of trimethylamine is demonstrated through the assessment of the biodegradation pathway. Trimethylamine is degraded by methylotrophic bacteria through successive cleavage of the methyl groups (Large, 1971; Meiberg and Harder, 1978). Consortia of microorganisms degrading the alkyl chain of alkyltrimethylammonium salts and trimethylamine are therefore capable of complete (ultimate) degradation of alkyltrimethylammonium salts. Complete degradation of alkyltrimethylammonium salts using a mixed culture has been demonstrated by Nishiyamaet al.(1995). More recently, Nishiyama and Nishihara (2002) have isolated aPseudomonas spcapable of degrading both the alkyl chain and trimethylamine.  Both the pure and mixed culture studies showed that the degradation of the alkyl chain of alkyltrimethylammonium salts results in the formation of water, carbon dioxide and ammonium (see Figure 1).

For figure 1:Biodegradation pathway of alkyltrimethylammonium salts- please refer to the attachment under 'attached background material'

In conclusion, estimation of biodegradation based on the ThOD_NH3 is therefore considered to be a more appropriate choice for assessment for biodegradation of C18 TMAC.

References:

• Ginkel CG van, Dijk JB van, and Kroon AGM (1992). Metabolism of hexadecyltrimethylammonium chloride in Pseudomonas strain B1. Appl. Env. Microbiol. 58:3083-3087.L
• arge PJ (1971). The oxidative cleavage of alkyl-nitrogen bonds in micro-organisms. Xenobiotica, 1:457-467.
• Meiberg JBM, and Harder W (1978). Aerobic and anaerobic metabolism of trimethyl¬amine, dimethylamine and methylamine in Hyphomicrobium X. J. Gen. Microbiol. 106:265-276.Nishiyama N, Toshima Y and Ikeda Y (1995). Biodegradation of alkyltrimethylammonium salts in activated sludge. Chemosphere 30:593-603.
• Meiberg JBM, and Harder W (1978). Aerobic and anaerobic metabolism of trimethyl¬amine, dimethylamine and methylamine in Hyphomicrobium X. J. Gen. Microbiol. 106:265-276.
• Nishiyama N, Toshima Y and Ikeda Y (1995). Biodegradation of alkyltrimethylammonium salts in activated sludge. Chemosphere 30:593-603.
• Nishiyama N and Nishihara T (2002). Biodegradation of dodecyltrimethylammonium bromide byPseudomonas fluorescensF7 and F2 isolated from activated sludge. Microbes Environments 17:164-169.
• Pitter P and Chudoba J (1990). Biodegradability of organic substances in the aquatic environment. CRC Press, Boca Raton, USA p 191.
• Takenaka S, Tonoki T, Taira K, Murakami S and Aoiki K (2007). Adaptation ofPseudomonas spstrain 7-6 to quaternary ammonium compounds and their degradation via dual pathways. Appl. Environ. Microbiol. 173:1797-1802.

.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the biodegradation of the test substance was determined to be 77% and the test substance was therefore considered readily biodegradable (activated sludge, domestic).

Executive summary:

A study was conducted to determine the biodegradation in water of the read across substance, C18 TMAC (99.5% active) according to OECD guideline 301D, EU Method C.6 and ISO 10707 (Closed Bottle test), in compliance with GLP. The test was performed with activated sludge, domestic in 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with the read across substance. The concentrations of the read across substance, and sodium acetate in the bottles were 1.0, and 6.7 mg/L, respectively. (A slight inhibition of the endogenous respiration of the inoculum by the read across substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected. This toxicity was the reason for testing at an initial test compound concentration of 1.0 mg/L). The read across substance was biodegraded by 77% and 73% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. The test was valid, as shown by an endogenous respiration of 1.1 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 66% of its theoretical oxygen demand after 14 day. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the read across substance can be considered readily biodegradable (van Ginkel, 2005). Based on the results from this longer chain alcohol free quaternary ammonium substance, which would represent a worst case – as longer chains tend to biodegrade more slowly than shorter chains, the test substance, which is mix of C16 and C18 alkyl chains, can be expected to be degrading faster than the read across substance.

Description of key information

Overall, considering all the above information together, the test substance is considered to be readily biodegradable undergoing complete mineralization.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Study 1: A preliminary non-GLP study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the test substance, C16-18 ADBAC (95.2% active), according to the OECD Guideline 301D. Due to the well-known toxicity of the quaternary substances, the test substance was evaluated using detoxification methods through the addition of the sorbents such as silica gel and humic acid at two different concentrations. Activated sludge or river water were used as t inoculum .In addition, a sorbent free test group without any deviations from the guideline was also included as a ‘negative control’ , 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 inoculum concentration in the bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively. The tests were performed in triplicates using 0.3 L BOD bottles with glass stoppers. In the tests ‘without sorbent’ use was made of 3 bottles with the test substance (at 2 mg/L) and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 μg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the test substance (at 2 mg/L), the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 μg/L isopropanol, and silica gel or humic acid. Silicagel and humic acid concentrations in the bottles (test and control) were 1 and 2 g /bottle and 1 and 2 mg acid/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 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. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The theoretical oxygen demand (ThOD) of test substance was calculated from its molecular formula and molecular weight. The BOD (mg/mg) of the test substance was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle. The ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.89 g/g and 3.05 g/g, respectively. The biodegradation percentages at Day 28 using activated sludge as inoculum were slightly higher compared to results achieved with river water. Using the conservative ThODNO3 to calculate the biodegradation of test substance still >60% biodegradation was achieved within 28 days using activated sludge as inoculum and 1 g silica gel / bottle for detoxification. 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 7.4 and 7.2±0.1 (activated sludge) and 8.2 and 8.0±0.1 (river water) at the start and end of Day 42 of the test respectively. 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 and this was clearly detected until day 21-28 in the sorbent free ready biodegradation tests. Silica gel and humic acid were added as sorbent for detoxification of the test substance. Detoxification by the sorbents in the closed bottle tests was successful except for the 1 mg/L humic acid sorbent which still showed an inhibition of the endogenous respiration at day 7. No inhibition of the biodegradation due to the “high” initial test substance concentration is therefore expected in the presence of the sorbents: silica gel (1 and 2 g/bottle) and humic acid (2 mg/L). Under the study conditions, the test substance was determined to be readily biodegradable and the use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the test substance was considered further for the main study (Geerts, 2020).   

The main study was conducted to determine the ready biodegradability of the test substance, C16-18 ADBAC (95.2% active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the domestic wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. The measured dry weight of the inoculum was 3.2 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles. The inoculum concentration in the BOD bottles determined by colony count was 1E+6 CFU/L. 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 1 g silica gel/bottle as sorbent for detoxification of the test substance, and incubated in the dark at 22.7 to 22.9°C for 28 days. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. The concentration isopropanol added to the control bottles with silica gel was 35 µg/L which is comparable to the isopropanol content present in the test bottles. Each of the prepared solutions was dispensed into the respective group of biochemical oxygen demand (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. Endogenous respiration, theoretical oxygen demand (ThOD), biochemical oxygen demand (BOD) and biodegradation were calculated. The degradation of the test substance was assessed by the measurement of oxygen consumption. The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.89 and 3.05 g oxygen/g active ingredient, respectively. The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate. 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. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected. The validity of the test is demonstrated by an endogenous respiration of 1.10 mg/L at Day 28. 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 75%. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period. Solvent free test substance was biodegraded by 65% (based on ThODNH3) at Day 28 in the Closed Bottle test. 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. Under the study conditions, the test substance was determined to be readily biodegradable with 61% biodegradation after 28 days (Geerts, 2020).

Study 2: A study was conducted to determine the ready biodegradability of the test substance, C16-18 ADBAC (78% active), in water according to OECD Guideline 301, in compliance with GLP.The test was performed with river water receiving domestic waste water as inoculum into 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 10 bottles containing river water and humic acids (4 mg/L), 10 bottles containing river water with the test substance and humic acid 6 bottles containing river water and sodium acetate. The concentrations of the test substance and sodium acetate in the bottles were 2 mg/L (1.6 mg a.i./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. The test was found to be valid as shown by an endogenous respiration of 1.4 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 day. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability was determined to be 64% and 60% within 28 days using ThODNH3 and ThODNO3 equations respectively. Therefore the substance can be considered readily biodegradable in water. Furthermore, the test substance did not cause a reduction in the endogenous respiration under the chosen test conditions; as such, hence was considered to be non-inhibitory to the inoculum. The 10-day window pass criterion is not applicable for UVCB surfactant substances. Under the study conditions, the test substance can be considered to be readily biodegradable (van Ginkel, 2010).

Study 3: A study was conducted to determine the biodegradation in water of the read across substance, C18 TMAC (99.5% active) according to OECD guideline 301D, EU Method C.6 and ISO 10707 (Closed Bottle test), in compliance with GLP. The test was performed with activated sludge, domestic in 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with the read across substance. The concentrations of the read across substance, and sodium acetate in the bottles were 1.0, and 6.7 mg/L, respectively. (A slight inhibition of the endogenous respiration of the inoculum by the read across substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected. This toxicity was the reason for testing at an initial test compound concentration of 1.0 mg/L). The read across substance was biodegraded by 77% and 73% at Day 28 by the end of the 28 days using ThODNH3 and ThODNO3 equations respectively. The test was valid, as shown by an endogenous respiration of 1.1 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 66% of its theoretical oxygen demand after 14 day. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the read across substance can be considered readily biodegradable (van Ginkel, 2005). Based on the results from this longer chain alcohol free quaternary ammonium substance, which would represent a worst case (as longer chains tend to biodegrade more slowly than shorter chains), the test substance, which is mix of C16 and C18 alkyl chains, can be expected to be degrading faster than the read across substance.

The use of silica gel in the key study on biodegradation is supported by the findings from van Ginkel 2008, which showed that silica gel was the best adsorbent as compared to lignosulphonic acid and humic acid (see Figure 1 in the CSR).

Overall, the results obtained with the test substance are in agreement with what is reported in the literature, as summarized below inTable 4.4.

Table 4.4. Compilation of ready biodegradability test results obtained with quaternary ammonium salts (adapted van Ginkel, 2007)

Substance	Test	Results at Day 28 (%)
Tetradecylbenzyldimethylammonium Chloride (C14 ADBAC)	MITI	>80
Decylbenzyldimethylammonium Chloride (C10 ADBAC)	Closed bottle	>60
Hexadecyltrimethylammonium Chloride (C16 TMAC)	Headspace Carbon Dioxide	75*
Octadecyltrimethylammonium Chloride (C18 TMAC)	Sturm test	>70
Cocotrimethylammonium (Coco TMAC)	Closed bottle	>60
Octylbenzyldimethylammonium chloride (C18 ADBAC)	MITI	>80

*Mean from 10 laboratories; also cited in OECD TG 310 (adopted on 23 March 2006)

In addition, several literature data are available to clarify the metabolic basis of degradation by micro-organisms.Benzyldimethylamine was found as first metabolite of alkylbenzyldimethylammonium salts degradation byAeromonas hydrophila, activated sludge and aPseudomonas spp. community (Patrauchan and Oriel, 2003; van Ginkel 2004; Tezelet al.,2012). Alkyl-N fission is therefore the most probable strategy to gain access to the carbon of the alkyl chain. The next step in the biodegradation of these quaternary ammonium compounds was the successive removal of the two methyl groups. Benzylamine formed, in turn, is converted into benzaldehyde and ammonium (Patrauchan and Oriel, 2003). In contrast to the alkylbenzyldimethylammonium salts biodegradation pathway reported by Patrauchan and Oriel, (2003), neither benzylmethylamine nor benzylamine were identified as metabolites by two enrichment cultures. Exposure of activated sludge to decylbenzyldimethyl­ammonium chloride probably selects for three microorganisms that utilize the alkyl chain, the aromatic moiety, and dimethylamine (van Ginkel, 2004). Kinetic assays demonstrated that benzylmethylamine and benzylamine were not intermediates of alkylbenzyldimethylammonium salts transformation by the enrichedPseudomonasspp. community (Tezelet al., 2012). Thus, benzyldimethylamine is thought to be transformed to dimethylamine and benzoic acid via debenzylation. Dimethylamine is degraded by the successive removal of both methyl groups resulting in the formation of ammonium (Large, 1971). Both the pure and mixed culture studies showed that the degradation of the alkyl chain of alkylbenzyldimethylammonium chloride results in the formation of water, carbon dioxide and ammonium (see Figure 2 in the CSR).

Further, according to the evidence presently available on the biodegradation rate, microorganisms readily oxidize the hydrophobic alkyl chains of the cationic surfactants, which is followed by a slower oxidation of the hydrophilic moiety (the corresponding amines) (van Ginkel, 2004). The above biodegradation process for the two moieties plays a key role in the differences in the results between the different cationic surfactants. However, based on the available experimental data and literature evidence, the alkyl chains and the trimethylamine of the test substance is readily biodegradable.

Overall, considering all the above information together, the test substance is considered to be readily biodegradable undergoing complete mineralization.