Quaternary ammonium compounds, di-C12-18-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

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:

not specified

GLP compliance:

no

Remarks:

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

Specific details on test material used for the study:

Chemical name: Quaternary ammonium compounds, di C12-C18 alkyldimethyl, chlorides (solvent free)
CAS no: 61789-77-3; 68391-05-9
EC no: 269-924-1
Trade name Arquad 2C-75 solvent dry
(dried Arquad 2C-75, batch number 1796263)
Batch no.: SPA19005
Composition: Active ingredient 95.7 % m/m
Water 3.4 % m/m
Isopropanol 0.9 % m/m

Oxygen conditions:

aerobic

Inoculum or test system:

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

Details on inoculum:

(a) Activated sludge was obtained 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). 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. 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 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:

22

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:

24

Sampling time:

28 d

Remarks on result:

other: 33% 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:

24

Sampling time:

28 d

Remarks on result:

other: 33% 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:

21

Sampling time:

28 d

Remarks on result:

other: 27% 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:

19

Sampling time:

28 d

Remarks on result:

other: 25% 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:

22

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:

20

Sampling time:

28 d

Remarks on result:

other: 24% 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:

23

Sampling time:

28 d

Remarks on result:

other: 27% 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:

24

Sampling time:

28 d

Remarks on result:

other: 31% 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:

27

Sampling time:

28 d

Remarks on result:

other: 36% 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 at day 7 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 silica gel sorbents in the closed bottle tests was most successful in combination with the activated sludge inoculum. Humic acid sorbent still showed an inhibition of the endogenous respiration at day 7. No or very little inhibition of the biodegradation due to the “high” initial test substance concentration is therefore expected in the presence of the sorbent silica gel (1 and 2 g/bottle) using activated sludge as inoculum.

The Closed Bottle test results

The ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 3.02 g/g and 3.16 g/g, respectively.Biodegradation percentages in the range of 20 – 28% were found at day 28 for both inocula (Table I). The test item is therefore considered to be not readily biodegradable. In the prolonged Closed Bottle test (day 42) the biodegradation slowly further increased. The achieved result in these tests in accordance with results described already in the literature. Adsorption and low solubility of long-chain dialkyldimethylammonium salts make them less bioavailable thus reducing the rate of biodegradation in ready biodegradation tests (van Ginkel 2004). Based on results obtained in ready biodegradation screening tests didecyldimethylammonium chloride is classified as readily biodegradable and C14 up to C18 dialkyldimethylammonium salts are not readily biodegradable (van Ginkel 2003, Swisher 1987). In the environment however competent microorganisms, mineralizing C10 up to C18 dialkyldimethylammonium salts, do however exist (van Ginkel, 2003). The lack of complete biodegradation in the Closed Bottle test does therefor not mean that the test substance is recalcitrant in nature because the stringency of the ready biodegradation test procedures most likely accounts for the observed recalcitrance in the Closed Bottle test.In this ready biodegradation screening study, it was not possible to find the optimal test conditions to achieve a ready biodegradability test result for quaternary ammonium compounds, di C12-C18 alkyldimethyl, chlorides (solvent free). Therefore, there is for the set-up of the final GLP test only a slight preference, based on the detoxification achieved, to use silica gel in combination with activated sludge.

Table I Percentages biodegradation of test substance (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
		-6	4	19	22	-
Activated sludge	No sorbent*	-1	13	19	24	33
	2 g silica gel /bottle	0	7	17	24	33
	1 g silica gel / bottle	-3	13	19	21	27
	2 mg/L humic acid	-4	3	14	19	25
	1 mg/L humic acid	-8	4	17	22	-
River water	No sorbent*	-2	14	20	20	24
	2 g silica gel /bottle	-2	17	21	23	27
	1 g silica gel / bottle	-3	19	23	24	31
	2 mg/L humic acid	-6	14	23	27	36
	1 mg/L humic acid	-6	4	19	22	-

* 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 (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*	-6	4	19	22	-
	2 g silica gel /bottle	-1	13	19	24	33
	1 g silica gel / bottle	0	7	17	24	33
	2 mg/L humic acid	-3	13	19	21	27
	1 mg/L humic acid	-4	3	14	19	25
River water	No sorbent*	-8	4	17	22	-
	2 g silica gel /bottle	-2	14	20	20	24
	1 g silica gel / bottle	-2	17	21	23	27
	2 mg/L humic acid	-3	19	23	24	31
	1 mg/L humic acid	-6	14	23	27	36

* 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:

not readily biodegradable

Remarks:

24% after day 28 (based on ThODNO3)

Conclusions:

Under the study conditions, the test substance was determined to be not readily biodegradable.

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, C12 -18 DAQ (95.7% 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 silica gel and humic acid at two different concentrations. 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. 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. 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 3.02 g/g and 3.16 g/g, respectively. The biodegradation percentages at Day 28 using activated sludge as inoculum were nearly same to results achieved with river water. Using the conservative ThODNO3 to calculate the biodegradation of test substance still 24% 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 7-14 in the sorbent free ready biodegradation tests. The humic acid sorbent still showed an inhibition of the endogenous respiration (negative biodegradation percentages) at Day 7. Detoxification was most successful by the silica gel sorbents and no inhibition of the biodegradation due to the “high” initial test substance concentration is expected in the presence of silica gel (1 and 2 g/bottle). Under the study conditions, the test substance was determined to be not 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 20 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. 60 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:

acetic 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:

19

Sampling time:

28 d

Remarks on result:

other: not readily biodegradable

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

20

Sampling time:

28 d

Remarks on result:

other: not readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

39

Sampling time:

60 d

Remarks on result:

other: inherently biodegradable

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

41

Sampling time:

60 d

Remarks on result:

other: inherently biodegradable

Results

Theoretical oxygen demand (ThOD)

The ThOD_NH3 and ThOD_NO3 of the test substance used to calculate the biodegradation percentages is 2.86 and 3.06 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 at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

 

Test conditions

The pH of the media for the 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 67% (based on ThOD_NH3) at day 28 in the Closed Bottle test. Assuming complete nitrification, and calculating the biodegradation based on the ThOD_NO3 the test substance was biodegraded by 63% 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	8.6
Mean (M)	8.50	4.55	8.35	8.60
14	8.0	4.2	8.0	8.1
	8.1	4.1	8.0	8.0
Mean (M)	8.05	4.15	8.00	8.05
21	8.0		8.0	7.3
	8.0		8.0	7.2
Mean (M)	8.00		8.00	7.25
28	7.8		7.8	6.6
	7.8		7.8	6.7
Mean (M)	7.80		7.80	6.65
42	7.7		7.7	6.0
	7.7		7.7	5.9
Mean (M)	7.70		7.70	5.95
60	7.5		7.6	5.3
	7.5		7.6	5.2
Mean (M)	7.50		7.60	5.25

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 = 1.92 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	-0.25	3.95	-4	-4	76
14	-0.05	3.90	-1	-1	75
21	0.75		13	12
28	1.15		20	19
42	1.75		30	29
60	2.35		41	39

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Remarks:

(19% after 28 days); but (39 % after 60 days- prolonged exposure): further degradation expected because a plateau was not been reached

Conclusions:

Under the study conditions, the test substance was determined to be inherently biodegradable with 39% biodegradation after 60 days.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, C12 -18 DAQ (95.7% 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 60 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.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 BODbottle. 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 3.02 and 3.17 g oxygen/g active ingredient, respectively. According to the results of this study, the test substance caused a reduction in the endogenous respiration at Day 14. The test substance in the presence of silica gel is therefore considered to be inhibitory to the inoculum in the test. The test substance was biodegraded by 20% (based on ThODNH3), at Day 28. Assuming a complete nitrification of the organic nitrogen present in the test substance and using a correction for the oxygen consumption by the nitrification, the test substance was biodegraded by 19% at day 28 (based on ThODNO3). Whereas, in the prolonged Closed Bottle test substance was biodegraded 39% at day 60 (based on ThODNO3). Biodegradation could increase further as a plateau phase was not reached at Day 60. The obtained linear growth curve suggests that the biodegradation rates are limited by the bioavailability of the test substance (limited by the desorption rate). The biodegradation reached at Day 60 demonstrates that this substance is partially degraded. The lack of complete biodegradation in the Closed Bottle test does not mean that part of 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 1.10 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 75% 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. Under the study conditions, the test substance was determined to be inherently biodegradable with 39% biodegradation after 60 days (Geerts, 2020)

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From February 15, 2006 to July 14, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test concentration below the minimum concentration set out in OECD 301B

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

yes

Remarks:

Test concentration lower than the recommended range; humic acid is added to reduce toxicity;

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal sewage treatment plant, D-31137 Hildesheim
- Storage conditions: in an aerobic condition by aeration with CO2 free air.
- Storage length: 7 d
- Pretreatment: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration with CO2-free air for 7 days. 10ml/L were used to initiate inoculation.
- Initial cell/biomass concentration: 10E7 - 10E8 CFU/L
- Water filtered: no

Duration of test (contact time):

ca. 28 d

Initial conc.:

10 mg/L

Based on:

test mat.

Remarks:

corresponding to 7.3 mgC/L

Initial conc.:

20 mg/L

Based on:

other: humic acid

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 301 B/CO2 Evolution Test.
- Test temperature: 22±2°C
- Aeration of dilution water: 30 - 100 mL/min
- Continuous darkness: no (low-light conditions)
- Other:
- Dispersion treatment: Continuously stirring.

TEST SYSTEM
- Culturing apparatus: 5000 mL brown glass bottles
- Number of culture flasks/concentration: 2 for test item concentration, 1 for the reference item, 2 for the inoculum control, 1 for the toxicity control and 2 for the humic acid control.
- Measuring equipment: pH-meter, Thermohygrograph, type 3.015/3 K, Flow meter, Analytical balance, Balance, Ultrasound.

SAMPLING
- Sampling frequency: The room temperature was measured continuously by a thermohygrograph. Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in an alkaline solution (0.0125 mol/L Ba(OH)2). Back titration of the residual Ba(OH)2 with 0.05 N HCl was carried out three times a week during the first ten days and thereafter twice weekly. On day 28, the pH value of all solutions was measured prior to acidification.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Mineral salts medium
- Abiotic sterile control: Mineral salts medium +20 mg/L humic acid
- Toxicity control: Test item and reference item in test concentration + 20 mg/L humic acid.

STATISTICAL METHODS: No data

TYPE AND FREQUENCY OF MEASUREMENTS
The room temperature was measured continuously by a thermohygrograph

Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in an alkaline solution (0.0125 mol/L Ba(OH)2).

Backtitration of the residual Ba(OH)2 with 0.05 N HCl was carried out three times a week during the first ten days and thereafter twice weekly.
On day 28 the pH-value of all solutions was measured prior to acidification.

Equipment
pH-Meter, Multilab 340i, WTW
Thermohygrograph, type 3.015/3 K, fabr.-no. 9003146
Flow meter, KROHNE DUISBURG TYP DK 800 PV
Analytical balance, SARTORIUS
Balance, PJ Precisa junior 2000 C, DIGITANA
Ultrasound, SONOREX

VALIDITY CRITERIA
- The percentage degradation of the functional control must reach the pass level of 60 % by day 14.
- The total CO2 evolution in the blank at the end of the test should be lower than 40 mg/L and not exceed 70 mg/L.
- The difference of extremes of replicate values of removal of the test item at the end of the test, at the plateau or at the end of the 10-d-window as appropriate must be less than 20 %.
- The percentage degradation of the toxicity control must reach the pass level of 25 % by day 14. If the degradation is lower than 25 % the test item must be assumed to be inhibitory and the study must be repeated with a lower test concentration.

EVALUATION
The theoretical production of carbon dioxide (ThCO2) of the test item and functional control is calculated by the sum formula:

ThCO2 [mgCO2 /mg] = C - Atoms molecular weight of CO2 / molecular weight of test or reference item

The produced CO2 was calculated by:
1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2

The net amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the control groups (humic acid control for the test item, control for the functional control).

The biodegradation is calculated from the ratio theoretical CO2 production to net CO2 production in the following equation:
Degradation [%] = net CO2*100 / ThCO2 [mgCO2 /3L]

Reference substance:

benzoic acid, sodium salt

Preliminary study:

No data

Test performance:

No data

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 61

Sampling time:

28 d

Remarks on result:

other: The value reported is the mean biodegradation, calculated from the 2 replicates of test substance.

Details on results:

- In the toxicity control 50 % biodegradation occurred within 14 d and came to 67 % after 28 d. The biodegradation of the reference item was not inhibited by the test item.
- The mean biodegradation of the test item was 61 %. The 10 % level (beginning of biodegradation) was reached by the 1st test item replicate after 12 d, by the 2nd replicate after 6 d. The 2nd test item replicate reached the pass level of 60% after 26 d and came to 66% after 28 d. The 1st replicate came to 56% after 28 d.
- The test material is regarded as biodegradable after 28 d. The test item complies with the biodegradability criteria as laid down in Annex III of Regulation (EC) No 648/2004 on detergents.

Results with reference substance:

The adaptation phase of the functional control changes after 2 d into the degradation phase (degradation ≥ 10 %). The course of the degradation phase is rapid and reaches a degradation rate of 60 % after 7 d. The degradation came to 100 % after 14 d. The validity criterion degradation ≥ 60 % after 14 d is fulfilled. In the toxicity control 50 % biodegradation occurred within 14 days and came to 67 % after 28 days. The biodegradation of the reference item was not inhibited by the test item. The biodegradation of the test item is shown graphically in comparison to the readily degradable functional control and the toxicity control. The 10 % level (beginning of biodegradation) was reached by the 1st test item replicate after 12 days, by the 2nd replicate after 6 days. The 2nd test item replicate reached the pass level of 60 % after 26 days and came to 66 % after 28 days. The 1st replicate came to 56 % after 28 days. The mean biodegradation came to 61 % after 28 days. The test item must be regarded as biodegradable after 28 days. The test item complies with the biodegradability criteria as laid down in Annex III of Regulation (EC) No 648/2004 on detergents.

Results

Carbon Content of the Test Item

Based on the carbon content a ThCO2 of 2.68 mg CO2/mg test item was calculated. A test concentration of 10 mg/L, corresponding to a carbon content of 7.3 mg C/L in the test vessels was selected.

Colony Forming Units of the Inoculum

Colony forming units (CFU) of the inoculum for the Modified Sturm Test are checked every 3 months by standard dilution plate count. Last determination: 2.3 × 108 CFU/L (May 2006)

CO2 -Production and Biodegradation

The total amount of CO2 produced in 28 d was analysed by titration in 12 measurements. The 28 d values are shown in comparison to the readily degradable functional control in summarized form in Table 1.

Table 1: CO2-Production and Biodegradation after 28 d 

CO2 production After 28 d	Control Mv	Functional Control 20 mg/L	Humic acid control	Test item 10 mg/L No 1	Test item 10 mg/L No. 2	Toxicity Control 20 + 10 mg/L
Gross [mg/ 3 L]           [mg/L]	169.0 56.3	-	172.8 57.6	-	-	-
Net     [mg/ 3L]           [mg/L]	- -	151.4 50.5	- -	45.3 15.1	53.2 17.7	140.0 46.7
Theor. [mg/ 3L]           [mg/L]	- -	127.8 42.6	- -	80.4 26.8	80.4 26.8	208.2 69.4
Degradation [%] After 28 d	-	100	-	56	66	67

In the control a maximum of 56.3 mg CO2/L and in the humic acid control 57.6 mg CO2/L were formed after 28 d (validity criterion: <70 mg CO2 /L after 28 d).

Table 2: CO₂-Production and Biodegradation in the Control and Functional Control

study day	date	control     [mg CO2/3L] mv	functional control 20 mg/L
			[mg CO2/3L]		degr. [%]
			gross	net sum
1	16.06.	3.1	7.3	4.2	3
4	19.06.	20.5	53.1	36.9	29
6	21.06.	15.5	53.2	74.6	58
8	23.06.	12.3	38.7	101.0	79
11	26.06.	15.8	38.7	123.9	97
14	29.06.	18.8	32.2	137.3	100
18	03.07.	22.0	25.5	140.9	100
21	06.07.	17.2	17.7	141.4	100
25	10.07.	18.0	21.6	144.9	100
28	13.07.	13.4	17.7	149.2	100
29*	14.07.	12.4	14.5	151.4	100

degr.=degradation mv=mean value

*) results of last two gas wash bottles

Table 3: CO₂-Production and Biodegradation in the Humic Acid Control, Test Item and Toxicity Control Samples

study day	date	humic acid control	test item 10 mg/L						toxicity control   20 mg/L reference item + 10 mg/L test item
			replicate 1			replicate 2
		[mg CO2/3L]	[mg CO2/3L]		degr.	[mg CO2/3L]		degr.	[mg CO2/3L]		degr.
		mv	gross	net sum	[%]	gross	net sum	[%]	gross	net sum	[%]
1	16.06.	7.3	5.4	0.0	0	8.9	1.7	2	11.8	4.5	2
4	19.06.	21.7	20.6	0.0	0	23.5	3.5	4	53.0	35.9	17
6	21.06.	16.8	20.4	3.5	4	21.9	8.6	11	51.8	70.8	34
8	23.06.	12.9	13.3	3.9	5	15.5	11.2	14	28.5	86.4	42
11	26.06.	16.8	18.4	5.4	7	21.1	15.5	19	23.1	92.7	45
14	29.06.	16.2	25.1	14.4	18	24.3	23.6	29	27.5	104.0	50
18	03.07.	19.1	28.6	23.8	30	32.3	36.8	46	35.1	120.0	58
21	06.07.	15.5	17.6	25.9	32	21.7	43.0	53	25.5	130.0	62
25	10.07.	18.4	22.4	29.9	37	21.6	46.1	57	22.7	134.2	64
28	13.07.	13.4	26.0	42.5	53	20.0	52.7	66	19.3	140.0	67
29*	14.07.	14.5	17.4	45.3	56	14.5	53.2	66	14.1	140.0	67

degr. = degradation mv = mean value

*) results of last two gas wash bottles

Validity Criteria

The study was performed according to OECD 301 B / CO2 Evolution Test and GLP Guidelines. The validity criteria were fulfilled according to the guideline:

- The total CO2 evolution at the end of the test was 56.3 mg CO2/L in the control and

57.6 mg CO2/L in the humic acid control.

- The degradation of the functional control reached the pass level of  60 % by day 14.

- The degradation of the toxicity control reached the pass level of 25 % after 14 days.

- The difference of extremes of replicate values of removal of the test item at the end of the test was < 20 %.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the test substance was considered as readily biodegradable.

Executive summary:

A study was conducetd to determine the ready biodegradability of the test substance, C12 -18 DAQ (98% active), according to the OECD guideline 301 B using CO2 evolution test, in compliance with GLP. The biodegradability was determined with a non-adapted activated sludge for the test substance over a test period of 28 days in the Modified Sturm Test. The test material was tested with a concentration of 10 mg/L in duplicates, corresponding to carbon content (TOC) of 7.3 mg C/L in the test vessels. To reduce the toxicity of the test material 20 mg/L humic acid was added to the test substance and toxicity control replicates. The biodegradation of the test material was followed by titrimetric analyses of the quantity of CO2 produced by the respiration of bacteria. The degradation was finished on Day 28 by acidification and the last titration was made on Day 29, after the soluble CO2 was turned out over a period of 24 h. The percentage CO2 production was calculated in relation to the theoretical CO2 (ThCO2) of the test material. Sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % after 7 d. The degradation came to 100 % after 14 d. In the toxicity control containing both test and reference item a biodegradation rate of 50 % occurred within 14 d and came to 67 % after 28 d. The biodegradation of the reference item was not inhibited by the test substance in the toxicity control. The 10 % level (beginning of biodegradation) was reached by the 1st replicate after 12 d, by the 2nd replicate after 6 d. The 2nd replicate reached the pass level of 60 % after 26 d and came to 66 % after 28 d. The 1st replicate came to 56 % after 28 d. The mean biodegradation came to 61 % after 28 d. The validity criteria according to the guideline were fulfilled. Under the study conditions, the test substance was considered as readily biodegradable (Fiebig, 2006).

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From March 14, 1990 to September 27, 1990

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:

The closed bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of Day 28 using a special funnel

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Secondary activated sludge was obtained from the RWZI Nieuwgraaf in Duiven. The plant treats predominately domestic wastewater. The sludge was preconditioned to reduce the endogenous respiration rates. To precondition, the sludge (200 mg Dry Weight (DW)/L) was aerated for a period of one week. The sludge was diluted to a concentration in the BOD bottles of 2 mg DW/litre. Ammonium chloride was omitted from the medium to prevent nitrification.

Duration of test (contact time):

ca. 214 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
The test compound is toxic to microorganisms and therefore test material was tested in the presence of silica gel to reduce the concentration in the water phase. The test substance was first dissolved in dicloromethane. The test substance in dichloromethane (0.56 mL) was added to 2 g silica gel (100 – 200 mesh) weighed in a glass petri dish. The solvent was allowed to evaporate by pacing the petri dish in a ventilated hood for 3 h, and the entire contents were then transferred to the BOD bottle. Although no additional oxygen consumption was expected, controls with silica gel were carried out as well.

TEST SYSTEM
- Culturing apparatus: The test was performed in 280 mL BOD bottles

STATISTICAL METHODS:
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The ThOD of the test compound is 2.6 g O2/g test substance. The BOD of the test substance and sodium acetate were calculated from the oxygen concentrations in the bottles without and with test substance (B, C) and the bottles without and with sodium acetate gel (B, D). The BOD of Arquad 2C-75 in the presence of silica gel was calculated from the oxygen concentrations in the bottles with and without test substance but with silica gel.

Reference substance:

acetic acid, sodium salt

Remarks:

Applied on silica gel for control

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 0

Sampling time:

28 d

Remarks on result:

other: not readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 56

Sampling time:

214 d

Remarks on result:

other: inherently biodegradable

Details on results:

- Test material is not biodegraded in the closed bottle test after 28 d. However, in the prolonged closed bottle test test material is biodegraded.
- Test material is toxic at 2 mg/L. After 90 d, bacteria are adapted to test material and biodegradation was observed. In the presence of silica gel no inhibition of the endogenous respiration was observed. In this test, 2-propanol was biodegraded within 28 d whereas dicocodimethylammonium chloride was not biodegraded.

Results with reference substance:

The test is valid as shown by the oxygen consumption in the control bottle with sodium acetate and endogenous respiration of 0.4 and 0.5 mg/L

Results

The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The ThOD of the test compound is 2.6 g 02/g test substance. The BOD of the test substance and sodium acetate were calculated from the oxygen concentrations in the bottles without and with test substance and the bottles without and with sodium acetate gel respectively The BOD of test substance in the presence of silica gel was calculated from the oxygen concentrations in the bottles with and without test substance but with silica gel . See attached report for full details.

Time Days	5	15	28	42	70	98	126	182	214
Test substance	0	0	0	0	0	6	19	50	56
Sodium Acetate	77	83	88	-	-	-	-	-	-

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Remarks:

(inherently biodegradable)

Conclusions:

Under the study conditions, the test substance can be considered to be inherently biodegradable.

Executive summary:

A study was conducted to determine the biodegradability of the test substance, C12-18 DAQ (76.4% active) using closed bottle test according to OECD guideline 301D, in compliance with GLP.  In the closed bottle test, the test substance was added to an aqueous solution of mineral salts and exposed to relatively low numbers of microorganisms under aerobic conditions for a period of 28 days. Few deviations were introduced in the protocol. Instead of an effluent, activated sludge was used as the inoculum. The 200 mg DW/L of sludge was preconditioned by aerating for one week, to reduce the endogenous respiration rates and was diluted to a concentration of 2 mg DW/L in the BOD bottles. Ammonium chloride was omitted from the medium to prevent nitrification. Due to well-known toxicity to micro-organisms, the test substance was tested in the presence of silica gel to reduce the concentration in the water phase. In addition, the closed bottle test was prolonged by measuring the oxygen the course of the oxygen decrease in the bottles of Day 28 using a special funnel.The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter. The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The test substance did not biodegrade in the closed bottle test within 28 days. However, after prolongation of the test to 214 days, the test substance biodegraded to 56%. The test substance was toxic at 2 mg/L as shown by the inhibition of the endogenous respiration. After 90 d, bacteria were adapted to test material and biodegradation was observed. In the presence of silica gel no inhibition of the endogenous respiration was observed. Silica reduced the bioavailability of the test substance and therefore no toxicity was observed but also biodegradation was limited. The validity of the tests were shown by the oxygen consumption in the control bottle with sodium acetate and endogenous respirations of 0.4 and 0.5 mg/L. The pH's of the medium at Day 28 were 7.0 and 7.3. Under the study conditions, the test substance was determined to be not readily biodegradable (Van Ginkel, 1990).

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No data on GLP compliance. Seawater used as inoculum no rationale given for testing conditions

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(Small Media Modification and extension of test length.)

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Oxygen conditions:

aerobic

Remarks:

determined electrochemically using an oxygen electrode

Inoculum or test system:

other: sea water

Details on inoculum:

Seawater was collected from coastal water near the Oosterscheldedam (Banjaard, The Netherlands) at high tide. The seawater was sampled approximately 10 cm below the water surface. The seawater was aged to reduce the concentration of biodegradable compounds present in the seawater. To this end, the seawater was aerated for two weeks.

The seawater in the closed bottle test contained no additional nutrients. The test compound was added to the bottles using stock suspension of 1.0 g/L. A stock suspension of test material was prepared by ultrasonic dispersion at 375W for 5 min. The concentration of this stock was 1 g/L. This suspension was added to the bottle.

Duration of test (contact time):

ca. 112 d

Initial conc.:

2 mg/L

Based on:

test mat.

Details on study design:

TEST CONDITIONS
The seawater in the closed bottle test contained no additional nutrients. The test compound was added to the bottles using stock suspension of 1.0 g/L. A stock suspension of Arquad 2C was prepared by ultrasonic dispersion at 375W for 5 mins. The concentration of this stock was 1 g/L. This suspension was added to the bottle.
10 bottles contained only inoculum and 10 bottles contained test substance and inoculum. The concentration of the test compound in the bottles was 2.0 mg/L respectively. The prepared solution in seawater was dispensed into the respective groups of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analysed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated at 18 °C ± 2 °C 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.

TEST SYSTEM
The closed bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of Day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle.

STATISTICAL METHODS:
Calculation of ThOD:
ThODNH3(mgO2/mg)= 16(2C +1/2(H-Cl-3N)+3S+21/2Na-O)/MW

Calculation of BOD:
Provided that the O2 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 consumption (mg/L)=Mc-Mt or a

Mt or a = the mean oxygen concentration in the bottle containing the test compound (t) or the reference compound, sodium acetate (a) and inoculated with seawater n-days after the start of the test
Mc = the mean oxygen level in the control bottle inoculated with seawater n-days after the start of the test
The biological oxygen demand (BOD) mg/mg of test compound was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle, respectively.

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

Reference substance:

other: Potassium hydrogen phthalate

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 37

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 79

Sampling time:

112 d

Remarks on result:

other: Extended closed bottle.

Details on results:

The calculated ThOD of test material is 2.5 mg/mg. The COD of the test substance was 2.2 mg/mg. The ThOD was used to calculate the biodegradation percentages

Results

The test substance was for 37 % biodegraded in the closed bottle test after 28 d. However, the test substance contained 15 % isopropanol which is readily biodegradable. After prolongation of the test till 112 d, 79% biodegradation was observed demonstrating that the active ingredient in the test substance is also completely biodegradable. The presence of both isopropanol and suspended matter will have influenced the bioavailability and due to that the biodegradation rate of the active during the test.

Table 1: Oxygen consumption (mg/L) and the percentage biodegradation (BOD: ThOD) of test material in the closed bottle test inoculated with seawater

Time (Days)	Oxygen consumption (mg/L)	Biodegradation (%)
0	0.0	0
7	0.0	0
14	0.5	12
21	0.9	21
28	1.6	37
42	2.1	49
56	2.3	53
84	2.6	60
112	3.4	79

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Remarks:

(inherently biodegradable)

Conclusions:

Under the study conditions, the test substance can be considered to be inherently biodegradable.

Executive summary:

A study was conducted to determine the biodegradability of the test substance, C12-18 DAQ (75% active) in sea water using closed bottle test according to OECD guideline 306. Use was made of 10 bottles containing only inoculum, and 10 bottles containing test substance and inoculum. The concentration of the test compound in the bottles was 2.0 mg/L, respectively. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The test was prolonged because the pass level was not reached at Day 28. Inhibition of the endogenous respiration of the inoculum by the test substance was not detected. No inhibition of the biodegradation due to the "high initial concentration of the test compound was expected. In the study, the test substance was biodegraded 37 % at Day 28 in the closed bottle test inoculated with seawater. This was not considered readily biodegradable. However, 79% of the test compound biodegraded at Day 112. This demonstrated that test substance does biodegrade in seawater. The test was valid as shown by an endogenous respiration of 1.0 mg/L. The blank respiration therefore does not exceed 30% of the oxygen in the test bottles. The most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period. Under the study conditions, the test substance was determined to be not readily biodegradable (Van Ginkel and Garttener, 1999).

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1974

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Report consists of one page from 1974 stating that biodegredation was observed. Not sufficient for validation.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The biodegradability of the test substance was tested in 48 h biodegradation test.

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

other: bacterial culture

Duration of test (contact time):

ca. 48 h

Initial conc.:

ca. 10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

The test substance at 10 mg/L concentration was added to a bacterial culture and kept mixed and aerated. The chnage in concentration of test substance was measured as function of time using standard colorimetry procedures.

Reference substance:

not specified

Key result

Parameter:

% degradation (test mat. analysis)

Value:

ca. 80.3

Sampling time:

48 h

Result

- 42.7% Biodegradation at 12 h time period

- 53.1% Biodegradation at 18 h time period

- 71.1% Biodegradation at 24 h time period

- 80.3% Biodegradation at 48 h time period

The test substance was found to be 80.3% biodegraded in 48 h test period.

Validity criteria fulfilled:

no

Interpretation of results:

readily biodegradable

Conclusions:

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

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, C12-18 DAQ (75% active) using 48 h test period. The test substance at 10 mg/L concentration was added to a bacterial culture and kept mixed and aerated. The chnage in concentration of test substance was measured as function of time using standard colorimetry procedures. The test substance was found to be 80.3% biodegraded in 48 h test period. Under the study conditions, the test substance was determined to be readily biodegradable (Guidry, 1974).

Description of key information

Overall, based on the available studies, the test substance can be considered to be inherently biodegradable. 

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

other: freshwater and marine water

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, C12-18 DAQ (95.7% active), according to 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 silica gel and humic acid at two different concentrations. 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. 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. 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, which could not be removed without compromising the stability/quality of 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. Silica gel 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 the 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 3.02 g/g and 3.16 g/g, respectively. The biodegradation percentages at Day 28 using activated sludge as inoculum were nearly same to results achieved with river water. Using the conservative ThODNO3 to calculate the biodegradation of test substance still 24% biodegradation was achieved within 28 days, using activated sludge as inoculum and 1 g silica gel / bottle for detoxification. Biodegradation could increase further as a plateau phase was not reached at Day 60. The obtained linear growth curve suggests that the biodegradation rates are limited by the bioavailability of the test substance (limited by the desorption rate). The biodegradation reached at Day 60 demonstrates that this substance is partially degraded. The lack of complete biodegradation in the Closed Bottle test does not mean that part of 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 1.10 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 75% 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. Under the study conditions, the test substance was determined to be not 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).  

Study 2:The main study was conducted to determine the ready biodegradability of the test substance, C12-18 DAQ (95.7% active), using closed bottle test, according to 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 60 days. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol[1], 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. 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. 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 3.02 and 3.17 g oxygen/g active ingredient, respectively. According to the results of this study, the test substance caused a reduction in the endogenous respiration at Day 14. The test substance in the presence of silica gel is therefore considered to be inhibitory to the inoculum in the test. The test substance was biodegraded by 20% (based on ThODNH3), at Day 28. Assuming a complete nitrification of the organic nitrogen present in the test substance and using a correction for the oxygen consumption by the nitrification, the test substance was biodegraded by 19% at Day 28 (based on ThODNO3). Whereas, in the prolonged Closed Bottle test substance was biodegraded 39% at Day 60 (based on ThODNO3). 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 most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period Under the study conditions, the test substance was determined to be inherently biodegradable with 39% biodegradation after 60 days (Geerts, 2020).  

The use of silica gel in the key study on biodegradation is supported by the findings from several publications available on quaternary ammonium salts (QAS):

A publication from van Ginkel 2008, showed that silica gel was the best adsorbent as compared to lignosulphonic acid and humic acid (see Figure 1 in CSR):

In addition, recent publications from Timmer et al., 2019 and Nabeoka et al., 2020 indicate that use of appropriate concentrations of moderate adsorbent carriers like silica gel has the ability to reduce the microbial toxicity of quaternary ammonium substances (by lowering their concentrations). These publications also show that there is a delicate balance between mitigation of the toxicity and sufficient bioavailability for biodegradation. For some more toxic QAS the amount of carrier needed to mitigate the toxicity is too high to allow a sufficient biodegradation rate to fulfil the CLP criteria for ready biodegradability. In such cases the dissolution rate limits the biodegradation rate which results in more linear shaped biodegradation curves. In addition, the use of silica gel was found to have no effect on highly persistent substances with specific chemical structures, e.g., branched alkyl chain containing substances as in benzethonium chloride (Nabeoka et al., 2020). This is a critical observation as it demonstrates that use of silica gel in the studies with the linear alkyl chain containing quaternary substances like the test substance does not create false positive biodegradability results and hence does not lead to overestimation of the biodegradation.

Study 3: A study was conducted to determine the ready biodegradability of the test substance, C12-18 DAQ (98% active), according to OECD guideline 301 B using CO2 evolution test, in compliance with GLP. The biodegradability was determined with a non-adapted activated sludge for the test substance over a test period of 28 days in the Modified Sturm Test. The test material was tested at a concentration of 10 mg/L in duplicates, corresponding to carbon content (TOC) of 7.3 mg C/L in the test vessels. To reduce the toxicity of the test material 20 mg/L humic acid was added to the test substance and toxicity control replicates. The biodegradation of the test material was followed by titrimetric analyses of the quantity of CO2 produced by the respiration of bacteria. The degradation was finished on Day 28 by acidification and the last titration was made on Day 29, after the soluble CO2 was turned out over a period of 24 h. The percentage CO2 production was calculated in relation to the theoretical CO2 (ThCO2) of the test material. Sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60% after 7 d. The degradation came to 100% after 14 d. In the toxicity control containing both test and reference item a biodegradation rate of 50% occurred within 14 d and came to 67% after 28 d. The biodegradation of the reference item was not inhibited by the test substance in the toxicity control. The 10% level (beginning of biodegradation) was reached by the 1st replicate after 12 d, by the 2nd replicate after 6 d. The 2nd replicate reached the pass level of 60% after 26 d and came to 66% after 28 d. The 1st replicate came to 56% after 28 d. The mean biodegradation came to 61% after 28 d. The validity criteria according to the guideline were fulfilled. Under the study conditions, the test substance was considered as readily biodegradable (Fiebig, 2006). 

Study 4:A study was conducted to determine the biodegradability of the test substance, C12-18 DAQ (76.4% active) using closed bottle test according to OECD guideline 301D, in compliance with GLP.  In the closed bottle test, the test substance was added to an aqueous solution of mineral salts and exposed to relatively low numbers of microorganisms under aerobic conditions for a period of 28 days. Few deviations were introduced in the protocol. Instead of an effluent, activated sludge was used as the inoculum. The 200 mg DW/L of sludge was preconditioned by aerating for one week, to reduce the endogenous respiration rates and was diluted to a concentration of 2 mg DW/L in the BOD bottles. Ammonium chloride was omitted from the medium to prevent nitrification. Due to well-known toxicity to micro-organisms, the test substance was tested in the presence of silica gel to reduce the concentration in the water phase. In addition, the closed bottle test was prolonged by measuring the oxygen the course of the oxygen decrease in the bottles of Day 28 using a special funnel. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter. The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The test substance did not biodegrade in the closed bottle test within 28 days. However, after prolongation of the test to 214 days, the test substance biodegraded to 56%. The test substance was toxic at 2 mg/L as shown by the inhibition of the endogenous respiration. After 90 d, bacteria were adapted to test material and biodegradation was observed. In the presence of silica gel no inhibition of the endogenous respiration was observed. Silica reduced the bioavailability of the test substance and therefore no toxicity was observed but also biodegradation was limited. The validity of the tests were shown by the oxygen consumption in the control bottle with sodium acetate and endogenous respirations of 0.4 and 0.5 mg/L. The pH’s of the medium at Day 28 were 7.0 and 7.3. Under the study conditions, the test substance was determined to be not readily biodegradable (Van Ginkel, 1990).   

Study 5:A study was conducted to determine the ready biodegradability of the test substance, C12-18 DAQ (75% active) using 48 h test period. The test substance at 10 mg/L concentration was added to a bacterial culture and kept mixed and aerated. The change in concentration of the test substance was measured as function of time using standard colorimetry procedures. The test substance was found to be 80.3% biodegraded in 48 h test period. Under the study conditions, the test substance was determined to be readily biodegradable (Guidry, 1974).  

Study 6: A study was conducted to determine the biodegradability of the test substance, C12-18 DAQ (75% active) in sea water using closed bottle test according to OECD guideline 306. Use was made of 10 bottles containing only inoculum, and 10 bottles containing test substance and inoculum. The concentration of the test compound in the bottles was 2.0 mg/L, respectively. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The test was prolonged because the pass level was not reached at Day 28. Inhibition of the endogenous respiration of the inoculum by the test substance was not detected. No inhibition of the biodegradation due to the “high initial concentration of the test compound was expected. In the study, the test substance was biodegraded 37% at Day 28 in the closed bottle test inoculated with seawater. This was not considered readily biodegradable. However, 79% of the test compound biodegraded at Day 112. This demonstrated that test substance does biodegrade in seawater. The test was valid as shown by an endogenous respiration of 1.0 mg/L. The blank respiration therefore does not exceed 30% of the oxygen in the test bottles. The most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period. Under the study conditions, the test substance was determined to be not readily biodegradable (Van Ginkel and Garttener, 1999). 

Apart from the above studies, several studies have also been published in the literature, where comparison of biodegradation potential of quaternary ammonium substances (QAS) have been investigated under different test conditions. Some of the interesting observations from these publications are summarised below:

In a publication by van Ginkel (1991) where the relation between structure of the QAS substances and their biodegradability was discussed noted that that the influence of alkyl chains in close bottle test is more pronounced. In this study, he compared the degradation potential between monoalkyl and dialkyl QAS (DAQ) containing eitherC12 or C18 alkyl chains, under the closed bottle test conditions. The biodegradation curves which gives an impression of the biodegradation rates has been shown in the belowfigure 2. Based on the study results, the monoalkyl QAS were found to be more rapidly biodegraded than the dialkyl QAS, but the latter was considered to be inherently biodegradable. The lower degradation potential of the dialkyl QAS was considered to be due to higher toxicity to the inoculum and limited availability due to adsorption.

Another publication from van Ginkel et al., (2003), demonstrated that the dissolved fractions of a DAQ substances are rapidly mineralised. Flow-through column experiments were used in an attempt to measure biodegradation without the limitation of the solubility and adsorption of the substrate. Strain DD1 (isolated from domestic water treatment plant), in flow-through columns using sterile mineral salts medium were found to be metabolise the dissolved didecyldimethylammonium chloride (C10 DAQ) and dioctadecyldimethylammonium chloride (C18 DAQ) rapidly. These micro-organisms were also found to degrade dicocodimethyl ammonium salt sorbed onto silica gel fed with river water. This was evident from the marked decline in the test concentration in the effluent of the column after few days (see belowFigure 3 in the CSR).

Based on these findings, the reason for the long biodegradation times in the standard closed bottle tests was attributed to the poor bioavailability of the substances (see Figure 3), which was due to poor desorption and re-solubilisation rates. This is also the reason for the “poor results” in the Geerts, 2020 study with the test substance. Therefore, despite of the inherent biodegradability classification of the test substance, the water-soluble fraction of the test substance (±80%) is not persistent. Therefore, as the substance is either sorbed or biodegraded, it is not considered to be hazardous to the aquatic environment.

Biodegradation pathways of quaternary substances

The pathway of dialkyldimethylammonium salts has been studied with pure cultures. The pure culture, strain DD1, capable of growing on didecyldimethylammonium salt as sole carbon and energy source was isolated from activated sludge. Decyldimethylamine, decanoate, and acetate also served as growth substrates. Dimethylamine was stoichiometrically accumulated during growth on didecyldimethylammonium chloride. These results strongly indicate that the alkyl chains are metabolized sequentially (van Ginkel et al, 2003). Another bacterium is required to degrade the dimethylamine formed (Large, 1971). Nishihara et al (2000) isolated aPseudomonas fluorescensstrain TN4 with didecyldimethylammonium chloride as carbon and energy source. Decyldimethylamine and dimethylamine were identified as intermediates in the biodegradation pathway. Both pure culture studies demonstrate that the degradation of the alkyl chains of dialkyldimethylammonium salts precedes the breakdown of the dimethylamine (Figure 4). Pseudomonas fluorescensstrain TN4 also degraded other quaternary ammonium salts i.e., alkyltrimethylammonium salts and alkylbenzyldimethylammonium compounds (Nishihara et al, 2000). Strain DD1 was also capable of growing on didodecyldimethylammonium and ditetradecyldimethylammonium salts showing broad substrate specificity towards the alkyl chain lengths (van Ginkel et al, 2003). Broad substrate specificities with respect to alkyl chain were demonstrated more comprehensively for other fatty amine derivatives (van Ginkel, 2007).

The Figure 4 (see CSR) shows the ability of microorganisms to catalyze C-alkyl-N fissions, thereby forming alkanals that can enter the common pathways of metabolism via β-oxidation(van Ginkel, 2004).

Overall, based on the available information, the test substance is considered to be inherently biodegradable and therefore not likely to bepersistent or present a risk to the environment.   

[1]To correct for the small amount of isopropanol still present in the test substance, which could not be removed without compromising the stability/quality of the test substance.