Cetrimonium chloride

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:

weight of evidence

Study period:

June 19, 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)

Deviations:

not specified

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 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.
(c) 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 7.105 CFU/L and 6.105 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 nitrifiication. 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. Use was made of 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. For the test substance 3 bottles were used containing the respective inoculum 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. 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:

53

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:

57

Sampling time:

28 d

Remarks on result:

other: (60% after 42 d)

Remarks:

(activated sludge as inoculum and 2 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

62

Sampling time:

28 d

Remarks on result:

other: (65% after 42 d)

Remarks:

(activated sludge as inoculum and 1 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

59

Sampling time:

28 d

Remarks on result:

other: (68% after 42 d)

Remarks:

(activated sludge as inoculum and 2 mg/L humic acid / 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)

Remarks:

(activated sludge as inoculum and 1 mg/L humic acid / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

24

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:

31

Sampling time:

28 d

Remarks on result:

other: (41% after 42 d)

Remarks:

(river water as inoculum and 2 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

30

Sampling time:

28 d

Remarks on result:

other: (39% after 42 d)

Remarks:

(river water as inoculum and 1 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

49

Sampling time:

28 d

Remarks on result:

other: (71% after 42 d)

Remarks:

(river water as inoculum and 2 mg/L humic acid / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

49

Sampling time:

28 d

Remarks on result:

other: (67% after 42 d)

Remarks:

(river water as inoculum and 1 mg/L humic acid / bottle for detoxification)

Results

Test conditions

The validity of the test was demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of the media was 7.3 (activated sludge) and 8.2 (river water) at the start of the test. The pH was 7.2±0.2 (activated sludge) and 8.2±0.2 (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 -14 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 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 ThOD_NH3and ThOD_NO3of the active ingredient used to calculate the biodegradation percentages was 2.85 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 (Table I, II). From the four different detoxification methods used humic acid at a concentration of 1 and 2 mg/L were less effective compared to the other methods as demonstrated by negative biodegradation percentages at Day 7 (Table I, II).

 

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_NO3assumes 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 the test substance is ~19:1. Due to the omission of ammonium nitrogen from the nutrient medium in the sorbent modified tests this organic nitrogen is the only nitrogen present for growth. 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_NH3can be justified if the low nitrate/nitrite concentrations in the CBT can be analyzed.

However, the analysis is probably not accurate enough to demonstrate the small possible increase in nitrate/nitrite in the closed bottle test medium. Maximum theoretical amount of NO₃formed from organic nitrogen present in the test substance is 0.4 mg/L and the “background” NO₃concentration in CBT blank with active sludge and river water is ~0.7 mg/L and ~10 mg/L, respectively.

Using the conservative ThOD_NO3to calculate the biodegradation of the test substance, still >60% biodegradation was achieved within 28 days (Table II). The solvent free test substance should therefore be classified as readily biodegradable. For the final GLP test it was 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 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*	-5	37	46	57
	2 g silica gel /bottle	11	43	54	61	64
	1 g silica gel / bottle	13	44	56	66	70
	2 mg/L humic acid	-4	20	60	64	73
	1 mg/L humic acid	-4	11	53	62	66
River water	No sorbent*	-9	-1	15	26
	2 g silica gel /bottle	1	12	27	33	44
	1 g silica gel / bottle	-1	10	25	32	42
	2 mg/L humic acid	-2	5	45	52	76
	1 mg/L humic acid	-3	11	43	52	71

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

 

Table II Percentages biodegradation of test substance 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*	-4	35	43	53
	2 g silica gel /bottle	11	41	51	57	60
	1 g silica gel / bottle	12	41	52	62	65
	2 mg/L humic acid	-3	19	56	59	68
	1 mg/L humic acid	-3	10	49	58	62
River water	No sorbent*	-9	-1	14	24
	2 g silica gel /bottle	1	11	25	31	41
	1 g silica gel / bottle	-1	9	24	30	39
	2 mg/L humic acid	-2	4	42	49	71
	1 mg/L humic acid	-3	11	40	49	67

* NH₄Cl (0.5 mg/L) is 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 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

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 TMAC (98.5 % 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’, 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 7.105 CFU/L and 6.105 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. 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 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 2.86 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 7.3 and 7.2±0.2 (activated sludge) and 8.2 and 8.2±0.2 (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 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:

weight of evidence

Study period:

From May 08, 2020 to June 10, 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:

One minor deviation from the guideline is that ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, 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 103.7 °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 3.1 g/L.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992). The Colony forming units (CFU) of the preconditioned and diluted inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned and diluted inoculum as used in the closed bottles (2 mg/L dry weight) was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added.
The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar. Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.7 – 22.8 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 1.106 CFU/L.

Duration of test (contact time):

ca. 28 d

Initial conc.:

2 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Reference substance and chemicals
Sodium acetate anhydrous was used as a reference substance in the Closed Bottle test. This compound was purchased from Sigma-Aldrich, St Louis, US. The substance data were submitted by the supplier.
Reference compound - acetic acid, sodium salt
CAS reg. No. - 127-09-3
Purity - 99.9%
Batch/lot number - BCBP8197V
Appearance - white powder
The silica gel Davisil grade 636, pore size 60A, 35-60 mesh particle size (MKCH4201) was purchased from Sigma-Aldrich. 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.
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 and sodium acetate were added to the bottles using an aqueous stock solution of 1 g/L. Silica gel was added as a 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 procedure
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994)10 bottles containing only inoculum. 10 bottles containing inoculum and silica gel, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentration of the test substance and sodium acetate in the bottles was 2.0 mg/L and 6.7 mg/L, respectively.
Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were 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

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(Based on ThODNH3

Value:

ca. 65

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3

Value:

ca. 61

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Theoretical oxygen demand (ThOD) 

The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.85 and 3.05 g oxygen/g active ingredient, respectively. These ThODs were calculated with the molecular formula of the active ingredient (of the solvent free cetrimonium chloride). 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 at the start of the test was 7.0 for the control and reference substance and was 6.9 for the test substance and control with silica gel. The pH of the medium in the reference bottles measured at day 14 was 7.1. The pH of the medium at day 28 was 6.8 for the control and 6.7 for the test substance and control with silica gel. The temperature ranged from 22.7 to 22.8 °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 1.15 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 80. 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 cetrimonium chloride 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 cetrimonium chloride is classified as readily biodegradable based on the >60% biodegradation reached on 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	8.4	4.6	8.5	7.5
	8.4	4.6	8.5	7.7
Mean (M)	8.40	4.60	8.50	7.60
14	8.2	4.0	8.2	6.2
	8.2	4.0	8.2	6.3
Mean (M)	8.20	4.00	8.20	6.25
21	8.0		8.2	5.4
	7.9		8.2	5.4
Mean (M)	7.95		8.20	5.40
28	7.7		8.0	4.3
	7.8		7.9	4.3
Mean (M)	7.75		7.95	4.30

 

Oc Mineral nutrient solution with only inoculum. 

Ocs Mineral nutrient solution with inoculum and silica gel 

Ot Mineral nutrient solution with inoculum, test substance (2.0 mg/L test substance =  1.97 mg/L active ingredient) and silica gel 

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

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

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance		Acetate
			ThODNH3	ThODNO3
0	0.00	0.00	0	0	0
7	0.90	3.80	16	15	73
14	1.95	4.20	35	33	80
21	2.80		50	47
28	3.65		65	61

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

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 TMAC (98.5 % active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, Netherlands. The measured dry weight of the inoculum was 3.1 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 1.106 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 and silica gel, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentration of the test substance and sodium acetate in the bottles was 2.0 mg/L and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were 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 2.85 and 3.05 g oxygen/g active ingredient, respectively. According to the results of this study, the test substance did not cause a reduction in the endogenous respiration at Day 7.The test substance in the presence of silica gel is therefore considered to be non-inhibitory to the inoculum in the test. The test substance was biodegraded by 65% (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 61% at Day 28 (based on ThODNO3). The validity of the test is demonstrated by an endogenous respiration of 1.15 mg/L at day 28. Furthermore, the differences in the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 80. Finally, the validity of the test is shown 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 readily biodegradable with >60% biodegradation after 28 days, but failing the 10 -day window criteria (Geerts, 2020).

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 08 June, 2005 to 24 February, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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: Municipal sewage treatment plant, D-31137 Hildeshheim
- Initial cell/biomass concentration: 1.0E+7 to 1.0E+8 CFU/L

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral nutrient solution according to ORCD 301 B/CO2 evolution test.
- Test temperature: 22 ± 2°C.
- pH:
- Aeration of dilution water: 30 to 100 mL/min.
- Replicates: Duplicate
- TOC: 21.9%
- ThCO2: 0.8 mg CO2/mg test substance
- Carbon content in the vessel: 6.6 mg C/L
- pretreatment: Ultrasound, 10 min. at room temperature

TEST SYSTEM
- Culturing apparatus: 5 litre brown glass bottles.
- Number of culture flasks: 2 for test substance and 1 for reference material.
- Details of trap for CO2: 100 mL flasks to absorb CO2 in the Ba(OH)2 solution.

SAMPLING
- Sampling frequency: Three times a week during the first 10 d and thereafter twice a week until the Day 28.
- Other: On the Day 28, 1 mL of 37% HCI added to flasks in order to eject all the remaining CO2 and last sampling was carried out on the Day 29.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, in duplicates (nutrient solution)
- Toxicity control: Yes, single (test substance and reference substance in test concentration)
- Functional control: Yes; single (sodium acetate (test conc: 35 mg/L; ThCO2: 1.07 mg CO2 /mg; ThTOC: 0.29 mg C/mg; Carbon content in the vessel: 10.2 mg C/L)

Reference substance:

acetic acid, sodium salt

Remarks:

at concentration of 30 mg/L = 10.2 mg carbon/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 93.5

Sampling time:

28 d

Details on results:

With the test substance, the 10% level (beginning of biodegradation) was reached by one replicate after 7 d and the pass level of 60% was reached after 17 d. The other replicate reached the 10% level after 12 d and 60% after 19 d. The biodegradation came to 92% and 95% respectively after 28 d. Hence, the mean biodegradation extent of the test substance can be calculated to be 93.5% within 28 d after acidification (mean value of two test vessels). For further details - please refer to Tables and Figures provided in the below sections.

Results with reference substance:

The functional control reached a biodegradation rate of more than 60% after 10 d.

Results:

Table 1: Biodegradation of the test substance in comparison to the functional control and toxicity control.

	Biodegradation (%) Study day (d)
	6 d	14 d	21 d	28 d
Test substance, 1^streplicate 30 mg/L	0	53	72	92
Test substance, 2^nd replicate 30 mg/L	0	37	68	95
Functional control 35 mg/L	41	74	79	91
Toxicity control 30 mg/L test substance + 35 mg/L reference substance	21	43	54	73

Details on results:

Table 2: CO2-Production and Biodegradation after 28 Days

 

CO2-production     after 28 d		control     mv	functional control 35 mg/L	test substance		30	mg/L	toxicity control     35 + 30 mg/L
				No.1	No. 2
gross	[mg/3 L]	119.9	222.0	186.3	188.3			255.4
	[mg/L]	40.0	74.0	62.1	62.8			85.1
net	[mg/3 L]	-	102.1	66.4	68.4			135.5
	[mg/L]	-	34.0	22.1	22.8			45.2
theor.	[mg/3 L]	-	112.4	72.0	72.0			184.4
	[mg/L]	-	37.5	24.0	24.0			61.5
Degradation [%] after 28 d		-	91	92	95			73

mv = mean value

Table 3: CO2-Production and Biodegradation in the Control, Functional Control and Toxicity Control Samples

 

studyday	date	control           [mg CO2/3L] mv	functional control 35 mg/L				toxicity control 35 mg/Lreference substance + 35 mg/L test substance
			[mg	CO2/3L]		degr. [%]	[mg	CO2/3L]			degr. [%]
			gross		net		gross		net
1	27.01.	3.9	7.9		4.0	4	4.8		0.9		0
4	30.01.	13.7	46.2		32.5	29	32.2		18.5		10
6	01.02.	21.5	68.0		46.5	41	59.6		38.1		21
8	03.02.	28.0	88.1		60.1	53	79.2		51.2		28
11	06.02.	37.2	110.9		73.7	66	102.4		65.2		35
14	09.02.	50.2	133.0		82.8	74	129.2		79.0		43
18	13.02.	66.4	152.8		86.4	77	158.1		91.7		50
21	16.02.	80.9	169.9		89.0	79	180.9		100.0		54
25	20.02.	96.3	188.7		92.4	82	206.1		109.8		60
28	23.02.	108.3	201.4		93.1	83	225.7		117.4		64
29*	24.02.	119.9	222.0		102.1	91	255.4		135.5		73

degr.=degradation         mv=meanvalue

*) results of last two gas wash bottles

Table 4: CO2-Production and Biodegradation in the Control and Test substance Samples

studyday	date	control   [mg CO2/3L] mv	test substance 30 mg/L
			replicate 1			replicate 2
			[mg CO2/3L]		degr. [%]	[mg CO2/3L]		degr. [%]
			gross	net		gross	net
1 4 6 8 11 14 18 21 25 28 29*	27.01. 30.01. 01.02. 03.02. 06.02. 09.02. 13.02. 16.02. 20.02. 23.02. 24.02.	3.9 13.7 21.5 28.0 37.2 50.2 66.4 80.9 96.3 108.3 119.9	3.4 11.7 20.5 41.0 66.9 88.5 112.9 132.9 156.8 173.7 186.3	-0.5 -2.0 -1.0 13.0 29.7 38.3 46.5 52.0 60.5 65.4 66.4	0 0 0 18 41 53 65 72 84 91 92	4.4 13.2 18.5 23.9 43.7 76.9 108.6 129.5 153.9 170.7 188.3	0.5 -0.5 -3.0 -4.1 6.5 26.7 42.2 48.6 57.6 62.4 68.4	1 0 0 0 9 37 59 68 80 87 95

degr.=degradation    mv = meanvalue

*) results of last two gas wash bottles

Table 5: pH-Values on Day28

 

control		functional control	test item		toxicity control
No. 1	No.2		No. 1	No.2
7.40	7.44	7.79	7.48	7.46	7.76

Criteria met for the validity of the study:

- The total CO₂ evolution in the inoculum blank at the end of the test was 40 mg/L.

 

- The biodegradation of the reference substance reached the pass level of ≥60% by Day 14.

 

- The biodegradation of the toxicity control reached the pass level of 25% after 14 d.

 

- The difference of extremes of replicate values of removal of the test substance at the end of the 10-d-window was less than 20%.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the test substance was readily biodegradable after 28 d.

Executive summary:

A study was conducted to determine the ready biodegradability of C16 TMAC (28.9% active in water) according to the OECD Guideline 301 B (CO2-evolution test method), in compliance with GLP. In this study, non-adapted activated domestic sludge was exposed to 30 mg/L of test substance (corresponding to carbon content of 6.6 mg/L) in duplicates for 28 days. Test and reference substances (functional control: sodium acetate and toxicity control; test substance plus sodium acetate) were added to the bottles containing the inoculum and mineral components followed by sampling three times a week during the first 10 days and thereafter twice a week until the Day 28, to measure the CO2 evolution by titrimetric analysis. The percentage degradation of the functional control reached the pass level of 60% after 10 days. In the toxicity control, a biodegradation rate of 43% occurred within 14 days which increased up to a maximum of 73% after 28 days. This indicated that the biodegradation of the reference substance was not inhibited by the test substance in the toxicity control group. For the biodegradation of the test substance, a 10% level (beginning of biodegradation) was reached by one replicate after 7 days and the pass level of 60% was reached after 17 days. The other replicate reached the 10% level after 12 days and 60% after 19 days. The total biodegradation in the two replicate samples reached 92 and 95% respectively after 28 days with a mean value as 93.5%. The validity criteria according to the guideline were all fulfilled. Under the study conditions, the test substance was readily biodegradable (Fiebig, 2006).

Description of key information

Based on the available weight of evidence, the test substance can be considered to be readily biodegradable undergoing complete mineralisation.

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 TMAC (98.5 % 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’, 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 7.105 CFU/L and 6.105 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. 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 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 2.86 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 7.3 and 7.2±0.2 (activated sludge) and 8.2 and 8.2±0.2 (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 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, C16 TMAC (98.5 % active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, Netherlands. The measured dry weight of the inoculum was 3.1 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 1.106 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 and silica gel, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentration of the test substance and sodium acetate in the bottles was 2.0 mg/L and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were 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 2.85 and 3.05 g oxygen/g active ingredient, respectively. According to the results of this study, the test substance did not cause a reduction in the endogenous respiration at Day 7.The test substance in the presence of silica gel is therefore considered to be non-inhibitory to the inoculum in the test. The test substance was biodegraded by 65% (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 61% at Day 28 (based on ThODNO3). The validity of the test is demonstrated by an endogenous respiration of 1.15 mg/L at day 28. Furthermore, the differences in the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 80. Finally, the validity of the test is shown 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 readily biodegradable with >60% biodegradation after 28 days, but failing the 10 -day window (Geerts, 2020).

Study 3: A study was conducted to determine the ready biodegradability of C16 TMAC (28.9% active in water) according to the OECD Guideline 301 B (CO2-evolution test method), in compliance with GLP. In this study, non-adapted activated domestic sludge was exposed to 30 mg/L of test substance (corresponding to carbon content of 6.6 mg/L) in duplicates for 28 days. Test and reference substances (functional control: sodium acetate and toxicity control; test substance plus sodium acetate) were added to the bottles containing the inoculum and mineral components followed by sampling three times a week during the first 10 days and thereafter twice a week until the Day 28, to measure the CO2 evolution by titrimetric analysis. The percentage degradation of the functional control reached the pass level of 60% after 10 days. In the toxicity control, a biodegradation rate of 43% occurred within 14 days which increased up to a maximum of 73% after 28 days. This indicated that the biodegradation of the reference substance was not inhibited by the test substance in the toxicity control group. For the biodegradation of the test substance, a 10% level (beginning of biodegradation) was reached by one replicate after 7 days and the pass level of 60% was reached after 17 days. The other replicate reached the 10% level after 12 days and 60% after 19 days. The total biodegradation in the two replicate samples reached 92 and 95% respectively after 28 dayswith a mean value as 93.5%. The validity criteria according to the guideline were all fulfilled. Under the study conditions, the test substance was readily biodegradable, fulfilling the 10 -day window criteria (Fiebig, 2006).

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):

In addition, recent publications from Timmeret al.,2019 and Nabeokaet 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) and hence increasing their biodegradation. However, 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 overestimate the biodegradation.

Further, the results obtained with the test substance are in agreement with what is reported in the literature for other quaternary ammonium substances, 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 (%)
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
Tetradecylbenzyldimethylammonium Chloride (C14 ADBAC)	MITI	>80
Decylbenzyldimethylammonium Chloride (C10 ADBAC)	Closed bottle	>60

*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. Bacteria identified as Pseudomonas sp capable 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 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.