Trimethyloctadecylammonium chloride

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 4, 2005 to November 02, 2005

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:

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Degradation-biotic degradation: Closed Bottle test

Deviations:

yes

Remarks:

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

Qualifier:

according to guideline

Guideline:

ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)

Deviations:

yes

Remarks:

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

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

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

Duration of test (contact time):

ca. 28 d

Initial conc.:

1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

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

Reference substance:

acetic acid, sodium salt

Remarks:

concentration in the bottles: 6.7 mg/L

Preliminary study:

Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant. A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test substance is expected. This toxicity was the reason for testing at an initial test substance concentration of 1.0 mg/L.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

ratio BOD/ThoD

Value:

77

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Details on results:

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

Key result

Parameter:

ThOD

Value:

ca. 2.9 other: mg O2/mg

Remarks on result:

other: (NH3)

Key result

Parameter:

ThOD

Value:

ca. 3.06 mg O2/g test mat.

Remarks on result:

other: (NO3)

Results with reference substance:

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

Validity of the test:

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

Lower test concentrations than the guideline:

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

Omission of ammonium from the test medium:

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

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

Nitrification corrections:

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

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

 

 

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

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

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

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

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

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

References:

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

.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

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

Executive summary:

A study was conducted to determine the biodegradation in water of the test substance, C18 TMAC (99.5% active) according to OECD guideline 301D, EU Method C.6 and ISO 10707 (Closed Bottle test), in compliance with GLP. The test was performed with activated sludge, domestic in 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with the test substance. The concentrations of the test substance, and sodium acetate in the bottles were 1.0, and 6.7 mg/L, respectively. (A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected. This toxicity was the reason for testing at an initial test compound concentration of 1.0 mg/L). The test substance was biodegraded by 77% at Day 28 in the Closed Bottle test. The test was valid, as shown by an endogenous respiration of 1.1 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 66% of its theoretical oxygen demand after 14 day. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the test substance can be considered readily biodegradable, but failing 10 -day window (van Ginkel, 2005).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From March 18, 1997 to April 29, 1997

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:

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 (e.g. location, sampling depth, contamination history, procedure): community wastewater treatment plant Hildesheim, Germany

Duration of test (contact time):

ca. 28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Initial conc.:

2.4 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

For details, kindly refer to the attached background material section of the IUCLID.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

18

Sampling time:

28 d

Details on results:

Reference substance: 86% degradation in 28 days
Toxicity control: 67% degradation in 28 days

For result tables, kindly refer to the attached background material section of the IUCLID.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

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

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C18 TMAC (80% active) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. Non-adapted activated sludge was exposed to 3 mg/L test substance (corresponding to 2.4 mg a.i./L based on a purity of ca. 80%), equivalent to a theoretical oxygen demand of 6.96 mg O2/L, for 28 days. A reference substance and a toxicity control were run in parallel. The test substance degraded to 18% within 28 days. Degradation of the reference substance and the toxicity control were 86 and 67%, respectively. Under the study conditions, the test substance was not readily biodegradable (Noack, 1997).

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 12, 1992 to September 15, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

acceptable deviations

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Secondary activated sludge from WWTP Nieuwgraaf In Duiven, The Neatherlands. It was an activated sludge plant treating predominantly domestic wastewater.
- The sludge was preconditioned to reduce endogenous respiration rates. To this end, the sludge (200 mg dry wt/L) was aerated for a period of 7d. The sludge was diluted to a concentration in the biochemical oxygen demand (BOD) bottles of 2 mg dry wt/L.

Duration of test (contact time):

ca. 35 d

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Medium:
- Composition of the medium:
The nutrient medium of the closed bottle test contained per litre 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.
- Test temperature: 21+/- 1°C (not reported)
- pH: 7.1
- pH adjusted: no
- Aeration of mineral medium: yes
- Suspended solids concentration: sludge DW in BOD bottles = 2 mg/L
- Continuous darkness: yes

Test system:
- Culturing apparatus: incubator
- Number of culture flasks/concentration:
10 bottles containing only mineral nutrient solution
10 bottles containing only minteral nutrient solution and inoculum = inoculum blank
10 bottles containing mineral nutrient solution, test substance and inoculum = test
10 bottles containing mineral nutrient solution sodium acetate and inoculum = reference control
10 bottles containing only minteral nutrient solution, silica gel and inoculum = inoculum blank (silica gel)
10 bottles containing mineral nutrient solution, test substance coated on silica gel and inoculum =test
- Method used to create aerobic conditions: aeration with pressured air
- Measuring equipment: oxygen meter

Sampling:
- Sampling frequency: Day 0, 7, 14, 21 , 28 and 35

Control and blank system:
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: no
- Reference control: yes
- Other: inoculum blank with silica gel (to prove the non-biodegradability of silica gel).

Calculations:
THOD test substance = 2.3 mg/mg
THOD sodium acetate = 0.8 mg/mg
Oxygen consumption (mg/L) (BOD) = mean oxygen concentration (mg/L) inoculum blank - mean oxygen concentration (mg/L) test (or reference)

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

53

Sampling time:

35 d

Results

Toxicity

Inhibition of the degradation of a sodium acetate by the test compound in the closed bottle test was not determined because possible toxicity of test substance to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance in the presence of silica gel was not detected. Therefore, no inhibition of the biodegradation due to the "high” initial concentration of the test compound is expected.

Validity

The validity of the test is demonstrated by an endogenous respiration of 0.95 mg/I at day 28. Furthermore, the differences of the replicate values of the control at day 28 were Jess than 20%. The biodegradatlon percentage of the reference compound. sodium acetate, at day 14 was 77. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/I in all bottles during the test period.

Closed bottle test

Test substance was tested in the closed bottle in the presence of silica gel. Silica gel was added to reduce the concentration of the test compound in the water phase, thereby reducing the toxicity of test substance in the test. In the presence of silica gel, test substance was biodegraded 48% at day 28. In the prolonged closed bottle test with silica gel the biodegradation percentage reached 53 at day 35. Hence, test substance should be classified as biodegradable.

Validity criteria fulfilled:

yes

Remarks:

endogenous respiration of 0.95 mg/L, total mineralization of sodium acetate >0.5 mg/L O2 in bottles during the test; differences between replicates at Day 28 < 20%

Interpretation of results:

inherently biodegradable

Conclusions:

Under the study conditons, the test substance was determined to be inherently biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C18 TMAC (49% active in hydroalcoholic solution) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. The test was performed with activated sludge, domestic and non-adapted, exposed to 4.0 mg/L test substance (49% active substance and 36% 2-propanol) for 35 days. Silica gel was added in the study setup to reduce the concentration of test substance in the water phase, thereby reducing its toxicity. The test substance in the presence of silica gel caused no reduction in the endogenous respiration. In the presence of silica gel, test substance was biodegraded 48% at day 28. In the prolonged closed bottle test with silica gel the biodegradation percentage reached 53% at day 35. Hence, test substance should be classified as biodegradable.The test is valid as shown by an endogenous respiration of 0.95 mg/I and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 77% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/I in all bottles during the test period. Due to the large fraction of 2-propanol in the test substance (approximately 36%) it could not be concluded that the active test substance was biodegradable. Under the study conditons, the test substance was determined to be inherently biodegradable (van Ginkel, 1993).

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 19, 1990 to October 18, 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:

acceptable deviations

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge plant RWZI Nieuwgraaf in Duiven, treating predominantly domestic wastewater
- Preparation of inoculum for exposure: The sludge was preconditioned to reduce endogenous respiration rates. To this end, the sludge (200 mg dry wt/L) was aerated for a period of 7d. The sludge was diluted to a concentration in the biochemical oxygen demand (BOD) bottles of 2 mg dry wt/L.

Duration of test (contact time):

ca. 175 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test conditions:
- Composition of medium: The nutrient medium of the closed bottle test contained per litre 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, 0.25 mg FeCl3.6H2O.
- pH: 6.9 - 7.4
- pH adjusted: no
- Aeration of mineral medium: yes
- Suspended solids concentration: sludge DW in BOD bottles = 2 mg/L
- Continuous darkness: yes

Test system:
- Culturing apparatus: incubator
- Number of culture flasks/concentration:
8 bottles containing only mineral nutrient solution
8 bottles containing only minteral nutrient solution and inoculum = inoculum blank
8 bottles containing mineral nutrient solution, test substance and inoculum = test
8 bottles containing mineral nutrient solution sodium acetate and inoculum = reference control
8 bottles containing only minteral nutrient solution, silica gel and inoculum = inoculum blank (silica gel)
8 bottles containing mineral nutrient solution, test substance coated on silica gel and inoculum =test
- Method used to create aerobic conditions: aeration with pressured air
- Measuring equipment: oxygen meter

Sampling
- Sampling frequency: Day 0, 5, 15, 28 , 55, 92, 119, 175 (without silica gel); Day 0, 5, 15, 28 ,42, 56, 84 (with silica gel)
- Sampling method: the bottles from before Day 28 were discarded after the oxygen measurement and the bottles from Day 28 were used also for measuring Days > Day 28 (by using a special funel to collect the disipated medium)

Control and blank system:
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: no
- Reference control: yes
- Other: inoculum blank with silica gel (to prove the non-biodegradability of silica gel).

Calculations:
THOD test substance = 2.8 mg/mg
THOD sodium acetate = 0.8 mg/mg
Oxygen consumption (mg/L) (BOD) = mean oxygen concentration (mg/L) inoculum blank - mean oxygen concentration (mg/L) test (or reference)
Biodegradation (%) = BOD/THOD *100

Reference substance:

acetic acid, sodium salt

Remarks:

6.7 mg/L

Key result

Parameter:

% degradation (O2 consumption)

Value:

77

Sampling time:

175 d

Remarks on result:

other: without silica gel

Key result

Parameter:

% degradation (O2 consumption)

Value:

30

Sampling time:

30 d

Remarks on result:

other: with test substance coated on silica gel

Key result

Parameter:

% degradation (O2 consumption)

Value:

57

Sampling time:

84 d

Remarks on result:

other: with test substance coated on silica gel

Table 1. Percentages biodegradation of the test subsance and sodium acetate in the closed bottle test

Time (Days)	5	15	28	55	92	119	175
Test substance(%BOD/ThOD)	0	0	0	0	28	65	77
sodium acetate (%BOD/ThOD)	75	88	85

Table 2. Percentages biodegradation of the test subsance on silica gel and sodium acetate in the closed bottle test

Time (Days)	5	15	28	42	56	84
Test substance (%BOD/ThOD)	0	17	30	45	55	57
sodium acetate (%BOD/ThOD)	73	83	90

The test substance was not biodegraded in the closed bottle test (28 days). However in the prolonged closed- bottle tests with and without silica gel test substance was biodegraded and therefore should be classified as biodegradable. The validity of the test was shown by the oxygen consumption in the control bottle with sodium acetate and endogenous respirations of 0.4 and 0.5 mg/litre. The oxygen depletion in the bottle without inoculation did exceed the values laid down in the guidelines slightly, The pH-values of the media at day 28 were 6.9 and 7.4.

Validity criteria fulfilled:

yes

Remarks:

endogenous respiration of 0.4-0.5 mg/L, total mineralization of sodium acetate within 14d >0.5 mg/L O2 in bottles during test, differences of replicates <20% at Day 28

Interpretation of results:

inherently biodegradable

Conclusions:

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

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C18 TMAC (92% active) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. The test was performed with activated sludge, domestic and non-adapted, exposed to 2.0 mg/L test substance for 175 days. The test was realised with and without silica gel. In the prolonged closed bottle test without silica gel, the substance degraded up to 77% by Day 175. Toxicity of test substance results in a long lag phase of 55d. After 55d biodegradation starts and reaches 77% at Day 175 showing complete minteralization of test substance. In the test with silica gel, (added to ensure a slow release of the test substance into the water phase), the substance was 30% biodegraded at Day 28 and reached 57% biodegradation at Day 84. In this test the lag phase is reduced to 5d. The test substance has to desorp from the silica gel into the water phase to be bioavailable for the micro-organisms. Desorption and herewith bioavailability of test substance is probably the liming factor in the experiment with silica, resulting in a biodegradation percentage that levels just around the 60%. Both tests were valid as shown by endogenous respirations of 0.4 and 0.5 mg/L and the total mineralization of the reference substance, sodium acetate. Under the study conditions, the test substance was considered to be inherently biodegradable (van Ginkel, 1990).

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 study was conducted to determine the biodegradation in water of the test substance, C18 TMAC (99.5% active) according to OECD guideline 301D, EU Method C.6 and ISO 10707 (Closed Bottle test), in compliance with GLP. The test was performed with activated sludge, domestic in 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with the test substance. The concentrations of the test substance, and sodium acetate in the bottles were 1.0, and 6.7 mg/L, respectively. (A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected. This toxicity was the reason for testing at an initial test compound concentration of 1.0 mg/L). The test substance was biodegraded by 77% at Day 28 in the Closed Bottle test. The test was valid, as shown by an endogenous respiration of 1.1 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 66% of its theoretical oxygen demand after 14 day. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the test substance can be considered readily biodegradable, but failing 10 -day window (van Ginkel, 2005).

Study 2: A study was conducted to determine the biodegradation of the test substance, C18 TMAC (80% active) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. Non-adapted activated sludge was exposed to 3 mg/L test substance (corresponding to 2.4 mg a.i./L based on a purity of ca. 80%), equivalent to a theoretical oxygen demand of 6.96 mg O2/L, for 28 days. A reference substance and a toxicity control were run in parallel. The test substance degraded to 18% within 28 days. Degradation of the reference substance and the toxicity control were 86 and 67%, respectively. Under the study conditions, the test substance was not readily biodegradable (Noack, 1997).

Study 3: A study was conducted to determine the biodegradation of the test substance, C18 TMAC (49% active in hydroalcoholic solution) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. The test was performed with activated sludge, domestic and non-adapted, exposed to 4.0 mg/L test substance (49% active substance and 36% 2-propanol) for 35 days. Silica gel was added in the study setup to reduce the concentration of test substance in the water phase, thereby reducing its toxicity. The test substance in the presence of silica gel caused no reduction in the endogenous respiration. In the presence of silica gel, test substance was biodegraded 48% at day 28. In the prolonged closed bottle test with silica gel the biodegradation percentage reached 53% at day 35. Hence, test substance should be classified as biodegradable.The test is valid as shown by an endogenous respiration of 0.95 mg/I and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 77% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/I in all bottles during the test period. Due to the large fraction of 2-propanol in the test substance (approximately 36%) it could not be concluded that the active test substance was biodegradable. Under the study conditons, the test substance was determined to be inherently biodegradable (van Ginkel, 1993).

Study 4: A study was conducted to determine the biodegradation of the test substance, C18 TMAC (Purity: 92%) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. The test was performed with activated sludge, domestic and non-adapted, exposed to 2.0 mg/L test substance (92% purity) for 175 days. The test was realised with and without silica gel. In the prolonged closed bottle test without silica gel, the substance degraded up to 77% by Day 175. Toxicity of test substance results in a long lag phase of 55d. After 55d biodegradation starts and reaches 77% at Day 175 showing complete minteralization of test substance. In the test with silica gel, (added to ensure a slow release of the test substance into the water phase), the substance was 30% biodegraded at Day 28 and reached 57% biodegradation at Day 84. In this test the lag phase is reduced to 5d. The test substance has to desorp from the silica gel into the water phase to be bioavailable for the micro-organisms. Desorption and herewith bioavailability of test substance is probably the liming factor in the experiment with silica, resulting in a biodegradation percentage that levels just around the 60%. Both tests were valid as shown by endogenous respirations of 0.4 and 0.5 mg/L and the total mineralization of the reference substance, sodium acetate. Under the study conditions, the test substance was considered to be inherently biodegradable (van Ginkel, 1990).

In general, the use of silica gel in biodegradation studies 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 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 (Nabeokaet 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 (%)
Octadecyltrimethylammonium Chloride (C18 TMAC)	Sturm test	>70
Hexadecyltrimethylammonium Chloride (C16 TMAC)	Headspace Carbon Dioxide	75*
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 asPseudomonas spcapable of degrading alkyltrimethylammonium salts were isolated from activated sludge (van Ginkelet al., 1992; Takenakaet al., 2007). Alkyltrimethylammonium salts with octadecyl, hexadecyl, tetradecyl, dodecyl, decyl, octyl, hexyl and coco alkyl chains supported growth of the isolates, showing the broad substrate specificity with respect to the alkyl chain length. Alkanals, and fatty acids can also serve as a carbon and energy source (van Ginkelet al., 1992; Takenakaet al., 2007). In simultaneous adaptation studies,1H nuclear magnetic resonance spectrometry (1H-NMR) and GC-MS showed that acetate, alkanals and alkanoates are the main intermediates of alkyltrimethylammmonium salt degradation, indicating that the long alkyl chain is utilized for microbial growth (van Ginkelet al., 1992; Nishiyama and Nishihara, 2002; Takenakaet al., 2007). Trimethylamine is stoichiometrically produced by pure cultures of microorganisms growing with the alkyl chain of alkyltrimethylammonium chloride as the sole source of carbon. The cleavage of the C-alkyl-N bond of alkyltrimethylammonium salts resulting in the formation of trimethylamine is initiated by a mono-oxygenase (van Ginkelet al., 1992). Additional evidence of the cleavage of the C-alkyl-N bond as the initial degradation step of alkyltrimethylammonium salts was presented by Nishiyamaet al. (1995) and Takenakaet al. (2007). 

Dehydrogenase activity present in cell-free extract of hexadecyltrimethylammonium chloride-grown cells catalysed the oxidation of alkanal to fatty acids. The route of the fatty acid degradation is by β-oxidation. Trimethylamine, a naturally occurring compound is readily biodegradable (Pitter and Chudoba 1990). Complete degradation of trimethylamine is demonstrated through the assessment of the biodegradation pathway. Trimethylamine is degraded by methylotrophic bacteria through successive cleavage of the methyl groups (Large, 1971; Meiberg and Harder, 1978). Consortia of microorganisms degrading the alkyl chain of alkyltrimethylammonium salts and trimethylamine are therefore capable of complete (ultimate) degradation of alkyltrimethylammonium salts. Complete degradation of alkyltrimethylammonium salts using a mixed culture has been demonstrated by Nishiyamaet al.(1995). More recently, Nishiyama and Nishihara (2002) have isolated aPseudomonas sp. capable 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 (seeFigure 2).

Figure 2: Biodegradation pathway of alkyltrimethylammonium salts (van Ginkel, 2004, 2007)

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.