Benzyl-C12-14-alkyldimethylammonium chlorides

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

January, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Not GLP

Justification for type of information:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

>106 CFU/mL; consisting of micro-organisms in mud collected from Station Epuration Wavre 2nd Stage, a household water treating plant.
Although the inoculum concentration is unbounded, however, this is not expected to affect the overall result as can be demonstrated based on fulfillment of the validity criteria for the control group. That is, the total CO2 evolution in the inoculum blank at the end of the test is 39.2 mg which is <40 mg/L medium, which is an evidence of the inoculum having a low concentration of microorganisms (not being excessive).

Duration of test (contact time):

28 d

Initial conc.:

5 other: mgC/L

Based on:

test mat.

Remarks:

Testing at low concentrations, was required due to the toxicity of the test substance towards the inoculum at higher concentrations (see section 6.1.7 of IUCLID)

Details on study design:

Test conditions:
- Test temperature: 22 ± 2°C
- pH: 7.3 at start and 7.8 at the end of study
- Aeration of dilution water: Air free from carbon dioxide was passed through the solutions using a flow of 50 to 100 mL/min
- Continuous darkness: Yes
- Other: Total organic carbon content: 62.8%
Test system:
- Culturing apparatus: 3-litre flasks mounted with an aeration tube on a magnetic stirrer
- Details of trap for CO2 and volatile organics if used: the CO2 produced in each flask was precipitated with Ba(OH2). The amount of CO2 produced was determined by titration.

Reference substance:

benzoic acid, sodium salt

Remarks:

20 mgC/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

95.5

Sampling time:

28 d

Details on results:

- The biodegradability in the test flask was determined to be 95.5% after 28 days (See Table 1 under 'Any other information on results inc. tables).
- The CO2 production in the blank (inoculum control) was 39.2 mg. The pH measured at the start and end of the test period was 7.3 and 7.8, respectively. The temperature throughout the test ranged from 20.8 to 21.4°C.

Results with reference substance:

The biodegradability in the reference flask was determined to be 88.9% after 28 days (See Table 1 under 'Any other information on results inc. tables and the graph under 'Illustration (picture/graph)'.

Table 1. Biodegradability values:

Day	Reference substance		Test substance
	% CO2Produced	% CO2 Total	% CO2Produced	% CO2 Total
0	0.00	0.00	0.0	0.0
1	14.16	14.16	1.2	1.2
3	28.54	42.69	1.0	2.2
6	21.20	63.89	9.5	11.6
8	8.77	72.67	16.0	27.7
10	3.72	76.39	20.2	47.9
13	3.26	79.65	17.4	65.3
15	2.53	82.18	7.5	72.8
17	1.47	83.65	5.1	77.9
20	2.31	85.95	3.9	81.8
22	1.15	87.11	1.6	83.5
24	0.25	87.36	4.4	87.8
28	0.16	87.52	1.8	89.6
29	1.35	88.87	5.8	95.5

For details on results of replicates of test substance and the graphs of test and reference substances, please refer to the attachment under 'Attached background material'.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions, the biodegradation of the read across substance in water was determined to be 95.5 after 28 days (CO2 evolution). The read across substance was considered to be readily biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the read across substance, C12-16 ADBAC (80% active in hydroalcoholic solution), in water according to OECD Guideline 301B (CO2 evolution test). Flasks containing inoculum from a household water-treating plant dosed with the equivalent of 5 mg C/L test or 20 mg C/L reference substances were maintained for 28 d. Testing at low concentrations, was required due to the toxicity of the read across substance towards the inoculum at higher concentrations. Biodegradability was calculated from the released CO2 over time in the test and reference flasks compared to the blank control (a flask prepared without test or reference substance). CO2 production in the blank (inoculum control) was 39.2 mg. Biodegradability in the reference flask was determined to be 88.9% after 28 d. Under the test conditions, the biodegradation of the read across substance in water was determined to be 95.5% after 28 d (CO2 evolution). The read across substance was considered to be readily biodegradable (van Dievoet, 2005). Based on the results of the read across study, the test substance is considered to be readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

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

Adequacy of study:

key study

Study period:

From March 16, 1992 to April 20, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

slight inoculum modification

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: Secondary activated sludge was used

Details on inoculum:

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 the sludge (200 mg Dry Weight (DW)/L) was aerated for one week. The sludge was diluted to a concentration of 2 mg DW/L in the BOD bottles. This method was described in a proposal for harmonizing ready biodegradability test protocols.

Duration of test (contact time):

28 d

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- The closed bottle test was performed according to the EEC, OECD and ISO Test Guidelines. Ten bottles containing only inoculum, 10 bottles containing test substance and inoculum, and 10 bottles containing sodium acetate and inoculum were used. The concentrations of the test compound and sodium acetate in the bottle were 4.0 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. The zero time bottles were immediately analysed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated at 21°C in the dark.
- Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14 , 21 and 28. One extension from the protocol of the closed bottle test was introduced.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

63

Sampling time:

28 d

Details on results:

The test was conducted in the presence of silica gel due to toxicity to inoculum.
The validity of the test was demonstrated by an endogenous respiration of 1.3 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 78.
The validity of the test was shown by oxygen concentrations being >0.5 mg/L in the bottles.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 78.

Table 1. Biodegredation percentages (%)

	Test substance	Reference substance
0	0	0
7	1	69
14	50	78
21	66	86
28	63	88

Nitrification corrections:

Ammonium chloride is omitted from the medium to prevent oxygen consumption bynitrifying bacteria increasing the endogenous oxygen consumption in the BOD bottles. The reason for this omission is the goal to lower the endogenous respiration thereby increasing the accuracy of the assessment of the biodegradability. This goal 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 should not hamper the biodegradation of organic compounds in the Closed Bottle test. The biodegradation of the reference substance (sodium acetate) does demonstrate that nitrogen is not limiting growth. The nitrogen introduced with the inoculum is sufficient to fulfill the nitrogen requirement of the microorganisms.

Nitrification of the nitrogen present in test substance itself could occur. This could be a reason for using the ThOD_NO3. Using the ThOD_NO3of 1.52 g/g would result in a biodegradation percentage of 57.4, not allowing classification as readily biodegradable.

	Molecular formula	MW	ThODNH3 (g/g)	ThODNO3(g/g)	Weight (%)
C12-16 ADBAC ( n= C12)	C21H38NCl	340	2.78	2.96	0.501
Water	H2O	18	0	0	0.488
Amines	C16H35N	241.46	3.18	3.45	0.011
The ThODNH3of the test substance is =					1.43
The ThODNO3of the test substance is =					1.52

 

Day	O2 consumption	BOD	ThODNH3	% biodegradation	ThODNO3	% biodegradation
7	0.1	0.025	1.43	1.8	1.52	1.6
14	2.8	0.7		49.1		46.0
21	3.7	0.925		64.9		60.7
28	3.5	0.875		61.4		57.4

Test conc: 4 mg/L

However, it is not obligatory to use ThOD_NO3for all nitrogen containing test substances. The choice of the ThOD used to calculate biodegradation percentages 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_NH3do therefore 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 occurring 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.

Biodegradration of the C12 -16 ADBACs and in general:

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

 

Figure:Biodegradation pathway of alkylbenzyldimethylammonium salts

Alkylbenzyldimethylammonium salts are biodegraded by microorganisms first utilizing the alkyl chain. Subsequently the methyl and benzyl groups are removed. In conclusion calculation of the biodegradation percentages with the ThODNH3 is thought to be a more appropriate choice.Further, the registrant would like to point out that this study has been evaluated as a key and valid study under the biocide dossier by the RMS - Italy, which has been published by ECHA (RMS: Italy) in June 2015 (ECHA, 2015; refer to page 29). As per the assessment report, C12-16 ADBAC has been concluded to be readily biodegradable.The registrant will include the above details in the next dossier update.

References

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

·        ECHA (RMS: Italy) 2015: Assessment Report: Alkyl (C12-16) dimethylbenzyl ammonium chloride, Product-type 8 (Wood preservative); Regulation (EU) No 528/2012 concerning the making available on the market and use of biocidal products, Evaluation of active substances. (Biocides assessment report), Approval ID: 0063-08; Asset No: EU-0005415-0000; Rapporteur Member State: Italy. Report date: June, 2015.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of the study, the biodegradation of the read across substance was determined to be 63% at Day 28. The read across substance was considered readily biodegradable.

Executive summary:

A study was conducted to determine the biodegradation of the read across substance, C12-16 ADBAC (50.1% active in water) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. Secondary activated sludge was used in this experiment and the percentage of degradation (O2 consumption) was measured. Since the substance was toxic to microorganisms, it was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the substance was released slowly from the silica gel. The validity of the test was demonstrated by an endogenous respiration of 1.3 mg/L at Day 28. Furthermore, the differences between the replicate values at Day 28 were less than 20%. The biodegradation of the reference substance, sodium acetate, at Day 14 was 78%. Finally, the validity of the test was shown by oxygen concentrations being > 0.5 mg/L in the bottles. Under the conditions of the study, the biodegradation of the substance was determined to be 63% at Day 28. The substance was considered readily biodegradable (van Ginkel and Stroo, 1992). Based on the results of the read across study, the test substance is considered to be readily biodegradable.

Description of key information

Based on the results of the read across studies and in line with the biocides assessment report, the test substance is also considered to be readily biodegradable.

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 of the read across substance, C12-16 ADBAC (80% active in hydroalcoholic solution), in water according to OECD Guideline 301B (CO2 evolution test). Flasks containing inoculum from a household water-treating plant dosed with the equivalent of 5 mg C/L test or 20 mg C/L reference substances were maintained for 28 d. Testing at low concentrations, was required due to the toxicity of the read across substance towards the inoculum at higher concentrations. Biodegradability was calculated from the released CO2 over time in the test and reference flasks compared to the blank control (a flask prepared without test or reference substance). CO2 production in the blank (inoculum control) was 39.2 mg. Biodegradability in the reference flask was determined to be 88.9% after 28 d. Under the test conditions, the biodegradation of the read across substance in water was determined to be 95.5% after 28 d (CO2 evolution). The read across substance was considered to be readily biodegradable (van Dievoet, 2005).

Study 2: A study was conducted to determine the biodegradation of the read across substance, C12-16 ADBAC (50.1% active in water) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. Secondary activated sludge was used in this experiment and the percentage of degradation (O2 consumption) was measured. Since the substance was toxic to microorganisms, it was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the substance was released slowly from the silica gel. The validity of the test was demonstrated by an endogenous respiration of 1.3 mg/L at Day 28. Furthermore, the differences between the replicate values at Day 28 were less than 20%. The biodegradation of the reference substance, sodium acetate, at Day 14 was 78%. Finally, the validity of the test was shown by oxygen concentrations being > 0.5 mg/L in the bottles. Under the conditions of the study, the biodegradation of the substance was determined to be 63% at Day 28. The substance was considered readily biodegradable (van Ginkel and Stroo, 1992).

Further, the C12-16 ADBAC Biocides assessment report available published by the Italian authorities in June 2015, reported the two key studies summarised above and stated that “The reliability factor of US ISC study (van Dievoet, 2005)is 1. Therefore, the study by US ISC should be considered for the environmental risk assessment at product authorization stage. In conclusion, ADBAC/BKC is ready biodegradable being the 10-day window criterion met (OECD 301B). On the other hand, the EQC study (van Ginkel and Stroo, 1992) has a reliability factor of 2 because it cannot distinguish between the degradation of ADBAC/BKC and Propan-2-ol (solvent). If we follow the argument that Propan-2-ol is readily biodegradable and might contribute more to the oxygen consumption. This results in an overestimation of ADBAC/BKC, and the 14-day window criteria were not met (OECD 301D). Alkyl (C12-16) dimethylbenzyl ammonium chloride is readily biodegradable.”  

Based on the results of the read across studies and in line with the biocides assessment report, the test substance is also considered to be readily biodegradable.

In general, 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 belowFigure 1):

Figure 1:Biodegradation of C18 TMAC in the Closed Bottle tests without additions () and with 2 g silica per bottle (  ), 2.0 mg/L lignosulphonic acid ( ) and 2.0 mg/L humic acid ( ) (adapted from van Ginkel, 2008).

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

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

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

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

In addition, several literature data are available to clarify the metabolic basis of degradation by micro-organisms.Benzyldimethylamine was found as first metabolite of alkylbenzyldimethylammonium salts degradation byAeromonas hydrophila, activated sludge and aPseudomonas spp. community (Patrauchan and Oriel, 2003; van Ginkel, 2004; van Ginkel, 2007; Tezelet al.,2012). Alkyl-N fission is therefore the most probable strategy to gain access to the carbon of the alkyl chain. The next step in the biodegradation of these quaternary ammonium compounds was the successive removal of the two methyl groups. Benzylamine formed, in turn, is converted into benzaldehyde and ammonium (Patrauchan and Oriel, 2003). In contrast to the alkylbenzyldimethylammonium salts biodegradation pathway reported by Patrauchan and Oriel, (2003), neither benzylmethylamine nor benzylamine were identified as metabolites by two enrichment cultures. Exposure of activated sludge to decylbenzyldimethyl­ammonium chloride probably selects for three microorganisms that utilize the alkyl chain, the aromatic moiety, and dimethylamine (van Ginkel, 2004). Kinetic assays demonstrated that benzylmethylamine and benzylamine were not intermediates of alkylbenzyldimethylammonium salts transformation by the enrichedPseudomonasspp. community (Tezelet al., 2012). Thus, benzyldimethylamine is thought to be transformed to dimethylamine and benzoic acid via debenzylation. Dimethylamine is degraded by the successive removal of both methyl groups resulting in the formation of ammonium (Large, 1971). Both the pure and mixed culture studies showed that the degradation of the alkyl chain of alkylbenzyldimethylammonium chloride results in the formation of water, carbon dioxide and ammonium (seeFigure 2).

Alkylbenzyldimethylammonium salts are biodegraded by microorganisms first utilizing the alkyl chain. Subsequently, the methyl and benzyl groups are removed. 

 

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