Hexadecyltrimethylammonium methyl sulphate

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is readily biodegradable

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

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

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Study 1: A study was conducted to determine the ultimate ready biodegradability of the read across substance, C12-14 ADMAES (98.80% active), according to OECD 310 Guideline and ISO 14593:1999 using the headspace CO2 biodegradation test. The read across substance at 20.15 mg/L was incubated with sludge from an activated sludge plant treating predominantly domestic waste. The extent of biodegradation was expressed as a percentage of the theoretical maximum inorganic carbon (IC) production (ThIC), based on the quantity of the read across substance (as organic carbon) added initially over a period of 28 days. The read across substance reached a biodegradation of 67.77% at Day 28. Mean net CO2 values from the read across substance vessels on Days 3, 7, 14, 21 and 28 were 10.25, 30.31, 56.30, 63.98, and 67.77%, respectively, of the theoretical amount. The 10-day window pass criterion is not applicable for UVCB surfactant substances. Under the study conditions, the read across substance is readily biodegradable (De Nadai, 2007). Based on the results of the read across study, the test substance is considered to be readily biodegradable.

Study 2: A preliminary non-GLP study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the read across substance, C16 TMAC (98.5 % active), according to the OECD Guideline 301D. Due to the well-known toxicity of the quaternary substances, the read across 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 read across 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 read across 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 read across substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the read across 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 read across substance was calculated from its molecular formula and molecular weight. The BOD (mg/mg) of the read across substance was calculated by dividing the oxygen consumption by the concentration of the read across 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 read across 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 read across 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 read across substance concentration is expected in the presence of silica gel (1 and 2 g/bottle). Under the study conditions, the read across substance was determined to be readily biodegradable and the use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the read across substance was considered further for the main study (Geerts, 2020). Based on the results of the read across study, the test substance is considered to be readily biodegradable.

The main study was conducted to determine the ready biodegradability of the read across 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 fromthe 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 read across 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 read across 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 read across substance, 6 bottles containing inoculum and sodium acetate. The concentration of the read across 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 read across substance was assessed by the measurement of oxygen consumption. The ThODNH3 and ThODNO3 of the read across 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 read across substance did not cause a reduction in the endogenous respiration at Day 7.The read across substance in the presence of silica gel is therefore considered to be non-inhibitory to the inoculum in the test. The read across substance was biodegraded by 65% (based on ThODNH3), at Day 28. Assuming a complete nitrification of the organic nitrogen present in the read across substance and using a correction for the oxygen consumption by the nitrification, the read across 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 read across substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020). Based on the results of the read across study, the test substance is considered to be readily biodegradable.

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 read across 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; read across 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 read across substance in the toxicity control group. For the biodegradation of the read across 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 read across substance was readily biodegradable (Fiebig, 2006). Based on the results of the read across study, the test substance is considered to be readily biodegradable.

The use of silica gel in the biodegradation study conducted with C16 TMAC (study 2), 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 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 influence the biodegradability potential of the and hence 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)

Biodegradation pathways of quaternary substances

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 (seeFigure 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.