Registration Dossier

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Administrative data

Endpoint:
sediment toxicity: long-term
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
data waiving: supporting information
Reference

Overall, considering all the above 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 preliminary non-GLP study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the test substance, C16-18 and C18-unsatd. TMAC (96% 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 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively. The tests were performed in triplicates using 0.3 L BOD bottles with glass stoppers. In the tests ‘without sorbent’ use was made of 3 bottles with the test substance (at 2 mg/L) and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 μg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the test substance (at 2 mg/L), the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 μg/L isopropanol, and silica gel or humic acid. Silicagel and humic acid concentrations in the bottles (test and control) were 1 and 2 g /bottle and 1 and 2 mg acid/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The theoretical oxygen demand (ThOD) of test substance was calculated from its molecular formula and molecular weight. The BOD (mg/mg) of the test substance was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle. The ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.86 g/g and 3.06 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 7.4 and 7.2±0.1 (activated sludge) and 8.2 and 8.0±0.1 (river water) at the start and end of Day 42 of the test respectively. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption and this was clearly detected until day 7-14 in the sorbent free ready biodegradation tests. The humic acid sorbent still showed an inhibition of the endogenous respiration (negative biodegradation percentages) at Day 7. Detoxification was most successful by the silica gel sorbents and no inhibition of the biodegradation due to the “high” initial test substance concentration is expected in the presence of silica gel (1 and 2 g/bottle). Under the study conditions, the test substance was determined to be 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).

The main study was conducted to determine the ready biodegradability of the test substance, C16-18 and C18-unsatd. TMAC (96% active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the domestic wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. The measured dry weight of the inoculum was 3.2 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles. The inoculum concentration in the BOD bottles determined by colony count was 1E+6 CFU/L. The test substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles supplemented with 1 g silica gel/bottle as sorbent for detoxification of the test substance, and incubated in the dark at 22.7 to 22.9°C for 28 days. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. The concentration isopropanol added to the control bottles with silica gel was 35 µg/L which is comparable to the isopropanol content present in the test bottles. Each of the prepared solutions was dispensed into the respective group of biochemical oxygen demand (BOD) bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. 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 2.86 and 3.06 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 67% (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 63% at day 28 (based on ThODNO3). The validity of the test is demonstrated by an endogenous respiration of 1.10 mg/L at Day 28. Furthermore, the differences of the replicate values at Day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 75%. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period Under the study conditions, the test substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020).

Study 2:A study was conducted to determine the ready biodegradability of the test substance, C16-18 and C18-unsatd. TMAC (49% active in isopropanol and water), in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. The test substance at 2 mg/L was incubated with inoculums from river water and O2 consumption was followed over a period of 28 days. The test was performed using 10 bottles containing only river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle), 10 bottles containing river water, silica gel and test substance, and 6 bottles containing sodium acetate and river water. The concentrations of the test substance and sodium acetate in the bottles were 2.0 and 6. 7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of 0.3 L BOD bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. The test was found to be valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 76% of its theoretical oxygen demand after 14 day. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability was determined to be 71% and 69% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. Therefore, the substance can be considered readily biodegradable in water. Furthermore, the test substance did not cause a reduction in the endogenous respiration in presence of silica gel, hence was considered to be non-inhibitory to the inoculum. Under the conditions of the study, the test substance is considered to be readily biodegradable (van Ginkel CG, 2010).

Study 3:A study was conducted to determine the biodegradation in water of the read across 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 read across substance. The concentrations of the read across 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 read across 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 read across substance was biodegraded by 77% and 73% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. 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 read across substance can be considered readily biodegradable (van Ginkel, 2005). Based on the results from this longer chain alcohol free quaternary ammonium substance, which would represent a worst case – as longer chains tend to biodegrade more slowly than shorter chains, the test substance, which is mix of C16 and C18 alkyl chains, can be expected to be degrading faster than the read across substance.

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 (seeFigure 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)

Biodegradation pathways of quaternary substances

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 (seeFigure 2in 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

Reason / purpose for cross-reference:
data waiving: supporting information
Reference

The results of the read across study, supported with the estimated BCF value for the test substance together with its ionic nature indicates a low bioaccumulation and biomagnification potential. The higher experimental BCF value of 79 L/kg wt-wt from the read across study with C12-16 ADBAC and the growth corrected kinetic biomagnification factor (BMFkg) value of 0.0463 based on read across to C18 TMAC, has been considered further for hazard/risk assessment. 

BCF (aquatic species):
79 L/kg ww
BMF in fish (dimensionless):
0.046

Study 1:A study was conducted to determine the aquatic bioaccumulation of the read across substance, C12-16 ADBAC (30.64% active; 98.9% radiolabeled purity) inLepomis macrochirus(bluegill fish) under flow-through conditions, according to EPA OPP 165-4, in compliance with GLP. The blue gill fish were continuously exposed to a nominal concentration of 0.050 mg/L of the read across substance (equivalent to a measured concentration of 0.076 mg/L) in well water for 35 days, followed by transfer of 35 fish into flowing uncontaminated water for a 21-d depuration period. Sampling was carried out on Days 0, 1, 3, 7, 9, 10, 14, 21, 23, 28 and 35 for the exposure period and Days 1, 3, 7, 10, 14 and 21 for the depuration period. Water samples were collected on Day 8 of the exposure period and Day 16 of the depuration for analytic determination of the read across substance concentration. Radiometric analyses of the water and selected fish tissues revealed that the mean steady state bioconcentration factor (BCF) in the edible, non-edible and whole-body fish tissue during the 35 days of exposure to be 33, 160 and 79 L/kg. The half-life for non-edible tissue was attained between Days 14 and 21, while it could not be reached for the edible and whole-body fish tissues by the end of 21-d depuration period. By Day 21 of the depuration period, the 14C residues present on the last day of exposure in the edible, non-edible and whole-body fish tissues had been eliminated by 29, 60 and 44% respectively. Analysis of skin tissue after 35 d of exposure showed residue levels somewhat higher than those observed for edible tissue at the same sampling period, indicating that there is likely significant binding of 14C-ADBAC to the skins and scales of exposed bluegill, as expected behaviour of cationic surfactants. Under the conditions of the study, the whole body BCF of the read across substance was determined to be 79, indicating low potential to bioaccumulate (Fackler, 1989).  

Study 2: A study was conducted to determine the biomagnification (BMF) potential of the read across substance, C18 TMAC (purity 95%), following the principles of OECD TG 305. For the main study rainbow trout (Oncorhynchus mykiss) with an average weight of 5.42 g were fed test diets enriched with read across substance (23.6 mg/kg read across the substance in feed. The resulting treatment and one control group (each 40 animals) were tested simultaneously. The uptake phase of 14 days was followed by a depuration phase lasting 14 days. All animals were fed the non-spiked feed during the depuration phase. The concentrations of the read across substance in fish samples were determined by chemical analysis and all tissue concentrations were calculated based on a wet weight basis. Chemical analysis of the read across substance was performed by liquid chromatography with coupled mass spectrometry (LC-MS/MS). In the main study five animals of each group were sampled randomized on Day 7 and Day 14 of the uptake phase and after 10 h, 24 h, 2 days, 3 days, 7 days and 14 days of depuration. Biomagnification factor (BMF) and distribution factor were calculated based on the tissue concentrations measured at the end of the uptake phase. No mortality or abnormal behaviour of the test animals was observed during the main study. The experimental diets were accepted by the test animals and showed a decent digestibility as confirmed by the texture and appearance of the feces. One fish was euthanized at Day 25 due to injuries. The specific growth rates of the animals ranged from 1.95 to 2.71 %/d over the entire experiment. During the study, the feed conversion ratio (FCR) was 0.69 to 0.95. Fish were measured and weighed at the beginning of the experiment as well as at respective sampling time points to monitor growth and associated growth-dilution effects during the feeding study. Growth rate constants were determined separately for the uptake and depuration phases, for the treatments and the control group, using the ln-transformed weights of the fish. A subsequent parallel line analysis (PLA, as suggested by the OECD Guideline) resulted in no statistical differences between the uptake and the depuration phase among the treated groups with the read across substance. No statistically significant difference was detected with regard to the growth of the treated groups. Hence it was deduced that neither adverse nor toxic effects were caused by the enriched diets. As steady state seemed to be reached after 14 days of exposure, steady state biomagnification factors (BMFss) could be calculated as 0.02709 g/g, which showed that read across substance did not biomagnify after dietary exposure. In general, the GIT and the liver showed the highest values for the BMFk and BMFkg. The kinetic BMF (BMFk) and growth-corrected biomagnification factor (BMFkg) were calculated for the read across substance to be 0.0404 and 0.0463, respectively. Overall, it was concluded from the screening that ionization lowers the tendency of a chemical to bioaccumulate, compared to non-ionized chemicals. Aside from the well-known lipophobicity of ionized groups, fast depuration seems to be a major reason for the observed low biomagnification of ionic compounds, in particular anions. Fast depuration may happen due to rapid metabolism or conjugation of charged compounds, and future studies should test this hypothesis. Under the study conditions, the read across substance BMFss, BMFk and BMFkg values on whole body wet weight basis in rainbow trout were determined to be 0.02709, 0.0404 and 0.0463 g/g, respectively, suggesting low biomagnification potential (Schlechtriem, 2021). Based on the results of the read across study, a similar low biomagnification potential is expected for the test substance. 

Study 3:A study was conducted to determine the tissue distribution of two cationic surfactants mixtures in Rainbow Trout (Oncorhynchus mykiss) following exposure via water for seven days and analysis of different fish tissues. The test chemicals were grouped into two mixtures of six containing 10 alkyl amines and 2 quaternary alkylammonium surfactants: C10 TMAB (as part of MIX 2) and C14 TMAC (as part of MIX 1). Studying chemical mixtures has the advantage that differences in behavior between chemicals are not obscured by biological variability or experimental variables. Bioconcentration studies with mixtures have been shown to provide similar results to studies with single chemicals. The experiments were conducted in 300 L fiberglass aquaria with a water renewal rate of 1.3 L min−1 (MIX 1) and 1.45 L min−1 (MIX 2). A solution of the test chemical mixture in methanol was infused continuously (3.5 and 3.8 μL min−1 for MIX 1 and MIX 2, respectively) into the water inflow using a syringe pump. The intended concentrations of C10 TMAB and C14 TMAC were 59 and 1.3 μg/L (measured). The water temperature was 10 °C and the pH 7.5. The water hardness was estimated to be 1.1 mM Ca2+. For each mixture, the syringe pump was started in an aquarium containing no fish. After 16 h, to allow the concentrations to stabilize, 12 rainbow trout were added. After 7 d of exposure, the fish in the exposure aquaria as well as several unexposed (control) fish were sacrificed followed by blood collection. The surface of the fish posterior of the gills was rinsed with 100% methanol to remove read across substance residues adsorbed to the outer surface of the skin and absorbed in the skin mucus. The fish were then dissected and the liver, the kidney, the gills, and the remaining contents of the abdominal cavity were taken and weighed. Skin and muscle samples were prepared from the upper dorsal region on semi-frozen fish after the methanol rinse had removed the mucus. For 6 fish from each aquarium and 3 control fish, samples of muscle, skin, liver, and gills were homogenized in a bullet blender (muscle and liver) or in a cryo-mill (skin and gill). A sub-sample of 0.5−1.2 g of the homogenate was extracted twice in methanol, employing centrifugation at 4000 rpm for phase separation. Isotope labeled standards of C10 TMAB and C14 TMAC were added to a portion of the extract corresponding to 12−75 mg of the sample. Whole blood was analyzed rather than plasma because of the small quantity of sample available and the anticipated low concentrations. The test chemical concentrations generally increased in the order muscle <blood < skin < gills < liver. Because the mass of extracted mucus was not determined, the concentrations in mucus were normalized to the estimated fish’s total surface area excluding the head, which was not rinsed. The concentration in mucus was on average 3.9 (range 0.9−11.6) times lower than the surface area-normalized concentration in gills. To calculate the quantity of the test chemical in the different tissues, the amount of each tissue in the fish was estimated and multiplied by the concentration in that tissue. The test chemical quantities in the different tissues were then summed to give the body burden in each fish. The apparent BCFs (BCFapp) values at the end of the 7-day exposure were calculated by dividing the surfactant body burden (blood, muscles, liver, gills, skin, mucus) by the fish mass, and dividing this by the average measured concentration in water samples taken during the exposure phase. Under the study conditions, the BCFapp for the two quaternary substances C10 TMAB and C14 TMAC were determined to be 0.1 and 31 L/kg ww, respectively. Mucus, skin, gills, liver, and muscle each contributed at least 10% of body burden for the majority of the test chemicals. In contrast to the analogue alkylamine bases, the permanently charged quaternary ammonium compounds accumulated mostly in the gills and were nearly absent in internal tissues, indicating that systemic uptake of the charged form of cationic surfactants is very slow (Kierkegaard, 2020).  

Study 4:The Bioconcentration factor (BCF) value of test substance, C16-18 and C18-unsatd TMAC was predicted using regression-based and Arnot-Gobas BAF-BCF models of BCFBAF v3.02 program (EPI SuiteTMv4.11). The Arnot-Gobas method, takes into account mitigating factors, like growth dilution and metabolic biotransformations, therefore the BCF values using this method is considered to be more realistic or accurate. Therefore, except for ionic, pigments and dyes, perfluorinated substances, for which it is not recommended (as of now), the Arnot-Gobas method is used preferentially used for BCF predictions. Considering that the test substance is an UVCB containing majorly ionic (e.g., (e.g., the quaternary ammonium salts) and few non-ionic constituents (e.g., amines), the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter. The BCF values for the constituents ranged from 3.16 to 162.4 L/kg ww (log BCF: 0.50 to 2.21), indicating a low bioaccumulation potential. On comparing with domain descriptors, all constituents were found to meet the MW, log Kow and/or maximum number of correction factor instances domain criteria as defined in the BCFBAF user guide of EPISuite. Further, given that the major constituents are structurally very similar and vary only in the carbon chain length, a weighted average value, which takes into account the percentage of the constituent in the substance, has been considered to dampen the errors in predictions (if any). Therefore, the weighted average BCF value was calculated as 70.3 L/Kg ww (Log BCF = 1.85). Overall, considering either the individual BCF predictions for the constituents or the weighted average values, the test substance is expected to have a low bioaccumulation potential. However, taking into consideration the model’s training set and validation set statistics and the fact that the training set only contains 61 ionic compounds, the BCF predictions for the individual constituents are considered to be reliable with moderate confidence. 

This is further supported by the no bioaccumulation potential evidence observed in in the two toxicokinetic studies in mammals with the read across substance, C12-16 ADBAC (Selim, 1987 and Appelqvist, 2006). 

Also, the biocides assessment reports available from RMS Italy on Coco TMAC and C12-16 ADBAC, concluded the substances to show low potential for bioaccumulation, based on the results from the above study (Fackler, 1989) and an additional read across to DDAC for the Coco TMAC’s assessment ((ECHA biocides assessment report, 2015, 2016). The report concluded the following in the Coco TMAC assessment report:“Coco alkyltrimethylammonium chloride is readily biodegradable, is rapidly excreted and does not accumulate in mammals, and it adsorbs onto the fish surface where its irritating action is expressed (therefore accumulation is more related to the concentration of the administered solution). Based on these properties’ bioaccumulation is not expected to be of concern for ATMAC/TMAC. An experimental BCFwhole body of 81 L/kg was determined in a flow-through test with Lepomis machrochirus and the read across substance DDAC (Lonza Cologne GmbH and Akzo Nobel Surface Chemistry AB, same study). A very similar result was obtained for the other quaternary ammonium compound benzyl-C12-16-alkyldimethyl ammonium chloride (C12-16-BKC/ADBAC) in a fish bioconcentration test, which gave a BCFwhole body = 79 L/kg (Akzo Nobel Surface Chemistry AB, access to Lonza Cologne GmbH study). Being both studies equally reliable, the BCFwhole body = 81 L/kg is chosen because related to the lead read across substance (DDAC) and it is slightly higher than the C12-16 BKC/ADBAC endpoint.”  

Overall, the results of the read across study, supported with the estimated BCF value for the test substance together with its ionic nature indicates a low bioaccumulation and biomagnification potential. The higher experimental BCF value of 79 L/kg wt-wt from the read across study with C12-16 ADBAC and the growth corrected kinetic biomagnification factor (BMFkg) value of 0.0463 based on read across to C18 TMAC, has been considered further for hazard/risk assessment. 

Reason / purpose for cross-reference:
data waiving: supporting information
Reference

Based on the most recent and radiolabelled aerobic biodegradation study in soil with the read across substance, C12-16 ADBAC, the transformation of the substance was considered to be rapid with DT50 values ranging from 2.2-28.7 days with the SFO model and 1.6 – 23.3 days with the FOMC model at 20°C.​ Further, in the biocides dossier, a weighted estimate of the DT50 value at 12°C was extrapolated for C12-16 ADBAC by assuming the highest allowable concentrations for the major chains. These calculations resulted in the estimated FOMC DT50 of 17.1 days at 12°C and SOF DT50 of 19.2 days at 12°C. The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower error (x2)compared to the SFO model was used further for risk assessment. 

Half-life in soil:
17.1 d
at the temperature of:
12 °C

Study 1: A study was conducted to determine the aerobic transformation/dissipation in the soil of the read across substance, C12 -16 ADBAC (radiochemical purity: 98.5%), according to the OECD Guideline 307, in compliance with GLP. Four different standard soils (LUFA 2.2, 2.3, 2.4 and 5M, field fresh sampled), varying in their organic carbon content, pH, clay content, cation exchange capacity and microbial biomass, were treated with [ring-U-14C] Benzalkonium chloride. Soil samples were incubated in the dark under aerobic conditions for up to 128 days under controlled laboratory conditions. After appropriate time intervals, soil samples were extracted, and the extracts were analysed for read across substance and transformation products to calculate DT50 and DT90 values. The mineralization was determined by trapping and analysis of the evolved 14CO2. Non-extractable residues (NER) were determined after combustion of the extracted soil samples. The total radioactivity of the soil extracts, the extracted soil (NER) and evolved 14CO2 was determined by LSC. Read across substance and transformation products in the soil extracts were analysed by LC-FSA (radio-HPLC). Evaluation of the transformation pathway was done by LC-HRMS. Transformation of the C12 chain of the read across substance [ring-U-14C]Benzalkonium chloride was rapid in all four soils. The transformation of the C14 chain started after a short adaptation phase but was thereafter rapid as well. Within 7 - 21 days the concentration of the C12 chain decreased from initially 67.2 – 69.6% of applied radioactivity (AR) to < 20 % of AR. The concentration of the C14 chain decreased from initially 23.8 – 24.6 % of AR to < 10 % of AR within 10 – 36 days. Formation of NER started directly after application of the read across substance. Further formation of NER increased in parallel to the start of increased mineralisation, indicating that a major amount of NER is comprised by radioactivity incorporated in microbial biomass. At the test end, the biomass concentration was in the range of 1.46 – 2.62 % of soil organic carbon content in all four soils, indicating that viable microbial biomass was present throughout the incubation time. The mass balance was in the range 99.9 – 103.0 % at test start and 90.4 – 94.0 % at test end.The predominant initial degradation step was the oxidative removal of the alkyl chain. Dimethylbenzylamine was determined as the major metabolite, the highest concentrations of dimethylbenzylamine were determined until Day 22, thereafter the concentrations deceased continuously until test end. Methylbenzylamine was transient and only present in traces. Benzylamine, a suspected metabolite, was not detected. Further metabolites containing partly degraded alkyl chains were all transient and were not detected or only <0.2 % of AR (soil 2.3) at the test end. With regard to the kinetics, the transformation showed a slight bi-phasic pattern, therefore the ‘Single First Order Model’ (SFO) and the ‘First-Order Multi-Compartment Model’ (FOMC) were compared. Based on the visual fit and x2 error, the transformation of [ring-U-14C]Benzalkonium chloride met the requirements for both models well for all four soils. The calculated DT50 values with the Single-First-Order Model (SFO) for the dissipation of [ring-U-14C]Benzalkonium chloride were 2.2 – 8.7 days (C12 chain) and 6.1 – 28.7 days (C14 chain), the DT90 values were 7.2 – 28.8 (C12 chain) days and 20.2 – 95.4 days (C14 chain). The calculated DT50 values with the FOMC model for the dissipation of [ring-U-14C]Benzalkonium chloride were 1.6 – 7.2 days (C12 chain) and 5.5 – 23.3 days (C14 chain), the DT90 values were 15.0 – 48.8 days (C12 chain) and 35.8 – 164.3 days (C14 chain).

The read across substance is predominantly C12-ADBAC and C14-ADBAC, with low to negligible amounts of C16-ADBAC. The chain length distribution is defined as follows:C12 (35-80%), C14 (20-55%), C16 (0-15%). C16-ADBAC was not included in this study because it is present in very low amounts; there are technical difficulties with having sufficient radioactivity for substances present in small amounts relative to other constituents. C16-ADBAC would be expected to degrade by the same route but at a slower rate than its C12 and C14 counterparts, as degradation rate tends to decrease with increasing chain lengths.Under the study conditions, transformations of both C12 and C14 carbon chains of the read across substance were determined to be rapid in all four soils and the DT50 values were determined to be 2.2 – 8.7 days [C12 chain] and 6.1 – 28.7 days [C14 chain] with the SFO model and 1.6 – 7.2 days [C12 chain] and 5.5 – 23.3 days [C14 chain] with the FOMC modelat 20°C (Fiebig, 2019).

 

Further, in the biocides dossier, to account for the potential contribution of C16 ADBAC to the overall DT50 of ADBAC, a geometric mean of SFO and FOMC DT50s for C12 and C14 ADBAC in the four soils (as recommended in BPR Vol IV Part B and C) was calculated and converted to 12° using the following equation (DT50 (12°) = DT50 (20°) * e(0.08*(20-12)). This was followed by linear extrapolation of the geometric mean DT50s for C12 and C14 ADBAC, to estimate the DT50 for C16 ADBAC. See table below:

 

Soil 2.2

Soil 2.3

Soil 2.4

Soil 5M

Geo. Mean

Adj. to 12° C

SFO DT50s

C12 ADBAC

2.2

3.3

6.2

8.7

4.4

8.4

C14 ADBAC

6.1

8.9

12.9

28.7

11.9

22.6

C16 ADBAC

--

--

--

--

--

36.7

FOMC DT50s

C12 ADBAC

1.6

3.2

5.8

7.2

3.8

7.3

C14 ADBAC

5.5

8.3

12.1

23.3

10.7

20.2

C16 ADBAC

--

--

--

--

--

33.1

A weighted estimate of the DT50 of ADBAC (C12-C16) at 12°C was calculated by assuming the highest allowable concentrations of C14- and C16- ADBAC and the balance of C12-ADBAC (i.e., 12% C16, 52% C14 and 36% C12), which resulted in the following estimated DT50s:

SFO DT50 = 19.2d at 12°C; FOMC DT50 = 17.1d at 12°C

However, due to the relatively low levels of C16-ADBAC, the overall estimated DT50s were considered rather insensitive to the assumed DT50 for C16-ADBAC.The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower errorwas used further for risk assessment.

Based on the results of the read across study, similar degradation potential and half-life is considered for the test substance.

 

Study 2:A study was conducted to determine the aerobic biodegradation of the read across substance, C12-16 ADBAC (50% active in water) in loamy soil, according to the US FDA Environmental Assessment Handbook, Technical Assistance Document 3.12 (1987). The study comprised two treatments: test and chemical blank control group, each with three replicates. The read across substance was added into biometers at a concentration of 10 mg carbon per 50 g soil using appropriate amount of deionised water required for bringing the soils to 50-70% of the moisture capacity. Loam was added to the biometers after the test solutions to facilitate uniform moistening of the soils by capillary action. The test was then incubated at 22 ± 3°C and run for approximately 90 d. The side tube of the biometer contained 20 mL 0.2 M KOH for absorbing carbon dioxide produced by the microorganisms. The theoretical CO2 production of the read across substance was calculated from its carbon content. The amounts of carbon dioxide were calculated by subtracting the mean carbon dioxide production in the test systems containing the read across substance and the mean carbon dioxide production level in the control blank. Biodegradation was calculated as the ratio of experimental carbon dioxide production to theoretical carbon dioxide production [ThCO2P]. Under the study conditions, there was 64% degradation of the read across substance after 70 days. This percentage of the theoretical carbon dioxide production presumes complete mineralization. The DT50 was estimated to be 40 days (Ginkel, 1994). Based on the results of the read across study, similar degradation potential and half-life is considered for the test substance.  ​

Based on the most recent and radiolabelled aerobic biodegradation study in soil with the read across substance, C12-16 ADBAC, the transformation of the C12 and C14 carbon chains of the substance was considered to be rapid with DT50 values ranging from 2.2-28.7 days with the SFO model and 1.6 – 23.3 days with the FOMC model at 20°C.​ Further, in the biocides dossier, a weighted estimate of the DT50 value at 12°C was extrapolated for C12-16 ADBAC by assuming the highest allowable concentrations for the major chains. These calculations resulted in the estimated FOMC DT50 of 17.1 days at 12°C and SOF DT50 of 19.2 days at 12°C. The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower error (x2)compared to the SFO model was used further for risk assessment. Therefore, in line with the biocides dossier, the DT50 of 17.1 days at 12°C derived for the read across substance based on the biphasic model (FOMC) also has been considered further for hazard/risk assessment of the test substance.  

Reason / purpose for cross-reference:
data waiving: supporting information
Reference

Based on the results of the read across study, a similar complete removal of the test substance from the domestic wastewater treatment plants can be expected.

Further, based on the results of the soil biodegradation study with the read across substance, the DT50 value for degradation in the sediment compartment can be considered to be approximately 171 days.  

Half-life in freshwater sediment:
171 d
at the temperature of:
12 °C

Surface water simulation testing:

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

Sewage treatment simulation testing

A continuous activated sludge (CAS) study was conducted to determine the biodegradation of the read across substance, C12-16 ADBAC (49.2% active in water), in domestic wastewater according to OECD Guideline 303A, in compliance with GLP. In this study, the domestic waste microorganisms were exposed to the read across substance, by spiking at a nominal influent concentration of 49 mg/L (36 mg/L carbon) for a period of 58 days. An additional unit fed only with the domestic wastewater was maintained as the control group. All samples were analysed for NPOC. A strong increase in the concentration of NPOC was noted on Day 2 in the test units. This was probably caused by toxicity of the read across substance. The activated sludge acclimatised to the read across substance within a few days, resulting in a decrease of the NPOC concentrations. After 3 weeks, very high carbon removal percentages were achieved. The mean removal percentage in the test unit assessed using a HLPC-MS/MS was determined to be 99.998%, indicating ultimate biodegradation. Removal of the read across substance from the influent through adsorption onto sludge was only 0.023% on Day 58, showing that the main mechanism of elimination was biodegradation. Based on the results of the study, the read across substance was removed from wastewater at a very high percentage (approximately 99.998%) in the continuous activated sludge test. Removal of the read across substance from the influent through adsorption onto sludge was only 0.016 to 0.023% at two sampling times, demonstrating that the read across substance was removed almost completely and biodegraded. This suggests that the read across substance biodegrades almost completely in conventional biological wastewater treatment plants (Ginkel, 2007). Based on the results of the read across study, a similar complete removal of the test substance from the domestic wastewater treatment plants can be expected.  

Sediment:simulation testing

The study does not need to be conducted because the substance is readily biodegradable.Nevertheless, as per the ECHA E.16 guidance, the half-life in the sediment compartment will be a factor 10 higher than the half-life in soil. Therefore, the sediment half-life value of 171 days has been considered further for risk assessment.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion