Benzalkonium chloride metabolites – in moving bed biocarrier reactors and WWTPs

Yrsa Larsson; Andrea Mongelli; Kai Bester

Abstract

Abstract: Biodegradation was observed in moving bed biofilm reactors, revealing the presence of 45 metabolites and leading to the identification of two degradation pathways. Concentrations of two quaternary ammonium compounds (QUATs), namely BAC-12 and -14, were determined in both the wastewater treatment plant (WWTP) influent and effluent and in a sludge. BAC metabolites were detected in the sludge and a WWTP effluent.
Introduction
Benzalkonium chlorides (BACs) are biocides most commonly used in disinfectants, household and industrial cleaners and detergents (Fortunato et al., 2019; Knauf et al., 2018). Despite their common use, BACs pose environmental risks as they are directly toxic to microorganisms and detrimental to aquatic life (ECHA, 2023; Sütterlin et al., 2008). Efficient degradation of BACs in wastewater treatment plants (WWTPs) is crucial to limit the environmental spread of these compounds. However, as these compounds are surfactants, it is not easy to determine their physio-chemical data and predictions for their behavior in WWTPs are difficult to make.
Given the complexity of the microbial community in WWTPs, it is important to understand how BACs are degraded and what they are metabolized into. This study intends to reveal the BAC degradation pathway in aerobic moving bed biofilm reactors (MBBRs) with a heterogeneous microbial community and to investigate which BAC metabolites can be found in WWTPs.
Material and Methods
Degradation and metabolization pathways were studied using lab-MBBRs with a multispecies biofilm grown on plastic carriers. Influent and effluent concentrations of three WWTPs in Sjælland were determined using HPLC-MS/MS using 24-hour composite sampling. Metabolite identification was performed using an API6600 high-resolution mass spectrometer and the MarkerView, MetabolitePilot and ChemSketch software. The full method is documented in Larsson et al., 2024.
Results and Discussion
By the findings of 45 metabolites in the micro-MBBR experiment two biodegradation pathways were determined (Fig 1). The initial degradation was an ω-oxidation which involves hydroxylation of the terminal carbon followed by two oxidation steps forming a carboxyl group (Sandhir, 2014). Successively, the metabolite with a carboxyl group is either further oxidized by α-oxidation or β-oxidation releasing either an acetyl or a formyl equivalent. It was most probable, that these two pathways are not only degrading individually (linearly) but are in reality intertwined. These pathways further followed the degradation of β-oxidation until the metabolites of the mass 208 Da and 194 Da were detected.
Two metabolites from this pathway were detected in the outlet of one WWTP, one being the 208 Da and another of the mass 250 Da. One other metabolite detected in the lab-MBBRs was also detected in the WWTP effluent (152 Da) together with two BAC-metabolites known from literature (136 and 186 Da) (Table 1). With the discovery of these five metabolites in a WWTP effluent, this study underscores the importance of a comprehensive metabolite database.
Conclusions
BACs are biodegraded and metabolized in WWTPs. Possibly persistent end products were identified in the degradation pathway and five BAC metabolites were found in the WWTPs. As BACs contribute to antimicrobial resistance, maintaining low environmental concentrations is crucial and knowledge about the persistence and toxicological properties is needed for metabolites from biocides such as BACs.

References
ECHA. (2023). Registration dossier. Quaternary ammonium compounds, benzyl C12-C16 (even
numbered)-alkyldimethyl chlorides. https://echa.europa.eu/da/registration-dossier/-/registered
dossier/13152/6/2/3
Fortunato, M. S., Baroni, S., González, A. J., Álvarez Roncancio, J. D., Storino, A., Parise, C., Planes,
E., Gallego, A., & Korol, S. E. (2019). Biodegradation and Detoxification of Benzalkonium Chloride in
Synthetic and Industrial Effluents in Upflow Biofilm Aerobic Reactors. Water, Air, & Soil Pollution,
230(4), 79. https://doi.org/10.1007/s11270-019-4126-9
Knauf, G. A., Cunningham, A. L., Kazi, M. I., Riddington, I. M., Crofts, A. A., Cattoir, V., Trent, M.
S., & Davies, B. W. (2018). Exploring the Antimicrobial Action of Quaternary Amines against
Acinetobacter baumannii. mBio, 9(1), e02394-17. https://doi.org/10.1128/mBio.02394-17
Larsson, Y., Mongelli, A., Kisielius, V., & Bester, K. (2024). Microbial biofilm metabolization of
benzalkonium compounds (benzyl dimethyl dodecyl ammonium & benzyl dimethyl tetradecyl
ammonium chloride). Journal of Hazardous Materials, 463, 132834.
https://doi.org/10.1016/j.jhazmat.2023.132834
Sandhir, R. (2014). Metabolic Pathways | Lipid Metabolism. In Encyclopedia of Food Microbiology
(pp. 520–534). Elsevier. https://doi.org/10.1016/B978-0-12-384730-0.00200-7
Sütterlin, H., Alexy, R., & Kümmerer, K. (2008). The toxicity of the quaternary ammonium compound
benzalkonium chloride alone and in mixtures with other anionic compounds to bacteria in test systems
with Vibrio fischeri and Pseudomonas putida. Ecotoxicology and Environmental Safety, 71(2), 498
505. https://doi.org/10.1016/j.ecoenv.2007.12.015

Original language	English
Publication date	2024
Publication status	Published - 2024
Event	YWP European Conference 2024 - DI Building, Copenhagen, Copenhagen, Denmark Duration: 16 Jun 2024 → 19 Jun 2024

Conference

Conference	YWP European Conference 2024
Location	DI Building, Copenhagen
Country/Territory	Denmark
City	Copenhagen
Period	16/06/2024 → 19/06/2024

Cite this

@conference{7b0370c7463b4379a0bd72d75c8b3dee,

title = "Benzalkonium chloride metabolites – in moving bed biocarrier reactors and WWTPs",

abstract = "Abstract: Biodegradation was observed in moving bed biofilm reactors, revealing the presence of 45 metabolites and leading to the identification of two degradation pathways. Concentrations of two quaternary ammonium compounds (QUATs), namely BAC-12 and -14, were determined in both the wastewater treatment plant (WWTP) influent and effluent and in a sludge. BAC metabolites were detected in the sludge and a WWTP effluent.Introduction Benzalkonium chlorides (BACs) are biocides most commonly used in disinfectants, household and industrial cleaners and detergents (Fortunato et al., 2019; Knauf et al., 2018). Despite their common use, BACs pose environmental risks as they are directly toxic to microorganisms and detrimental to aquatic life (ECHA, 2023; S{\"u}tterlin et al., 2008). Efficient degradation of BACs in wastewater treatment plants (WWTPs) is crucial to limit the environmental spread of these compounds. However, as these compounds are surfactants, it is not easy to determine their physio-chemical data and predictions for their behavior in WWTPs are difficult to make.Given the complexity of the microbial community in WWTPs, it is important to understand how BACs are degraded and what they are metabolized into. This study intends to reveal the BAC degradation pathway in aerobic moving bed biofilm reactors (MBBRs) with a heterogeneous microbial community and to investigate which BAC metabolites can be found in WWTPs. Material and MethodsDegradation and metabolization pathways were studied using lab-MBBRs with a multispecies biofilm grown on plastic carriers. Influent and effluent concentrations of three WWTPs in Sj{\ae}lland were determined using HPLC-MS/MS using 24-hour composite sampling. Metabolite identification was performed using an API6600 high-resolution mass spectrometer and the MarkerView, MetabolitePilot and ChemSketch software. The full method is documented in Larsson et al., 2024.Results and Discussion By the findings of 45 metabolites in the micro-MBBR experiment two biodegradation pathways were determined (Fig 1). The initial degradation was an ω-oxidation which involves hydroxylation of the terminal carbon followed by two oxidation steps forming a carboxyl group (Sandhir, 2014). Successively, the metabolite with a carboxyl group is either further oxidized by α-oxidation or β-oxidation releasing either an acetyl or a formyl equivalent. It was most probable, that these two pathways are not only degrading individually (linearly) but are in reality intertwined. These pathways further followed the degradation of β-oxidation until the metabolites of the mass 208 Da and 194 Da were detected. Two metabolites from this pathway were detected in the outlet of one WWTP, one being the 208 Da and another of the mass 250 Da. One other metabolite detected in the lab-MBBRs was also detected in the WWTP effluent (152 Da) together with two BAC-metabolites known from literature (136 and 186 Da) (Table 1). With the discovery of these five metabolites in a WWTP effluent, this study underscores the importance of a comprehensive metabolite database. ConclusionsBACs are biodegraded and metabolized in WWTPs. Possibly persistent end products were identified in the degradation pathway and five BAC metabolites were found in the WWTPs. As BACs contribute to antimicrobial resistance, maintaining low environmental concentrations is crucial and knowledge about the persistence and toxicological properties is needed for metabolites from biocides such as BACs. ReferencesECHA. (2023). Registration dossier. Quaternary ammonium compounds, benzyl C12-C16 (evennumbered)-alkyldimethyl chlorides. https://echa.europa.eu/da/registration-dossier/-/registereddossier/13152/6/2/3Fortunato, M. S., Baroni, S., Gonz{\'a}lez, A. J., {\'A}lvarez Roncancio, J. D., Storino, A., Parise, C., Planes,E., Gallego, A., & Korol, S. E. (2019). Biodegradation and Detoxification of Benzalkonium Chloride inSynthetic and Industrial Effluents in Upflow Biofilm Aerobic Reactors. Water, Air, & Soil Pollution,230(4), 79. https://doi.org/10.1007/s11270-019-4126-9Knauf, G. A., Cunningham, A. L., Kazi, M. I., Riddington, I. M., Crofts, A. A., Cattoir, V., Trent, M.S., & Davies, B. W. (2018). Exploring the Antimicrobial Action of Quaternary Amines againstAcinetobacter baumannii. mBio, 9(1), e02394-17. https://doi.org/10.1128/mBio.02394-17Larsson, Y., Mongelli, A., Kisielius, V., & Bester, K. (2024). Microbial biofilm metabolization ofbenzalkonium compounds (benzyl dimethyl dodecyl ammonium & benzyl dimethyl tetradecylammonium chloride). Journal of Hazardous Materials, 463, 132834.https://doi.org/10.1016/j.jhazmat.2023.132834Sandhir, R. (2014). Metabolic Pathways | Lipid Metabolism. In Encyclopedia of Food Microbiology(pp. 520–534). Elsevier. https://doi.org/10.1016/B978-0-12-384730-0.00200-7S{\"u}tterlin, H., Alexy, R., & K{\"u}mmerer, K. (2008). The toxicity of the quaternary ammonium compoundbenzalkonium chloride alone and in mixtures with other anionic compounds to bacteria in test systemswith Vibrio fischeri and Pseudomonas putida. Ecotoxicology and Environmental Safety, 71(2), 498505. https://doi.org/10.1016/j.ecoenv.2007.12.015 ",

author = "Yrsa Larsson and Andrea Mongelli and Kai Bester",

year = "2024",

language = "English",

note = "YWP European Conference 2024 ; Conference date: 16-06-2024 Through 19-06-2024",

}

TY - ABST

T1 - Benzalkonium chloride metabolites – in moving bed biocarrier reactors and WWTPs

AU - Larsson, Yrsa

AU - Mongelli, Andrea

AU - Bester, Kai

PY - 2024

Y1 - 2024

N2 - Abstract: Biodegradation was observed in moving bed biofilm reactors, revealing the presence of 45 metabolites and leading to the identification of two degradation pathways. Concentrations of two quaternary ammonium compounds (QUATs), namely BAC-12 and -14, were determined in both the wastewater treatment plant (WWTP) influent and effluent and in a sludge. BAC metabolites were detected in the sludge and a WWTP effluent.Introduction Benzalkonium chlorides (BACs) are biocides most commonly used in disinfectants, household and industrial cleaners and detergents (Fortunato et al., 2019; Knauf et al., 2018). Despite their common use, BACs pose environmental risks as they are directly toxic to microorganisms and detrimental to aquatic life (ECHA, 2023; Sütterlin et al., 2008). Efficient degradation of BACs in wastewater treatment plants (WWTPs) is crucial to limit the environmental spread of these compounds. However, as these compounds are surfactants, it is not easy to determine their physio-chemical data and predictions for their behavior in WWTPs are difficult to make.Given the complexity of the microbial community in WWTPs, it is important to understand how BACs are degraded and what they are metabolized into. This study intends to reveal the BAC degradation pathway in aerobic moving bed biofilm reactors (MBBRs) with a heterogeneous microbial community and to investigate which BAC metabolites can be found in WWTPs. Material and MethodsDegradation and metabolization pathways were studied using lab-MBBRs with a multispecies biofilm grown on plastic carriers. Influent and effluent concentrations of three WWTPs in Sjælland were determined using HPLC-MS/MS using 24-hour composite sampling. Metabolite identification was performed using an API6600 high-resolution mass spectrometer and the MarkerView, MetabolitePilot and ChemSketch software. The full method is documented in Larsson et al., 2024.Results and Discussion By the findings of 45 metabolites in the micro-MBBR experiment two biodegradation pathways were determined (Fig 1). The initial degradation was an ω-oxidation which involves hydroxylation of the terminal carbon followed by two oxidation steps forming a carboxyl group (Sandhir, 2014). Successively, the metabolite with a carboxyl group is either further oxidized by α-oxidation or β-oxidation releasing either an acetyl or a formyl equivalent. It was most probable, that these two pathways are not only degrading individually (linearly) but are in reality intertwined. These pathways further followed the degradation of β-oxidation until the metabolites of the mass 208 Da and 194 Da were detected. Two metabolites from this pathway were detected in the outlet of one WWTP, one being the 208 Da and another of the mass 250 Da. One other metabolite detected in the lab-MBBRs was also detected in the WWTP effluent (152 Da) together with two BAC-metabolites known from literature (136 and 186 Da) (Table 1). With the discovery of these five metabolites in a WWTP effluent, this study underscores the importance of a comprehensive metabolite database. ConclusionsBACs are biodegraded and metabolized in WWTPs. Possibly persistent end products were identified in the degradation pathway and five BAC metabolites were found in the WWTPs. As BACs contribute to antimicrobial resistance, maintaining low environmental concentrations is crucial and knowledge about the persistence and toxicological properties is needed for metabolites from biocides such as BACs. ReferencesECHA. (2023). Registration dossier. Quaternary ammonium compounds, benzyl C12-C16 (evennumbered)-alkyldimethyl chlorides. https://echa.europa.eu/da/registration-dossier/-/registereddossier/13152/6/2/3Fortunato, M. S., Baroni, S., González, A. J., Álvarez Roncancio, J. D., Storino, A., Parise, C., Planes,E., Gallego, A., & Korol, S. E. (2019). Biodegradation and Detoxification of Benzalkonium Chloride inSynthetic and Industrial Effluents in Upflow Biofilm Aerobic Reactors. Water, Air, & Soil Pollution,230(4), 79. https://doi.org/10.1007/s11270-019-4126-9Knauf, G. A., Cunningham, A. L., Kazi, M. I., Riddington, I. M., Crofts, A. A., Cattoir, V., Trent, M.S., & Davies, B. W. (2018). Exploring the Antimicrobial Action of Quaternary Amines againstAcinetobacter baumannii. mBio, 9(1), e02394-17. https://doi.org/10.1128/mBio.02394-17Larsson, Y., Mongelli, A., Kisielius, V., & Bester, K. (2024). Microbial biofilm metabolization ofbenzalkonium compounds (benzyl dimethyl dodecyl ammonium & benzyl dimethyl tetradecylammonium chloride). Journal of Hazardous Materials, 463, 132834.https://doi.org/10.1016/j.jhazmat.2023.132834Sandhir, R. (2014). Metabolic Pathways | Lipid Metabolism. In Encyclopedia of Food Microbiology(pp. 520–534). Elsevier. https://doi.org/10.1016/B978-0-12-384730-0.00200-7Sütterlin, H., Alexy, R., & Kümmerer, K. (2008). The toxicity of the quaternary ammonium compoundbenzalkonium chloride alone and in mixtures with other anionic compounds to bacteria in test systemswith Vibrio fischeri and Pseudomonas putida. Ecotoxicology and Environmental Safety, 71(2), 498505. https://doi.org/10.1016/j.ecoenv.2007.12.015

AB - Abstract: Biodegradation was observed in moving bed biofilm reactors, revealing the presence of 45 metabolites and leading to the identification of two degradation pathways. Concentrations of two quaternary ammonium compounds (QUATs), namely BAC-12 and -14, were determined in both the wastewater treatment plant (WWTP) influent and effluent and in a sludge. BAC metabolites were detected in the sludge and a WWTP effluent.Introduction Benzalkonium chlorides (BACs) are biocides most commonly used in disinfectants, household and industrial cleaners and detergents (Fortunato et al., 2019; Knauf et al., 2018). Despite their common use, BACs pose environmental risks as they are directly toxic to microorganisms and detrimental to aquatic life (ECHA, 2023; Sütterlin et al., 2008). Efficient degradation of BACs in wastewater treatment plants (WWTPs) is crucial to limit the environmental spread of these compounds. However, as these compounds are surfactants, it is not easy to determine their physio-chemical data and predictions for their behavior in WWTPs are difficult to make.Given the complexity of the microbial community in WWTPs, it is important to understand how BACs are degraded and what they are metabolized into. This study intends to reveal the BAC degradation pathway in aerobic moving bed biofilm reactors (MBBRs) with a heterogeneous microbial community and to investigate which BAC metabolites can be found in WWTPs. Material and MethodsDegradation and metabolization pathways were studied using lab-MBBRs with a multispecies biofilm grown on plastic carriers. Influent and effluent concentrations of three WWTPs in Sjælland were determined using HPLC-MS/MS using 24-hour composite sampling. Metabolite identification was performed using an API6600 high-resolution mass spectrometer and the MarkerView, MetabolitePilot and ChemSketch software. The full method is documented in Larsson et al., 2024.Results and Discussion By the findings of 45 metabolites in the micro-MBBR experiment two biodegradation pathways were determined (Fig 1). The initial degradation was an ω-oxidation which involves hydroxylation of the terminal carbon followed by two oxidation steps forming a carboxyl group (Sandhir, 2014). Successively, the metabolite with a carboxyl group is either further oxidized by α-oxidation or β-oxidation releasing either an acetyl or a formyl equivalent. It was most probable, that these two pathways are not only degrading individually (linearly) but are in reality intertwined. These pathways further followed the degradation of β-oxidation until the metabolites of the mass 208 Da and 194 Da were detected. Two metabolites from this pathway were detected in the outlet of one WWTP, one being the 208 Da and another of the mass 250 Da. One other metabolite detected in the lab-MBBRs was also detected in the WWTP effluent (152 Da) together with two BAC-metabolites known from literature (136 and 186 Da) (Table 1). With the discovery of these five metabolites in a WWTP effluent, this study underscores the importance of a comprehensive metabolite database. ConclusionsBACs are biodegraded and metabolized in WWTPs. Possibly persistent end products were identified in the degradation pathway and five BAC metabolites were found in the WWTPs. As BACs contribute to antimicrobial resistance, maintaining low environmental concentrations is crucial and knowledge about the persistence and toxicological properties is needed for metabolites from biocides such as BACs. ReferencesECHA. (2023). Registration dossier. Quaternary ammonium compounds, benzyl C12-C16 (evennumbered)-alkyldimethyl chlorides. https://echa.europa.eu/da/registration-dossier/-/registereddossier/13152/6/2/3Fortunato, M. S., Baroni, S., González, A. J., Álvarez Roncancio, J. D., Storino, A., Parise, C., Planes,E., Gallego, A., & Korol, S. E. (2019). Biodegradation and Detoxification of Benzalkonium Chloride inSynthetic and Industrial Effluents in Upflow Biofilm Aerobic Reactors. Water, Air, & Soil Pollution,230(4), 79. https://doi.org/10.1007/s11270-019-4126-9Knauf, G. A., Cunningham, A. L., Kazi, M. I., Riddington, I. M., Crofts, A. A., Cattoir, V., Trent, M.S., & Davies, B. W. (2018). Exploring the Antimicrobial Action of Quaternary Amines againstAcinetobacter baumannii. mBio, 9(1), e02394-17. https://doi.org/10.1128/mBio.02394-17Larsson, Y., Mongelli, A., Kisielius, V., & Bester, K. (2024). Microbial biofilm metabolization ofbenzalkonium compounds (benzyl dimethyl dodecyl ammonium & benzyl dimethyl tetradecylammonium chloride). Journal of Hazardous Materials, 463, 132834.https://doi.org/10.1016/j.jhazmat.2023.132834Sandhir, R. (2014). Metabolic Pathways | Lipid Metabolism. In Encyclopedia of Food Microbiology(pp. 520–534). Elsevier. https://doi.org/10.1016/B978-0-12-384730-0.00200-7Sütterlin, H., Alexy, R., & Kümmerer, K. (2008). The toxicity of the quaternary ammonium compoundbenzalkonium chloride alone and in mixtures with other anionic compounds to bacteria in test systemswith Vibrio fischeri and Pseudomonas putida. Ecotoxicology and Environmental Safety, 71(2), 498505. https://doi.org/10.1016/j.ecoenv.2007.12.015

M3 - Conference abstract for conference

T2 - YWP European Conference 2024

Y2 - 16 June 2024 through 19 June 2024

ER -

Benzalkonium chloride metabolites – in moving bed biocarrier reactors and WWTPs

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