Cleaning Up after the Pandemic: How Old and New Quaternary Ammonium Compounds Affect Anaerobic Digestion

Summary of Abstract Quaternary ammonium compounds (QACs) have been widely used as disinfectants. The COVID-19 pandemic led to a rise in the usage of QACs, which has increased the load to wastewater treatment plants. QACs partition to solids and therefore enter anaerobic digesters. The objective of this research was to understand how QACs, which are designed to inhibit microbes, impact the biological process of anaerobic digestion. Specifically, different chemical classes were studied, including the widely used and widely studied benzylalkyldimethylammonium compounds (BACs) and the more recently used, but less studied, ethylbenzylalkyldimethylammonium compounds (EtBACs). Both short-term anaerobic toxicity assays and long-term chronic exposure experiments were conducted. Results revealed that the acute and chronic toxicity of QACs are closely related to their core chemical structure. The toxicity of QACs decreased with the growth of their alkyl chain. Meanwhile, dialkyldimethylammonium compounds (DADMACs) were more toxic than BACs at the same effective concentration. Additional experiments revealed that anaerobic digesters were more susceptible to functional collapse (i.e., biogas production and volatile solids reduction decrease) by other inhibitors after exposure to QACs. Volatile fatty acid profiles provided insights into the mechanisms of inhibition. Finally, QACs were removed from the feed to chronically exposed anaerobic digesters to elucidate the reversibility of decreased functional performance. This is the first research reported to comprehensively investigate the role of EtBACs along with BACs and DADMACs on anaerobic digestion. 1. Background, Originality and Status of Subject Quaternary ammonium compounds (QACs) have been used for decades as antimicrobials and preservatives as well as for other functions in personal care products and consumer goods (Figure 1).1 Especially during the SARS-CoV-2 pandemic, the usage of QACs increased dramatically as the predominant ingredients in disinfecting products to limit disease transmission.2 Currently over 200 disinfectants recommended by the US Environmental Protection Agency for use against COVID-19 contain QACs. The global QACs market was valued at $963.7 million in 2019, and it is expected to grow at a rate of 6.8% and reach $1.63 billion by 2027 due to increased demand for disinfectant products from hospitals, clinics, the food industry, and households.3 Thus, it should be anticipated that the amounts of QACs used and released into the environment will increase. Approximately 75% of QACs used enter into sewers and consequently water resource recovery facilities (WRRFs).4 The majority of QACs in influent wastewater adsorb to sludge and are then transferred to solids handling processes, such as anaerobic digestion (AD), where QACs levels are as high as 500 mg/kg (dry weight).1 AD is a critical biological treatment technology for solids stabilization and energy recovery. Different types of bacteria and archaea work together to carry out sequential tasks including hydrolysis, acidogenesis, acetogenesis, and methanogenesis. However, precisely because anaerobic digestion is highly dependent on the synergistic action of multiple microorganisms, it is more sensitive to toxic substances than aerobic treatment. QACs are a class of organic, cationic chemicals that contain one or more positively charged quaternary amine groups and at least one hydrophobic alkyl chain. The most commonly used and studied groups of QACs are mono-cationic compounds including benzyl alkyl dimethyl ammonium compounds (BACs, also known as benzalkonium compounds), dialkyl dimethyl ammonium compounds (DADMACs), and alkyl trimethyl ammonium compounds (ATMACs) (Fig. 1). Additionally, ethyl benzyl ammonium compounds (EtBACs) usage has been increasing in recent years.5-7 Because QACs are designed to inhibit microorganisms,8 it is expected that QACs could adversely affect the performance of AD. However, it remains unknown how the chemical core structure of QACs (i.e. the chemical group of QACs) is linked to acute and chronic impacts on AD performance. It is important to understand the potential impacts of QAC groups, concentration, and exposure time to AD function and microbial community, as well understand if these impacts are reversible following removal of QACs from a system. Overall, as the amount, and types, of QACs in consumer usage continues to grow, so too must our understanding of environmental impacts. In this study, we evaluated the acute toxicity of ten different structural QACs and conducted chronic exposure experiments on eight of these QACs commonly found in wastewater. 2. Presentation Objectives The overall objective of this presentation is to present findings from the acute and chronic QAC exposure experiments using lab-scale anaerobic digesters. The first specific objective will be to present the acute exposure toxicity of 10 commonly used QACs with various chemical structures. IC50 results will be reported and correlated to chemical structure. The second objective is to reveal the impact of QACs on methane production and volatile fatty acid profiles. The third objective is to reveal the reversibility of these impacts. Lab results for the first two objectives are completed. Lab results for the reversibility objective will be completed by February 2024. 3. Approach, Key Results, and Discussion Anaerobic toxicity assay style testing was performed to determine inhibitory concentrations (acute toxicity) of the different QACs to methane production. In addition, 60 digesters (triplicate sets of each group) with different QACs amendment including individual QACs and mixed QACs were operated simultaneously to assess the impact of chronic exposure. To monitor functional health, biogas and pH were measured daily. Meanwhile, methane and volatile fatty acids (VFA) were measured weekly; total solids (TS) and volatile solids (VS) were measured every solids retention time (SRT). The chronic exposure experiments are set to last approximately 25 SRT values (375 days). The results of the short-term ATAs indicated that the toxicity of QACs varies by their core structure (Figure 2a). For the same class of QACs, the toxicity decreases as the alkyl chain length increases. Meanwhile, exposure to a group of mixed QACs had synergistic effects. Compared with BAC, DADMAC is more toxic to anaerobic digestion at the same effective concentration (Figure 2b). High level QACs exposure caused VFAs to accumulate (Figure 3), indicating that methanogens were more sensitive to QACs compared to acidogens/acetogens. The results from the chronic QAC exposure experiments revealed that exposure to low level (60 mg/kg TS) QACs did not inhibit the performance of anaerobic digestion (Figure 4), and the effect were not affected by the chemical structure of the QAC or the duration of exposure. However, low level QACs exposure significantly altered the functional stability of the anaerobic digester after exposure to other potential inhibitors such as ammonia, Benzethonium chloride, and Rumensin (Figure 5). With the exposure concentration gradually increasing, QACs with short hydrophobic alkyl chain (like C8DADMAC and C12BAC) first started to inhibit methane production. Accumulation of VFAs led to a rapid decrease in pH, which further inhibited the activity of methanogens, and eventually the anaerobic digester was completely disabled under the combined stress of QAC and low pH (Figure 6). Interestingly, during the chronic amendment of QAC, one lab-scale anaerobic digester became more tolerant to QAC and maintained normal function even when the exposure concentration was gradually increased above IC50. The functional health under high level QACs chronic exposure is being continuously monitored and the experiment will end in February, 2024. Finally, the reversibility of QACs' impacts will be assessed. These results will be generated prior to the conference. At the time of the conference, we will be able to conclude how old (BACs, DADMACs), and new (EtBACs) QACs affect anaerobic digestion performance. Broader Impacts and Environment Significance This study will connect acute/ chronic exposure simulation experiments to test the comprehensive effect and reversibility of a more realistic scenario of gradually loading QACs with different core structures on the performance of anaerobic digester. The findings of this study will contribute to advancing broader understanding on why process performance is affected based on the type of QACs in a particular wastewater stream. This work will provide guidance on potential environmental impacts and information on how to balance use recommendations with improvements in WRRFs such that health and environmental protection are balanced