Understanding the Role of Deconjugation of Phase II Metabolites in Wastewater: Implications for Wastewater-Based Epidemiology.

Metabolism is a critical bodily function that facilitates the removal of toxic chemical buildup within the body. In wastewater-based epidemiology (WBE), it is crucial to understand the metabolism of biochemical indicators (BCIs) because metabolites are indicative of consumption (e.g., illicit drugs, pharmaceuticals) or unintentional exposure (e.g., pesticides, endocrine disruptors). Phase I metabolites are more widely studied in WBE due to a combination of factors, including, but not limited to, stability and analyte cost. Phase II metabolites are often assumed to deconjugate within the sewer network due to high native concentrations of enzymes. This work deconstructs this assumption and demonstrates how the in-sewer stability of phase II metabolites is dependent on both the parent structure and the conjugate type. In total, 79 BCIs were assessed and compared to urinary metabolism studies via time-variable enzymatic deconjugation using two enzymes, β-glucuronidase and arylsulfatase. The concentrations of free analytes excreted as N-glucuronides, O-glucuronides, and sulfates increased following deconjugation, reinforcing the persistence of these BCIs during transport throughout the sewer network. Conversely, no concentration increase was observed for acylglucuronides, demonstrating complete in-sewer glucuronide cleavage. In-freezer stability of conjugates was also assessed over 6 months, where it was observed that the stability of the parent structure is the driver of stability rather than the conjugates themselves, indicating minimal enzymatic activity upon storage. Overall, this paper presents a framework that can be deployed to gain a more comprehensive understanding of phase II metabolism and improve the accuracy of WBE workflows as well as environmental risk assessment approaches.