Our long-term goal is to improve sustainability of water reuse and inform concomitant exposure risk by elucidating plant and contaminant interactions. The overall objective for this proposal is to determine the bioaccessibility, fate, and formation of conjugated CEC metabolites in plants, using representative compounds. Our central hypothesis is that direct in-plant conjugation reactions, which depend on compound structure, alter CEC bioavailability to consumers and can undergo subsequent back-transformation by bacteria in the gut. The rationale for the proposed research is that a mechanistic determination of conjugated CEC metabolite bioavailability will provide new opportunities for improving the safety and resilience of water recycling using nontraditional sources. In addition to our group's foundational past work and current preliminary data, our team is particularly well-prepared to undertake the proposed research. We hold proven expertise, equipment, and experience in high-resolution mass spectrometry metabolomics for discovering novel CEC plant transformation products in the lab and field with plant/CEC fate interactions, and are located in an agriculturally-intensive region, at the University of Iowa. We will achieve the overall objective through three Specific Aims:Determine bioaccessibility of conjugated CEC plant metabolites in the mammalian gut. Working Hypothesis: CECs present as undetected conjugated plant metabolites increase exposure potential during ingestion due to back-transformation in the mammalian gut. Using simulated gastric systems, we will determine exposure of benzotriazole (representative CEC) following plant consumption.Task 1.1: Establish the bioaccessibility of plant-conjugated BT in simulated stomach and gastric fluids.Task 1.2: Quantify product-to-parent revision potential under simulated gastric conditions.Quantify product-to-parent reversion of plant-excreted conjugated CEC metabolites. Working Hypothesis: Bacteria will readily back-transform conjugated CECs excreted from plants to their parent form. Glycosylated conjugates will more readily back-transform than amino acid conjugates. Using synthesized standards and hydroponic experiments, we will quantify plant excretion of conjugated benzotriazole metabolites and re-generation of parent CEC metabolites via soil bacteria cultures.Task 2.1: Biotransformation of excreted BT using soil bacteria.Task 2.2: Determine relevance of hydrolysis and photolysis as abiotic transformation mechanisms.Relate CEC chemical structure to propensity for direct in-plant conjugation. Working Hypothesis: Specific and predictable chemical characteristics, such as primary/ secondary amines or hydroxyl groups, will determine novel CEC direct conjugation pathways and active rates in plants. We will elucidate novel CEC transformation products and pathways using high-resolution mass spectrometry metabolomics approaches. We will focus on representative chemical structures to maximize broad applicability of findings, while also studying compounds relevant to recycling municipal and agricultural wastewater.Task 3.1: Quantify the impact of specific functional groups on CEC plant uptake kinetics and direct conjugation.Task 3.2: Quantify predicted plant transformation products resulting for substituted BT compounds using a semi-untargeted metabolomics approach.