Bioaccessibility and Transformation of Conjugated Benzotriazole Phytometabolites during In Vitro Digestion: Implications for Exposure from Recycled Irrigation Water

Recycled water for agricultural irrigation helps to address freshwater scarcity but can contain trace levels of contaminants of emerging concern (CECs), which pose potential human exposure risks. Traditional risk assessments, focused primarily on the parent compound, overlook CECs as conjugated phytometabolites. The objective of this study was to determine the bioaccesibility and transformation of conjugated phytometabolites of benzotriazole, a prevalent micropollutant that serves as a model contaminant. Arabidopsis thaliana seedlings, a model Brassica plant that can be representative of other plants, including food crops, were grown hydroponically and then exposed to benzotriazole to facilitate benzotriazole uptake and phytotransformation. Plant tissues containing benzotriazole phytometabolites underwent in vitro digestion, simulating stages of human digestion (mouth, stomach, intestine). Three known phytometabolites of benzotriazole were quantified through digestion stages, and ¹⁴C-benzotriazole tracked mass balances. Plant-accumulated ¹⁴C-benzotriazole was bioaccessible with only 7% remaining in the plant tissue following in vitro digestion. The plant-accumulated mass of the individual benzotriazole phytometabolites was 2.2-3.5-fold and 4-8 times lower after gastric and intestine phases, respectively, in digested compared to nondigested plants. Gastric and intestinal phases drive bioaccessibility. High resolution mass spectrometry analysis revealed 10 novel digestion phase transformation products with distinct differences in predicted pharmacokinetic and toxicity compared to the benzotriazole phytometabolites. This study enhances understanding of bioaccessibility and transformation of CEC phytometabolites with implications for recycled water.