The use of recycled water for agricultural irrigation comes with the concern of exposure to crops by contaminants of emerging concerns (CECs). The concentration of CECs in plant tissues will depend on uptake, translocation and metabolism in plants. However, relatively little is known about plant metabolism of CECs, particularly under chronic exposure conditions. In this study, metabolism of the pharmaceutical diazepam was investigated in Arabidopsis thaliana cells and cucumber (Cucumis sativus) and radish (Raphanus sativus) seedlings grown in hydroponic solution following acute (7 d)/high concentration (1 mg L-1), and chronic (28 d)/low concentration (1 μg L-1) exposures. Liquid chromatography paired with mass spectrometry, 14C tracing, and enzyme extractions, were used to characterize the metabolic phases. The three major metabolites of diazepam - nordiazepam, temazepam and oxazepam - were detected as Phase I metabolites, with the longevity corresponding to that of human metabolism. Nordiazepam was the most prevalent metabolite at the end of the 5 d incubation in A. thaliana cells and 7 d, 28 d seedling cultivations. At the end of 7 d cultivation, non-extractable residues (Phase III) in radish and cucumber seedlings accounted for 14% and 33% of the added 14C-diazepam, respectively. By the end of 28 d incubation, the non-extractable radioactivity fraction further increased to 47% and 61%, indicating Phase III metabolism as an important destination for diazepam. Significant changes to glycosyltransferase activity were detected in both cucumber and radish seedlings exposed to diazepam. Findings of this study highlight the need to consider the formation of bioactive transformation intermediates and different phases of metabolism to achieve a comprehensive understanding of risks of CECs in agroecosystems.

Contamination of agricultural soils by pharmaceutical and personal care products (PPCPs) resulting from the application of treated wastewater, biosolids and animal wastes constitutes a potential environmental risk in many countries. To date a handful of studies have considered the phytotoxicity of individual PPCPs in crop plants, however, little is known about the effect of PPCPs as mixtures at environmentally relevant levels. This study investigated the uptake and transport, physiological responses and detoxification of a mixture of 17 PPCPs in cucumber seedlings. All PPCPs were detected at higher concentrations in roots compared to leaves, with root activity inhibited in a dose-dependent manner. At 5-50 μg/L, the mature leaves exhibited burnt edges as well as a reduction in photosynthesis pigments. Reactive oxygen species (ROS) production and lipid peroxidation increased with increasing PPCP concentrations; and their contents were greater in roots than in leaves for all PPCP treatments. Enzymes involved in various functions, including oxidative stress (superoxide dismutase and ascorbate peroxidase) and xenobiotic metabolism (peroxidase and glutathione S-transferase), were elevated to different levels depending on the PPCP concentration. Glutathione content gradually increased in leaves, while a maxima occurred at 0.5 μg L^-1 PPCPs in roots, followed by a decrease thereafter. This study illustrated the complexity of phytotoxicity after exposure to PPCP mixtures, and provided insights into the molecular mechanisms likely responsible for the detoxification of PPCPs in higher plants.

Many pharmaceutical and personal care products (PPCPs) enter agroecosystems during reuse of treated wastewater and biosolids, presenting potential impacts on plant development. Here, acetaminophen, one of the most-used pharmaceuticals, was used to explore roles of glutathione (GSH) conjugation in its biotransformation in crop plants. Acetaminophen was taken up by plants, and conjugated quickly with GSH. After exposure to 5 mg L^-1 acetaminophen for 144 h, GSH-acetaminophen conjugates were 15.2 ± 1.3 nmol g^-1 and 1.2 ± 0.1 nmol g^-1 in cucumber roots and leaves, respectively. Glutathione-acetaminophen was also observed in common bean, alfalfa, tomato, and wheat. Inhibition of cytochrome P450 decreased GSH conjugation. Moreover, the GSH conjugate was found to further convert to cysteine and N-acetylcysteine conjugates. Glutathione S-transferase activity was significantly elevated after exposure to acetaminophen, while levels of GSH decreased by 55.4% in roots after 48 h, followed by a gradual recovery thereafter. Enzymes involved in GSH synthesis, regeneration and transport were consistently induced to maintain the GSH homeostasis. Therefore, GST-mediated conjugation likely played a crucial role in minimizing phytotoxicity of acetaminophen and other PPCPs in plants.