The homogenate was centrifuged at 13,000 g and 4 °C for 20 min. The activity of glycosyltransferase was measured immediately by mixing 100 µL of supernatant with 0.95 mL of the reaction mixture containing 50 mM PBS , 2 mM MgCl2, 2 mM uridine 5′-diphosphoglucose, 3.125 mM 4- nitrophenyl β-D-glucuronide, 3.125 salicin and 0.95 mL of 1 mM 2,4,5-trichlorophenol . The assay mixture was incubated at 30 °C for 30 min, and then stopped by adding 10 µL of phosphoric acid. After centrifugation at 13,000 g for 5 min, the supernatant was collected and diluted with HPLC grade acetonitrile and 0.1% trifluoroacetic acid . The enzyme activity was determined using an Agilent 1200 series HPLC paired with UV detector and a Thermo Scientific Acclaim™ 120 C18 5-µm column . An isocratic flow was set with 1 mL min-1 70:30 mobile phase A and mobile phase B for 10 min. The TCP-glucoside was detected at 205 nm. A six-point TCP standard calibration curve was used to determine activity. All treatments in the A. thaliana cell incubation experiment were conducted in triplicate, and all hydroponic cultivations were conducted using four replicate jars containing individual plants to account for potential loss of plants. Calibration curves with standards of diazepam, diazepam-d5, nordiazepam, temazepam, oxazepam and oxazepam-glucuronide were used for quantification with the r 2 values of at least 0.99 for all analytes. A limit of detection of 1 ng mL-1 and a limit of quantification of 3 ng mL-1 for diazepam and its metabolites were determined through preliminary experiments. For oxazepam-glucurnonide the LOD was 3 ng mL-1 and the LOQ was 5 ng mL-1 . LODs and LOQs were calculated based on a signal to noise ratio of 3 and 10, respectively. Individual peaks were detected and integrated using TargetLynx XS software from MassLynx platform . Data were analyzed with StatPlus and graphed using Prism 6 GraphPad software . Results were calculated as the mean ± standard deviation . The Student’s t-test was used to test significant differences in the extractable and non-extractable radioactivity and glycosyltransferase activity at α = 0.05. Systematic differences in the concentration of diazepam in plant tissues were assessed using one-way ANOVA with Fisher’s Least Significant Difference post-hoc .Active plant metabolism of diazepam was validated using a range of controls.

No diazepam was detected in the non-treated media or the cell blanks,hydroponic grow kit and there was no significant degradation of diazepam in the cell-free media, suggesting no contamination or significant abiotic transformation. Moreover, no significant difference was seen in cell mass between the chemical-free control and the treatments, indicating that diazepam did not inhibit the growth of A. thaliana. Furthermore, no significant amount of diazepam was adsorbed to the cell matter in the non-viable cell control. In contrast, diazepam dissipated appreciably from the media containing viable cells, with the average concentration decreasing from 698 ± 41.5 to 563 ± 8.93 ng mL-1 after 120 h of incubation, a decrease of nearly 20% . Parallel with the dissipation in the medium, diazepam was detected in the A. thaliana cells, with the highest level appearing after 48 h and a substantial decrease thereafter . The decrease in diazepam level in the cell fit a first-order decay model and yielded a half-life of about 68 h . This half-life was in comparison to a biological half-life of 48 h in humans , indicating a moderate persistence in plant cells. Out of the four known diazepam metabolites only nordiazepam and temazepam were detected in the A. thaliana cells over the 120 h incubation. Temazepam was detected first, with the highest concentration being observed at 12 h, which was followed by a decrease to 58.6 ± 17.0 ng g-1 at the end of the 120 h cultivation. Nordiazepam gradually increased over the 120 h incubation time from 128 ± 61.0 ng g-1 at 6 h to 535 ± 92.0 ng g-1 at the end of incubation . These results correlated with their behavior in the human body, as nordiazepam displayed one of the most prolonged biological half-lives of the benzodiazepine family , while temazepam had a significantly shorter half-life . The parallels observed between human and plant metabolism in this study and others is intriguing, as it indicates that we may be able to use the knowledge of biologically active metabolites formed during human metabolism as a guide to study their formation and longevity in environmental compartments such as higher plants. The complementary use of 14C labeled diazepam facilitated the determination of the fraction of diazepam and its metabolites that were incorporated into the cell matter , which could not be determined using traditional extraction and analytical methods. We observed that the radioactivity in the media decreased while the extractable and bound residue fractions increased over the 120 h incubation .

The extractable radioactivity in the viable cells increased to 113 ± 31 dpm g-1 at 120 h . The bound residues increased steadily to a final level of 1120 ± 224 dpm g-1 , indicating that A. thaliana cells were capable of metabolizing and then sequestering diazepam and its metabolites, likely invacuoles and cell walls. The formation of these bound residues is commonly regarded as a detoxification pathway of xenobiotics in higher plants. Diazepam was found in the cucumber and radish seedlings following a 7 d cultivation at the higher concentration and a 28 d of cultivation following treatment at the lower concentration . After treatment with 1 mg L-1 with diazepam for 7 d, a significantly higher concentration of diazepam was observed in the roots as compared to the shoots in radish seedlings whereas diazepam was more evenly distributed throughout the entire plant of cucumber seedlings . However, after the 28 d cultivation following the lower concentration treatment, this pattern appeared to be different for both plant species. In the radish plants, diazepam was more evenly distributed in the roots, but was significantly lower in the shoots . In the cucumber plants there was a significantly higher concentration in roots and a significantly lower concentration in the shoots . These differences may be due to variations in metabolism between the two species, as well as dynamic changes as a function of contact time in both plant growth and its ability to metabolize and translocate diazepam. Similar metabolites to those in A. thaliana cells were found in seedlings grown in the nutrient solution spiked with diazepam, with nordiazepam being predominant . In the 7 d and 28 d cultivation experiments, temazepam was found to be the second major metabolite in the leaves of the cucumber seedlings, and the level was higher in the 7 d cucumber seedlings than the 28 d plants . Oxazepam was detected in the leaves of both plant species after the 7 d cultivation . The higher accumulation of diazepam and the biologically active metabolites in the leaves may have ecotoxicological ramifications; for example, many insects consume leaves, even if they are not edible tissues for humans . Our results were in agreement with recent findings in Carter et al. , in which they observed the formation of nordiazepam,hydroponic indoor growing system temazepam and oxazepam in radish and silver beet plants exposed to diazepam and chlordiazepoxide. They similarly showed nordiazepam to be the major metabolite with oxazepam and temazepam constituting a much smaller fraction at the end of 28 d cultivation in soil. However, in that study, the authors did not track the formation of these metabolites over time or influence of treatment concentrations.

Phase III metabolism appeared to increase from the 7 d to 28 d cultivation for both radish and cucumber seedlings . Between the plant species, the cucumber seedlings had a greater fraction of non-extractable radioactivity in comparison to the radish seedlings . In the 7 d cultivation experiment, the mass balances came to 99.3% for the cucumber plants but only 58.1% for the radish seedlings . Due to the multiple water changes , a complete mass balance was not attainable for the 28 d cultivation experiment. However, when a proxy mass balance was calculated for both species, a similar pattern was observed. A total of 83.0% of the added 14C radioactivity was calculated for the cucumber treatments while the fraction was 61.3% for the radish plants. This could be due to increased mineralization in the growth media and respiration of 14CO2 through plant in the radish cultures. As mineralization is viewed as the final stage of detoxification , it is likely that the radish plant was more efficient in their ability to detoxify diazepam than cucumber plants. The Brassicaceae family, which includes the common radish, has been shown to be effective for phytoremediation due to their possession of genes that increase tolerance to stressors and activation of enzymes capable of extensive bio-transformations .No detectable level of oxazepam-glucuronide was observed in radish or cucumber seedlings for either the 7 d or 28 d cultivation. However, there was a significant difference in the glycosyltransferase activity in radish seedlings treated with diazepam for 7 d and 28 d, although a distinct pattern in the changes of the enzyme activity was absent . For the 7 d cultivation experiment, a significant decrease in glycosyltransferase activity was observed in the shoots of radish seedlings when compared to the control . In contrast, no significant change in glycosyltransferase activity was observed in the shoots of cucumber seedlings when exposed to diazepam . In the 28 d cultivation experiment, only the cucumber seedlings exhibited significant differences in the enzyme activity, with an increase in activity detected in the shoots and a decrease in the cucumber buds . Even though we did not detect oxazepam-glucuronide in the exposed plants, changes in the glycosyltransferase activity indicated that conjugation might have occurred with the parent and its metabolites, including those not examined in this study, or at levels below our detection capability. In addition, it may be postulated that rapid phase III metabolism may have limited the accumulation of such conjugates in the plant tissues, making the conjugates transient metabolites. In previous studies, glycosyltransferase was observed to catalyze the detoxification of ibuprofen in Phragmite australis during a 21 d exposure . Further, the formation of a glucose conjugate has been considered to be a major detoxification pathway for several environmental contaminants . These studies together suggest the importance of phase II metabolism in the metabolic fate of pharmaceuticals in higher plants. Water scarcity has led to continuously increasing use of municipally treated wastewater in agro-environments, especially in arid and semi-arid regions . Similarly, the use of municipal bio solids to improve soil health is increasing . The land application of TWW and bio solids can introduce contaminants of emerging concern into terrestrial environments . Consequently, a range of CECs have been detected in agricultural soils . Literature pertaining to the effects of CECs in terrestrial ecosystems is, however, limited . The majority of previous research has been concerned with the fate and effects of CECs in plants , and only a few studies have considered CECs in terrestrial invertebrates . One of the most important invertebrates in agricultural fields is earthworm. Earthworms ameliorate agricultural soil structure through the formation of new aggregates and macropores; improving soil tilth, aeration, infiltration, and drainage . Furthermore, earthworms consume plant litter, recycle organic matter and aid in nutrient cycling . Earthworms dominate soil fauna with an average biomass of 10 – 200 g m-2 . Due to their ecological importance and abundance, earthworms are a good candidate for ecotoxicity testing . Herein we sought to understand some of the potential consequences of CECs exposure in earthworms. For this study, we selected theearthworm species Eisenia fetida as the test organism due to their widespread use in the scientific literature and extensive habitat range. For CECs, we considered three pharmaceuticals, i.e., naproxen, diazepam, and sulfamethoxazole, and one cosmetic preservative, i.e., methyl paraben. These four compounds were selected due to their range of physicochemical properties and frequent detections in the environment . Naproxen is a commonly consumed nonsteroidal anti-inflammatory drug that has been often found in TWW and bio solids . Diazepam is a psychoactive compound from one of the most commonly prescribed classes of pharmaceuticals of wastewater treatment plants. Diazepam has also been frequently detected in TWW due to poor removal efficiency . Sulfamethoxazole is an antibiotic and has garnered significant scientific interest due to the growing concern over antibiotic resistance .