The mechanisms for plant accumulation of neutral organic compounds have been well studied for pesticides and herbicides, but relatively little work has been reported for PPCP/EDCs. Neutral compounds are thought to be taken up by passive diffusion through the root cell membrane, which is hampered by strong polarity or hydrophobicity . For neutral PPCP/EDCs in this study, a positive linear correlation with log Dow was observed for BCF leaf or BCF root . The effect of hydrophobicity Translocation of compounds from root to aerial tissues may lead to their accumulation in edible leaves or fruits. A translocation factor , the concentration in leaf tissue divided by that in root tissue, was calculated for PPCP/EDCs in each treatment . In this study, atorvastatin, ibuprofen, and sulfamethoxazole were the least translocated , while carbamazepine, meprobamate, and dilantin were the most translocated . The mean TF value was the highest for tomato at 2.90, with a range of 0 – 18.40, followed by carrot at 1.47, with a range of 0 – 13.58, while lettuce showed the least translocation with an average TF of 0.84 and a range of 0 – 5.50. The warm-dry treatment, which induced higher transpiration , also showed greater TF values than the cool-humid treatment. This observation suggested that increased mass flow due to transpiration enhanced the movement of PPPC/EDCs from roots to leaves in this study. To assess the effect of transpiration on TFs of anionic, cationic, and neutral PPCP/EDCs,vertical farms the TF values in each treatment were compared to the mass of nutrient solution transpired by each treatment .
For cationic and neutral PPCP/EDCs, significant positive correlation was observed between TF values and the transpired mass , suggesting that translocation of cationic and neutral compounds from root to leaves was influenced by transpiration. The impact of transpiration on TF was similar for both cationic and neutral compounds, as evident from their similar slopes of the regression lines . In contrast, a similar relationship was not found for anionic PPCP/EDCs . Cationic compounds also had significantly greater TF values than neutral compounds or anionic compounds , which suggests that cationic compounds were more likely than the other compounds to translocate from root to leaf tissues. This behavior may be due to the partitioning behavior of the cation molecules; charged molecules of cationic species tend to be sequestered in plant compartments with high pH, such as phloem . On the other hand, TF values for anionic compounds were generally low, which may be due to the ion trap effect in roots that are known for other anionic compounds . The ion trap effect occurs when the neutral fraction moves into root cells and become partly dissociated due to the change in pH inside the cells. The dissociated anions would not be able to quickly diffuse out of the cell into xylem and other plant parts, due to electrical repulsion, causing limited translocation. Global climate change has resulted in shifts in precipitation patterns, causing stress on freshwater resources, especially in arid and semi-arid regions . In many of these areas, demand for water has led to increasing use of municipally treated wastewater . Agriculture has been one of the primary targets for TWW reuse with water districts and governments promoting the adoption of recycled water for irrigation . However, the use of TWW for irrigation may come with potential risks, as TWW is known to contain a wide variety of human pharmaceuticals . The use of pharmaceutical compounds has increased with population growth and economic development, resulting in over 1500 compounds currently in circulation .
Their widespread consumption has led to their occurrence in TWW as well as in TWW impacted surface water . For many of these pharmaceuticals, there is limited knowledge about their potential chronic effects in the environment . Further, many of these compounds can transform in the environment, resulting in the formation of transient or recalcitrant transformation products, many with unknown fates and effects in environmental compartments . Diazepam belongs to the class of psychoactive compounds known as benzodiazepines, one of the most prescribed classes of pharmaceuticals . Diazepam is one of the most commonly detected pharmaceuticals in TWW, with concentration ranging from ng L−1 to low μg L−1 . This is likely due to its extensive use and low removal efficiency during secondary wastewater treatment . In humans, diazepam is primarily metabolized via phase I oxidative metabolism by demethylation to nordiazepam , or hydroxylation to temazepam , and then further oxidized to oxazepam . Oxazepam undergoes phase II metabolism via rapid glucuronidation and then excretion via urine . The three primary metabolites of diazepam are psychoactive compounds, and each is a prescribed pharmaceutical for treating psychological conditions and alcohol withdrawal symptoms . Both oxazepam and nordiazepam have been commonly detected in TWW, often at μg L−1 levels . However, there is little knowledge about the occurrence, formation, and fate of such metabolites outside the wastewater treatment systems . Several studies have focused on the uptake and accumulation of pharmaceuticals in agricultural plants as a result of TWW irrigation . These studies have demonstrated the capacity of higher plants to take up these compounds; however, until recently, relatively little consideration has been given to their metabolism in plants .
Recent studies have shown that higher plants can metabolize xenobiotics similarly to humans with phase I modification reactions followed by phase II conjugation reactions using detoxification enzymes that function as a ‘green liver’ . In higher plants, phase I and phase II reactions are followed by a phase III sequestration, resulting in the formation of bound residues . Many of these studies have also highlighted a chemical-specific and species-specific nature of plant metabolism of pharmaceuticals. In this study, we examined the uptake and biotransformation of diazepam in higher plants.Cucumber and radish seedlings were then used under hydroponic conditions to understand metabolism of diazepam and its effect on selected metabolic enzymes in whole plants.PSB-D A. thaliana cell line was purchased from the Arabidopsis Biological Resource Center at Ohio State University and cultured in a liquid culture suspension at 25 °C and 130 rpm in the dark. Cell cultures were maintained in accordance with the ARBC maintenance protocol . The A. thaliana seed culture was produced by inoculating 7 mL of cell culture into 43 mL fresh growth media, followed by 96 h cultivation at 25 °C on a rotary shaker in the dark. After 96 h, 3 mL of the seed culture was inoculated into 27 mL fresh growth media to create an approximate initial cell density of 3.3 g . Flasks were spiked with 30 μL of a stock solution of diazepam and 10 μL of a 14Cdiazepam stock solution to yield an initial concentration of 1 μg mL−1 and a specific radioactivity of 7.4 × 103 dpm mL−1 with an initial methanol content of 0.13% . Simultaneously,vertical plant growing control treatments were prepared by auto claving cell suspension flasks before chemical spiking , flasks containing diazepam without cells , and flasks containing living cells without diazepam . Control treatments were used to determine adsorption, abiotic degradation, and potential toxicity to cells. Flasks were incubated for 120 h in triplicate and sacrificed at 0, 6, 12, 24, 48 and 96 h for sampling and analysis. At each sampling time point, samples were collected and centrifuged at 13,000g for 15 min in 50 mL polypropylene tubes. The supernatant was collected and stored at −20 °C until further analysis. Cells were immediately stored at −80 °C and then freeze-dried for 72 h. After drying, each sample was spiked with 50 μL of 10 mg L−1 diazepam-d5 as a surrogate for extraction-recovery calibration and extracted using a method from Wu et al. , with minor modifications. Briefly, cells were sonicated for 20 min with 20 mL methyl tert-butyl ether and then 20 mL of acetonitrile and centrifuged at 13,000g for 15 min.
The supernatants were combined and concentrated to near dryness under nitrogen at 35 °C and then reconstituted in 1 mL of methanol. The cells were then extracted with 20 mL acidified deionized water and the supernatant was combined with the methanol extract for cleanup. Prior to clean-up, 100 μL of cell material extract and growth media were combined with 5 mL liquid scintillation cocktail I to measure the radioactivity in the extractable form on a Beckman LS500TD Liquid Scintillation Counter . Clean-up was carried out using solid phase extraction with 150 mg Waters Oasis© HLB cartridges that were preconditioned with 7 mL methanol and 14 mL deionized water. Samples were loaded onto cartridges and then eluted with 20 mL methanol under gravity. The eluate was dried under nitrogen and further recovered in 1.5 mL methanol:water . After re-suspension extracts were transferred to micro-centrifuge tubes and centrifuged at 12,000g in a tabletop d2012 Micro-Centrifuge . Samples were further filtered through a 0.22-μm polytetrafluoroethylene membrane into 2 mL glass vials and stored at −20 °Cwas greater for root tissues as compared to leaves , likely due to the contribution of adsorption to the accumulation in root tissues. Other studies have suggested that the optimum log Kow value for plant uptake is around 1 – 3.5 . In this study, diazepam, with a log Dow value of 2.82, exhibited the largest BCF values among the neutral compounds considered in this study, which was in agreement with previous observations.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 the7 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, temazepam and oxazepam in radish and silverbeet 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 nonextractable 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 .The activity of glycosyltransferase was measured in the control seedlings as well as seedlings exposed to diazepam for the 7 d and 28 d cultivation experiments . Glycosyltransferase catalyzes the transfer of sugars, such as glucuronic acid, to many types of acceptor molecules, including xenobiotics . The conjugation of glucuronic acid with oxazepam is the major detoxification pathway of diazepam in humans . No detectable level of oxazepam-glucuronide was observed in radish or cucumber seedlings for either the 7 d or 28 d cultivation.