Methanol, and then water, 7 mL each, were added to each HLB cartridge for precondition, followed by the addition of the sample and then 5 mL of 5% methanol in water for clean-up. For elution and collection of the target analytes, a final pass-through of 15 mL methanol was performed. The resulting methanol eluent was collected in a glass tube, dried using a nitrogen evaporator, reconstituted in 1 mL methanol-water mixture , and filtered through a 2 mm PTFE filter into a 1.5 mL HPLC vial for instrumental analysis. Plant cell matter and wheat tissues were freeze dried at -50 ˚C for at least 72 h to remove moisture and weighed. Before extraction, 50 μL of a depurated compound was added to each sample as the recovery surrogate. Samples were firstly extracted with 10 mL MTBE via sonication for 30 min. The sonication process was then repeated with 10 mL fresh MTBE one additional time and 10 mL acetonitrile twice. Extracts from the extraction were combined and dried by a nitrogen evaporator, followed by reconstitution in 1 mL methanol and dilution with 20 mL water. The resulting liquids were cleaned up with HLB cartridges using a similar protocol as described above. The final extracts were dried under a gentle nitrogen gas flow, reconstituted in 1 mL methanol: water ,vertical grow shelf and filtered through a 2 mm PTFE filter before instrument analysis. No significant difference was found in the biomass between the treated groups and control groups for experiments using A. thaliana cells. No target analytes were detected in the method blanks.
The concentrations of target analytes in control groups with no cells, or with nonviable cells, varied in the range of 93.4-116.0% of the spiked concentration at the end of exposure as compared to the initial concentrations, suggesting stability of these compounds under abiotic conditions. The individual compounds were found to be taken up by live A. thaliana cells. The levels in the plant cell matter reached maxima within 3 h for all compounds except acetaminophen, which exhibited the highest accumulation at 6 h into the incubation .At the end of 96-h cultivation, the level in the cell matter was < 0.3 μg/g for most compounds, suggesting rapid metabolism in viable plant cells and likely excretion into the aqueous medium. Among the different compounds, DM-diazepam and diazepam appeared to be accumulated to higher levels than the other compounds and were also more recalcitrant to metabolism. After 96 h of incubation, 4.78 ± 0.90 μg/g of DM- diazepam or 3.63 ± 1.74 μg/g of diazepam still remained in the A. thaliana cells. In previous studies, CECs including acetaminophen, diazepam and naproxen were found to be readily metabolized in different plant species 40–42 . The CECs and their methylated/demethylated derivatives showed different accumulation potentials in A. thaliana cells. For example, acetaminophen was detected in the A. thaliana cells at significantly higher concentrations than M-acetaminophen at any given sampling time point . After 6 h of incubation, 6.10 ± 1.57 μg/g of acetaminophen was found in the A. thaliana cells, while the level was only 2.52 ± 0.57 μg/g for M-acetaminophen . In comparison, methylparaben was found to accumulate more than DM-methylparaben at all sampling time points.
For example, at 1 h, methylparaben was found at 11.6 ± 2.81 μg/g in the cell matter, while DMmethylparaben at only 0.06 ± 0.00 μg/g. The difference in accumulation by A. thaliana cells between DM-methylparaben and methylparaben may be partly attributed to the fact that DM-methylparaben was present mostly in an ionized form in the nutrient media . Negatively charged chemicals are known to not easily cross the negatively charged cell walls and membranes and are limited in their plant uptake 43,44. Like methylparaben, higher concentrations of naproxen than DM-naproxen were also detected in the cell matter throughout the exposure time . After 1 h of incubation, 12.31 ± 2.46 μg/g of naproxen was found to be in the cell matter, while the level was only 2.10 ± 0.40 μg/g for DM-naproxen. However, no statistically significant difference was observed between diazepam and DM-diazepam in their levels in A. thaliana cells duringthe exposure experiment. This may be attributed to the fact that log Kow of DM-diazepam is similar to that of diazepam . As the test chemicals were taken up by A. thaliana cells, the levels of CECs and their methylated or demethylated derivatives in the culture media concurrently decreased. In the culture media, the concentration of DM-methylparaben and methylparaben, and DM-naproxen and naproxen all decreased rapidly, and their level fell below the detection limit after just a few hours into the incubation . In comparison, the decrease of acetaminophen and M-acetaminophen, and DM-diazepam and diazepam was relatively slower, with 0.12-0.40 mg/L, or 12-40% still remaining in the cell culture media after 48 h of exposure. The dissipation of CECs and their methylated or demethylated derivatives in the culture media was further fitted to the first-order decay model, and the fit was generally good, with R2 > 0.63. The half-life T1/2 was then calculated from the first-order rate constant . The estimated T1/2 values were very small for methylparaben, DM-methylparaben, and naproxen. The dissipation of DM-naproxen was so rapid that T1/2 could not be derived.
Methylation appeared to increase T1/2 for acetaminophen and DM-diazepam, with statistically significant difference . A mass balance approach was not followed in this study, as subsequent transformation products in the A. thaliana cells were not characterized. Given that the compounds considered in this study were stable under abiotic conditions, the rapid dissipation in the culture media and limited accumulation in the A. thaliana cellssuggested that the CECs and their methylated or demethylated counterparts underwent rapid metabolism in the A. thaliana cells. In the case of acetaminophen, methylparaben, and naproxen, demethylation introduced a hydroxyl or carboxyl group into the molecule. As shown in previous studies, compounds with a hydroxyl or carboxyl functional group can undergo rapid conjugation with various biomolecules in plants. The conjugated intermediates are substantially larger in molecular size and may become “immobilized” once formed in the A. thaliana cells. Future research should consider the formation of conjugates for demethylated compounds and understand the fate and risks of such plant-origin conjugates. Uptake and translocation of the paired compounds were further measured in wheat plants grown hydroponically in nutrient solutions. Roots and shoots of wheat seedlings were collected and analyzed separately to understand the in-plant translocation. Target CECs and their methylated or demethylated counterparts showed great stability in hydroponic solution without wheat seedlings, with recoveries ranging from 99.1-125.7% of the initial spiked concentration after 240 h incubation. No compounds of interest were detected in the untreated hydroponic solution or wheat seedlings. In general, the level of chemicals in the plant tissues first increased and then decreased, suggesting uptake into the roots from the hydroponic media, followed by translocation from roots into shoots and/or metabolism in the plant. All CECs and their methylated or demethylated TPs were detected in wheat roots, and the concentrationswere much higher than those in shoots, indicating generally limited translocation . Among the different CECs, acetaminophen and DM-naproxen were not detected in wheat shoots, while DM-methylparaben was only found occasionally at trace levels. The accumulation of acetaminophen was also limited in the roots,vertical hydroponic which may explain its absence in the shoots. From a previous study, after formation from naproxen through demethylation, DM-naproxen was found to metabolize readily through phase II and phase III pathways in A. thaliana cells. The rapid metabolism of DM-naproxen in plants may have contributed to its absence in the shoots. Higher concentrations were consistently detected for M-acetaminophen than acetaminophen in both wheat roots and shoots . In wheat shoots, only Macetaminophen was detected, suggesting that methylation rendered acetaminophen more mobile and a greater potential to translocate from roots to shoots. In general, DMmethylparaben was found to be taken up more rapidly than methylparaben into wheat roots and reached 20.66 ± 2.78 μg/g at 6 h after the treatment . In comparison, the highest level of methylparaben in roots was observed at 12.34 ± 1.33 μg/g after 96 h of exposure. However, methylparaben consistently exhibited much higher concentrations than DM-methylparaben in the shoots, suggesting a greater potential for translocation for methylparaben . Both compounds were found to undergo rapid metabolism, and their levels after 10 d of incubation were considerably lower than at earlier time points in the roots, while essentially no DM-methylparaben was found in the shoots. As the demethylated derivative of naproxen, although DM-naproxen was taken up quickly and reached 33.32 ± 8.41 μg/g in wheat roots after 24 h, it appeared to be rapidly metabolized , as only 0.33 ± 0.02 μg/g DM-naproxen was detected in the roots after 10 d.
In comparison, naproxen was accumulated in both roots and shoots at consistently higher concentrations than DM-naproxen throughout the experiment . In wheat shoots, DM-naproxen was consistently below the detection limit, suggesting limited translocation, and/or rapid transformations in the roots via pathways such as conjugation. Both DM-diazepam and diazepam showed significant accumulation in wheat plant . At the end of 10-d exposure, 32.74 ± 0.64 μg/g and 13.12 ± 2.79 μg/g of diazepam were detected in roots and shoots, respectively, while the corresponding values were 15.36 ± 1.51 μg/g and 11.81 ± 0.40 μg/g for DM-diazepam, suggesting active translocation after entry in the roots. Among the four pairs of compounds considered in this study, diazepam and DM-diazepam have the largest log Kow . Between diazepam and DM-diazepam, the root accumulation of DM-diazepam was greater than diazepam during the first few sampling time points; however, an opposite trend was observed after 48 h of incubation, where the level of diazepam appeared to be significantly greater than DM-diazepam . Levels of both diazepam and DMdiazepam in the shoots increased over time, and there was no statistically significant difference between their concentrations at the same time points. It must be noted that unlike the other compounds considered in this study, demethylation of diazepam does not introduce a hydroxyl group into the structure and therefore, the almost identical accumulation of diazepam and DM-diazepam may be attributed to their similar physicochemical properties . As CECs and their methylated or demethylated derivatives were taken up by wheat seedlings, their levels in the nutrient solution decreased . The rate of dissipation was similar between acetaminophen and M-acetaminophen, and between methylparaben and DM-methylparaben. However, diazepam and naproxen appeared to decline at a slower rate than their demethylated counterparts . Consequently, the estimated T1/2 values were also significantly longer for diazepam and naproxen than their demethylated derivatives . The prolonged availability of diazepam and naproxen in the nutrient solution may have contributed to their relatively high accumulation in wheat seedlings . No significant correlation was found between log TF and log Dow in this study , likely due to the limited number of compounds considered in this study. Wu et al. observed a generally negative correlation for pharmaceuticals and personal care products in lettuce, spinach, cucumber and pepper. Another study conducted by Li et al. did not show any significant correlation between log TF and log Kow for neonicotinoids in Japanese mustard. Different treatments, plant species and compounds were used in those studies, suggesting that the translocation of xenobiotics in plants may be affected by not only the physicochemical properties of the xenobiotics, but also the inherent characteristics of plants. In addition, plants have a cascade of enzymes that are capable of facilitating metabolic transformations, and metabolism affects TF, as rapid metabolism in the root would translate into a diminished TF. Also, weak acidic CECs dissociated in the cytosol could be repelled by the negatively charged cell membranes, and therefore, become “trapped” in root cells, which may also limit their translocation. Active metabolism, such as conjugation with endogenous plant biomolecules, and the possible “ion trap” in root cells, likely contributed to the lack of apparent translocation for acetaminophen, DM-naproxen and DM-methylparaben in this study. Tools like ChemAxon could help predict basic properties of organic compounds, including TPs that do not always have experimentally derived values. It is feasible to incorporate changes in physicochemical properties, using either experimentally derived or estimated values, into well-established empirical relationships to evaluate the potential influence of common transformation reactions such as methylation and demethylation on plant uptake for a large range of CECs in the scenarios of beneficial reuse of treated wastewater effluent and bio-solids.