No target analytes were detected in the method blanks, matrix blanks, or solvent blanks, indicating no carry-over contamination during extraction or instrument analysis. Data in this study were calculated as mean ± standard deviation . The data were analyzed using SPSS Statistics 27 and graphed by Prism 9 . To explore the potential interconversion of selected CECs and their methylated or demethylated TPs in A. thaliana cells, we exposed A. thaliana cells to acetaminophen, M-acetaminophen, DM-diazepam, diazepam, DM-methylparaben, methylparaben, DMnaproxen and naproxen, individually. The changes in the level of the parent compound, the formed TP, and the unidentified portion were considered in estimating the mass balance . No methylated or demethylated products were found in the non-viable A. thaliana cell culture control groups spiked with their corresponding counterparts, suggesting that when methylation or demethylation was observed, it was due to biologically mediated transformations in live A. thaliana cells. Shortly exposure to the methylated compounds M-acetaminophen, diazepam, methylparaben or naproxen, their demethylated counterparts started to appear in the A. thaliana cells . After reaching a peak level, the concentration of the demethylated products generally decreased as the incubation time further increased, likely due to subsequent metabolism of the demethylated intermediates in A. thaliana cells. After 11 h of cultivation, A. thaliana cells demethylated a small fraction of Macetaminophen to acetaminophen, with 14.9 ± 1.9 ng/g of acetaminophen found in A. thaliana cell matter,vertical grow system and the concentration further increased to 39.4 ± 27.0 ng/g at 48 h. The molar equivalent of acetaminophen to M-acetaminophen was approximately 0.05 in A. thaliana cells at 48 h. Demethylation of diazepam, methylparaben or naproxen in A. thaliana cells appeared to take place immediately after the treatment.

The concentration of DM-diazepam in A. thaliana cells spiked with diazepam increased to 394.5 ± 59.8 ng/g during the first 24 h and then decreased slightly. At the end of 96-h cultivation, the level of DM-diazepam in the cells was still at 320.5 ± 199.1 ng/g. Demethylation of methylparaben was found to occur extensively in this study. Noticeably, at 0 h, 7195.6 ± 434.9 ng/g of DM-methylparaben was found in A. thaliana cell matter. This was likely caused by the conversion during the sample preparation process after chemical spiking, including centrifugation, which lasted for about 1 h. When calculated as molar equivalent, it was approximately 0.68 for DMmethylparaben to methylparaben in the cell matter at 0 h. The level of DMmethylparaben in A. thaliana cells decreased thereafter, likely due to the rapid metabolism of DM-methylparaben.At the end of exposure, only 49.7 ± 42.1 ng/g of DM-methylparaben remained in the cell matter. In contrast, demethylation of naproxen was found to be less substantial under similar conditions, with the highest concentration of DM-naproxen detected at 0 h. At the end of the 96-h cultivation, 10.8 ± 5.5 ng/g of DM-naproxen was found in the A. thaliana cell matter. This observation was in agreement with previous studies where DM-naproxen was found at lower levels than other metabolites in A. thaliana cells exposed to naproxen, or was not detected in Garden cress Lepidium sativum exposed to naproxen.Naproxen conjugates in plants were reported in previous studies, suggesting that DM-naproxen is an intermediate metabolite of naproxen that can be further transformed through phase II pathways such as conjugation. Similarly, acetaminophen and DM-methylparaben were also found to be conjugated with biomolecules in plants. The common occurrence of conjugation implies that the actual degree of demethylation of the CECs may be substantially greater than what was experimentally measured in this study. Methylation of the demethylated compounds in A. thaliana cells was concurrently evaluated under similar conditions . Generally, methylation was less extensive as compared to the corresponding demethylation. For example, during the first 11 h of incubation, methylation of acetaminophen was limited, and M-acetaminophen was below detection. At 24 h into the incubation, 27.4 ± 3.7 ng/g M-acetaminophen was detected in the cell matter, which further increased to 38.0 ± 10.1 ng/g at 48 h. The methylated acetaminophen was then metabolized and was not detectable at the end of 96-h cultivation.

Therefore, methylation of acetaminophen in A. thaliana cells was mostly negligible under the experimental conditions. Methylation of DM-diazepam to diazepam was not observed in A. thaliana cells throughout the 96-h incubation duration. Similarly, methylation of DM-methylparaben was also found at much slower rates than demethylation of methylparaben. Methylparaben was found at 68.0 ± 45.0 ng/g in A. thaliana cells at 48 h in the DM-methylparaben treatment. Naproxen was detected at 29.8 ± 23.3 ng/g at 0 h in A. thaliana cells treated with DM-naproxen and decreased thereafter. Overall, for the four demethylated CECs considered in this study, their methylation was more limited in relation to the demethylation of their counterparts. Outside of the A. thaliana cells, trace levels of the methylated or demethylated TPs were occasionally found in the aqueous culture media . Among the different CECs, DM-naproxen was below the detection limit in the culture media spiked with naproxen. The absence of DM-naproxen in the culture media was consistent with the limited formation of DM-naproxen in A. thaliana cells. A similar pattern was also observed for acetaminophen and DM-diazepam, where their methylated products were not found in the cell culture media. The demethylated products of methylparaben and Macetaminophen, and the methylated TP of DM-naproxen, were found in the range of 0-3.0 μg/L in the cell media. In contrast, the demethylated product of diazepam, i.e., DMdiazepam, was found at relatively high levels in the cell media. After 48 h of incubation, DM-diazepam reached 40.1 ± 0.3 ng/mL in the cell media treated with diazepam. The accumulation of DM-diazepam in the cell media may be attributed to the persistence of DM-diazepam in A. thaliana cell cultures, and was in agreement with the finding that DM-diazepam was readily formed in A. thaliana cells. The changes in the level of the parent compound, the formed TP, and the unidentified portion in hydroponic wheat seedling systems were considered to estimate the mass balance .

The interconversion between CECs and their methylated or demethylated counterparts displayed different patterns in wheat roots and shoots . Results showed that demethylation of methylparaben and naproxen, and methylation of acetaminophen, DM-diazepam and DM-methylparabenwere significantly more extensive in roots than in shoots . In addition, demethylation took place at a significantly greater extent as compared to the corresponding methylation in both wheat roots and shoots , except for the demethylation of acetaminophen and methylparaben in wheat roots , which aligns with the results in A. thaliana cells.Demethylation and methylation of the test CECs in the wheat roots exhibited molecular specificity . Demethylation of naproxen in wheat roots was more pronounced , with 18744.8 ± 2869.2 ng/g of DM-naproxen detected in wheat roots at 48 h in the naproxen-treated system. The level of DM-naproxen decreased with time but remained at 6697.0 ± 4404.7 ng/g at 240 h. In contrast, methylation of DM-naproxen was not detected in wheat roots . These results suggested that naproxen in wheat roots was rapidly metabolized and/or translocated, and its demethylation to DM-naproxen was a substantial metabolism pathway in wheat roots. This was consistent with previous studies where demethylation of naproxen was found to take place in A. thaliana cells and seedlings,vertical farming companies followed by subsequent conjugation reactions.The limited methylation of DM-naproxen may be partly attributed to further metabolism and potential translocation of the derived naproxen, rendering it non-detectable. The demethylation product of diazepam, DMdiazepam, was also observed in wheat roots and the level of DM-diazepam reached2707.7 ± 826.0 ng/g at the end of the 10-d exposure . In contrast, methylation of DM-diazepam was relatively negligible, and diazepam was detected at 43.1 ± 30.5 ng/g at 12 h in the roots treated with DM-diazepam and became non-detectable thereafter . This pattern was similar to that in A. thaliana cells, suggesting again that demethylation was substantially more active than methylation for the diazepam and DMdiazepam pair in plants. In wheat roots exposed to 1 mg/L methylparaben, DM-methylparaben was found at trace levels for the first 168 h but increased thereafter, reaching 321.9 ± 16.5 ng/g at the end of experiment . Demethylation of methylparaben in the roots was found to be more limited than that in A. thaliana cells. This may be attributed to the more rapid dissipation in wheat roots caused by active metabolism, translocation out of the roots, and/or microbial degradation in the rhizosphere. The concentration of methylparaben, on the other hand, increased to 589.7 ± 20.9 ng/g at 48 h in wheat roots grown in the DMmethylparaben spiked hydroponic solution, and then decreased to 36.5 ± 13.9 ng/g at the end of experiment . No appreciable demethylation of M-acetaminophen was observed in the roots exposed to M-acetaminophen . In comparison, methylation of acetaminophen to M-acetaminophen was more substantial , with M-acetaminophen detected at 316.5 ± 20.8 ng/g after 12 h and 112.4 ± 25.8 ng/g at the end of experiment. Among the four pairs of CECs and their corresponding methylated or demethylated TPs, naproxen and diazepam showed a greater degree of demethylation, while theirdemethylated products showed little back conversion in wheat roots. In contrast, acetaminophen and DM-methylparaben exhibited notable methylation, while demethylation of M-acetaminophen and methylparaben appeared to be limited.

This observation indicates that even though methylation and demethylation could take place simultaneously in plants, the interconversion may be somewhat directional for individual CECs, with one transformation favored over the back transformation. The methylated or demethylated TPs of CECs found in wheat shoots could potentially have two sources, i.e., in situ transformation from the parent CEC in the shoots, and translocation of the TP from the roots. The interconversion between Macetaminophen and acetaminophen took place at similar levels in wheat shoots . After 48 h of incubation in hydroponic solution spiked with 1 mg/L Macetaminophen, acetaminophen was detected at 161.1 ± 74.0 ng/g, which decreased to 51.8 ± 25.7 ng/g at the end of exposure . It should be noted that acetaminophen was not found in wheat roots exposed to M-acetaminophen, and previous studies showed that the translocation of acetaminophen was negligible in wheat and cucumber seedlings.Therefore, it is likely that the occurrence of acetaminophen in wheat shoots grown in the hydroponic solution spiked with M-acetaminophen was a result of demethylation taking place in the shoots, rather than translocation from the roots. After 12 h of incubation in media spiked with 1 mg/L acetaminophen, 107.2 ± 0.2 ng/g of M-acetaminophen was found in wheat shoots . Similar to wheat roots, demethylation of methylparaben in wheat shoots was relatively limited as compared to the other CECs, with DM-methylparaben detected at 166.7 ± 28.7 ng/g in the shoots at the end of experiment . Methylation of DM-methylparaben was negligible, with methylparaben found at only 8.3 ± 3.5 ng/g in the shoots at 6 h . The accumulation of DM-methylparaben was previously found to be very limited in wheat shoots, which may explain the absence of its methylation in the shoots. Demethylation of naproxen in wheat shoots was found at lower levels compared to the roots , which may be attributed to the limited translocation and/or rapid metabolism of DM-naproxen in wheat shoots.Like in the roots, methylation of DMnaproxen was not observed in wheat shoots. Since DM-naproxen was not detected in the shoots of wheat seedlings exposed to DM-naproxen,the absence of naproxen in wheat shoots exposed to DM-naproxen may be mainly due to limited plant uptake and translocation of DM-naproxen. In contrast, the formation of DM-diazepam was substantial in the shoots of wheat seedlings exposed to diazepam, with the level increasing quickly and reaching 8839.0 ± 2275.1 ng/g at the end of exposure .In a previous study, DM-diazepam was found to be metabolized faster in wheat roots than diazepam, while their levels in shoots were similar.Therefore, after demethylation, the formed DM-diazepam may undergo further metabolism, especially in roots. When considered in molar equivalents, about 8.0% of the total spiked diazepam was demethylated to DM-diazepam in wheat shoots, which was substantial given that some of the formed DM-diazepam was likely not extractable by solvent and/or had undergone further metabolism. By comparison, methylation of DM-diazepam was not significant in the shoots, with the highest level at only 46.0 ± 32.5 ng/g at 48 h and non-detectable at the later time points .