The rhizosphere is usually considered an important player in the overall metabolism of xenobiotics by whole plants, as root exudates generally enhance the richness of microbial communities in the root zone, leading to a greater microbial abundance and accelerated microbial degradation.Although the rhizosphere in a hydroponic system may differ greatly from that in soil in terms of microbial community abundance, it was likely that some of the transformations of the target CECs or their TPs occurred in the solution due to rhizosphere-mediated microbial degradation.This could result in the occurrence of methylated or demethylated metabolites in the hydroponic solution and their subsequent uptake into the plant. In addition, previous studies also showed that some xenobiotics may be excreted from plant roots into their bathing solution. Analysis for the target CECs in the nutrient solution in this study, however, generally showed an absence of the corresponding methylation or demethylation products in the nutrient solution, except for acetaminophen and M-acetaminophen . The calculated compliance constants of the methylated CECs are summarized in Table 1, along with the calculated R-CH3 relaxed force constants. A stronger chemical bond is harder to break as it requires more energy, while it is easier to form as more energy may be released. The computation results of the relaxed force constants showed that the chemical bond strength between the methyl group and the major molecular fragment in the methylated CECs followed a general order of methylparaben < diazepam < naproxen < M-acetaminophen. Therefore,vertical garden indoor demethylation may be expected to occur more readily for methylparaben, but more slowly for M-acetaminophen.

Conversely, methylation of DM-methylparaben may be expected to be the hardest, while it is relatively easy for acetaminophen. The trends observed for the four pairs of CECs in A. thaliana cells generally followed the prediction from the bond strengths. For example, the demethylation of methylparaben in A. thaliana cells was the most extensive among the test compounds, followed by diazepam. In contrast, demethylation of M-acetaminophen or naproxen was negligible under the same conditions. Methylation from acetaminophen to M-acetaminophen was found to proceed more readily than the conversion from DMnaproxen to naproxen, while methylation of DM-diazepam was not observed. Due to the limited number of compounds considered in this study, a quantitative correlation between the calculated bond strength and transformation rates was not carried out. However, future studies may consider ascertaining such a relationship, with information from more compounds, in order to better understand the impacts of molecular structures on bio-transformation in plants. The demethylation and methylation processes involve distinct subfamilies of CYP450s, esterases and methyltransferases, which may depend on plant species specific enzyme activities, as well as the chemical structure of xenobiotics. The generally good agreement between the experimental results and bond strength-based predictions in this study suggests that evaluation of chemical characteristics such as the bond strength of R-CH3 may be used to identify CECs with a high tendency for specific transformation reactions. Given the large number of CECs, such a first-cut screening approach may be invaluable for developing a priority list of CECs that may undergo such conversions. The usefulness of such predictions may be further improved by considering more compounds and different plant species, and by developing and refining quantitative structural-activity relationships.To ensure confident identification and quantitative measurement of CECs and their TPs, an artificially high concentration was used in the growth media for A. thaliana and wheat seedlings.In addition, hydroponic cultivation was a simplified system, and the absence of soil should impart significant influences on the adsorption and hence the availability of CECs for plant uptake. Microorganisms in rhizosphere soil under field conditions likely play a great role in facilitating transformations of CECs, and therefore, the interconversion of CECs and their TPs in the soil-plant continuum may exhibit patterns different from observations from this study.

Nevertheless, results from the controlled experiments in this study clearly showed that plants can mediate transformations of CECs such as methylation and demethylation. In some cases, demethylated products were found at relatively high levels under experimental conditions. Given that a large fraction of TPs was likely non-extractable or conjugated, the actual occurrence of such transformations in plants may be much more pronounced than that detected in this study. Conjugated metabolites may become deconjugated upon ingestion, for example, by enzymes in the gastrointestinal tract, releasing bio-active molecules.The methylated or demethylated TPs likely retain or have even increased biological activity. For example, DM-diazepam , although a demethylated TP of diazepam, is itself a drug for treating anxiety. The addition or loss of a methyl group alters the physicochemical properties of a compound, leading to different environmental behaviors such as bio-accumulation, metabolism, and toxicity. For example, diclofenac methyl ether showed greater acute toxicity to aquatic invertebrates than diclofenac.Bisphenol A mono- and di-methyl ether also displayed greater developmental toxicity to zebrafish embryos than bisphenol A.Therefore, when considering the whole life cycle of CECs, e.g., along the entire human-wastewater-soil-plant-human continuum, such circular interconversions may effectively prolong the persistence of CECs and contribute to enhanced human and ecotoxicological risks, underscoring an urgent need to consider such interconversions for more comprehensive risk assessment. For the four pairs of CECs considered in this study, demethylation appeared to proceed more readily than methylation, and there were also differences among different compounds. A preliminary analysis showed a dependence of the methylation or demethylation rate on the bond strength of R-CH3 of the compounds. As CYP450s, esterases and methyltransferases are involved in the metabolism of many xenobiotics, CECs with similar functional groups like -OH, -OCH3, -NH-, and -NCH3- may also undergo the methylation and demethylation cycle. With more experimental observations, it is feasible to predict the likelihood of such transformations using basic chemical structures and molecular descriptors.

This is particularly valuable given that CECs and their TPs are numerous in numbers and identifying compounds or structural features conducive to interconversions constitutes an important first step to better understand the significance of this phenomenon for the overall environmental fate and risks of CECs.The occurrence of numerous contaminants of emerging concern in the effluent from wastewater treatment plants and impacted aquatic environments has been extensively reported.However, most research has focused on the parent form of CECs while generally neglecting their transformation products that are often in co-existence. Many CECs contain reactive functional groups, such as hydroxyl, carboxyl and amide groups, making them susceptible to various biotic and abiotic transformation reactions.Simple transformations, such as methylation and demethylation, have been observed in various environmental matrices for many CECs.For example, previous studies showed the presence of methylated TPs of triclosan and bisphenol A in wastewater effluents and receiving streams.The methyl ethers of tetrabromobisphenol A were formed in aquatic environments in the presence of background methyl iodide.Methylation of acetaminophen was observed in soil.On the other hand, demethylation is a major metabolism pathway for CECs in organisms. For example, after oral administration in humans, naproxen and diazepam are demethylated to 6-O-desmethyl naproxen and nordiazepam , respectively.Despite the fact that TPs seem to occur readily and co-exist with their parent forms in the environment,vertical garden indoor system the ecotoxicological consequences of such transformations have not been adequately considered. Transformations such as the addition or loss of a methyl group can significantly change a compound’s physicochemical properties, such as Kow that is known to influence its fate and bioaccumulation.Methylated products of diclofenac, BPA, and triclosan all displayed enhanced toxicity or bioaccumulation potential in aquatic organisms. In this study, we comparatively explored the behaviors of four typical CECs, i.e., acetaminophen, diazepam, methylparaben, and naproxen, and their methylated or demethylated TPs in Daphnia magna, by considering their bio-accumulation, acute toxicity, and interconversions. Quantitative structure-activity relationship models were further developed and used to describe the experimental results. The study findings highlight the importance of simple transformation reactions such as methylation and demethylation in understanding the overall ecological risks posed by CECs in aquatic environments. To further understand the effect of methylation and demethylation on the acute toxicity to D. magna, bio-accumulation of the CECs and their methylated or demethylated counterparts was measured in adult organisms. The concentrations of target compounds remained relatively constant in the aqueous media during the 24 h uptake phase, with RSDs ranging from 2.8% to 18.4% . Therefore, the mean measured concentrations of target compounds in the water phase were used as to fit Equations and to derive BCF values. The bio-accumulation kinetics of target compounds are shown in Figure 2. The concentrations of CECs and their methylated or demethylated TPs generally showed an increasing trend at the beginning of the uptake phase and reached an apparent equilibrium in 24 h.

Upon transferring the exposed D. magna to clean AFW to initiate the depuration phase, the concentration of test compounds gradually declined over time. With the exception of diazepam, methylated derivatives consistently showed much higher concentrations in D. magna than their demethylated counterparts. For example, after 2 h of exposure, the concentrations of acetaminophen and M-acetaminophen in D. magna were found at 308.7 ± 42.6 ng g-1 and 8730.7 ± 2900.9 ng g-1 , respectively, a 28-fold difference . This was consistent with the fact that methylated acetaminophen has a higher log Dlipw than acetaminophen . In addition, at pH 8.5, acetaminophen was expected to be partially ionized in the aqueous media, while M-acetaminophen should be completely in its neutral state . Methylparaben also displayed a much higher accumulation than DM-methylparaben in D. magna at the end of the uptake phase . The 3-fold change also coincided with the difference in log Dlipw between DM-methylparaben and methylparaben . The level of DM-naproxen in D. magna was below LOD, and therefore its bioaccumulation may be deemed negligible . In contrast, significant accumulation of naproxen in D. magna was observed, again suggesting a pronounced effect by hydrophobicity induced by methylation. It is also likely that DM-naproxen was rapidly metabolized due to the presence of an exposed hydroxyl group . The presence of the hydroxyl group in DM-naproxen may facilitate its conjugation with an amino acid or glucose in D. magna, contributing to its rapid metabolism and reduced bio-accumulation. Unlike the other three pairs, there was no significance in the bio-accumulation between DMdiazepam and diazepam in D. magna , with 6792.5 ± 1215.8 ng g-1 and 7599.7 ± 1470.3 ng g-1 detected in D. magna after 24 h, respectively. This may be attributed to the fact that methylation or demethylation does not result in a great change in their physicochemical properties and that both compounds have similar log Kow or log Dlipw values . The derived kinetic parameters of target compounds are given in Table S3. In general, the methylated derivative in each pair had a larger ku than the corresponding demethylated counterpart. The dynamic BCF values, calculated as the ratio of ku and kd, showed a strong correlation with the BCF values derived from the steady state , suggesting enhanced bioaccumulation for most methylated CECs. For example, the dynamic BCF of M-acetaminophen was 10.0 ± 0.0 in D. magna, which was significantly higher than the dynamic BCF of acetaminophen . For DMdiazepam and diazepam, however, the BCF values in D. magna were not significantly different from each other, which again coincided with their generally similar physicochemical properties. For aquatic organisms, increased bioaccumulation of contaminants is often attributed to a compound’s hydrophobicity, as bio-accumulation is driven by lipids in an organism and is positively related to hydrophobicity or log Kow for neutral compounds. Increased bioaccumulation after methylation was previously observed for diclofenac in aquatic invertebrates. Bioaccumulation of methylated diclofenac was found to be 25-110-fold that of diclofenac in H. azteca and G. pulex. In this study, methylation generally increased log Kow of CECs, and further log Dow and log Dlipw, although the relative increases are specific to the individual compounds. The generally enhanced bioaccumulation in D. magna was also in agreement with the effect of methylation on CEC bio-accumulation in plants.Methylation of CECs could occur in natural water bodies due to the presence of methyl iodide,during wastewater treatment, and during biological transformations in soil, plants ,and earthworms.Therefore, methylated derivatives of CECs may be prevalent in the environment and should be considered in a holistic risk assessment because of their different behaviors and biological activities, such as increased bio-accumulation potentials.