Simple reactions such as methylation and demethylation are common abiotic and biotic transformations, which contribute to the co-occurrence of many TPs of man-made chemicals in the environment. As demonstrated in this study, methylation and demethylation could result in changes in a chemical’s physicochemical properties, and the magnitude of change is specific to the molecule and the types of functional groups undergoing the conversion. The changes in a chemical’s physicochemical properties could subsequently lead to different environmental behaviors and risks, such as accumulation and translocation in higher plants. Moreover, a methylated or demethylated derivative may have increased or decreased biological activity as compared to the parent compound. Given that there are numerous CECs in sources such as treated wastewater and bio-solids, the co-existence of additional TPs presents another layer of challenge to the risk assessment of man-made chemicals. Although not explored in this study, differences may be similarly expected in microbial degradation and hence persistence of TPs, and further, bio-accumulation and toxicity to non-target organisms,vertical farming supplies such as aquatic and terrestrial invertebrates. For instance, methylated diclofenac showed a 430-fold increase in acute toxicity to Hyalella azteca than diclofenac. BPA mono- and dimethyl ethers were also found to result in enhanced mortality and developmental toxicity in zebrafish embryos than BPA. Methyl triclosan was shown to exhibit greater bioaccumulation in snails but reduced bioaccumulation in algae compared to triclosan.
The potential influence of methylation and demethylation on the phytotoxicity of CECs was not explored in this study; further research should consider this aspect by evaluating changes in enzyme activities and photosynthetic efficiency, among other endpoints.It must be noted that the experiments in this study were conducted under simplistic conditions. More processes and variables are involved in the soil-plant system under field conditions and their interactions likely determine the ultimate fate and risks of a chemical. For instance, methylation or demethylation may alter a compound’s stability in the rhizosphere as well as its adsorption to the soil solid phase, which in turn influence the chemical’s availability for plant uptake. As a chemical’s log Kow increases, its adsorption to soil increases while its presence in the soil pore water decreases, leading to a reduced availability for uptake into plant roots. The interactions of these fate and transport processes in the soil-plant system may therefore amplify or diminish the effects brought upon by the transformations and should be further studied under field-relevant conditions. A significant bottleneck to the holistic assessment of environmental risks is the sheer number of CECs and the fact that experimentally determined physicochemical properties are often not available for their transformation intermediates. It is likely that for many CECs, transformation reactions consistently lead to reduced biological availability and lower non-target toxicity. In this case, only certain transformation reactions for a subset of CECs may pose an increased risk. Predicting essential physicochemical parameters such as pKa and log Dow using well-established chemical calculation tools may generate the first line of information for identifying TPs with enhanced potential for bio-accumulation or non-target toxicity.
This approach may be used to effectively direct future research efforts to better understand the environmental significance of common transformation reactions for CECs. The use of treated wastewater and bio-solids promotes environmental and agricultural sustainability and is increasingly practiced around the world.However, numerous contaminants of emerging concern are present in the wastewater treatment plant effluent and bio-solids.Reuse of these resources introduces CECs into agroecosystems, where some CECs may be taken up by plants and enter terrestrial food chains.Even though the transfer of CECs from WWTP effluent or bio-solids to higher plants has been increasingly reported, in most cases only the parent form of CECs is considered.Many CECs, unlike legacy contaminants such as PCBs and organochlorine pesticides, possess reactive functional groups like hydroxyl and carboxyl groups, making them more susceptible to abiotic and biotic transformations. Such transformations have been reported for CECs after human consumption,during treatment at WWTPs,and in other biologically-mediated processes.For instance, methyl triclosan was often detected alongside triclosan in WWTP effluent and bio-solids, sometimes at even higher levels.Acetaminophen can be methylated by microorganisms in soil.Therefore, with the use of treated wastewater and bio-solids, CECs are often introduced into the agroecosystems together with their transformation products , such as methylated or demethylated TPs, before they come into contact with plants.For example, demethylation is a common transformation for xenobiotics as phase I metabolism catalyzed by cytochrome P450 enzymes, and esterase catalyzed or nonenzymatic hydrolysis for esters.Diazepam was previously found to be demethylated to nordiazepam in Arabidopsis thaliana cell cultures, cucumber and radish seedlings.Naproxen was demethylated to 6-Odesmethylnaproxen in A. thaliana cells.
Methylation, as a phase II metabolism catalyzed by methyltransferases,was also reported for a broad spectrum of substrates ranging from nucleic acids, lipids to xenobiotics.For example, tetrabromobisphenol A was converted to TBBPA mono- and di-methyl ethers in pumpkin seedlings.Therefore, in-plant transformations such as methylation or demethylation may occur after CECs or their TPs are taken up into plants, influencing the environmental cycling of CECs. It is further plausible that methylation and demethylation happen simultaneously and form a metabolic cycle within plants. For example, exposure of pumpkin plants to TBBPA dimethyl ether showed that the methylated TBBPA metabolite was demethylated back to TBBPA.This interconversion between CECs and their methylated or demethylated TPs may effectively prolong the environmental persistence of CECs, leading to uncertainties in our understanding of their ecological and human exposure and risks. To date, there has been little research on interconversions of CECs in plants, even though plants play a critical role in terrestrial ecosystems including agricultural systems. Here we examined the interconversion between four pairs of CECs, i.e., acetaminophen, diazepam, methylparaben and naproxen, and their methylated or demethylated counterparts in A. thaliana cells and wheat seedlings. These compounds were selected because of their ubiquitous occurrence in the environment.Additionally, TPs of these CECs are known to possess biological activity. For instance, DM-diazepam is not only a TP resulting from demethylation of diazepam but also a pharmaceutical in its own right.Similarly, DM-methylparaben also serves as a raw material in various industrial applications.A. thaliana cell suspensions were selected in this study due to the easiness for cultivation, high metabolic activity, and their common use as a fast-screening tool for evaluating plant metabolism.Whole plants, on the other hand, are more complex in structures as they have differentiated organs as well as associated microorganisms.Whole plants are therefore complementary to cell models as they provide more environmental relevance.Results from this comparative evaluation provide knowledge on the occurrence of such plant-mediated inter conversions and the potential significance of this process to the environmental fate and risks of CECs. Wheat seedlings were germinated from seeds in a seed germination tray kit in the dark at room temperature. When the seedlings grew to around 5 cm in height,vertical lettuce tower five seedlings were transplanted into a 50-mL polypropylene centrifuge tube containing 30 mL Milli-Q water. The wheat seedlings were cultivated in a growth chamber at 24 ℃ with a 16:8 h light:dark schedule. The water solution in the tubes was replaced in 2-d intervals to 1/4 strength and then 1/2 strength Hoagland® nutrient solution to allow gradual acclimation for the seedlings. After seedlings were acclimated for another 2 d in the 1/2 strength Hoagland® nutrient solution, the hydroponic solution was replaced with 30 mL fresh 1/2 strength Hoagland® nutrient solution spiked with individual compounds at an initial concentration of 1 mg/L.
Fresh Milli-Q water was added to each tube to replenish the water lost through evapotranspiration every other day. Control groups, including wheat seedlings growing in clean culture solution, and spiked culture solution in tubes without wheat seedlings, were used for quality control and assurance. Triplicate containers were sacrificed after 0, 3, 6, 12, 24, 48, 96, 168 and 240 h of cultivation for each of the treatment groups. Control groups were sampled only after 240 h. Seedlings were rinsed, dried and separated into roots and shoots. The nutrient solution and the plant tissues were stored at -80 ℃ until analysis.Extraction methods were adopted from previous studies, with minor modifications.A. thaliana cell culture media and wheat seedling hydroponic culture solution were processed using solid phase extraction . Prior to the extraction, deuterated compounds were added to 5 mL nutrient solution as recovery surrogates. HLB cartridges purchased from Waters were preconditioned with 7 mL methanol and 7 mL water in sequence. The aqueous sample was passed through the preconditioned SPE cartridge, followed by the addition of 5 mL 5% methanol in water for clean-up. A final elution using 15 mL methanol was carried out and the resulting eluent was collected in a glass vial, dried under a gentle nitrogen gas flow, and then reconstituted with 1 mL 1:1 methanol:water. The final extracts were filtered through 0.2-μm PTFE filters into 1.5 mL glass vials for instrument analysis. Plant tissues, including A. thaliana cell matter, wheat seedling roots and shoots, were freeze-dried at -50 ℃ for 3 d to remove moisture. Wheat roots and shoots were then cut into small pieces. Before extraction, a 50-μL aliquot of depurated compounds was added to the tissue samples as recovery surrogates. The samples were extracted with 15 mL MTBE in a sonication water bath for 30 min. The sonication extraction process was repeated with 15 mL fresh MTBE once and then 15 mL fresh acetonitrile twice. Extracts from all steps were combined and dried on a nitrogen evaporator, and then recovered using 1 mL methanol. The resulting samples were diluted with 20 mL Milli-Q water and then passed through HLB cartridges following a similar process to that given above for the aqueous samples. The eluent was then dried under a gentle stream of nitrogen gas, reconstituted in 1 mL 1:1 methanol:water, and filtered through a 0.2-μm PTFE filter before instrument analysis. Quantitative analysis of all target compounds in this study was conducted on a Waters ACQUITY TQD tandem quadrupole UPLC-MS/MS . Chromatographic separation was performed using a Waters ACQUITY BEH C18 column at 40 ℃. The mobile phase A and B were 0.01 % formic acid in LC-grade water and pure optimal-grade methanol, respectively, with a flow rate of 0.3 mL/min. The flowing gradient was set as: 0-1 min, 5% to 40% B; 1-2 min, 40% to 90% B; 2-4 min, 90% to 95% B; and 4-6 min, re-equilibrate with 5% B. The injection volume was 5 μL. The MRM transitions of all target compounds were optimized and summarized in the Supporting Information . Quantification was completed using the TargetLynx XS software . To better understand the effect of molecular structures on methylation or demethylation transformations of CECs in plants, the strength of the chemical bond between the major fragment and the methyl group in the methylated compounds was estimated. Although indirect methods like bond-dissociation energies are frequently used to characterize the bond strength by considering enthalpy change when the bond is cleaved, they often do not accurately describe the intrinsic strength of a particular bond.The calculation of compliance constants offers an alternative way to directly determine bond strength without referring to arbitrary or poorly defined states, therefore leading to more reliable results.Compliance constants address the question of which displacement is caused by a given force on a single coordinate, while all other forces thereby introduced are allowed to relax. The relaxed force constants , as the reciprocal of individual compliance constants, measure the force required to distort a coordinate by a unit amount while allowing all other coordinates to relax. The relaxed force constants of R-CH3 in the methylated TPs were computed using the software Compliance ,and the configurations of target compounds were optimized with density functional theory calculations at B3LYP/6-31G* level by the software Gaussian 16 prior to computation. A larger value of relaxed force constant would indicate a stronger chemical bond strength of R-CH3. Recoveries of all target compounds for extraction efficiency and limits of quantification are given in SI . Method blanks and matrix blanks were included during extraction to check for possible contamination. One solvent blank and one check standard were injected after every 10 samples to check cross-contamination and for continued calibration during analysis .