Biologically mediated transformations such as methylation and demethylation may also occur in organisms such as D. magna after their uptake of CECs, which may further influence their toxicity. Methylation and demethylation in D. magna were investigated after exposing D. magna to the individual compounds. Methylation of the selected demethylated CECs was negligible, as no methylated product was detected in D. magna after its exposure to the corresponding demethylated counterpart. However, demethylation of diazepam, methylparaben and naproxen in D. magna was evident , while acetaminophen was not detected in D. magna exposed to Macetaminophen. The demethylation of methylparaben was limited, with a peak concentration of DM-methylparaben at 0.5 ± 0.0 nmol g -1 in D. magna after 12 h of exposure to 1 mg L-1 methylparaben. This represented only about 2.0% of the molar equivalent of methylparaben in D. magna. The demethylation of diazepam was found at similar levels, with DM-diazepam at 4.4% molar equivalent of diazepam. Interestingly, the molar equivalents of the demethylated derivatives increased over time during the depuration phase, even though the overall concentrations generally decreased over time. For example, the molar equivalents of DM-diazepam and DM-methylparaben reached 33.5% and 54.8% at the end of depuration, respectively. This may be attributed to the fact that demethylation continued during the depuration phase, which may have influenced the apparent depuration of these compounds .The demethylation of naproxen in D. magna was the most pronounced among the four methylated compounds,vertical garden growing with DM-naproxen generally detected at levels higher than naproxen itself during both the uptake and depuration phases . DM-naproxen was formed quickly in D. magna after exposure to naproxen, with 21.5 ± 2.7 nmol g-1 after 12 h into the uptake phase, which was significantly higher than that of the parent naproxen .
Similar to DM-diazepam and DMmethylparaben, the molar equivalent of DM-naproxen also continued to increase during the depuration phase. At the end of depuration, DM-naproxen accounted for approximately 88.9% of the total naproxen and DM-naproxen residues in D. magna. The high proportion of DM-naproxen in D. magna also suggested that demethylation was the primary metabolism pathway of naproxen in D. magna. To better understand the demethylation of CECs in D. magna, the formation rates of DM-diazepam, DM-methylparaben and DM-naproxen were estimated by simulating their formation over the initial 12-h period, during which good linear relationships between their formation and time were present . Formation rates showed no significant differences between DM-diazepam and DM-methylparaben. However, the formation rate of DM-naproxen was significantly greater than DM-diazepam or DM-methylparaben. Based on their respective chemical structure , the demethylation of diazepam and naproxen appears to differ slightly from that of methylparaben. While the demethylation of methylparaben involves the removal of a methyl group from a carboxyl group, which may be catalyzed bycarboxylesterases,CYP450s,or through non-enzymatic hydrolysis,the demethylation of M-acetaminophen, diazepam and naproxen reflects the removal of a methyl group from an amide or hydroxyl group, which likely is catalyzed mainly by CYP450s.Previous studies showed that carboxylesterases play a more important role in drug metabolism in invertebrates due to the lower activity of CYP450s.The more significant demethylation observed for naproxen in comparison to methylparaben suggests that CYP450s may also play an important role in the metabolism of such substrates in aquatic invertebrates. The observed significant differences in the demethylation rates of diazepam and naproxen imply that CYP450s in aquatic invertebrates like D. magna may exhibit different levels of activity towards different CECs.In vivo half-lives of the test compounds were derived from the depuration rate during the 24-h depuration phase in D. magna. The in silico half-life was estimated from the primary bio-transformation rate in fish and normalized to a 10 g fish at 15 °C based on the inherent characteristics of the QSAR model.
Similar to BCF values, in vivo and in silico half-lives could not be compared directly between the different organisms. Hence, the relative persistence of test compounds was calculated for evaluation. As shown in Table 2, in silico predictions suggest that methylation may prolong the persistence of CECs in fish. This was in contrast to the in vivo results in D. magna, which showed that methylation generally shortened the persistence of CECs. As mentioned above, methylated CECs generally accumulated faster with a larger ku value during the uptake phase, but dissipated rapidly during the depuration process. Considering that biota residing in wastewater effluent-dominated streams often experience pseudo-persistent exposure to CECs due to the constant discharge of effluents from WWTPs, uptake rates may be more important in regulating the accumulation of CECs in aquatic organisms dwelling in the impacted system. The prolonged bio-transformation half-lives of methylated CECs should be validated under field conditions. Overall, in silico predictions and experimental measurements were in agreement for the influences introduced by methylation or demethylation. This highlights the feasibility of incorporating QSAR models to evaluate the potential influence of common transformations such as methylation and demethylation on the environmental risks of CECs to non-target organisms in impacted ecosystems. Simple transformations such as methylation and demethylation contribute to the proliferation of the numbers of CECs and diverse structures in environmental compartments impacted by e.g., wastewater effluent.This study showed that these transformations can alter the physicochemical properties of CECs, resulting in changes in their environmental processes such as bio-accumulation and acute toxicity in aquatic organisms. These transformations of man-made chemicals may also take place within a non-target organism after their accumulation from the ambient environment.
Certain transformations, like methylation, likely result in enhanced bio-accumulation and increased toxicity in non-target organisms. Although not considered in this study, halogenation of man-made chemicals, such as gemfibrozil, 4-nonylphenol and naproxen, during the disinfection process in WWTPs, has also been reported, and the halogenated products generally exhibited increased bio-accumulation and toxicity to aquatic invertebrates.Due to the presence of numerous CECs in sources such as wastewater effluents and sediments, the co-existence of various TPs presents an additional challenge in addressing the overall environmental risks of man-made chemicals. It is important to note that high concentrations of test CECs and their corresponding methylated or demethylated TPs were used in this study in order to derive the LC50values and examine conversions in D. magna; these concentrations were above environmentally relevant levels. However, previous studies suggested that BCFs may be greater at lower exposure concentrations.Therefore, the effect of methylation or demethylation on bio-accumulation of CECs may be more pronounced than what was observed in this study. The environmental occurrence and concentrations of methylated or demethylated TPs are largely unknown for most CECs. Further research into the occurrence of TPs in different environmental compartments is needed to gain knowledge about the realistic exposure levels and to refine risk assessment. A major challenge in comprehensively assessing environmental risks is the sheer number of CECs and their TPs. It is unrealistic to experimentally evaluate transformation-induced changes in their environmental behaviors and toxicological profiles for all CECs.The incorporation of well-established QSAR models to predict essential chemical properties and environmental risk markers, such as hydrophobicity and lipophilicity, bio-accumulation potential, and acute toxicity, may help prioritize TPs with enhanced biological activities.This approach can be used to more effectively direct future research efforts to better understand the environmental significance of common transformation reactions for CECs. Four CECs and their methylated or demethylated TPs were comparatively evaluated for their uptake into A. thaliana cells or by wheat seedlings. The methylated compounds, generally more hydrophobic with a greater log Kow and log Dow,equipment for vertical farming often displayed a greater accumulation potential in both plant models as compared to their demethylated counterparts, with the exception of acetaminophen/M-acetaminophen in A. thaliana cells. The influence of methylation and demethylation on the translocation of CECs in wheat plants was molecular-specific. Methylation caused a significant increase in the translocation of acetaminophen, but a significant decrease for DM-diazepam. Methylation also generally prolonged the persistence of CECs in both A. thaliana cell culture media and wheat seedling hydroponic solution. A significant linear relationship was observed between log Dow and log BCF, indicating that the generally increased accumulation of methylated compounds may be attributed to their higher hydrophobicity. Results from this study suggested that common transformations such as methylation and demethylation may affect the persistence and accumulation of CECs in plants, and their role should be considered to obtain a more comprehensive understanding of the risks of CECs in the terrestrial environment including agro-food systems.
The interconversions between CECs and their methylated or demethylated TPs were evaluated in A. thaliana cells and wheat seedlings after their uptake. The methylation demethylation cycle was observed in both plant models, with demethylation generally taking place at a greater degree than methylation. Computation results 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, a pattern reflective of experimental observations for demethylation in A. thaliana cells. Future studies considering more chemical structures would help strengthen such QSAR models so that the potential for simple transformations such as methylation and demethylation may be predicted in the absence of experimental data. The acute toxicity of selected CECs and their methylated or demethylated TPs was further assessed by exposing D. magna to individually compounds. Methylation or demethylation resulted in changes in the acute toxicity for most CECs, and the influence was compound-specific. Methylation led to a significant increase in the acute toxicity of DM-methylparaben and DM-naproxen, but a decrease for acetaminophen. A significant negative linear relationship was observed between log LC50 values and log Dlipw values, indicating that as log Dlipw increased, the acute toxicity generally increased. Methylation increased the bio-accumulation in D. magna for acetaminophen, DM-methylparaben and DM-naproxen, and the increased bio-accumulation likely underlined the increases in acute toxicity for methylated compounds. In D. magna, active demethylation of diazepam, methylparaben and naproxen was observed, with the demethylation of naproxen especially pronounced, suggesting that enzymes in D. magna exhibited different levels of activity towards different substrates. QSAR models were used to predict changes in acute toxicity and bio-accumulation as a result of methylation, and the predicted values were in good agreement with experimental observations. The exploratory research presented in this dissertation clearly showed that simple transformations such as methylation and demethylation can significantly change the physico chemical properties of CECs and subsequently cause changes in their environmental behaviors such as accumulation by plants and aquatic organisms, toxicity and persistence. Methylation generally leads to increased hydrophobicity and further greater bio-accumulation and acute toxicity. However, exceptions were also observed in this study, suggesting that specific molecular structures may respond differently to the impact of simple transformations. QSAR models using molecular descriptors have the capability to predict the easiness of transformation reactions such as methylation and demethylation, the subsequent changes in physicochemical properties from such transformations, and further, the ensuing changes in bio-accumulation, translocation, and toxicity. Such models should be calibrated with more experimental observations and by the inclusion of more diverse structures. Such predictive tools are extremely valuable, given the enormous number of CECs and their transformation products, which renders experimentation-based approaches largely infeasible. This dissertation research highlights the prevalence of simple transformations such as methylation and demethylation in the environment, and the need to consider such transformations in achieving a more comprehensive understanding of the environmental fate and risks of CECs.Results from this dissertation research and a few other studies showed that simple transformations can effectively influence the environmental behaviors of CECs, and the effect is specific to molecular structures. Changes in bio-accumulation and toxicity due to transformations should be further evaluated under environmentally relevant conditions. The greatest challenge to understanding the environmental risks of CECs is the sheer number of CECs and their metabolites. In the absence of experimental data, predictive tools such as QSAR models and computational chemistry should be used to predict the possibility for the occurrence of transformations as well as the changes in physicochemical properties accompanying these transformations. Likewise, modeling may be also used to estimate changes in environmental behaviors and risks for CECs that are susceptible to transformations. It must be noted that only methylation and demethylation were considered in this research.