The Accumulation Of Three Organic Pollutants And The Mechanism Of Caffeine Metabolism In Hydroponic Vegetable System

Organic contaminants and their active metabolites can enter the food chain through wastewater irrigation,and ultimately have adverse effects on the ecosystem and human health.Caffeine(log K_OW=-0.07)is regarded as the most common pollutant in freshwater and urban water.Carbamazepine(log K_OW=2.45)is persistent and highly bioaccumulative in the environment.Benzene kresoxim-methyl(log K_OW=4.54)has potential endocrine disrupting properties.The three organic pollutants are closelyrelated to human health,and there are few reports on their subcellular distribution in vegetables and the mechanism of caffeine metabolism.In this paper,combining isotope tracing technology with modern instrumental analysis technology,¹⁴C-caffeine,¹⁴C-carbamazepine,and ¹⁴C-benzene kresoxim-methyl were used as tracers to explore the removal efficiency,bioconcentration,subcellar distribution and metabolic mechanisms of these contaminants in hydroponic vegetable systems.The main results of this thesis were as follows:（1）The uptake,accumulation and subcellular distribution of ¹⁴C-caffeine,¹⁴C-carbamazepine and ¹⁴C-benzene kresoxim-methyl in the open system of hydroponic vegetables were clarified.Chinese flowering cabbage and water spinach can effectively absorb these three pollutants from the nutrient solution,and the removal efficiency was caffeine（98.0-98.4%）>carbamazepine（93.0-93.2%）>benzene kresoxim-methyl（81.2-94.2%）.These three pollutants absorbed by plant roots could be futher transferred to the shoots,where they mainly concentrated in bottom leaves.At 32 d,¹⁴C-caffeine,¹⁴C-carbamazepine and their metabolites were mainly distributed in the leaves,and the ¹⁴C concentration were 29.8μmol kg^-1 and 90.3μmol kg^-1,respectively.¹⁴C-benzene kresoxim-methyl and its metabolites were mainly distributed in the roots,and the ¹⁴C concentration was 156μmol kg^-1.Correspondingly,the radioautograph of¹⁴C distribution was consistent with its detection data.Results of the subcellular distribution showed that the three pollutants could pass through the cell wall and the cell membrane,and were distributed in various cellular components.The largest reservoir of hydrophilic contaminant caffeine was the soluble fraction(51.6-69.6μmol kg^-1).The lipophilic Benzene kresoxim-methyl were mainly distributed in the plastids of the roots(154-189μmol kg^-1).The medium-polar carbamazepine had little difference in the distribution of leaf and root cells.The log RCF of these three pollutants were highly linearly related to their log K_OW,and the correlation coefficient was greater than 0.800.In addition,the lipid content in water spinach root was 2.07%,which was significantly higher than that in Chinese flowering cabbage（p<0.05）.As a result,the pollutants were more likely to remain in the roots of water spinach and were difficult to transport upwards.Under the experimental conditions,the human exposure after consuming Chinese flowering cabbage and water spinach contaminated by these three compounds was still far below the limit dose,and the health risk of human consumption was relatively small.（2）The metabolites and metabolic pathways of caffeine in the closed hydroponic cabbage system were clarified,and the metabolic mechanism was revealed.The recovery of ¹⁴C-caffeine and its metabolites in the closed system was94.3-104%.The ¹⁴C-caffeine in the solution was continuously absorbed by the plants.At the end of the culture,the ¹⁴C content in the solution droped to 2.10%of the applied amount,the ¹⁴C content in the plant reached 40.0%,and the ¹⁴CO₂ in the system was50.9%.Through LC-LSC analysis,two radioactive substances（caffeine parent and metabolite M1）were detected in water,and three radioactive substances（parent,metabolite M1 and M2）were detected in vegetable plants.The metabolites were identified by LC-QTOF-MS,and the results showed that M1 was xanthine and M2 was theobromine.M1 and M2 were demethylated products of caffeine.The mineralized ¹⁴CO₂in the process of caffeine metabolism was derived from the degradation of caffeine in water by rhizosphere and sterile culture experiments.In the rhizosphere solution of flowering Chinese cabbage and water spinach,the mineralization of caffeine was 74.8%and 77.5%,respectively,but only 16.1%in the control,indicating that rhizosphere microorganisms played a major role in the caffeine mineralization.In the sterile system,the ¹⁴C recovery rate reached 93.6%-102%,indicating that caffeine was not basically mineralized under sterile conditions,further confirming the mineralization of microorganisms.We also proposed the metabolic pathways of caffeine in the hydroponic system.Three methyl groups were removed from caffeine under the action of microorganisms to generate xanthine,which continued to mineralize into carbon dioxide;in Chinese flowering cabbage,caffeine removed one methyl group to produce theobromine,and then removed two methyl groups to produce xanthine.In addition,compared with the metabolism of the whole plant,the proportion of caffeine in the cell suspensions culture of cabbage and water spinach were 90.8-98.2%and 95.2-96.9%,respectively,indicating that caffeine was difficult to metabolize in suspension cells and suspension cells cannot be used for rapid prediction of potential caffeine metabolites in plants.（3）Through microbial diversity analysis of the nutrient solution and transcriptome analysis of Chinese flowering cabbage leaves,the degradation bacteria and genes of caffeine in the hydroponic system were inferred.By the analysis of microbial diversity,the caffeine-degrading bacteria in the rhizosphere solution may be Burkholderia-Caballeronia-Paraburkholderia,Acidisoma,Asticcacaulis,Leifsonia,Pandorace,Acidipila,Terriglobus.Burkholderia-Caballeronia-Paraburkholderia and Acidisoma existed specifically in the rhizosphere system of Chinese flowering cabbage and water spinach,and the LDA value was greater than 4.00,which may play a leading role in caffeine degradation.In fungal analysis,Candida,Exophiala,Paracremonium,and Trichoderma were possible caffeine-degrading strains.Among them,Candida and Trichoderma existed specifically,with LDA values greater than 3.00,which may play a leading role.Based on the transcriptome analysis of Chinese flowering cabbage leaves,there were 13 up-regulated genes and 1 down-regulated gene treated with 1 mg L^-1 caffeine,and there were 18 up-regulated genes and no down-regulated genes treated with 5 mg L^-1 caffeine.Ten CYP genes were up-regulated in both two treatments,namely CYP97B3,CYP71A12,CYP707A3,CYP72A13,CYP81F2,CYP707A1,CYP75B138,CYP707A1,CYP75B137,CYP71B23,which was speculated to be involved in the metabolism of caffeine in Chinese flowering cabbage.Organic pollutants have great impacts on agricultural ecosystems and human health.In this paper,the transportation,accumulation,and metabolic mechanisms were discussed in aquatic ecosystems,which had important scientific significance for ecological environmental protection and life health,and provided a methodological basis for the metabolism research of other organic pollutants in the hydroponic-plant system.