Ecotoxicological Mechanism Of Sulfamethoxazole On Chinese Cabbage And Rice Seedlings

Sulfamethoxazole（SMZ）is a sulfonamide antibiotic widely used in agricultural production due to its low cost and broad-spectrum activity.However,the ecological and toxicological effects of its misuse and inadequate disposal have raised concerns.The accumulation of sulfamethoxazole in soil and water bodies can negatively affect plant growth and development and even disrupt the balance of the ecosystem.Therefore,investigating the toxic effects and mechanisms of sulfamethoxazole on plants is of great significance for assessing its potential risks to the environment and formulating scientifically reasonable management measures.This study screened sensitive varieties of two plants,Chinese cabbage（Brassica rapa var.chinensis）and rice（Oryza sativa L.）,by comparing the inhibitory effects of sulfamethoxazole on their biomass.The toxic effects of sulfamethoxazole on plants were explored from two aspects:oxidative stress and the photosynthetic system.Furthermore,proteomics and metabolomics were used to investigate the mechanism of action of sulfamethoxazole on plants.To gain a more comprehensive understanding of the potential risks posed by this antibiotic to the ecosystem,the combined effects of sulfamethoxazole with sulfonamide synergist trimethoprim（TMP）on plants were also studied.The main contents include:（1）Sulfamethoxazole was applied to Chinese cabbage and rice seedlings respectively according to the different concentrations,from which sensitive species were screened.The growth inhibition of sulfamethoxazole on Chinese cabbage and rice seedlings was positively correlated with the concentration,and low concentration of sulfamethoxazole had a certain promotion effect on plant growth,while high concentration of sulfamethoxazole had a growth inhibition on seedlings,which showed a decrease in biomass.The EC₅₀ value was used as the basis for judging the sensitive species.The relative degree of sensitivity to sulfamethoxazole in Chinese cabbage and rice was:Ruiguan Qingbang>Suzhou Qing>Changgeng Chickpea;Wu Yunjing 21>Yong You 9>Lian Jing 7.（2）The antioxidant system and photosynthesis-related indexes were measured for the sensitive species Ruiguan Qingbang and Wu Yunjing 21.The results showed that the contents of oxidative stress biomarkers,including cell membrane permeability,malondialdehyde（MDA）,reactive oxygen species（H₂O₂）,and soluble protein,increased with sulfamethoxazole concentration.The activities of superoxide dismutase（SOD）,catalase（CAT）and peroxidase（POD）showed a trend of first decreasing and then increasing.Integrated biomarker index assessment（IBR）indicated that higher sulfamethoxazole concentrations resulted in greater toxicity to plant seedlings.Chlorophyll a,chlorophyll b and carotenoids showed different trends in Chinese cabbage（cotyledons,true leaves）and rice（old and new leaves）.Chlorophyll and its synthetic substances in Chinese cabbage showed a positive correlation with sulfamethoxazole concentration in general;chlorophyll content decreased and then increased in old leaves of rice,while no significant difference was found in new leaves.The contents of the other four chlorophyll synthesis precursors increased and then decreased in Chinese cabbage,and were no less than the control（greater than the control or no significant difference）.In rice,the contents of chlorophyll synthesis precursorsδ-aminolevulinic acid（ALA）and porphobilinogen（PBG）increased and then decreased,and the contents of protoporphyrinogen IX（Proto IX）,Mg-protoporphyrin IX（Mg-Proto IX）,and protochlorophyllide（Pchlide）decreased and then increased.Photosynthetic rate increased and then decreased,and respiration rate decreased with increasing sulfamethoxazole concentration.Actual quantum yield Y（Ⅱ）,maximum photosynthetic efficiency Fv/Fm,and photochemical burst coefficient q P decreased and then increased with sulfamethoxazole concentration and the values of Y（NPQ）and Y（NO）were negatively correlated.There was no significant difference in the rapid light curves among the treatment groups.The phenomenon of photosynthesis-related indicators in Chinese cabbage leaves at high concentrations was different from the response of most plants under sulfonamide antibiotic stress,and there may be a special response mechanism.Transmission electron microscopy（TEM）showed a reduction in the volume of cells under high concentrations of sulfamethoxazole stress,with a greater number of chloroplasts per unit area.Sulfamethoxazole leads to cell plasmolysis.The chloroplasts were more closely arranged and caused more severe damage to the cell membrane.Moreover,on average,more chloroplasts were present in each cell,with more starch granule lumen formation.The chloroplasts were swollen,the number of grana thylakoids were significantly reduced,and the stroma thylakoids structure were no longer ordered but swollen.（3）The proteomic differential proteins were analyzed through GO and KEGG pathways to investigate the impact of high concentrations of sulfamethoxazole stress on Chinese cabbage.The results showed an upregulation in the abundance of proteins related to plastid,thylakoids,and photosystem,leading to an increased antennae proteins cross-sectional area of photosystem II（PSⅡ）and ultimately enhancing light absorption and photosynthesis.The abundance of proteins related to folate and ribosome enrichment pathways were down-regulated,resulting in decreased plant nutrient synthesis capacity,thus hindering plant growth.Further analysis through protein-protein interaction（PPI）network revealed that sulfamethoxazole inhibited folate synthesis,with the bifunctional dihydrofolate reductase-thymidine synthase protein being the most associated protein with other proteins.Sulfamethoxazole significantly altered the type and content of metabolites with a general promotion of sugar and amino acid metabolites.Sulfamethoxazole also affected sugar and amino acid synthesis,glycolysis,and citric acid cycle（TCA cycle）pathways,indicating that energy metabolic processes were impacted.Specifically,under sulfamethoxazole exposure,perturbations were observed in sugar metabolism such as sucrose and glucose,glyoxylate and dicarboxylic acid metabolism,TCA cycle processes,and amino acid anabolic processes such as serine and proline.Overall,these findings suggest that sulfamethoxazole has a significant impact on plant growth and metabolism.（4）The impact of sulfamethoxazole combined with the sulfonamide synergist trimethoprim on sensitive species of Chinese cabbage and rice was investigated.It was observed that methoprene potentiated the toxicity of sulfamethoxazole.Rice was more sensitive to the compound antibiotic treatment than Chinese cabbage.The antioxidant enzymes of Chinese cabbage responded more significantly and showed higher activity.Seedlings showed lower chlorophyll content under high concentrations of complex antibiotic treatment than the group treated with the same concentration of sulfamethoxazole.The complex antibiotic treatment increased respiration rate and decreased photosynthetic rate.The PSⅡchlorophyll fluorescence parameters were generally simillar to those of the sulfamethoxazole-treated group,with an increase in the opening of the PSⅡreaction center at higher concentrations.The rapid light curves suggested that the complex treatment increased the electron transfer efficiency of seedling leaves at high antibiotic levels.