Enantioselective Toxicity And Physiological And Biochemical Mechanism Of Imazamox To Duckweed(Lemna Minor)

Imazamox is a typical chiral imidazolinone herbicide,which is mainly used to control annual weeds in soybean and peanut field.Due to its long residual period of time and strong mobility in the soil,it is easy for the herbicide to enter the water systems via run off.Thus it would pose a threat to plants and animals in freshwater ecosystem.Duckweed（Lemna minor）is an important aquatic higher plant in aquatic ecosystem.Due to its small size,fast growth,easy reproduction,simple laboratory operation and low cost,duckweed is widely used in environmental toxicology researches.In the traditional safety evaluation research,only the effect of racemic imazamox on the non-target organism is usually considered,but the data obtained are often unreliable.There are few studies on the toxicity of imazamox to aquatic non-target organisms from the chiral level.In this study,the enantiomers of imazamox were separated and prepared.The effects of imazamox enantiomers and O-demethylated imazamox on the growth,target enzyme and antioxidant system of duckweed were studied.The physiological and biochemical mechanisms of enantioselective toxicity of imazamox to duckweed were investigated.And the effects of imazamox on major metabolic pathways in duckweed were further investigated.The results were shown as follows:（1）Two enantiomers of imazamox were separated and obtained by chiral OJ-H（0.46 cm×25 cm）,mobile phase of hexane:ethanol:trifluoroacetic acid=70:30:0.1,flow rate of 1.0 m L/min,ultraviolet detection wavelength of 214 nm,and temperature of 35℃.（2）In this study,the acute toxicity of imazamox and its metabolites（O-demethylated imazamox）to duckweed was studied.According to the test method in the duckweed growth inhibition test criteria of the agricultural industry standard of the People’s Republic of China（NY/T 3090-2017）,the number and fresh weight of duckweed were measured in 7 days.It was found that the toxicity of imazamox enantiomers and metabolites to duckweed was different.The EC₅₀（7d）of Rac-imazamox,R-imazamox,S-imazamox and O-demethylated imazamox on duckweed fresh weight was EC₅₀（7d）=0.036mg/L,EC₅₀（7d）=0.035 mg/L,EC₅₀（7d）=0.203 mg/L,EC₅₀（7d）=0.888 mg/L,respectively.The toxicity was R-imazamox>Rac-imazamox>S-imazamox>O-demethylated imazamox.（3）Duckweed exposure to Rac-imazamox,R-imazamox,S-imazamox and O-demethylated imazamox after 7d,the soluble protein content,malondialdehyde（MDA）and the activity of three antioxidant enzymes,including superoxide dismutase（SOD）,catalase（CAT）and peroxidase（POD）,all increased compared with the control group.With the increase of treatment concentration,soluble protein and MDA content increased and then decreased.The activity of CAT and SOD in duckweed increased and then decreased with the concentration of Rac-and R-imazamox increased.The activity of CAT and SOD in duckweed increased with the concentration of S-and O-demethylated imazamox increased.With the increase of Rac-,R-and O-demethylated imazamox concentration,POD content in duckweed showed a trend of increasing first and then decreasing.The activity of POD in duckweed increased with the concentration of S-imazamox increased.In this study,the imazamox may cause oxidative damage and membrane lipid peroxidation of duckweed.Duckweed may reduce the oxidative damage caused by the imazamox by synthesizing functional proteins such as SOD,CAT and POD.Based on the increase of MDA content,it was speculated that the imazamox might make the membrane damage more serious.（4）Duckweed exposure to Rac-imazamox,R-imazamox,S-imazamox and O-demethylated imazamox after 7d.Compared with the control group of duckweed,the experimental group has a significant decrease in acetolactic synthase（ALS）.And the content of ALS decreased with the increase of the treatment group concentration.The imazamox may damage duckweed by inhibiting the synthesis of its target enzyme ALS.（5）The metabolomics study on the toxicity of chiral imazamox to duckweed showed that compared with the control group,68 different metabolites were obtained in the duckweed treated with Rac-imazamox,which mainly affected the biosynthesis of secondary metabolites,biosynthesis of amino acids and biosynthesis of antibiotics.Compared with the control group,43 different metabolites were obtained from the duckweed treated with S-imazamox,which mainly affected the pathways including phenylalanine metabolism,ubiquinone and other terpenoid-quinone biosynthesis,and anthocyanin biosynthesis.Compared with the control group,71 different metabolites were obtained from the duckweed treated with R-imazamox,which mainly affected the pathways including aminoacyl-t RNA biosynthesis,metabolic pathways,and nicotinate and nicotinamide metabolism.（6）The transcriptome of the toxicity of the enantiomer of imazamox to duckweed showed that,compared with the control group,there were 2,050up-regulated genes and 1,057 down-regulated genes in R-imazamox.Compared with the control group,1475 up-regulated genes and 1105 down-regulated genes were found in Rac-imazamox.There were 70 up-regulated genes and 64down-regulated genes in S-imazamox（7）Metabonomics and transcribed correlation analysis of the toxicity of the enantiomers of imazamox on duckweed showed that R-imazamox and Rac-imazamox might affect the duckweed through Carbon fixation in photosynthetic organisms,Pentose phosphate pathway,Glutathione metabolism,Biosynthesis of secondary metabolites,Valine,leucine and isoleucine degradation.S-imazamox might affect the duckweed through Glutathione metabolism,Biosynthesis of secondary metabolites,Metabolic pathways.In this study,the toxicity of imazamox enantiomers and major metabolites to duckweed were investigated.The experimental results are helpful to reveal the potential toxicity mechanism to duckweed,and provide a basis of the scientific a use and safety evaluation of the chiral herbicide imazamox.