Toxic Effects And Molecular Mechanism Of Different Methyl-Substituted Ionic Liquids On Scenedesmus Obliquus

Ionic liquids?ILs?have a widespread use in numerous chemical reactions and industrial processes because of their unique properties.However,on account of the readily solubility and hard biodegradability,ILs can be leached into the water environment by breaking through the traditional sewage treatment unit,thereby accumulating and forming persistent organic pollutants?POPs?in the environment.The effects of three imidazolium-based ILs with different numbers of methyl substituents:1-decyl-limidazolium chloride([C₁₀IM]Cl),1-decyl-3-methylimidazolium chloride([C₁₀MIM]Cl),and 1-decyl-2,3-dimethylimidazolium chloride([C₁₀DMIM]Cl)on growth inhibition,cell structure,photosynthetic and respiratory rates,oxidative stress and proteomics of Scenedesmus obliquus were investigated.The growth inhibition rate was positively correlated with the concentrations of ILs under the same exposure time.At low ILs concentrations,the growth inhibition rate decreased with the augment of exposure time,indicating that algae had an ability of recovery.Conversely,ILs at high concentrations can promote the inhibition rate with the augment of exposure time,demonstrating that ILs has caused irreversible damage to S.obliquus.Comparing the IC₅₀values of three ILs,which first decreased and then rose with the prolongation of exposure time,implied that algae were most sensitive after 48 h exposure.The IC₅₀ value of[C₁₀IM]Cl was greater than that of[C₁₀MIM]Cl,and the IC₅₀ value of[C₁₀DMIMI]Cl was between two of them,which indicated that the addition of methyl substituents increased ILs toxicity,and one methyl substituent ILs have higher toxicity than two methyl substituents ILs.The toxicity of three ILs were ranked as following:[C₁₀MIM]Cl?[C₁₀DMIM]Cl>[C₁₀IM]Cl.In addition,the structure of S.obliquus were destroied and the damage degree increased with the increasing of ILs toxicity.The cells were deformed,the cell membrane was damaged and plasmolysis occured.The chloroplast expanded and thylakoids were loose,and the obvious increasing numbers of starch granules and vacuoles in the cells may related to the detoxification mechanism of algae.After 48 and 96 h treatment,chlorophyll a,chlorophyll b and total chlorophyll contents had negative relationship with concentrations,of which[C₁₀MIM]Cl had the greatest inhibition,followed by[C₁₀DMIM]Cl and[C₁₀IM]Cl.Besides,chlorophyll fluorescence parameters?F₀,F_m,F_v/F_m and F_v/F₀?and fast photoresponse curve?Y?II?,NPQ and Y?NO??were all changed significantly,and had concentration-effect relationship with ILs,the decrease in one pathway yield could lead to an increase of other pathways yield,the situation was alleviated with the augment of exposure time.The photosynthetic and respiration rate were decreased with the increasing of ILs concentration,and the inhibition to photosynthetic rate caused by ILs was greater than that of the respiration rate.Besides,the expression levels of photosynthetic electron transport chain genes:psaA,psa B,psbB,psbD and rbc L were down-regulated.After 48 h of ILs treatment,the inhibition of psaB and psb B were the most significant.The reactive oxide species?ROS?content in S.obliquus was positively correlated with the ILs concentration,and the green fluorescence was almost distributed in the whole algae cells under high ILs concentration.In addition,ILs inhibited the synthesis of soluble protein in S.obliquus.The endogenous ROS balance may be breaked,and the changing of ROS-scavening enzymes as the consequence of oxidative damage in S.obliquus.The key antioxidant enzymes:superoxide dismutase?SOD?,peroxidase?POD?and catalase?CAT?activities first increased and then decreased.SOD decomposes superoxide anion radical(O₂^·-)into hydrogen peroxide?H₂O₂?,excessive H₂O₂ then triggered an increase of antioxidant en-zyme activity and antioxidant content.Some of which collided with CAT molecules and oth-ers combined with glutathione?GSH?and ascorbic acid?AsA?molecules,producted H₂O under the catalysis of glutathione peroxidase?GPX?and ascorbate peroxidase?APX?.When the concentration exceeded the self-regulation ability of antioxidant system,excessive ROS can't be eliminated in time and eventually causes apoptosis.Furthermore,as a biomarker of plant stress,the content of proline?PRO?may be initiated in response to ILs exposure,which in turn destroyed the osmotic balance of the cells and the function of the mitochondria.The results of integrated biomarker response?IBR?index analysis showed that[C₁₀MIM]Cl had the highest IBR and[C₁₀IM]Cl had the lowest IBR,it is consistent with the conclusion of acute toxicity and photosynthesis.The identified proteins number of S.obliquus in[C₁₀IM]Cl treatment group was analogous to control group,and mutual proteins in[C₁₀MIM]Cl treatment group was significantly lower than control.Protein localization analysis revealed that 25.00%of proteins in control group located in the nucleus,and 17.39%was located in the chloroplast thylakoid membrane in[C₁₀MIM]Cl treatment group,revealed that nucleus and chloroplasts were the target organelles for ILs attack,reduced photosynthetic efficiency in cells.Cluster analysis revealed that[C₁₀IM]Cl treatment group had a slight down-regulation proteins to control group,the down-regulation degree intensified in[C₁₀MIM]Cl and[C₁₀DMIM]Cl treatment groups.Fur-hermore,GO analysis of differentlly expressed proteins in[C₁₀MIM]Cl groups indicated that the main changes were ATP binding in molecular function and metabolic processes in biological processes.The results of KEGG pathway enrichment analysis shown that ILs mainly lead to the down-regulation of carbon fixation pathway of algae,causing the up-regulation of biosynthesis of secondary metabolites,antibiotics and amino acids.The research demonstrated that ILs induced deficiency of normal physiological functions of plant,and the up-regulation of metabolites pathway may be a self-protection mechanism of algae in response to ILs expoaure to protect themselves from damage.