| Ionic liquids (ILs) are a novel class of green organic solvents which remain in the fluid state at room temperature, composed by large organic cations and small organic or inorganic anions. The extremely low vapour pressure is the most predominant features of ILs, and it makes them an alternative to replace traditional organic solvents as a "green solvents". However, the chemical and thermodynamic stability of ILs makes them difficult to degrade in environment, so the environmental behavior and ecological toxicity of ILs has received great attention. In this paper, five imidazolium chloride ILs with varying chain lengths are adopted for studying the acute toxicity of ILs to scenedesmus obliquus. Then, three of the above five ILs are selected for studying the ecotoxicological effects of ILs on scenedesmus obliquus. The main research includes four parts as follows: (1) The acute toxicity of [C6mim]Cl,[C8mim]Cl,[C10mim]Cl,[C12mim]Cl and [C16mim]Cl to scenedesmus obliquus was evaluated by the standard method of acute toxicity for chemicals. In the same exposure time, a positive correlation between the acute toxicity and concentration of ILs was observed. The growth inhibition decreased with increasing exposure time in treatments of low concentrations, increased first and then decreased in treatments of medium concentrations, while increased in treatments of high concentrations. The EC50value of the five imidazolium chloride ILs decreased and then increased with increasing exposure time, reached the minimum after exposure time of48h, suggesting that the alga was most sensitive after exposure time of48h. At a fixed exposure time, the EC50value decreased with the increasing chain lengths of ILs, it can be clearly seen that there’s a relationship of positive correlation between the acute toxicity and the chain lengths of ILs, that’s to say, the toxic order was as [C16mim]C1>[C12mim]Cl>[C10mim]Cl>[C8mim]Cl>[C6mim]C1.(2) The cellular membrane permeability of scenedesmus obliquus exposed to [C8mim]Cl,[C12mim]Cl and [C16mim]Cl for24,48,72and96h was measured by the method of FDA fluorescence staining, and the ultrastructure of alga was also observed. In a certain exposure time, a positive correlation between the cellular membrane permeability of alga and the concentration of ILs was observed, and the cellular membrane permeability of alga increased with increasing chain lengths of ILs, the toxic order with respect to cellular membrane permeability was as [C16mim]Cl>[C12mim]Cl>[C8mim]Cl. Ultrastructure studies revealed that all the adopted treatments had adverse effects on the cellular morphology and structure of alga. The cell wall of treated alga turned into wavy and separated from cell membrane, obscure and loose chloroplast grana lamellae, osmiophilic material in the chloroplast, swelled mitochondria and its cristae, black inclusions in vacuoles were observed, with respect to the treatments of high concentration, the cellular morphology changed and the overall arrangement of organelle in the cytoplasm became disordered. Compared the ultrastructure of2mg/L [C8mim]Cl,0.04mg/L [C12mim]Cl and0.02mg/L [C16mim]Cl treatments (growth inhibitions were all about70%) with that of the control group, we can see that there’s no obvious difference among the three treatments as to the cell wall, cell membrane, chloroplast, mitochondria and vacuoles. Compared the treatments of0.1mg/L [C16mim]Cl (growth inhibition was about70%) and0.02mg/L [C16mim]Cl (growth inhibition was about80%) with the control group, it can be seen that ILs of high concentration had greater influence on the cellular morphology and structure of alga.(3) The effects of [C8mim]Cl,[C12mim]Cl and [C16mim]Cl on the photosynthesis of alga at different exposure time was studied. There is a good linear correlation between the chlorophyll content and growth inhibition or cellular membrane permeability of alga, suggesting that the three ILs affected the normal photosynthesis of chloroplast by damaging the cellular membrane permeability of alga, sequentially leading to the growth inhibition of alga. With prolong of exposure time, the effects of the three ILs on chlorophyll content first increased and then decreased, reached the maximum at48h. Likewise, the effects of [C12mim]Cl and [C16mim]Cl on Fv/Fm, Y(Ⅱ) and rETR first increased and then decreased, reached the maximum at48h, while the effects of [C8mim]Cl on Fv/Fm, Y(Ⅱ) and rETR gradually increased. In addition, the effects of [C8mim]Cl,[C12mim]Cl and [C16mim]Cl on chlorophyll content, Fo, Fm, Fv/Fm, Fv/Fo, Y(Ⅱ), qP, NPQ and rETR increased with increasing concentration of ILs, presenting an obvious dose-effect relationship, and the toxic order as to the effect of the three ILs on chlorophyll content, Fo, Fm, Fv/Fm, Fv/F0, Y(Ⅱ), qP and NPQ was as [C16mim]Cl>[C12mim]Cl>[C8mim]Cl.(4) The interaction between [C8mim]Cl,[C12mim]Cl,[C16mim]Cl and ctDNA was investigated by UV-Vis absorption spectra. The results indicated that the binding mode between the three ILs and ctDNA is probably intercalative, however, the other mode of interaction hadn’t been ruled out. The interaction of the three ILs with ctDNA was further studied through melting temperature (Tm) curve, iodide quenching, the method of ethidium bromide (EB) fluorescence probe and the effects of ionic strength. The results showed that the binding mode of the three ILs with DNA is an intercalation binding, not groove binding or electrostatic interaction. Fluorescence spectra were used to study the effects of ctDNA on the fluorescence emission of the three ILs, the results indicated that the fluorescence emission of the three ILs can be quenched by ctDNA, and the Stern-Volmer plots at different temperatures proved that quenching mechanism of ILs-ctDNA was a static quenching type. The binding constant between [C8mim]Cl,[C12mim]Cl,[C16mim]Cl and ctDNA was1443,11169,67189at temperature of298K, respectively, and the number of binding sites was0.89,1.10,1.27at temperature of298K, respectively, suggesting that the intercalation binding between the three ILs and ctDNA increased with increasing chain lengths of ILs. |
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