| Ionic liquid ILs is a kind of organic salt which is completely composed of larger organic cations and smaller inorganic anions in the state of room temperature.Compared with traditional organic solvents,ionic liquids are called"green solvents"because of their low vapor pressure,stable chemical properties and strong conductivity.However,as ionic liquids are widely used in industrial production and laboratory,they will inevitably be discharged into the environment,and the refractory characteristics of ionic liquids will accumulate in the environment,resulting in environmental pollution and even harm human health.Therefore,ionic liquids as"green solvents"have been questioned,and their ecological toxicity to the environment has been widely concerned.In this study,we used Bombyx mori L as a model insect to evaluate the toxicity of ionic liquids.Bombyx mori L is an important economic insect with short growth cycle,high reproduction rate and sensitive to environmental pollutants.It can be used as a model organism to evaluate the toxicity of ionic liquids.In this study,three kinds of ionic liquids,1-octyl-3-methylimidazole chloride（[C8mim]Cl）,N-octyl-3-methylpyridinechloride（[C8mpy]Cl）,and1-octyl-3-methylimidazole tetrafluoroborate（[C8mim]BF4）were selected to carry out acute toxicity test on three strains of silkworm（Jingsong×Haoyue,P50,Yi16）.The toxic effects of ionic liquids on silkworm were studied at physiological,biochemical and genetic levels,and the effects of ionic liquids on silkworm growth and development were discussed.The main results are as follows:（1）The LC50 values of three silkworm strains exposed to ionic liquid[C8mim]Cl were compared to determine the sensitive strain.The results showed that the lethal rates of Jingsong×Haoyue,Yi16 and P50 were 95%,77.5%and 50%respectively after exposure to200μg g-1 ionic liquid for 12 h,which indicated that Jingsong×Haoyue was the most sensitive variety among the three strains and could be used to evaluate the biological effects of ionic liquid.In addition,when the silkworm larvae were exposed to 200μg g-1 ionic liquid[C8mim]Cl for 12 h,the silkworm appeared obvious poisoning symptoms,and the midgut tissues were found to be blackened,perforated and atrophied,which indicated that[C8mim]Cl had obvious toxic effect on the silkworm larvae,especially on the midgut tissues.According to these data,we found that ionic liquids have obvious toxic effects on silkworm larvae.Therefore,it is reasonable to use silkworm larvae to evaluate the toxicity of ionic liquids.（2）When comparing the toxicity of three kinds of ionic liquids with different structures to Jingsong×Haoyue silkworm strain,the results showed that the acute toxicity increased with the increase of exposure time,and the toxicity of imidazole ionic liquids was greater than that of pyridine ionic liquids,and the toxicity of ionic liquids containing BF4-was greater than that of ionic liquids containing Cl-.The relationship between the toxicity of the three ionic liquids is:[C8mim]BF4>[C8mim]Cl>[C8mpy]Cl.In addition,the activities of some antioxidant enzymes in Bombyx mori L exposed to ionic liquids increased,which can be considered as an active defense against ionic liquids exposure,and its order of action depends on the accumulation of ROS.At the same time,the content of MDA in the midgut and silk gland of Bombyx mori L increased under the exposure of ionic liquid.The exposure of ionic liquid may cause lipid peroxidation and cell damage,and eventually lead to cell apoptosis.（3）The midgut and silk gland of Bombyx mori L exposed to ionic liquid（[C8mim]Cl）for 6 h and 12 h were sequenced by transcriptome sequencing.The results showed that after6 h and 12 h exposure to ionic liquid,5118,2211 and 926,822 differentially expressed genes were identified in the midgut and silk gland tissues of the silkworm,respectively.The differentially expressed genes related to oxidative stress and apoptosis were screened out,and the results were verified by q RT-PCR,and the results were consistent with the sequencing results.In addition,GO and KEGG enrichment analysis showed that these genes were significantly enriched in the pathways related to oxidative stress and apoptosis.We speculated that oxidative stress reaction occurred in the midgut and silk gland tissues of Bombyx mori L after exposure to ionic liquid,and tissue damage was accompanied by apoptosis.（4）The toxic effects of ionic liquid on different instars of silkworm were studied,and the cocooning rate,pupation rate and dead cage rate of different instars of silkworm after exposure to ionic liquid were counted.The results showed that when the silkworm was exposed to ionic liquids for 24 h,the 2nd and 3rd instar larvae did not die,and the LC50values of 4th and 5th instar larvae were 213.15 and 91.38μg g-1 respectively,indicating that the 4th and 5th instar silkworm were more sensitive to ionic liquids than 2nd and 3rd instar larvae.In addition,with the increase of ionic liquid concentration,the cocooning rate and pupation rate of different instars of silkworm gradually decreased,and the dead cage rate gradually increased.However,cocooning did not appear after continuous exposure of silkworm larvae.（5）The midgut and silk gland tissues of Bombyx mori L exposed to ionic liquid for 12h were evaluated by histopathology.Through paraffin section and HE staining,the results showed that the morphology of midgut cells changed,the number of midgut cells decreased,the interstitial became thinner,the microvilli disappeared,the midgut nucleus pyknosis and other phenomena;the epithelial cells of silk gland tissue were sparse,the glandular membrane rupture was obvious,the glandular cavity appeared vacuoles,and the silk gland rupture and other phenomena.The results showed that ionic liquid had obvious tissue damage to the midgut and silk gland of Bombyx mori L.In conclusion,ionic liquids have obvious toxicity to silkworm,and different cation and anion will affect the toxicity of ionic liquids.At the same time,silkworm can be used as a model organism to evaluate the toxicity of ionic liquids.This study provides more favorable data for the rational assessment of the toxicity risk of ionic liquids and the discussion of the toxicity mechanism of ionic liquids to silkworm,and lays a foundation for the design and synthesis of truly non-toxic,green and degradable ionic liquids in the future. |
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