Toxicological And Biochemical Responses Of The Earthworm Eisenia Fetida Exposed To Phenolic Acids From Eucalyptus Grandis Litter

Eucalyptus has been introduced into many developing countries because of its rapid growth,short of rotation period,high economic efficiency and strong adaptability.But a large area of Eucalyptus also caused ecological problems such as low biodiversity,soil fertility decline and allelopathy,and the allelopathy of Eucalyptus is controversial.Eucalyptus can produce volatile and water-soluble allelochemicals,and the allelopathy of water-soluble allelochemicals is stronger.The research of water-soluble phenolic allelopathy of Eucalyptus paid more attention to the organ or soil water extract to the effect of plant physiology,but the lack of research on the effect of phenolic allelochemicals to soil organisms.Therefore,we selected a chronosequence of Eucalyptus grandis plantations（2,4,6,8,10 years old）as the research object;take the derelict land soil near the research site and the soil from various aged Eucalyptus grandis,and added the litter extracts of various aged E.grandis into these soils.The growth indicators（survival rate,growth index）,protein content,activity of antioxidant enzymes（MDA,SOD,GST）and enzyme activity related to the nervous system（AChE）of Eisenia fetida were evaluated according to the identifition of phenolic acid substances in litter.According to UPLC-MS,we found nine kinds of phenolic acids substances in soil and litter.And then through the preparatory experiment to choose two kind of phenolic acids substances（salicylic acid,6-methylsalicylate acid）and research the effect to growth,protein content,antioxidant enzyme（SOD,GST）and the nervous system enzyme（AChE）activity of E.fetida at the exposure time of 2,7 and 14 d and the influence of the microstructure in the gut at the exposure time of 2 d.This research will reveal the phenolic acids substances mechanism of Eucalyptus plantations to soil organisms,and served as the theoretical basis for scientific management and sustainable development.The results showed that:（1）The survival rate and weight of earthworm did not change significantly when the litter aqueous extracts were added into the abandoned soils at the exposure time of 7,14,21and 28 d.The protein content of the earthworm was promoted under the treatments of water extracts of litters with 2 or 8 plantation ages at the exposure time of 28 d.The MDA content of earthworm increased significantly when treated with 8 and 10 year-old litter extracts.Over all,the GST activities were inhibited and the effects were strengthened with the increasing concentrations,which also increased significantly when treated with 4 and 6 year-old litter aqueous extracts.The AChE activity decreased under the treatment of 4 year-old litter extracts.In addition,the results revealed that E.fetida exhibited significant avoidance behavior under the treatment of all the forest ages of the highest concentration of litter aqueous extracts and6-8 years of higher concentrations,and E.fetida exhibited significant attract behavior under the treatment of 2 years of the lowest concentration.（2）When the litter extracts of various aged E.grandis were added into the corresponding aged soils,it had no significant effects on survival rate of earthworm,the weight of earthworm decreased significantly under the treatments of higher dose litter extracts of elder tree at the exposure time of 7,14,21 and 28 d.The treatments had no effects on the protein of earthworm at the exposure time of 28 d;the SOD activity decreased significantly treated with8 aged litter extracts except for the C3 dose;GST activity decreased significantly with the increasing plantation age.The activity of AChE were promoted under the treatment of younger aged litters but were inhibited under the treatment of elder aged litter.（3）The treatments of soils from various aged E.grandis on E.fetida showed that the survival rate of earthworm did not change significantly,the weight decreased significantly under the treatment of 2 or 4 year-old,8 or 10 year old litter extracts at the exposure time of7,14,21 and 28 d.The protein content of the earthworm had no significant changes at the exposure time of 28 d;MDA content increased significantly under the treatment of10-year-old litter extracts;SOD were lower with the litter extracts of 4 or 10 year old.GST had no significant changes.AChE did not change significantly in the litter extracts of 2-8year-old trees but increased significantly in the litter extracts of 10 year-old trees.In addition,E.fetida exhibited significant avoidance behavior under the treatment of the 8-10 years of soils.（4）Seven phenolic acid metabolites were identified from litter and soil of Eucalyptus grandis using UPLC-MS,the content of phenolic acid metabolites in litter were significantly higher than in soil,and the content of metabolites were higher after the rotation period.Through preliminary screening experiment found that 6-methyl salicylicacid and salicylic acid had significantly toxicology effect on E.fetida,and there were obvious change related to the contents of 6-methylsalicylicacid and salicylic acid with the growth of E.grandis plantations.（5）Under the treatment of 6-methylsalicylic acid and salicylic acid after 7 d,with the increasing of concentration and exposure time,the survival rate of E.fetida decreased significantly.In the present study,the 7-and 14-day LC₅₀ values for salicylic acid were highest as 94.622 and 67.192 mg kg^-11 soil.The LC₅₀ value of 6-methylsalicylate at exposure time of 7 d was 405.912 mg kg^-1.The two kinds of phenolic acids have significant inhibitory effects on the weight of E.fetida,and the inhibitory effects gradually increased with the increasing dose and exposure time.（6）Under higher concentrations treatment after 2 d and the low concentration of6-methylsalicylic acid after 7 d treatments,the contents of protein were promoted,while others and all concentrations of salicylic acid treatments were inhibited.The SOD activities were promoted,but were inhibited on treatment of salicylic acid at 14 d.The AChE activities were stimulated for all doses treatment after 7 d and 14 d and higher concentrations treatment after 2 d of 6-methylsalicylate,while others and all concentrations of salicylic acid treatments were inhibited.The GST activities were promoted under low concentrations treatment of salicylic acid after 2 d and 6-methylsalicylate in all concentrations after treatment while others were inhibited.MDA contents were stimulated for high concentrations treatment after 7 d and all concentrations treatment of salicylic acid after 14 d,but were inhibited on the treatment of high concentration of 6-methylsalicylate acid.Among the two kind of phenolic acids,salicylic acid exerted strong effects on enzymatic activities.（7）Skin and intestinal epithelium tissue were damaged under two phenolic acids substances over the 2-d exposure periods.As concentrations increased,the earthworms showed progressively severe signs and symptoms of toxicity such as coiling,curling,mortality,body mass changes,and the damage to skin and intestinal tissue.In addition,intestinal epithelium tissue of E.fetida was damaged significantly under the treatment of low concentration of phenolic acids because the low sensitivity od earthworms and feeding directly,but skin of E.fetida was damaged significantly under the treatment of high concentration of phenolic acids because the earthworms refused food.In summary,the litter aqueous extracts of E.granidis exert a significant toxicological impact on E.fetida among which 6-methylsalicylicacid and salicylic acid have the stronger effects.The observations and related findings will be helpful to achieve a better understanding of the mechanism of allelopathy of water-soluble phenolic acid metabolites from E.grandis on soil fauna.