Study Of CNTs,PCP-Na And Their Complex Toxic Effect Based On Earthworm Coelomocytes

Carbon nanotubes(CNTs)was widely studied and utilized since it was discovered in 1991 because of its unique physical and chemical properties and good biocompatibility.At the same time,CNTs was also discharged into the environment a lot,its toxicological effects on ecosystem is increasing,its toxicity study is also being valued gradually.Pentachlorophenol(PCP)and Sodium Pentachlorophenol(PCP-Na)has been widely used before the 1990s as herbicides and pesticides,although it was discontinued and prohibited,but due to its stability in environment,there are still lots of PCP residued in the environment.CNTs combine with organic pollution PCP may produce new toxicological effects.As standard testing organism in soil ecosystem,there has been CNTs toxicity study on Eisenia foetida Andrei.But the test at the individual level is time and animal consuming,and the sensitivity is limited.In contrast,cell test is fast,consumed small and have high sensitivity.Further more,the positive correlation exists between cytotoxical indexs and toxical indexs in individual level,the cytotoxicity can reflect the toxicity in individual.This study,we explored the optimal condition to culture coelomocytes of Eisenia foetida andrei in vitro.And the coelomocytes was exposed to CNTs and PCP-Na to investigat the cytotoxicity via testing the lethal toxicity,oxiditve stress,membrane damage and DNA damage.The reproduction toxicity test was also made in individual level to help us explain the mechanism of toxicity of CNTs and PCP-Na.The main conlusion were as follows:(1)Reproduction test showed that CNTs may promoting the growing of earthworms.High concentrations of PCP-Na has a strong reproductive toxicity,it will be significantly reduced earthworm cocoon production.The low concentrations of PCP-Na significantly affect the growing of earthworms,but as compensation,the average cocoon production was improved.Reproductive toxicity of CNTs is low,and the reproductive toxicity of PCP-Na was also reduced when CNTs existing,indicating CNTs can absorbed the PCP-Na,and the concentration of free PCP-Na is lower,thereby reproductive toxicity is reducing.(2)The coelomocytes can be suspension cultured in vitro in primary.In 96-well plate culture,the optimal culture conditions are 25?,RPMI-1640 medium,no CO2,at a concentration of 2-4×104 cells/well(1-2×105 cells/mL),culture time was 24h.Cell survival rate almost 100%.(3)In acute toxicity test of coelomocytes.The LC50 of PCP-Na is 104.46mg/L?between the LC50 in artificial soil acute toxicity test at 7and14 days.The toxicity test of PCP-Na combined with 50mg/L CNTs was made,when the compounds added directly,LC50 of PCP-Na is 109.39mg/L,and at the condition that PCP-Na was absorbed by CNTs before added,the LC50 of PCP-Na was 112.92mg/L.The acute toxicity of PCP-Na slightly is reduced after combined with CNTs.Acute toxicity of CNTs is very low,we can not get the LC50 value,which is consistent with the acute toxicity test results on individual level.(4)Both CNTs and PCP-Na can cause oxidative damage on coelomocytes,and produce ROS,ROS can cause lipid peroxidation and generate MDA.Under low stress,cells can make stress response,SOD and CAT activity increased slightly,but at high stress,SOD and CAT activity were decreased significantly.Compared with SOD,CAT is less sensitive.This conclusion is similar to the conlusion in individual level.(5)High concentrations PCP-Na,50mg/L CNTs and composite groups could damage the membrane structure of the cell,increasing the permeability of membranes,and reduce the Mitochondrial Membrane Potential(MMP).PCP-Na can cause greater membrane damage,at the composite exposure groups,cell membrane is reduced,that indicate the two pollutants may be present as antagonism.The membrane structure damage isrelevant with MDA,indicating membrane damage is mainly caused by oxidative damage.(6)The DNA damage in each treated groups is significantly different compared with the control in the test,that indicates the comet assay is sensitive to evaluate the earthworm DNA damage caused by PCP-Na and CNTs.When exposed under CNTs,there is a significant dose-response relationship between CNTs and DNA damage.But at PCP-Na alone groups,the higher concentration group have lower DNA damage.In CNTs and PCP-Na composite exposure groups,DNA damage is greater than the groups exposed respectiveily,showed CNTs and PCP-Na have some synergy in genotoxicity.Test results with similar to the individual level comet assay.In summary,the cytotoxicity indexes are more sensitive than the individual level,and the cytotoxicity indexes have positive correlation with the indexes in individual level.Earthworm cytotoxicity test is expected to replace the individual level toxicity tests,and it can become a new fast,efficient and sensitive way to study the pollutants'toxicity on soil organisms.