A Study On The Toxic Effect Of Cu²⁺ On Periereis Nuntia Savigy

Tide flat is the transitional zone between marine and land.In recent years,coast waters were polluted by a large number of industrial and agricultural pollutants and sewage.Ecological vulnerability has become increasingly obvious in tide flats.Sustainable utilization of tide flats' resources was directly affected in severe polluted tide flats.Copper is one of the heavy metals whose contents are seriously above national standard for the marine environment.Therefore,it is very important to carry out the researches about the toxic effects of copper on the organisms at the tide flats so as to identify and evaluate potentially useful biomarkers for oxidative stress and DNA damage as early warning indices in the polychaete and explore the possible ways for bioremediation on heavy metal pollution in tide flats.Polychaetes live in the tide flat with wide distribution,large quantity and unique way of life,and play important roles in marine ecosystems.Nereis,as a family of ploychaete,has not typically representative organisms of the marine benthic polychaetes,but also the most dominant and abundant diversity.It plays an important role in the material recycle and energy flow of food webs in coastal waters,and has also high economic value.In this paper,the sandworm Periereis nuntia Savigy were used as the test organism with the typically representative of the heavy metal pollution copper as contaminants.According to the results of the LC50?96 h?of Cu2 +to the sandworm,4 different experimental groups?136?g/L?182?g/L?273?g/L?545?g/L?were designed with one control group to explore the toxic effect of Cu2 + on the sandworm by enzyme activity,real-time fluorescence quantitative technique,single cell gel electrophoresis and copper content analysis on the physiological and molecular level after 4 d and 14 d exposure,Following are the research results:1.The activity of superoxide dismutase?SOD?,catalase?CAT?,acetylcholine esterase?ACh E?and the content of glutathione?GSH?were measured.After 4 d exposure,Cu2 + at the concentration of 136?g/L group began to induce the increase of the activities of SOD,CAT and content of GSH.But the induced effect was different in biomarkers,for example,the activity of SOD and CAT was the largest at the Cu2 + concentration of 273?g/L,however,the content of GSH was the highest when exposed in 182?g/L group.Then the activity or content of each index decline,the activity of CAT was inhibited in 545?g/L group.During all the group,the activity of ACh E was inhibited,in 136?g/L group,the inhibition did not significant increase but there was significant increase in 545?g/L group.Compared with the results of 4d exposure,the enzyme activity and GSH content decreased after 14 d exposure.The inhibition effect of CAT and ACh E increased and GSH synthesis were inhibited.2.After 4d exposure,Cu2 + could induce the increase of metallothionein?MT?synthesis,and the inducing effect is the strongest in 273?g/L group with the highest MT content.The MT content declined to the level of the control group in the highest concentration group of 545?g/L.After 14 d exposure,MT content was increased in 136 and 182?g/L groups,but there were not significant differences between the two groups and the control group?P > 0.05?.The synthesis of MT were inhibited in the two group of 273 and545 ?g/L.Compared with the results of 4d exposure,the MT content of each group decreased with some different degrees.The MT content in the Nereis body decreased significantly in 273?g/L group after 14 d exposure?P <0.01?.The results suggested that the MT content in the Nereis body is sensitive to heavy metal copper in a certain scale and time,so it can be used as a biomarker for monitoring the pollution of Cu2 +.3.By selection and cloning,we obtained a 205 bp gene fragment.After blast analysis,it was presumed to belong to earthworms muscle hemoglobin family and confirmed to be MPII gene of the sandworm.Real-time PCR results showed that after 4d exposure,the level of MPII gene expression was significantly increased?P < 0.05?from the groups of 0,136,182 and273?g/L.The MPII gene relative expression level was the highest in the group of 273?g/L,which was 3.87 times of the control group.The expression level of MPII decreased in the545?g/L group.After 14 d exposure,in the 3 groups of 0,136 and 182?g/L,the expression level of MPII gene increased significantly?P < 0.05?.The expression level of MPII gene increased in the group of 273?g/L,but the increase is not obvious?P > 0.05?.The expression level of the highest concentration group significantly decreased?P < 0.05?.Compared with the 4d exposure,the expression levels of each experimental group decreased after 14 d exposure,but the change was not significant?P> 0.05?,except for the 273?g/L group which decreased significantly?P<0.05?.The result shows that the low concentration of Cu2 +was capable of inducing the expression increase of the MPII gene,but high concentrations could inhibited the expression of the gene.4.The DNA damage of the sandworm under the stress of different concentrations of Cu²⁺were detected by SGGE,the result showed that both the sandworm cell DNA comet tail length?TL?and Olive tail Moment?TM?of the control group and the low concentration group of136?g/L were small,tail DNA content?TD?were very low after 4d exposure,which could be almost neglected;but the cells of sandworm of the high concentration groups of 273,545?g/L had longer comet tail,Olive tail moment and more content of tail DNA.After 14 d exposure,in addition to,those three indexes of each treatment group were increased significantly?P<0.05?while those indexes of the control group and 136?g/L treatment group did not change significantly.The results show that as the increase of the time and concentrations of Cu2 +exposure,Cu2 + could cause the oxidative damage of the sandworm,while tail length,Olive tail moment and tail DNA content were related to the Cu2 + exposure time and concentration.5.The Cu²⁺bio accumulative content in the sandworm body of each experimental group were detected by electronic coupling method,the result were as follows : after 4d exposure,Cu²⁺bio accumulative content in the sandworm body were increased with the concentration of Cu²⁺increase and there were significant differences among the groups?P<0.01?.After 14 days,Cu²⁺bio accumulative concentration in the sandworm were increased significantly?P<0.05?in the136,182 and 273?g/L experimental groups compared to the control group,but there was no significant difference between these three groups;Cu²⁺bio accumulative content in the sandworm of the 545?g/L group rapidly increased to 221.471?g/g.dw,with very significant difference?P < 0.01?to other experimental groups;compared to the results of 4d exposure,Cu²⁺bioaccumulation in the sandworm after 14 d exposure were increased significantly,the groups difference was significantly?P < 0.05?.It suggested the sandworm has the strong tolerant and accumulative ability to copper.