The Influence Of HHCB And Cd Co-contaminated On The Ecological Effect Of Microbes In Soil

Synthetic musk as emerging pollutant from pharmaceuticals and personal care products(PPCPs),is highly yielded and wildly used with the characteristics of persistence,high fat-solubility and environmental stability.Heavy metal Cadmium(Cd)has become the primary pollutant in soil of China.Their ecological impacts are widely concerned by scholars domestic and abroad.Since sewage irrigation and sludge utilization is an important source of synthetic musk and Cd,the coexistence of them in soil environment is common,and they are becoming the new potentially harmful combined pollutants.So it has important practical significance to explore their impacts on the ecological effect of soil microorganism.In the present study,typical synthetic musk(HHCB)and Cd were chosen as the research objects.The effect of HHCB and Cd single and combined pollution on soil microbial quantity,soil enzyme activity,microbial community function,structural and genetic diversity were systematically studied by soil microcosm culture method.The combined pollution's eco-toxicological effects on soil microbial and the mode of action were roundly revealed,and it filled a domestic and overseas gap in this field.The results provide a fundamental data for the research of risk assessment and ecology diagnosis of HHCB,and also benefit to the establishment of eco-toxicological diagnosis indices,risk assessment and remediation benchmarks for combined pollution.The concentration of HHCB was 100,500 and 1000 mg-kg-1,and Cd was 10 mg-kg-1,respectively.The number of bacteria,fungi and actinomyces in soil were continuously determined by plate count for 10 weeks,and the joint toxicity of the combined pollution was analyzed.The results showed that,the soil of single HHCB pollution and HHCB-Cd combined pollution promoted the growth of soil bacteria after inhibiting and exhibited the promotion of fungi and significant inhibition of actinomycetes.Moreover,the inhibition rate of actinomycetes increased with the rising of HHCB concentration.The single toxic effect of HHCB on soil microbes from high to low was actinomycetes,bacteria,fungi in turn.Actinomycetes were found to be sensitive to HHCB,the number of actinomycetes was closely related to the soil HHCB pollution and they presented a logarithmic dose-effect relationship.The joint toxicity effect of HHCB and Cd co-contaminated was synergistic on bacteria at first and turned into antagonistic with the time while it was antagonistic on fungi and actinomycetes.The activities of urease,acid phosphatase and invertase in soil were determined,and the joint toxicity of combined pollution was analyzed in this study.The results showed that,both of the the soil of single HHCB pollution and HHCB-Cd combined pollution inhibited the urease activity after promoting,and promoted the acid phosphatase activity after inhibiting.Moreover,it had a significant inhibition on the invertase activity,and the inhibition rate of the invertase activity increased with the rising of HHCB concentration.The joint toxicity effect of HHCB and Cd co-contaminated on urease was synergistic,while that effect on acid phosphatase and invertase was mainly antagonistic.We found that invertase was more sensitive to HHCB pollution than urease and phosphatase.Thereby,invertase could be applied as a warning indicator of HHCB pollution in soil.Biolog-Eco microplate was used for the measurement of the utilization of carbon source by soil microbes.Additionally,the microbial community diversity index and metabolic function were analyzed.According to the experimental results,in the soil of single HHCB pollution and HHCB-Cd combined pollution,the Average Well Color Development(AWCD)were higher than them in the blank control group,furthermore,the ability of utilizing carbon source by soil microbes was promoted with the adding of HHCB.At the initial stage of culture(the 2nd week),the Shannon and Simpson indices did not change significantly.At the middle stage of culture(the 4th week),the Shannon,Simpson and McIntosh indices of the group in 500 and 1000 mg·kg-1 of single HHCB pollution and HHCB-Cd combined pollution soil were significantly higher than them in the blank control group,suggesting the species richness and the dominant population of the soil microbes were significantly promoted,and the homogeneity of the population was broken.At the end stage of culture(the 10th week),there was no significant change in the three indices.Soil microbes had different utilization intensities of the six kinds of carbon sources,the utilization of esters was the highest,while the utilization of carbohydrates and carboxylic acids were lower compared to the others carbon sources.In principal component analysis(PCA),the results showed that the metabolic function of soil microbes was significantly changed in the presence of 500 mg·kg-1 HHCB and Cd co-contamination.The phospholipids fatty acid(PLFA)was used to study the soil microbial community structure,measure the fatty acid contents of various strains in soil,and to analyze the principal component(PCA)and diversity index.As shown in the results,the fatty acid contents of bacteria increased with the increasing of the HHCB concentration,in contrast,the fatty acid contents of fungi and actinomycetes decreased.1000 mg· kg-1 HHCB pollution promoted bacterial growth,1000 mg-kg-1 HHCB and Cd co-contaminated significantly inhibited fungi growth.The single HHCB pollution and HHCB-Cd combined pollution all had significant inhibitory effect on actinomycetes,there was a close relationship between the fatty acid contents of actinomycetes and the soil HHCB pollution.1000 mg·kg-1 single HHCB pollution and HHCB-Cd combined pollution remarkably increased the B/F,GN/GP value and the pressure index,and greatly changed the soil microbial community structure.The PCA results showed that 500 mg·kg-1 HHCB and Cd co-contaminated,1000 mg· kg-1 single HHCB pollution and HHCB-Cd combined pollution had notable effect on fatty acid,i.e.17:0,17:Ocyclo,17:0anteiso,16:1?7c and 16:1?9c.The Shannon,Margalef and Pielou indices of the group in 1000 mg·kg-1 HHCB and Cd co-contaminated soil were substantially lower than in the blank control group.It suggested that the species diversity,evenness and of soil microbial community were significantly decreased.The PCR-DGGE and high-throughput sequencing were used for the study of the genetic diversity of soil microbial communities.As shown in the experimental results,a remarkable change took place in the number and the position of the strips in the PCR-DGGE profile of bacteria:some of the stripes disappeared,while others were rounded up;the effect of single HHCB pollution on microbial strain diversity was stronger than that of HHCB and Cd co-contaminated.The Shannon and Margalef indices decreased as the HHCB concentration rising in soil.The Shannon,Margalef and Pielou indices of groups in 500 mg·kg-1 HHCB single HHCB pollution,1000 mg·kg-1 single HHCB pollution and HHCB-Cd combined pollution soil were substantially lower than in the blank control group.The species diversity,evenness and soil microbial community were significantly decreased.The result of high through-put sequencing demonstrated that in the soil samples of different treatments,the main groups of microbial were Proteobacteria,Bacteroidetes,Actinobacteria,Acidobacteria and Gemmatimonadetes.The abundance of Proteobacteria increased with the increasing concentration of HHCB,while that of the other 4 phylum groups decreased.Single HHCB pollution and combined pollution promoted the growth of Proteobacteria,but inhibited the growth of the other 4 phylum.There was a strong relationship between the abundance of actinomycetes and soil HHCB pollution,which was consistent with the results of the plate count and PLFA.Therefore,it suggested that actinomycetes were sensitive to HHCB pollution,and since the latter had a strong toxic effect on the former,the former could be taken as an early warning indicator of HHCB pollution in soil.The clustering results of PCR-DGGE at phylum level and high throughput at genus level,showed that 500 and 1000 mg·kg-1 single HHCB pollution and HHCB-Cd combined pollution significantly changed the soil microbial community structure.