Effects Of New Pesticide Acetamiprid On Microbial Ecology In Upland Soil And Degradation Of Acetamiprid

The effects of a new neonicotinoid insecticide acetamiprid, (E)-N¹-[(6-chIoro-3-pyridyl)methyl]-N²-cyano-N¹-methylacetamidine, on soil microecosystem were studied in upland soil samples with a short-term treatment of acetamiprid at different concentrations. The culturable bacteria (plating counts), soil enzyme activities, and changes in microbial community structure (denaturing gradient gel electrophoresis (DGGE) analysis) were used to assess biological community in upland soil contaminated by acetamiprid. The activity response of the antioxidant enzymes, superoxide dismutase, catalase, ATP enzyme activities of Escherichia coli (G^-), Bacillus subtilis (G⁺) and acetamiprid-degrading bacterium, Pseudomonas sp. FH2 (G"), following exposure to acetamiprid was also investigated. Moreover, a bacterium capable of degrading acetamiprid was isolated, identified and phylogenetically analyzed. The metabolic pathway of acetamiprid by strain FH2 was detected and inferred basically. The results will be valuable to build up alert index systems in acetamiprid-contaminated upland soil, environmental quality evaluation and virtual utilization of acetamiprid-degrading bacterium in upland soil.The main results of this study are as follows:1. The influences of acetamiprid on the cultural microorganisms in upland soil, using traditional selective plating and direct viable counts methods, and soil enzymes activities were investigated. The results showed that the bacteria differed markedly in their response to acetamiprid. The concentration of acetamiprid applied is an important factor affecting populations of various microorganisms, except those characteristics of acetamiprid itself. The presence of 0.5mg kg^-1, 5mg kg^-1, 25mg kg^-1 and 50mg kg^-1 dry soil of acetamiprid could significantly increase numbers of bacteria and fungi. For N₂-fixing bacteria, acetamipriddecreased its numbers initially and then stimulated it two weeks later, which suggested metabolites of acetamiprid had no toxic to Ni-fixing bacteria. Acetamiprid, however, decreased actinomycetes population strongly and the mount of actinomycetes in treated soil was only 2-fold to that of the control within the first week after acetamiprid application. The populations of the two soil samples treated with 25 and 50 mg kg"' dry soil of acetamiprid were always inhibited during the whole 50-days incubation time.The various enzymes differed remarkably in their response to acetamiprid. Acetamiprid had no obviously influence on the urease and catalase activity, resulting no statistical difference in these enzymes activities between the treated soil samples and the controls during the whole experiment phase. Also, there had had obvious inhibition of acetamiprid on soil phosphatase activity. The four treatment concentrations could inhibit markedly the enzyme activity, and the higher the concentration was applied, the more the inhibition was observed at the first week after the pesticide was applied. The phosphatase activities in soil samples treated with acetamiprid in 25 and 50 mg kg"'dry soil were inhibited within the most time of experiment. The calculated EC]0 or EC50 at 7thd, 14th-d and 35th-ds for phosphatase were 28, 15 and 31 mg kg"1 or 146120, 14636 and 13414 mg kg''dry soil, respectively. The calculated EC]0 value was more than the value of normal field concentration (0.5 mg kg"1 dry soil), implying that acetamiprid at normal field dose would not pose a toxicological threat to soil phosphatase. The calculated EC50 value, however, was less than that of the highest experiment concentration, therefore, the possibility of acetamiprid posing a toxicological threat to soil phosphatase would become more obviously appeared when it accumulated or was misused in soil. Dehydrogenase activity was inhibited slightly at the first 2 weeks after acetamiprid treatment. From the third week, the activity of dehydrogenase in the soil samples treated with low acetamiprid concentration (0.5 and 5mg kg'1 dry soil) was stimulated and then that of all treated samples were simulated sequentially, and the simulating trend was in inverse order to the applied concentration. Changes of proteinase activities had no obviously relationship with the applied concentration during the initial three-week incubation time after acetamiprid application. Proteinase activities of soil samples underwent to be inhibited from the fourth week, and the activity in soil sample treated with 50 mg kg"' dry soil was statistically inhibited evidently (p<0.05). This inhibition should be related with the metabolism of acetamiprid in soil.The soil respiration was inhibited after acetamiprid treatment for two weeks, and the higher the applied concentration was, the stronger the inhibition was observed. The soil respiration was recovered at later time. Accounting to the nonlinear dose-effect regression curve, the calculated EC,oand EC50 at 21st d was 0.07 and 159 mg kg'1 dry soil, respectively. At this time, the EC10 value was significantly lower than that of the normal field application concentration, suggesting that acetamiprid would pose a certain threat to soil respiration toxicologically, even at field application concentration. Nevertheless, the value of calculated EC50 was much higher than that of the most concentrations selected in the experiment. Then, it was clear that acetamiprid would pose a toxicological threat to soil respiration even at normal field concentration, but the threat was not serious.2. DGGE method was applied to determine the relative genetic complexity of microbial communities in upland soil treated with acetamiprid. The result showed that there are a certain differences in bacterial communities diversity in upland soils treated with different concentrations of acetamiprid. However acetamiprid applied with normal field dose had no obvious effect on soil microbial diversity and there were no significant difference in DGGE patterns between 0.5 mg kg" dry soil of acetamiprid and the CK during the whole incubation time. This suggested aceatmirpid in normal field application dose would not pose threat to microbial communities, which was coincident to the conclusions obtained based on the traditional techniques, such as plating count and soil enzyme activities analysis. High concentration of acetamiprid had a certain effect on microbial diversity for a relatively short time and the difference in Jacarrd indexes among the different samples and between CK and samples became unconspicuous. Obviously, DGGE technique is a new and valuable method to search microbial gene diversity in environments contaminated by acetamiprid . At the same time, application-responsive bands from the acetamiprid treatments were sequenced and aligement. Clone 1 and clone 2 had very high similarities (100%) to many uncultured bacteria reported by different researchers. The strain was an uncultued bacterium which can be enriched at suitable concentration of acetamiprid. It could be regarded as reporting gene to indicate the contamination degree of acetamiprid.3. The activity response of the antioxidant enzymes superoxide dismutase, catalase, ATP enzyme activities of Escherichia coli (G"), Bacillus subtilis (G+) and Pseudomonas sp. FH2 (G") following exposure to acetamiprid was investigated. The bacterial strains were treated with thedifferent concentrations of acetamiprid (10, 100, and 1,000 mg L'1). Results obtained indicated that SOD and CAT activities of these bacteria were induced positively and obviously by acetamiprid. The inhibition of acetamiprid to ATPase activity in E.coli K.12, B.subtilis and Pseudomonas FH2 appeared stronger with the increase of application concentration, showing a striking dose-response relationship, which could, therefore, be used as an available bioindicator for acetamiprid pollution. Acetamiprid applied in 100 mg L'1 in this research had significant effects on these three bacteria at the early stage of incubation, but none of which was persistent. Native polyacrylamide gel electrophoresis and activity staining of SOD revealed that acetamiprid had effects on isoenzymes patterns of E.coli. No such change of the isoenzymes in Pseudomonas FH2 and B. subtilis was observed. In conclusion acetamiprid would have caused a certain oxidative stress for the three bacteria, which might not only elevate SOD and CAT activity but also generate new SOD isoenzymes to antagonize against oxidative stress. This oxidative stress, however, lasted for a relatively short time and did not cause a long-term damage.4. A bacterium, named strain FH2, capable of degrading acetamiprid, was isolated from a pesticide manufactory soil and identified to be Pseudomonas sp. based on morphology, physio-biochemical properties, G+C mol% in DNA and a partial sequencing of 16S rDNA. Strain FH2 could grow using acetamiprid as sole carbon and energy source with very low growth rate . And only 50% of acetamiprid was decomposed after incubating strain FH2 in acetamiprid inorganic salt medium for about 2 weeks.The growth rate was increased and nearly 98% of acetamiprid was degraded when 0.25% of yeast extract was added to inorganic salt medium. Data of GC/MS analysis suggested that strain FH2 could degrade acetamiprid to 6-chloro-3-nitro-2-picoline and methyl 5-hydroxynicotinate, but could not mineralize acetamiprid completely to CO2 and H2O and more work was needed for the pathway of biodegradation by strain FH2. Strain FH2 was insensitive to amikacin (40 ug mL"1), ampicillin (15 ug mL"'), penicillin (6 IU), erythromycin (15 ug mL'1) and gentamicin (50 ug mL"1). The optimal temperature and pH for its growth was 30°C and 7, respectively. Whole cell protein SDS-PAGE maps of strain FH2 induced by acetamiprid appeared a special band whereas had no on that without inducement , which means this band maybe relative with degradation and detoxification of strain FH2 to acetamiprid.