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Executive summary PL0526 The major environmental concern arising from the use of pesticides in agriculture is their potential to leach to surface and ground water and affect water quality. The susceptibility of a particular pesticide to leaching depends on complex interactions between the chemical structure of the pesticide and a variety of soil physical, chemical and microbiological properties, which control the pesticide sorption and rate of degradation. For several classes of pesticide, including phenoxyalkanoic acids and carbamate insecticides, repeated use on the same site over several years can results in development of enhanced biodegradation, where pesticide degradation occurs extremely rapidly, as a result of the proliferation of organisms adapted to utilise the compound as an energy source. Induction of enhanced degradation can reduce the eficacy of pesticides, creating uncertainty as to doses required for pest or weed control. Recently, there has been evidence that some pesticide classes previously though to be degraded slowly in the environment, including the phenyl-urea herbicides, may also be susceptible to enhanced degradation. The purpose of this project was to investigate the microbial mechanisms underlying enhanced degradation. The experiments used pesticides with contrasting susceptibility to the induction of enhanced degradation. Key pesticides used included carbofuran, for which enhanced degradation can be readily induced, isoproturon, for which 'hot spots' of rapid degradation have been found within isolated fields, and diuron, which has only very occasionally been found to degrade rapidly in soil. Studies investigated how the proliferation of pesticide degrading organisms is affected by the chemical and biological environment of the soil. Further experiments investigated spatial variability in biodegradation rates within top-soil and sub-soil. The characteristics and diversity of pesticide degrading organisms were also investigated. Experiments were conducted to examine degradation in the top- and sub-soil of fields that had received applications of the pesticide at varying times over the past 10 years. It was found that concentrations as low as 0.1 mg kg-1 soil could induce rapid degradation of a subsequent application. There was no relationship between top soil exposure to carbofuran and degradation rates in the sub-soil. In general, degradation rates in sub-soil were lower than in subsoil. However, there was evidence that some sub-soils had inherently high abilities to degrade the compound, with greater rates of degradation than in top-soils. Patterns of degradation in sub-soil samples indicated that there was a significant spatial variability in the distribution of carbofuran degrading organisms. In the case of isoproturon, it was shown that there was substantial variability in degradation rate within Deep Slade field at Wellesbourne, with the half life of the compound ranging from 6.5 to 30 days. Microsites at which isoproturon degraded rapidly had higher pH and larger biomass contents than slow degrading sites. Using 14C ring labelled isoproturon, it was shown that spatial variability in degradation rate had implications for the eventual fate of pesticide residues. Slow degradation promoted the formation of bound pesticide residues. In the case of both carbofuran and isoproturon, variation in the rate of pesticide degradation did not occur as a result of differences in the initial size of the pesticide degrading population. For both pesticides, the dynamics of growth and proliferation of the degradative community following pesticiide application was shown to control degradation kinetics. The extreme variability in isoproturon degradtion rate was found to arise from fundamental differences in the mode of degradation. Fast degradation rates were associated with the rapid proliferation of degrading organisms. However, in slow degrading sites, there was no evidence for proliferation of degrading organisms until at least 60 % of the pesticide had been degraded. This indicates that degradation had occurred by cometabolism, coincidentally to the general metabolic activities of the soil microbiota. In a number of slow degrading microsites, proliferation of isoproturon degrading organisms did subsequently occur. However in several sites, there had been no proliferation of degrading organisms even at the point of 90 % pesticide degradation. The characteristics of pesticide degrading communities were investigated. Bacteria showing abilities to degrade carbofuran were isolated from geographically distinct soils, from the UK and Greece. Partial sequence analysis of the 16S rRNA gene of the isolates revealed high diversity within the carbofuran degrading communities of both sites. While most UK isolates were Pseudomonas spp., the Greek isolates were predominantly members of the Flexibacter / Cytophaga / Bacteroides group. However, there was some overlap of species within the 2 communities, with Chrysobacterium sp. isolates from the two soils showing identical 16S rRNA gene sequences and protein profiles. A number of isoproturon degrading bacteria were isolated from Deep Slade field. Partial sequencing of the 16S rRNA gene of these isolates showed that they were Sphingomonas sp. In situ changes in soil microbial community structure during isoproturon degradation were investigated by denaturing gradient gel electrophoresis of 16S rRNA. It was found that 2 organisms proliferated during isoproturon degradation, both of which were closely related Sphingomonas sp. A further bacterium which was able to degrade diuron was isolated from Deep Slade field. 16S RNA sequencing showed that the isolate was an Arthrobacter sp. This organism could degrade a range of further phenyl-urea compounds, including isoproturon. The genes for phenyl-urea degradation were found to be located on a 22KD fragment within the plasmid pHRIM620. The project has highlighted the extreme spatial variability of pesticide degradation that can occur within a single field, in both sub-soil and top-soil. This has implications for the dynamics of pesticide leaching. Further work is needed to investigate whether the variability in carbofuran and isoproturon degradation rates are typical of other pesticide classes, and the scale over which the variability occurs.The work has provided tools that can be used to investigate the localisation, dynamics and survival of pesticide degrading organisms, which is necessary for an understanding of the mechansims underlying variability.