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Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium
PERGAMON
Soil Biology and Biochemistry 31 (1999) 677±686
Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium John E. Cullington *, Allan Walker Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Accepted 23 September 1998
Abstract An investigation into intra-®eld spatial variability in rates of degradation of phenylurea herbicides revealed the sudden onset of rapid diuron degradation in one laboratory-incubated soil sample after 43 d. Subsequent applications of diuron to this soil were degraded very rapidly (DT50<24 h). Through liquid enrichment culture, a bacterial isolate, that degraded diuron rapidly with no requirement for supplementary carbon and nitrogen sources, was obtained from this `enhanced' soil. Addition of this isolate at 9.3 106 cfu gÿ1 to soil containing `aged' diuron residues resulted in rapid diuron degradation in both pre-fumigated and non-fumigated soil. However, at an inoculum density of 9.3 103 cfu gÿ1, degradation only occurred in fumigated soil over the 16 d duration of the experiment. The isolate degraded a range of phenylureas in liquid culture, with rate of degradation in the order linuron>diuron>monolinuron>>metoxuronoisoproturon. However, the N 0 -monomethyl and demethylated derivatives of diuron were not degraded. Degradation of diuron and linuron resulted in accumulation of a single metabolite, which had the same retention time as 3,4-dichloroaniline. This, together with the lack of degradation of the monomethyl and demethylated derivatives of diuron, suggests that the molecules had been split directly at the carbonyl group, rather than undergoing step-wise demethylation. Isoproturon degradation by this isolate was greatly accelerated in the presence of additional carbon and nitrogen sources, resulting in the production of a metabolite with the same retention time as 4-isopropylaniline, again suggesting that degradation proceeded via carbonyl group cleavage. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted phenylurea herbicide used widely for long-term pre-emergence weed control in non-crop areas and also in a range of tree crops (Tomlin, 1994). It is relatively persistent in soil, with half-lives from 90 to 180 d (Hill et al., 1955), although extrapolated values of greater than 3000 d have been reported (Madhun and Freed, 1987). Microbial degradation is considered to be the primary mechanism for its dissipation from soil (Sheets, 1964; GeissbuÈhler et al., 1973). Under aerobic conditions, this is believed to occur by successive demethylation of the urea group, followed by hydrolysis to give the chlorinated aniline (Dalton et al., 1966; Tillmanns et al., 1978; Ellis and Camper, 1982). * Corresponding author. Tel.: +44-1789-470-382; fax: +44-1789470-552; e-mail: [email protected]. 0038-0717/99/$19.00 # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 8 ) 0 0 1 5 6 - 4
Occurrence of enhanced biodegradation has been reported for a wide range of pesticides in soils (Kaufman, 1987; Racke and Coats, 1990), including some substituted ureas (Walker and Welch, 1991, 1992; Roberts et al., 1993). However, to date, there have been no reports of accelerated biodegradation of diuron in soil. Ellis and Camper (1982) isolated several diuron-degrading mixed fungal±bacterial and mixed bacterial cultures from treated pond water±sediment systems which degraded diuron (at 10 mg lÿ1) completely within 7 d. Attaway et al. (1982) described rapid degradation of diuron in pond sediment maintained under anaerobic conditions, apparently by reductive dechlorination of the p-chlorine substituent. However, pure cultures of diuron-degrading microorganisms were not obtained in either of these studies. In a number of other studies, soil fungi have been shown to possess high activity against phenylureas, with occasional ability to degrade diuron. Tillmanns et al. (1978) described rapid demethylation of ®ve phenylurea herbicides, including diuron, by the fungus
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Table 1 Generalized structures of the compounds examined
Compound
Diuron Linuron Monolinuron Metoxuron Isoproturon 3-(3,4-Dichlorophenyl)-1-methylurea 3-(3,4-Dichlorophenyl)-urea
Substituent R1
R2
R3
R4
CH3 CH3 CH3 CH3 CH3 CH3 H
CH3 OCH3 OCH3 CH3 CH3 H H
Cl Cl H Cl H Cl Cl
Cl Cl Cl OCH3 CH(CH3)2 Cl Cl
Cunninghamella echinulata (Thaxter). Recently, Vroumsia et al. (1996) reported that a number of fungal isolates were able to degrade diuron to some extent. One isolate of Rhizoctonia solani was able to degrade diuron, isoproturon and chlorotoluron. Shelton et al. (1996) demonstrated some degradation of diuron by a Streptomyces strain, which was also active against linuron and a range of acetanilide and striazine herbicides. However, to date there has only been one report of an isolated soil bacterium showing some activity against diuron (Roberts et al., 1998). The present study was initiated after the serendipitous discovery of unusually rapid diuron degradation in one soil sample during a study of small-scale spatial variability in phenylurea herbicide degradation. The objectives of the experiments reported here were to isolate microorganisms capable of rapid diuron degradation, to determine their ability to degrade related phenylurea compounds and to investigate their capacity to degrade diuron residues when inoculated into soil. 2. Experimental methods and results 2.1. Soils and herbicides The soil used was a sandy-loam of the Wick series containing (w/w) 16% clay, 70% sand and 1.9% organic matter, with a moisture content of 9.8% (w/w) at an applied pressure of 33 kPa. Soil was collected in October 1996 from Deep Slade ®eld at Horticulture Research International, Wellesbourne. This ®eld had been under cereal cultivation for the previous 3 yr, with cereals or experimental ®eld vegetable plots
grown before this. According to records, the site had never been treated with diuron, or any phenylurea herbicide other than isoproturon, an autumn application of which had been made for at least the last 3 yr. Five samples were taken at 5 m intervals along each of two parallel transects, separated by approximately 20 m. Soil pH (soil:deionized water; 1:1) ranged from 7.8±8.1 in the samples from transect 1 and from 6.5±6.8 in samples from transect 2. Organic matter content of sub-samples of each soil varied from 2.6±3.1% (w/w). Aside from pH, there was no discernible dierence between samples. Soils were allowed to lose excess water by air-drying overnight at room temperature, after which they were sieved (3 mm). Soils were transferred to sealed polyethylene bags, with sub-samples dried overnight at 1108C to determine initial water content. Diuron was added to soils as a commercial wettable powder formulation (80% active ingredient). Analytical grade compounds were used in all liquid cultures: diuron, linuron, monolinuron and isoproturon (British Greyhound, Birkenhead, UK); 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3-(3,4-dichlorophenyl)-urea (DCPU) (Promochem, Herts); metoxuron (Novartis Agrochemicals, Basel, Switzerland). Structures of these chemicals are shown in Table 1. All solvents used were HPLC grade. 2.2. Soil incubations Diuron was added to 250 g samples of each soil as an aqueous suspension of the commercial formulation (5 ml, 750 mg a.i. mlÿ1), resulting in a ®nal concentration of 15 mg kgÿ1. Additional deionized water was added to adjust the moisture content to 9.8% (soil
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Fig. 1. Diuron degradation at 158C in soil from 10 sites in Deep Slade ®eld, showing occurrence of rapid degradation in soil from one of the sites (± R ±).
water potential, ÿ33 kPa). Samples were thoroughly mixed by hand and transferred to loosely capped polypropylene bottles. Incubations were performed at 158C, with bottles enclosed in partially sealed polyethylene bags containing wet paper towelling in order to minimise evaporative water loss. Soil moisture content was maintained by periodic additions of deionized water, as required. Samples (15 g) were removed immediately after preparation and then at 7 d intervals, with herbicide residues extracted with 20 ml methanol by shaking for 1 h on a wrist-action shaker. Both diuron concentration and presence of any degradation products in the clear supernatant were determined directly by HPLC. The column used was Lichrosorb RP18 (250 4.6 mm; Merck) and compounds were detected by UV absorbance at 240 nm. The HPLC mobile phase was acetonitrile:water:phosphoric acid (70:30:0.25 by volume), with a ¯ow rate of 1 ml minÿ1. Under these conditions, the retention time of diuron was 3.6 min. Diuron degradation was slow in all 10 samples for the ®rst 29 d of incubation (Fig. 1). However, when
Fig. 2. Degradation of diuron at 158C following retreatment of soil from the `rapid-degrading' site (R) and from a `slow-degrading' site (q).
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sampled on day 43, diuron residues in one soil sample (transect 1; pH 7.8) had declined to 2.75 mg kgÿ1, whereas in all other soils (pH 6.5±8.1), the residual concentration varied from 15±19 mg kgÿ1. After incubation for 57 d, soil from the treatment showing rapid degradation and from one of the treatments showing slow degradation, was retreated with the same amount of diuron, as above, resulting in ®nal concentrations of 17 and 36 mg kgÿ1, respectively. Samples (10 g) were taken over a period of 20 d and herbicide residues were determined as before. Diuron residues in the `active' soil were reduced by over 75% after incubation for 5 d at 158C, at which time little degradation had occurred in the retreated `non-active' soil (Fig. 2). All soil samples remaining from these preliminary incubations were stored separately in capped bottles at 58C. 2.3. Enrichment culture After storage for ca. 3 months, the soil that degraded diuron rapidly received three further diuron retreatments, at a concentration of 40 mg kgÿ1. The ®rst two incubations lasted for 6 d, and the third for 2 d. These successive diuron applications were degraded rapidly, with residues from the third retreatment reduced from 47.3 to 6.1 mg kgÿ1 in just 2 d (Fig. 3). After the third incubation, 0.5 g sub-samples of the soil were used to inoculate duplicate bottles (100 ml, Duran) containing 20 ml of mineral salts medium (after Roberts et al., 1993) plus diuron (20 mg lÿ1), in which the herbicide constituted the sole source of carbon and nitrogen in the medium. Mineral salts medium comprised (g lÿ1 deionized water): (KH2PO4, 2.27; Na2HPO412H2O, 5.97; NaCl, 1.0), (MgSO47H2O, 0.5; CaCl22H2O, 0.01; MnSO44H2O, 0.02), (FeSO4, 0.025); medium pH was 6.9. During preparation of the medium, the ®rst two groups of compounds were autoclaved separately (15 min at 1218C) and combined
Fig. 3. Degradation of diuron at 158C following successive retreatments of the `rapid-degrading' soil; vertical lines indicate when retreatments were made.
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Fig. 4. Diuron degradation in shaken bottles of MSM (mean of two replicates) inoculated with diuron-degrading soil and incubated at 208C; vertical lines denote transfer of culture samples to fresh media.
when cool, to limit precipitation of metal ions as insoluble phosphate compounds. The iron sulphate solution was ®lter sterilised prior to adding to the cool medium. Analytical grade diuron was added to sterile, dry bottles as 0.1 ml of a 4 mg mlÿ1 ®lter-sterilised methanol solution. The bottles were left uncapped on a laminar ¯owbench until the methanol had evaporated. Mineral salts medium (MSM) was added aseptically to the bottles, which were then shaken for 1 h on a wrist-action shaker, to ensure complete dissolution of the pesticide. Duplicate control bottles were prepared, to which no soil was added. Following addition of the soil inoculum, bottles were loosely capped and incubated at 208C on an orbital shaker (140 rpm). Bottles were sampled immediately after addition of the soil, and then daily, with 0.5 ml culture aseptically transferred to an HPLC vial and mixed thoroughly with 1 ml acetonitrile. Diuron concentration was determined by HPLC, as before. After incubation for 2 d, 0.5 ml of culture was transferred to further bottles containing MSM plus diuron, and the concentration of diuron remaining was measured every 1±2 d. This procedure was repeated after 3 d incubation of the second set of bottles, giving a third cycle of liquid culture from which, after 5 d incubation, 0.5 ml samples were taken and used to inoculate a fourth set of bottles. The results from these repeated sub-cultures are shown in Fig. 4. Rate of degradation was most rapid in the initial liquid culture receiving the soil inoculum, in which residues declined from 23.3 to <1 mg lÿ1 in three days. Diuron degradation was accompanied by a progressive increase in concentration of a major metabolite with the same HPLC retention time (ca. 4 min) as 3,4-dichloroaniline. 2.4. Isolation of diuron-degrading bacteria After 4 d incubation at 208C, serial 10-fold dilutions of the fourth-cycle liquid culture in the experiment
described above were prepared in deionized water, and spread (0.1 ml) over plates of mineral salts agar (MSM plus 15 g lÿ1 agar, Difco Bacto). The agar contained diuron at 20 mg lÿ1, added after autoclaving as 5 ml of a ®lter-sterilised methanol solution lÿ1 of agar. Plates were incubated at 208C for 8 d, at which time 20 individual well-separated colonies were streaked out onto plates of mineral salts agar (MSA) plus diuron and also onto nutrient agar (Difco) + diuron (20 mg lÿ1) to check for purity. After incubation for 6 d at 208C, a single well-separated colony was selected from 10 of the 20 MSA plates and spread separately over further MSA + diuron plates with 0.1 ml sterile deionized water. Degrading ability of isolates was assessed after 7 d incubation at 208C, when growth was washed from each of the 10 MSA plates with 1.5 ml sterile deionized water and 0.5 ml of each suspension used to inoculate bottles containing 20 ml MSM + diuron, as before. Incubation conditions and sampling methodology were as described previously, with diuron concentration determined after 2, 3, 8 and 14 d. Subsamples of the liquid cultures in which diuron degradation had occurred were streaked onto plates of Nutrient agar (NA) + diuron and MSA + diuron; culture samples were also frozen at ÿ768C, following addition of sterile glycerol (15% ®nal volume). In a parallel experiment, a further 12 colonies from the initial dilution plates were introduced directly into wells of a micro-titre plate, with each well containing 300 ml MSM + diuron (20 mg lÿ1). The plate was incubated in a sealed sterile plastic box in a static incubator at 208C. After 2, 5, 8 and 13 d, 50 ml volumes were removed from each well and mixed thoroughly with 100 ml acetonitrile in the wells of another micro-titre plate, prior to analysis by HPLC. Well contents in which diuron degradation had occurred were streaked onto plates of MSA + diuron and NA + diuron, to check for purity. Little growth occurred on MSA + diuron plates, therefore a single colony was selected from each of the NA + diuron plates and transferred to 5 ml MSM + diuron (20 mg lÿ1) in sterile plastic bottles (25 ml). These were incubated as before and sampled at hourly intervals. When diuron had degraded to below 50% of its initial concentration, samples were frozen as before. A total of ®ve individual colonies out of the 32 selected from the initial MSA + diuron dilution plates degraded diuron. Four of these were detected by direct introduction of colonies into the microplate wells. Only one colony out of the 10 puri®ed and sub-cultured colonies introduced into the shaken-bottle assay was found to degrade diuron. In this bottle, degradation was complete after 3 d. In two wells of the microplate assay, diuron concentration was reduced to zero after just 2 d, with total degradation in another two wells when sampled after 5 d. No degradation had
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occurred in the remaining wells on the ®nal sampling at 13 d. All ®ve isolates appeared pure on NA and also retained degrading ability after sub-culture on NA + diuron. 2.5. Characterisation of diuron-degrading isolates Colony growth and morphology were examined on NA, following incubation at 30 and 428C. Cell morphology, motility and Gram-reaction were examined under a light microscope. Production of ¯uorescent pigment was determined on Kings Medium B (King et al., 1954). Catalase and oxidase activities were determined as described by Gerhardt et al. (1994), with oxidation±fermentation reaction and substrate utilisation tests performed using API20NE test strips (bioMeÂrieux sa) according to the manufacturer's instructions. Culture growth and cell morphology was similar for all ®ve isolates, with yellow±cream, circular, mucoid colonies on NA incubated at 308C; no growth occurred at 428C and no ¯uorescent pigment production was observed. Cells of all isolates were nonmotile, appearing as non-pigmented short rods together with smaller spherical cells which commonly occurred as pairs or in short chains. Isolates were classed as Gram-negative, although cells were dicult to de-stain; all were oxidase negative and catalase positive. Results of the API20NE tests were the same for all ®ve isolates, suggesting that they were the same organism. The substrate utilization tests gave the following results (+ = positive, 2 = weakly positive, ÿ = negative result): glucose (+), arabinose (ÿ), mannose (2), mannitol (+), N-acetyl-glucosamine (ÿ), maltose (+), gluconate (+), caprate (ÿ), adipate (ÿ), malate (+), citrate (+) and phenyl-acetate (2). Results of additional tests were: reduction of NOÿ 3 to NOÿ 2 (+), indole production from tryptophan (ÿ), glucose fermentation (ÿ), arginine dihydrolase activity (ÿ), urease activity (ÿ), aesculin hydrolysis (+), gelatin hydrolysis (ÿ) and b-galactosidase activity (2). One isolate (coded as D47), obtained from the shaken-bottle assay, was used in all subsequent degradation experiments.
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with chloroform in a vacuum desiccator for 7 d at 308C, whilst the other was stored at 48C in a sealed polyethylene bag. Residual chloroform was removed from the fumigated soil by repeat evacuations in a vacuum desiccator on a laminar ¯ow bench. Fumigated and non-fumigated soils were dispensed aseptically into sterile polypropylene containers, giving a ®nal soil weight of 125 g per container, after addition of sucient sterile deionized water to bring moisture content back to 9.8%. Inoculum was prepared from a culture of isolate D47 grown on NA + diuron. Growth was washed from the plates with 2 ml MSM and the bacterial suspension adjusted with MSM to give a theoretical density of 1.3 109 cfu mlÿ1, as estimated by comparison of absorbance at 600 nm (A600) with a previously-prepared standard curve. Viable cell count of the inoculum was determined by plating six replicate 20 ml droplets of appropriate serial dilutions in MSM onto NA (Miles and Misra, 1933), which gave an actual density of 1.2 109 cfu mlÿ1. A lower density inoculum was also prepared, to contain 1.2 106 cfu mlÿ1. For both fumigated and non-fumigated soils, duplicate samples were inoculated with 1 ml of either bacterial suspension, resulting in a ®nal inoculum density of 9.3 106 or 9.3 103 cfu gÿ1 moist soil. Single control pots received 1 ml MSM in place of the bacterial inoculum. Inoculum was thoroughly mixed into the soil under aseptic conditions, together with the ad-
2.6. Degradation of `aged' diuron residues in soil Soil samples from a further 10 sub-sites (on a 10 10 m grid) in Deep Slade ®eld were amended with a commercial formulation of diuron (15 mg kgÿ1). After 88 d incubation at 158C and 9.8% moisture content (matric potential ÿ33 kPa), none of the sub-sites showed rapid degradation and ca. 60±70% of the applied diuron remained in all 10 samples. At this time, the samples were combined and thoroughly mixed. The combined soil was divided into two 750 g amounts, of which one was sterilised by fumigating
Fig. 5. Degradation of `aged' diuron residues at 208C, in Deep Slade soil samples (mean of two replicates) inoculated with isolate D47 at densities of 9.3 106 cfu gÿ1 (a) or 9.3 103 cfu gÿ1 (b), in fumigated (± q ±) and non-fumigated (± Q ±) soil; controls, fumigated (- - q - -) and non-fumigated (- - Q - -), received no bacterial inoculum.
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Fig. 6. Degradation of the phenylurea herbicides linuron (Q), diuron (r), monolinuron (.), metoxuron (), isoproturon (+), and the derivatives DCPMU (- - r - -) and DCPU (- - * - -), at 208C in bottles of MSM inoculated with isolate D47 at a density of 9.3 106 cfu mlÿ1 (mean of two replicates).
ditional sterile deionized water required for moisture content adjustments. Pots were then incubated overnight at 48C, allowing re-distribution of the applied inoculum and water throughout the soil, before being transferred to a 208C incubator. Samples (10 g) were removed, under aseptic conditions, immediately on transfer to the incubator and at intervals over 16 d. Diuron residues were determined as before. Diuron degradation was most rapid in fumigated soil receiving 9.3 106 cfu gÿ1, with residues reduced by 50% (from 7.0 to 3.5 mg kgÿ1) in both replicates after 2 d incubation (Fig. 5). In this treatment, the initial rate of degradation was rapid, but then declined once diuron concentrations had fallen below ca. 2 mg kgÿ1. The rate of degradation in non-fumigated soil receiving the same inoculum load was slower (DT50=4±6 d). Of treatments inoculated with 9.3 103 cfu gÿ1, only fumigated soils showed a slight increase in rate of degradation over the non-inoculated control soils. Two metabolites were detected in the soil extracts, of which one was present in soil of all treatments at the time of inoculation. This was produced during the slow microbial degradation of diuron that occurred over the initial 88 d incubation and had the same retention time as DCPMU. The second metabolite, with the same retention time as 3,4-dichloroaniline (3,4-DCA), was only found in inoculated treatments which showed rapid rates of degradation. Amounts of 3,4-DCA ¯uctuated, although at the end of the experiment, after 16 d incubation, only a very low concentration was detected in the inoculated treatments. 2.7. Degradation of other phenylureas Analytical grade diuron, isoproturon, linuron, monolinuron, metoxuron, DCPMU and DCPU
(Table 1) were added to sterile bottles as ®lter-sterile methanol solutions, to give ®nal concentrations of 20 mg lÿ1 MSM, as described above. Duplicate bottles, containing 20 ml MSM + test compound, were inoculated with 160 ml of the bacterial suspension used in the previous soil inoculation study (1.2 109 cfu mlÿ1), giving a ®nal inoculum density of 9.3 106 cfu mlÿ1. Controls, receiving 0.16 ml MSM in place of the bacterial suspension, were prepared for each compound. Bottles were incubated at 208C on an orbital shaker for 28 d, with 0.5 ml samples removed at regular intervals as before. Analysis was by HPLC, under the conditions described above for diuron, with the exception of monolinuron, for which the mobile phase was changed to acetonitrile:water:H3PO4 (60:40:0.25 by volume). Out of the seven compounds tested, all but DCPMU and DCPU were degraded by the bacterium. Linuron showed the most rapid rate of degradation, declining to 25% of the initial concentration after 24 h incubation. The rate of degradation of the other four compounds was slower, proceeding in the order: diuron>monolinuron>>metoxuronoisoproturon (Fig. 6). Isoproturon degradation was particularly slow, with ca. 50% of the initial amount remaining after 28 d incubation. As with diuron, linuron degradation was accompanied by an increase in concentration of a product with the same retention time as 3,4-DCA; unidenti®ed degradation products also accumulated during monolinuron and metoxuron degradation, with retention times of 3.47 and 3.34 min, respectively. Low concentrations of a metabolite with the same retention time as 4-isopropylaniline were detected during isoproturon degradation. 2.8. Isoproturon degradation, in the presence of an additional carbon and nitrogen source In order to determine whether isoproturon degradation rate could be increased by changes in the nutrient composition of the liquid medium, a sample of culture (0.5 ml) was removed from inoculated bottles containing MSM + isoproturon (in the experiment described above) after 24 d incubation and spread onto NA + isoproturon (20 mg lÿ1) plates. Following incubation for 2 d at 258C, growth was washed from a plate with 2 ml MSM and the bacterial suspension amended with further MSM to give a density of 4.1 108 cells mlÿ1, as estimated from A600 readings. Viable cell numbers, determined by colony counts on NA, gave a measured inoculum density of 3.2 108 cfu mlÿ1. Bottles were prepared with 20 ml of the following media: MSM + isoproturon (20 mg lÿ1), MSM + isoproturon + glucose (added as 0.1 ml ®lter-sterile solution in deionized water; ®nal concentration of 1 g lÿ1), MSM + isoproturon + NH4Cl
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Fig. 7. Isoproturon degradation in bottles (mean of two replicates) of MSM (w), MSM + glucose (Q), MSM + NH4Cl (R), and MSM + glucose + NH4Cl (q) inoculated with 7.7 106 cfu mlÿ1 of isolate D47 and an uninoculated MSM control (); degradation of diuron in MSM (W) by the same inoculum is shown for comparison.
(1 g lÿ1; replacing NaCl in the original MSM), MSM + isoproturon + glucose + NH4Cl. For comparison, bottles were also prepared with 20 ml MSM + diuron (20 mg lÿ1), but with no additional carbon or nitrogen source. Bottles not receiving 0.1 ml glucose solution were amended with 0.1 ml sterile deionized water. Duplicate bottles of each medium were inoculated with 0.5 ml bacterial suspension, giving a ®nal density of 7.7 106 cfu mlÿ1. A control bottle containing MSM + isoproturon received 0.5 ml MSM in place of the bacterial inoculum. Degradation of isoproturon was again slow when the pesticide provided the sole source of carbon and nitrogen, with over 50% remaining after 29 d incubation (Fig. 7). A similar rate was also seen with treatments receiving glucose or NH4Cl. However, in the presence of both glucose and NH4Cl, degradation was very rapid, with almost 90% degradation after just 6 d. During incubation, the inoculum density in this treatment increased markedly, rendering the culture opaque in comparison to the other, almost clear, cultures. Diuron degradation was again rapid, with only 12% remaining after just 2 d incubation. In the isoproturon treatment amended with glucose and NH4Cl, a single metabolite accumulated, with the same retention time as 4-isopropylaniline. Only trace amounts of this metabolite were detected in the other slow-degrading isoproturon treatments.
3. Discussion No previous reports of rapid biodegradation of diuron in ®eld soils have been reported; degradation is usually slow and follows ®rst order kinetics (Hill et al., 1955; Madhun and Freed, 1987). In our study, degradation in one soil sample out of the 10 examined was
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characteristically slow until some time between 29 and 43 d incubation, when a dramatic increase in rate occurred. It is dicult to attribute this rapid degradation of diuron to peculiar physical or chemical characteristics of this one soil sample. Soils from adjacent sites on transect 1, showing slow rates of diuron degradation, were of similar pH and, separated by only 5 m, were unlikely to have diered appreciably in other, unmeasured, properties. The ®eld site had, to all knowledge, never been treated with diuron, thus precluding the development of an adapted micro¯ora as a result of repeated exposure to the compound. One could perhaps speculate on whether the annual application of (structurally similar) isoproturon to this ®eld in¯uenced the diuron-degrading potential of the soil micro¯ora. However, failure to detect rapid diuron degradation in nearby, physically and chemically similar, soil samples suggests that this is unlikely. The rapid degradation that occurred in this soil sample was subsequently enhanced by retreatment with diuron, resulting in a DT50 of less than 24 h following the fourth and ®fth applications. The retreatment of a `slow-degrading' soil sample did not result in an increase in the rate of diuron degradation, over the 20 d duration of the experiment. It cannot be ruled out that, after more prolonged incubation, the rate of diuron degradation in this soil may have increased, as observed for the `active' soil in the initial incubation. However, there are no previous reports of acceleration of degradation in sites subjected to multiple diuron applications, indicating that eventual microbial adaptation to diuron degradation is not the norm. All ®ve diuron-degrading colonies were identical in appearance and in response to characterisation tests and would appear to be the same organism. Therefore it is likely that this bacterium constituted the major culturable, and possibly only, diuron-degrading component of the mixed culture. It was not possible to assign the bacterium to a de®nitive genus from the results of the characterisation tests alone; this would require further fatty acid- or nucleic acid-based procedures. Introduction of the higher density cell suspension of this isolate into fumigated soil containing `aged' diuron residues resulted in rapid degradation. Degradation in non-fumigated soil receiving the same inoculum load was somewhat slower. With the low density inoculum, detectable degradation occurred only in the fumigated soil. This could be attributed to a growth-promoting eect resulting from the liberation of nutrients from dead cells, or to a reduction in competition or antagonism by eradication of the indigenous soil micro¯ora. The bene®cial eect of soil fumigation on the ecacy of subsequently introduced inoculum has been shown by Gunalan and Fournier (1993) and Duquenne et al. (1997). Diuron residues had been present in the soil
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for over 12 weeks, during which time the overall rate of degradation had been very slow; yet on introduction of the diuron-degrading isolate, residue levels declined rapidly. Previous studies with a range of pesticides have demonstrated that increased residence time in soil results in stronger adsorption, with desorption to the soil solution becoming more dicult (Walker, 1987; Blair et al., 1990; Gaillardon et al., 1991; Walker et al., 1995). Working with sulphonylurea herbicides, Duy et al. (1993) provided evidence that degradation occurred primarily in the water phase of soil, with restricted rates of release from the adsorbed phase leading to reduced rates of degradation over time. These studies would suggest that `aged' residues are relatively unavailable to microbial attack. In the current experiment, aged diuron residues were degraded rapidly by the introduced bacterium. However, in the absence of parallel studies with freshly-added diuron, conclusions cannot be drawn on the possible in¯uence of increased pesticide residence time in soil on diuron degradation by this bacterium. This diuron-degrading bacterium was able to degrade both N 0 -methoxy phenylurea herbicides (linuron and monolinuron) and N 0 N 0 -dimethyl phenylureas (diuron, metoxuron), but had low activity against isoproturon. In contrast, in many previous investigations into bacterial degradation of phenylureas, activity has only been demonstrated against methoxysubstituted compounds (WallnoÈfer, 1969; Engelhardt et al., 1971; Roberts et al., 1993). An exception to this is the study of Roberts et al. (1998), where a bacterial isolate was obtained which was able to degrade both N 0 N 0 -dimethyl-substituted compounds (isoproturon, diuron) and also the methoxy-substituted compound linuron. However, in studies with phenylurea-degrading fungi, both dimethyl- and methoxy-substituted compounds have been shown to be readily metabolised by an individual isolate (Weinberger and Bollag, 1972; Tillmanns et al., 1978; Shelton et al., 1996). In the current study, degradation rate of phenylurea herbicides and derivatives would appear to be related to several structural features of the molecule. Linuron, with an N 0 -methoxy group, was degraded more rapidly than diuron, with a methyl in place of the methoxy group of linuron. However, substitution of one or two hydrogen atoms in place of the N 0 -methyl groups of diuron (DCPMU and DCPU, respectively) resulted in total inhibition of degradation. Substituents on the phenyl ring also greatly in¯uenced the rate of degradation. Removal of the 3-chlorine substituent from linuron, i.e. monolinuron, reduced the rate of degradation; substitution of a methoxy group for the 4-chlorine of diuron, i.e. Metoxuron, gave considerably slower degradation. The 4-isopropyl group of isoproturon, in place of the 3,4-dichloro substituents of diuron, caused a greater than 10-fold reduction in the rate of degra-
dation. These comparisons suggest that the size or nature of the urea N 0 -substituents is a determining factor in the occurrence of degradation and that ring chlorination, particularly at the 4-position, would appear to greatly enhance rates of degradation. In studies with several fungal isolates, Sharabi and Bordeleau (1969) observed a similar degradationenhancing in¯uence of 3,4-dichloro substitution with a range of acylanilides, although none of their isolates showed activity towards diuron. Roberts et al. (1993) also showed the apparent requirement for chlorination of the phenyl ring in studies with a mixed linurondegrading culture: chlorbromuron was degraded, but metobromuron, lacking the chlorine ring substituent, was not. In contrast, Vroumsia et al. (1996) found that isoproturon (no ring chlorination) was more readily degraded than diuron, in studies with a range of fungal isolates. In the current study, the rate of isoproturon degradation was greatly enhanced in the presence of a supplementary carbon and nitrogen source. This suggests that the degrading enzymes involved had low activity towards isoproturon, which was compensated for by the considerable increase in inoculum density (and hence enzyme production) which occurred under these conditions. Degradation of diuron and linuron by this isolate resulted in the accumulation of only one detectable break-down product, which would appear to be 3,4dichloroaniline; isoproturon degradation resulted in the apparent accumulation of 4-isopropylaniline. Detection of these metabolites would suggest that degradation proceeded by hydrolysis of the bond between the amino group of the aniline moiety and the carbonyl group of the substituted urea component. The degradation process may perhaps occur by a sequence of reactions, through a number of intermediate products, rather than by a direct hydrolysis; however, these may be short-lived (or polar) and hence were not detected by HPLC. There was no evidence of further breakdown of 3,4-DCA; in liquid culture, concentrations of this metabolite increased in line with the decrease in levels of parent compound and then remained stable. Degradation of monolinuron and metoxuron also resulted in accumulation of unidenti®ed metabolites; these were presumed to be the respective anilines. This pattern of degradation suggests that the bacterium was utilising the substituted urea component of the molecule as a carbon and nitrogen source. Studies with the carbamate insecticide carbofuran have also identi®ed bacterial isolates which, in a similar fashion, hydrolyse the methyl-carbamate side chain from the molecule, but do not metabolise the aromatic remainder, 7-carbofuran phenol (Karns et al., 1986; Chaudhry Rasul and Ali, 1988; Parekh et al., 1994). This hydrolysis yields methylcarbamic acid, which spontaneously decomposes to yield CO2 and
J.E. Cullington, A. Walker / Soil Biology and Biochemistry 31 (1999) 677±686
methylamine (Getzin, 1973), with the latter compound believed to be used by the bacteria as a carbon or nitrogen source (Karns et al., 1986; Chaudhry Rasul and Ali, 1988). Hydrolysis of diuron at the carbonyl group would produce dimethylcarbamic acid which, as for methylcarbamic acid, would decompose to CO2 and dimethylamine. It is possible that, as with the carbofuran degrading isolates, the latter compound is being utilised by the bacterium as a carbon and nitrogen source. However, in a previous investigation into bacterial metabolism of linuron (Engelhardt et al., 1972), both 3,4-dichloroaniline and N,O-dimethylhydroxylamine (from the urea side chain) were found to accumulate as degradation products, with CO2 also detected (from the urea carbonyl group). This earlier study would therefore suggest that, in contrast to the carbofuran-degrading isolates, the amine produced after hydrolysis of linuron is not being utilised by the bacterium. Further studies are required to determine which components of the urea side chain are being utilised by the diuron-degrading bacterium in our study. In other work with diuron, monomethyl and demethylated degradation products have been detected in addition to the aniline, suggesting that degradation in soil usually proceeds by successive N-demethylation, followed by hydrolysis of the urea to give the aniline (Dalton et al., 1966; Tillmanns et al., 1978; Ellis and Camper, 1982). In our experiments, these additional metabolites were detected in the initial soil incubations, but never during degradation mediated by isolate D47, in liquid culture, or when added to soil. The apparent inability of this isolate to degrade the monomethyl and demethylated breakdown products of diuron con®rms that diuron degradation did not proceed by N-demethylation. Bacteria isolated in the study of Roberts et al. (1998) appeared to degrade isoproturon by stepwise demethylation, with one isolate also showing degradation of diuron. In their study, diuron degradation by this isolate was accompanied by production of DCPMU (but not 3,4-dichloroaniline), suggesting occurrence of degradation by the usually observed demethylation pathway. In conclusion, this would appear to be the ®rst report of accelerated biodegradation of diuron in soil and only the second of a single bacterium able to degrade this herbicide. The apparent con®nement of rapid diuron-degrading activity to a single sub-site within a previously untreated ®eld is curious. It would be interesting to determine whether this was a fortuitous, chance occurrence, or if this isolate (or other diuron-degrading microorganisms) is in fact of wider distribution, albeit perhaps at very low population densities. The rapid diuron degradation observed on addition of this isolate to non-sterile soil suggests that, if introduced to sites treated with this or the other phenylureas against which it was active, equally rapid her-
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bicide degradation would occur in the ®eld. If the bacterium was suciently competitive to survive in soil after the phenylureas had disappeared, then it is also possible that future applications of these herbicides would undergo rapid degradation. The apparent con®nement of rapid diuron-degrading activity to a single sub-site in the ®eld, however, suggests that prior to laboratory treatments with diuron, the bacterium was occupying a very isolated niche. In this situation, it would be unlikely to have a signi®cant eect on herbicide ecacy.
Acknowledgements We are grateful to the UK Biotechnology and Biological Sciences Research Council for funding this work and also to Alun Morgan, Horticulture Research International, Wellesbourne, for characterisation of isolates.
References Attaway, H.H., Camper, N.D., Paynter, M.J.B., 1982. Anaerobic microbial degradation of diuron by pond sediment. Pesticide Biochemistry and Physiology 17, 96±101. Blair, A.M., Martin, T.D., Walker, A., Welch, S.J., 1990. Measurement and prediction of isoproturon movement and persistence in three soils. Crop Protection 9, 289±294. Chaudhry Rasul, G., Ali, A.N., 1988. Bacterial metabolism of carbofuran. Applied and Environmental Microbiology 54, 1414±1419. Dalton, R.L., Evans, A.W., Rhodes, R.C., 1966. Disappearance of diuron from cotton ®eld soils. Weeds 14, 31±33. Duy, M.J., Carski, T.H., Hanafey, M.K., 1993. Conceptually and experimentally coupling herbicide sorption and degradation in soil. In: Proceedings 9th Symposium on Pesticide Chemistry. Piacenza, Italy, pp. 295±308. Duquenne, P., Parekh, N.R., Catroux, G., Fournier, J.C., 1997. Eect of inoculant density, formulation, dispersion and soil nutrient amendment on the removal of carbofuran residues from contaminated soil. Soil Biology & Biochemistry 28, 1805±1811. Ellis, P.A., Camper, N.D., 1982. Aerobic degradation of diuron by aquatic microorganisms. Journal of Environmental Science and Health B17, 277±289. Engelhardt, G., WallnoÈfer, P.R., Plapp, R., 1971. Degradation of linuron and some other herbicides and fungicides by a linuroninducible enzyme obtained from Bacillus sphaericus. Applied Microbiology 22, 284±288. Engelhardt, G., WallnoÈfer, P.R., Plapp, R., 1972. Identi®cation of N,O-dimethylhydroxylamine as a microbial degradation product of the herbicide linuron. Applied Microbiology 23, 664±666. Gaillardon, P., Fauconnet, F., Jamet, P., Soulas, G., Calvet, R., 1991. Study of diuron in soil solution by means of a novel simple technique using glass micro®bre ®lters. Weed Research 31, 357± 366. GeissbuÈhler, H., Martin, H., Voss, G., 1973. The substituted ureas. In: Kearney, P.C., Kaufman, D.D. (Eds.), Herbicide Chemistry, Degradation and Mode of Action, vol. 1, 2nd ed. Marcel, Decker, New York, pp. 209±291.
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Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R., 1994. Methods for General and Molecular Biology. American Society for Microbiology, Washington. Getzin, L.W., 1973. Persistence and degradation of carbofuran in soil. Environmental Entomology 2, 461±467. Gunalan, Fournier, J.-C., 1993. Eect of microbial competition on the survival and activity of 2,4-D-degrading Alcaligenes xylosoxidans subsp. denitri®cans added to soil. Letters in Applied Microbiology 16, 178±191. Hill, G.D., McGahen, J.W., Baker, H.M., Finnerty, D.W., Bingeman, C.W., 1955. The fate of substituted urea herbicides in agricultural soils. Agronomy Journal 47, 93±104. Karns, J.S., Mulbry, W.W., Nelson, J.O., Kearney, P.C., 1986. Metabolism of carbofuran by a pure bacterial culture. Pesticide Biochemistry and Physiology 25, 211±217. Kaufman, D.D., 1987. Accelerated biodegradation of pesticides in soil and its eect on pesticide ecacy. Proceedings of the 1987 British Crop Protection Conference. Weeds. pp. 515±522. King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for the demonstration of pyocyanin and ¯uorescein. Journal of Laboratory Medicine 44, 301±307. Madhun, Y.A., Freed, V.H., 1987. Degradation of the herbicides bromacil, diuron and chlortoluron in soil. Chemosphere 16, 1003± 1011. Miles, A.A., Misra, S.S., 1933. The estimation of the bactericidal power of blood. Journal of Hygiene, Cambridge 38, 732±749. Parekh, N.R., Suett, D.L., Roberts, S.J., McKeown, T., Shaw, E.D., Jukes, A.A., 1994. Carbofuran-degrading bacteria from previously treated ®eld soils. Journal of Applied Bacteriology 76, 559±567. Racke, K.D., Coats, J.L. (Eds.), 1990. Enhanced Biodegradation of Pesticides in the Environment. American Chemical Society Symposium Series, vol. 426. p. 302. Roberts, S.J., Walker, A., Parekh, N.R., Welch, S.J., Waddington, M.J., 1993. Studies on a mixed bacterial culture from soil which degrades the herbicide linuron. Pesticide Science 39, 71±78. Roberts, S.J., Walker, A., Cox, L., Welch, S.J., 1998. Isolation of isoproturon- degrading bacteria from treated soil via three dierent routes. Journal of Applied Microbiology 85, 309±316.
Sharabi, N.E.-D., Bordeleau, L.M., 1969. Biochemical decomposition of the herbicide N-(3,4-dichlorophenyl)-2-methylpentanamide and related compounds. Applied Microbiology 18, 369±375. Sheets, T.J., 1964. Review of disappearance of substituted urea herbicides from soil. Journal of Agricultural and Food Chemistry 12, 30±33. Shelton, D.R., Khader, S., Karns, J.S., Pogell, B.M., 1996. Metabolism of twelve herbicides by Streptomyces. Biodegradation 7, 129±136. Tillmanns, G.M., WallnoÈfer, P.R., Engelhardt, G., Olie, K., Hutzinger, O., 1978. Oxidative dealkylation of ®ve phenylurea herbicides by the fungus Cunninghamella echinulata (Thaxter). Chemosphere 1, 59±64. Tomlin, C., 1994. The Pesticide Manual, 10th ed. BCPC, Farnham, Surrey. Vroumsia, T., Steinman, R., Seigle-Murandi, F., Benoit-Guyod, J.L., Khadrani, A., 1996. Biodegradation of three substituted phenylurea herbicides (chlortoluron, diuron and isoproturon) by soil fungi: a comparative study. Chemosphere 33, 2045±2056. Walker, A., 1987. Evaluation of a simulation model for prediction of herbicide movement and persistence in soil. Weed Research 27, 143±152. Walker, A., Welch, S.J., 1991. Enhanced degradation of some soilapplied herbicides. Weed Research 31, 49±57. Walker, A., Welch, S.J., 1992. Further studies of the enhanced biodegradation of some soil-applied herbicides. Weed Research 32, 19±27. Walker, A., Welch, S.J., Turner, I.J., 1995. Studies of time-dependent sorption processes in soils. In: Walker, A., Allen, R., Bailey, S.W., Blair, A.M., Brown, C.D., GuÈnther, P., Leake, C.R., Nicholls, P.H. (Eds.), British Crop Protection Council Monograph No. 62: Pesticide Movement to Water Farnham. pp. 13±18. WallnoÈfer, P., 1969. The decomposition of urea herbicides by Bacillus sphaericus, isolated from soil. Weed Research 9, 333±339. Weinberger, M., Bollag, J.-M., 1972. Degradation of chlorbromuron and related compounds by the fungus Rhizoctonia solani. Applied Microbiology 24, 750±754.
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Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium John E. Cullington *, Allan Walker Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Accepted 23 September 1998
Abstract An investigation into intra-®eld spatial variability in rates of degradation of phenylurea herbicides revealed the sudden onset of rapid diuron degradation in one laboratory-incubated soil sample after 43 d. Subsequent applications of diuron to this soil were degraded very rapidly (DT50<24 h). Through liquid enrichment culture, a bacterial isolate, that degraded diuron rapidly with no requirement for supplementary carbon and nitrogen sources, was obtained from this `enhanced' soil. Addition of this isolate at 9.3 106 cfu gÿ1 to soil containing `aged' diuron residues resulted in rapid diuron degradation in both pre-fumigated and non-fumigated soil. However, at an inoculum density of 9.3 103 cfu gÿ1, degradation only occurred in fumigated soil over the 16 d duration of the experiment. The isolate degraded a range of phenylureas in liquid culture, with rate of degradation in the order linuron>diuron>monolinuron>>metoxuronoisoproturon. However, the N 0 -monomethyl and demethylated derivatives of diuron were not degraded. Degradation of diuron and linuron resulted in accumulation of a single metabolite, which had the same retention time as 3,4-dichloroaniline. This, together with the lack of degradation of the monomethyl and demethylated derivatives of diuron, suggests that the molecules had been split directly at the carbonyl group, rather than undergoing step-wise demethylation. Isoproturon degradation by this isolate was greatly accelerated in the presence of additional carbon and nitrogen sources, resulting in the production of a metabolite with the same retention time as 4-isopropylaniline, again suggesting that degradation proceeded via carbonyl group cleavage. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted phenylurea herbicide used widely for long-term pre-emergence weed control in non-crop areas and also in a range of tree crops (Tomlin, 1994). It is relatively persistent in soil, with half-lives from 90 to 180 d (Hill et al., 1955), although extrapolated values of greater than 3000 d have been reported (Madhun and Freed, 1987). Microbial degradation is considered to be the primary mechanism for its dissipation from soil (Sheets, 1964; GeissbuÈhler et al., 1973). Under aerobic conditions, this is believed to occur by successive demethylation of the urea group, followed by hydrolysis to give the chlorinated aniline (Dalton et al., 1966; Tillmanns et al., 1978; Ellis and Camper, 1982). * Corresponding author. Tel.: +44-1789-470-382; fax: +44-1789470-552; e-mail: [email protected]. 0038-0717/99/$19.00 # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 8 ) 0 0 1 5 6 - 4
Occurrence of enhanced biodegradation has been reported for a wide range of pesticides in soils (Kaufman, 1987; Racke and Coats, 1990), including some substituted ureas (Walker and Welch, 1991, 1992; Roberts et al., 1993). However, to date, there have been no reports of accelerated biodegradation of diuron in soil. Ellis and Camper (1982) isolated several diuron-degrading mixed fungal±bacterial and mixed bacterial cultures from treated pond water±sediment systems which degraded diuron (at 10 mg lÿ1) completely within 7 d. Attaway et al. (1982) described rapid degradation of diuron in pond sediment maintained under anaerobic conditions, apparently by reductive dechlorination of the p-chlorine substituent. However, pure cultures of diuron-degrading microorganisms were not obtained in either of these studies. In a number of other studies, soil fungi have been shown to possess high activity against phenylureas, with occasional ability to degrade diuron. Tillmanns et al. (1978) described rapid demethylation of ®ve phenylurea herbicides, including diuron, by the fungus
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Table 1 Generalized structures of the compounds examined
Compound
Diuron Linuron Monolinuron Metoxuron Isoproturon 3-(3,4-Dichlorophenyl)-1-methylurea 3-(3,4-Dichlorophenyl)-urea
Substituent R1
R2
R3
R4
CH3 CH3 CH3 CH3 CH3 CH3 H
CH3 OCH3 OCH3 CH3 CH3 H H
Cl Cl H Cl H Cl Cl
Cl Cl Cl OCH3 CH(CH3)2 Cl Cl
Cunninghamella echinulata (Thaxter). Recently, Vroumsia et al. (1996) reported that a number of fungal isolates were able to degrade diuron to some extent. One isolate of Rhizoctonia solani was able to degrade diuron, isoproturon and chlorotoluron. Shelton et al. (1996) demonstrated some degradation of diuron by a Streptomyces strain, which was also active against linuron and a range of acetanilide and striazine herbicides. However, to date there has only been one report of an isolated soil bacterium showing some activity against diuron (Roberts et al., 1998). The present study was initiated after the serendipitous discovery of unusually rapid diuron degradation in one soil sample during a study of small-scale spatial variability in phenylurea herbicide degradation. The objectives of the experiments reported here were to isolate microorganisms capable of rapid diuron degradation, to determine their ability to degrade related phenylurea compounds and to investigate their capacity to degrade diuron residues when inoculated into soil. 2. Experimental methods and results 2.1. Soils and herbicides The soil used was a sandy-loam of the Wick series containing (w/w) 16% clay, 70% sand and 1.9% organic matter, with a moisture content of 9.8% (w/w) at an applied pressure of 33 kPa. Soil was collected in October 1996 from Deep Slade ®eld at Horticulture Research International, Wellesbourne. This ®eld had been under cereal cultivation for the previous 3 yr, with cereals or experimental ®eld vegetable plots
grown before this. According to records, the site had never been treated with diuron, or any phenylurea herbicide other than isoproturon, an autumn application of which had been made for at least the last 3 yr. Five samples were taken at 5 m intervals along each of two parallel transects, separated by approximately 20 m. Soil pH (soil:deionized water; 1:1) ranged from 7.8±8.1 in the samples from transect 1 and from 6.5±6.8 in samples from transect 2. Organic matter content of sub-samples of each soil varied from 2.6±3.1% (w/w). Aside from pH, there was no discernible dierence between samples. Soils were allowed to lose excess water by air-drying overnight at room temperature, after which they were sieved (3 mm). Soils were transferred to sealed polyethylene bags, with sub-samples dried overnight at 1108C to determine initial water content. Diuron was added to soils as a commercial wettable powder formulation (80% active ingredient). Analytical grade compounds were used in all liquid cultures: diuron, linuron, monolinuron and isoproturon (British Greyhound, Birkenhead, UK); 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3-(3,4-dichlorophenyl)-urea (DCPU) (Promochem, Herts); metoxuron (Novartis Agrochemicals, Basel, Switzerland). Structures of these chemicals are shown in Table 1. All solvents used were HPLC grade. 2.2. Soil incubations Diuron was added to 250 g samples of each soil as an aqueous suspension of the commercial formulation (5 ml, 750 mg a.i. mlÿ1), resulting in a ®nal concentration of 15 mg kgÿ1. Additional deionized water was added to adjust the moisture content to 9.8% (soil
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Fig. 1. Diuron degradation at 158C in soil from 10 sites in Deep Slade ®eld, showing occurrence of rapid degradation in soil from one of the sites (± R ±).
water potential, ÿ33 kPa). Samples were thoroughly mixed by hand and transferred to loosely capped polypropylene bottles. Incubations were performed at 158C, with bottles enclosed in partially sealed polyethylene bags containing wet paper towelling in order to minimise evaporative water loss. Soil moisture content was maintained by periodic additions of deionized water, as required. Samples (15 g) were removed immediately after preparation and then at 7 d intervals, with herbicide residues extracted with 20 ml methanol by shaking for 1 h on a wrist-action shaker. Both diuron concentration and presence of any degradation products in the clear supernatant were determined directly by HPLC. The column used was Lichrosorb RP18 (250 4.6 mm; Merck) and compounds were detected by UV absorbance at 240 nm. The HPLC mobile phase was acetonitrile:water:phosphoric acid (70:30:0.25 by volume), with a ¯ow rate of 1 ml minÿ1. Under these conditions, the retention time of diuron was 3.6 min. Diuron degradation was slow in all 10 samples for the ®rst 29 d of incubation (Fig. 1). However, when
Fig. 2. Degradation of diuron at 158C following retreatment of soil from the `rapid-degrading' site (R) and from a `slow-degrading' site (q).
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sampled on day 43, diuron residues in one soil sample (transect 1; pH 7.8) had declined to 2.75 mg kgÿ1, whereas in all other soils (pH 6.5±8.1), the residual concentration varied from 15±19 mg kgÿ1. After incubation for 57 d, soil from the treatment showing rapid degradation and from one of the treatments showing slow degradation, was retreated with the same amount of diuron, as above, resulting in ®nal concentrations of 17 and 36 mg kgÿ1, respectively. Samples (10 g) were taken over a period of 20 d and herbicide residues were determined as before. Diuron residues in the `active' soil were reduced by over 75% after incubation for 5 d at 158C, at which time little degradation had occurred in the retreated `non-active' soil (Fig. 2). All soil samples remaining from these preliminary incubations were stored separately in capped bottles at 58C. 2.3. Enrichment culture After storage for ca. 3 months, the soil that degraded diuron rapidly received three further diuron retreatments, at a concentration of 40 mg kgÿ1. The ®rst two incubations lasted for 6 d, and the third for 2 d. These successive diuron applications were degraded rapidly, with residues from the third retreatment reduced from 47.3 to 6.1 mg kgÿ1 in just 2 d (Fig. 3). After the third incubation, 0.5 g sub-samples of the soil were used to inoculate duplicate bottles (100 ml, Duran) containing 20 ml of mineral salts medium (after Roberts et al., 1993) plus diuron (20 mg lÿ1), in which the herbicide constituted the sole source of carbon and nitrogen in the medium. Mineral salts medium comprised (g lÿ1 deionized water): (KH2PO4, 2.27; Na2HPO412H2O, 5.97; NaCl, 1.0), (MgSO47H2O, 0.5; CaCl22H2O, 0.01; MnSO44H2O, 0.02), (FeSO4, 0.025); medium pH was 6.9. During preparation of the medium, the ®rst two groups of compounds were autoclaved separately (15 min at 1218C) and combined
Fig. 3. Degradation of diuron at 158C following successive retreatments of the `rapid-degrading' soil; vertical lines indicate when retreatments were made.
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Fig. 4. Diuron degradation in shaken bottles of MSM (mean of two replicates) inoculated with diuron-degrading soil and incubated at 208C; vertical lines denote transfer of culture samples to fresh media.
when cool, to limit precipitation of metal ions as insoluble phosphate compounds. The iron sulphate solution was ®lter sterilised prior to adding to the cool medium. Analytical grade diuron was added to sterile, dry bottles as 0.1 ml of a 4 mg mlÿ1 ®lter-sterilised methanol solution. The bottles were left uncapped on a laminar ¯owbench until the methanol had evaporated. Mineral salts medium (MSM) was added aseptically to the bottles, which were then shaken for 1 h on a wrist-action shaker, to ensure complete dissolution of the pesticide. Duplicate control bottles were prepared, to which no soil was added. Following addition of the soil inoculum, bottles were loosely capped and incubated at 208C on an orbital shaker (140 rpm). Bottles were sampled immediately after addition of the soil, and then daily, with 0.5 ml culture aseptically transferred to an HPLC vial and mixed thoroughly with 1 ml acetonitrile. Diuron concentration was determined by HPLC, as before. After incubation for 2 d, 0.5 ml of culture was transferred to further bottles containing MSM plus diuron, and the concentration of diuron remaining was measured every 1±2 d. This procedure was repeated after 3 d incubation of the second set of bottles, giving a third cycle of liquid culture from which, after 5 d incubation, 0.5 ml samples were taken and used to inoculate a fourth set of bottles. The results from these repeated sub-cultures are shown in Fig. 4. Rate of degradation was most rapid in the initial liquid culture receiving the soil inoculum, in which residues declined from 23.3 to <1 mg lÿ1 in three days. Diuron degradation was accompanied by a progressive increase in concentration of a major metabolite with the same HPLC retention time (ca. 4 min) as 3,4-dichloroaniline. 2.4. Isolation of diuron-degrading bacteria After 4 d incubation at 208C, serial 10-fold dilutions of the fourth-cycle liquid culture in the experiment
described above were prepared in deionized water, and spread (0.1 ml) over plates of mineral salts agar (MSM plus 15 g lÿ1 agar, Difco Bacto). The agar contained diuron at 20 mg lÿ1, added after autoclaving as 5 ml of a ®lter-sterilised methanol solution lÿ1 of agar. Plates were incubated at 208C for 8 d, at which time 20 individual well-separated colonies were streaked out onto plates of mineral salts agar (MSA) plus diuron and also onto nutrient agar (Difco) + diuron (20 mg lÿ1) to check for purity. After incubation for 6 d at 208C, a single well-separated colony was selected from 10 of the 20 MSA plates and spread separately over further MSA + diuron plates with 0.1 ml sterile deionized water. Degrading ability of isolates was assessed after 7 d incubation at 208C, when growth was washed from each of the 10 MSA plates with 1.5 ml sterile deionized water and 0.5 ml of each suspension used to inoculate bottles containing 20 ml MSM + diuron, as before. Incubation conditions and sampling methodology were as described previously, with diuron concentration determined after 2, 3, 8 and 14 d. Subsamples of the liquid cultures in which diuron degradation had occurred were streaked onto plates of Nutrient agar (NA) + diuron and MSA + diuron; culture samples were also frozen at ÿ768C, following addition of sterile glycerol (15% ®nal volume). In a parallel experiment, a further 12 colonies from the initial dilution plates were introduced directly into wells of a micro-titre plate, with each well containing 300 ml MSM + diuron (20 mg lÿ1). The plate was incubated in a sealed sterile plastic box in a static incubator at 208C. After 2, 5, 8 and 13 d, 50 ml volumes were removed from each well and mixed thoroughly with 100 ml acetonitrile in the wells of another micro-titre plate, prior to analysis by HPLC. Well contents in which diuron degradation had occurred were streaked onto plates of MSA + diuron and NA + diuron, to check for purity. Little growth occurred on MSA + diuron plates, therefore a single colony was selected from each of the NA + diuron plates and transferred to 5 ml MSM + diuron (20 mg lÿ1) in sterile plastic bottles (25 ml). These were incubated as before and sampled at hourly intervals. When diuron had degraded to below 50% of its initial concentration, samples were frozen as before. A total of ®ve individual colonies out of the 32 selected from the initial MSA + diuron dilution plates degraded diuron. Four of these were detected by direct introduction of colonies into the microplate wells. Only one colony out of the 10 puri®ed and sub-cultured colonies introduced into the shaken-bottle assay was found to degrade diuron. In this bottle, degradation was complete after 3 d. In two wells of the microplate assay, diuron concentration was reduced to zero after just 2 d, with total degradation in another two wells when sampled after 5 d. No degradation had
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occurred in the remaining wells on the ®nal sampling at 13 d. All ®ve isolates appeared pure on NA and also retained degrading ability after sub-culture on NA + diuron. 2.5. Characterisation of diuron-degrading isolates Colony growth and morphology were examined on NA, following incubation at 30 and 428C. Cell morphology, motility and Gram-reaction were examined under a light microscope. Production of ¯uorescent pigment was determined on Kings Medium B (King et al., 1954). Catalase and oxidase activities were determined as described by Gerhardt et al. (1994), with oxidation±fermentation reaction and substrate utilisation tests performed using API20NE test strips (bioMeÂrieux sa) according to the manufacturer's instructions. Culture growth and cell morphology was similar for all ®ve isolates, with yellow±cream, circular, mucoid colonies on NA incubated at 308C; no growth occurred at 428C and no ¯uorescent pigment production was observed. Cells of all isolates were nonmotile, appearing as non-pigmented short rods together with smaller spherical cells which commonly occurred as pairs or in short chains. Isolates were classed as Gram-negative, although cells were dicult to de-stain; all were oxidase negative and catalase positive. Results of the API20NE tests were the same for all ®ve isolates, suggesting that they were the same organism. The substrate utilization tests gave the following results (+ = positive, 2 = weakly positive, ÿ = negative result): glucose (+), arabinose (ÿ), mannose (2), mannitol (+), N-acetyl-glucosamine (ÿ), maltose (+), gluconate (+), caprate (ÿ), adipate (ÿ), malate (+), citrate (+) and phenyl-acetate (2). Results of additional tests were: reduction of NOÿ 3 to NOÿ 2 (+), indole production from tryptophan (ÿ), glucose fermentation (ÿ), arginine dihydrolase activity (ÿ), urease activity (ÿ), aesculin hydrolysis (+), gelatin hydrolysis (ÿ) and b-galactosidase activity (2). One isolate (coded as D47), obtained from the shaken-bottle assay, was used in all subsequent degradation experiments.
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with chloroform in a vacuum desiccator for 7 d at 308C, whilst the other was stored at 48C in a sealed polyethylene bag. Residual chloroform was removed from the fumigated soil by repeat evacuations in a vacuum desiccator on a laminar ¯ow bench. Fumigated and non-fumigated soils were dispensed aseptically into sterile polypropylene containers, giving a ®nal soil weight of 125 g per container, after addition of sucient sterile deionized water to bring moisture content back to 9.8%. Inoculum was prepared from a culture of isolate D47 grown on NA + diuron. Growth was washed from the plates with 2 ml MSM and the bacterial suspension adjusted with MSM to give a theoretical density of 1.3 109 cfu mlÿ1, as estimated by comparison of absorbance at 600 nm (A600) with a previously-prepared standard curve. Viable cell count of the inoculum was determined by plating six replicate 20 ml droplets of appropriate serial dilutions in MSM onto NA (Miles and Misra, 1933), which gave an actual density of 1.2 109 cfu mlÿ1. A lower density inoculum was also prepared, to contain 1.2 106 cfu mlÿ1. For both fumigated and non-fumigated soils, duplicate samples were inoculated with 1 ml of either bacterial suspension, resulting in a ®nal inoculum density of 9.3 106 or 9.3 103 cfu gÿ1 moist soil. Single control pots received 1 ml MSM in place of the bacterial inoculum. Inoculum was thoroughly mixed into the soil under aseptic conditions, together with the ad-
2.6. Degradation of `aged' diuron residues in soil Soil samples from a further 10 sub-sites (on a 10 10 m grid) in Deep Slade ®eld were amended with a commercial formulation of diuron (15 mg kgÿ1). After 88 d incubation at 158C and 9.8% moisture content (matric potential ÿ33 kPa), none of the sub-sites showed rapid degradation and ca. 60±70% of the applied diuron remained in all 10 samples. At this time, the samples were combined and thoroughly mixed. The combined soil was divided into two 750 g amounts, of which one was sterilised by fumigating
Fig. 5. Degradation of `aged' diuron residues at 208C, in Deep Slade soil samples (mean of two replicates) inoculated with isolate D47 at densities of 9.3 106 cfu gÿ1 (a) or 9.3 103 cfu gÿ1 (b), in fumigated (± q ±) and non-fumigated (± Q ±) soil; controls, fumigated (- - q - -) and non-fumigated (- - Q - -), received no bacterial inoculum.
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Fig. 6. Degradation of the phenylurea herbicides linuron (Q), diuron (r), monolinuron (.), metoxuron (), isoproturon (+), and the derivatives DCPMU (- - r - -) and DCPU (- - * - -), at 208C in bottles of MSM inoculated with isolate D47 at a density of 9.3 106 cfu mlÿ1 (mean of two replicates).
ditional sterile deionized water required for moisture content adjustments. Pots were then incubated overnight at 48C, allowing re-distribution of the applied inoculum and water throughout the soil, before being transferred to a 208C incubator. Samples (10 g) were removed, under aseptic conditions, immediately on transfer to the incubator and at intervals over 16 d. Diuron residues were determined as before. Diuron degradation was most rapid in fumigated soil receiving 9.3 106 cfu gÿ1, with residues reduced by 50% (from 7.0 to 3.5 mg kgÿ1) in both replicates after 2 d incubation (Fig. 5). In this treatment, the initial rate of degradation was rapid, but then declined once diuron concentrations had fallen below ca. 2 mg kgÿ1. The rate of degradation in non-fumigated soil receiving the same inoculum load was slower (DT50=4±6 d). Of treatments inoculated with 9.3 103 cfu gÿ1, only fumigated soils showed a slight increase in rate of degradation over the non-inoculated control soils. Two metabolites were detected in the soil extracts, of which one was present in soil of all treatments at the time of inoculation. This was produced during the slow microbial degradation of diuron that occurred over the initial 88 d incubation and had the same retention time as DCPMU. The second metabolite, with the same retention time as 3,4-dichloroaniline (3,4-DCA), was only found in inoculated treatments which showed rapid rates of degradation. Amounts of 3,4-DCA ¯uctuated, although at the end of the experiment, after 16 d incubation, only a very low concentration was detected in the inoculated treatments. 2.7. Degradation of other phenylureas Analytical grade diuron, isoproturon, linuron, monolinuron, metoxuron, DCPMU and DCPU
(Table 1) were added to sterile bottles as ®lter-sterile methanol solutions, to give ®nal concentrations of 20 mg lÿ1 MSM, as described above. Duplicate bottles, containing 20 ml MSM + test compound, were inoculated with 160 ml of the bacterial suspension used in the previous soil inoculation study (1.2 109 cfu mlÿ1), giving a ®nal inoculum density of 9.3 106 cfu mlÿ1. Controls, receiving 0.16 ml MSM in place of the bacterial suspension, were prepared for each compound. Bottles were incubated at 208C on an orbital shaker for 28 d, with 0.5 ml samples removed at regular intervals as before. Analysis was by HPLC, under the conditions described above for diuron, with the exception of monolinuron, for which the mobile phase was changed to acetonitrile:water:H3PO4 (60:40:0.25 by volume). Out of the seven compounds tested, all but DCPMU and DCPU were degraded by the bacterium. Linuron showed the most rapid rate of degradation, declining to 25% of the initial concentration after 24 h incubation. The rate of degradation of the other four compounds was slower, proceeding in the order: diuron>monolinuron>>metoxuronoisoproturon (Fig. 6). Isoproturon degradation was particularly slow, with ca. 50% of the initial amount remaining after 28 d incubation. As with diuron, linuron degradation was accompanied by an increase in concentration of a product with the same retention time as 3,4-DCA; unidenti®ed degradation products also accumulated during monolinuron and metoxuron degradation, with retention times of 3.47 and 3.34 min, respectively. Low concentrations of a metabolite with the same retention time as 4-isopropylaniline were detected during isoproturon degradation. 2.8. Isoproturon degradation, in the presence of an additional carbon and nitrogen source In order to determine whether isoproturon degradation rate could be increased by changes in the nutrient composition of the liquid medium, a sample of culture (0.5 ml) was removed from inoculated bottles containing MSM + isoproturon (in the experiment described above) after 24 d incubation and spread onto NA + isoproturon (20 mg lÿ1) plates. Following incubation for 2 d at 258C, growth was washed from a plate with 2 ml MSM and the bacterial suspension amended with further MSM to give a density of 4.1 108 cells mlÿ1, as estimated from A600 readings. Viable cell numbers, determined by colony counts on NA, gave a measured inoculum density of 3.2 108 cfu mlÿ1. Bottles were prepared with 20 ml of the following media: MSM + isoproturon (20 mg lÿ1), MSM + isoproturon + glucose (added as 0.1 ml ®lter-sterile solution in deionized water; ®nal concentration of 1 g lÿ1), MSM + isoproturon + NH4Cl
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Fig. 7. Isoproturon degradation in bottles (mean of two replicates) of MSM (w), MSM + glucose (Q), MSM + NH4Cl (R), and MSM + glucose + NH4Cl (q) inoculated with 7.7 106 cfu mlÿ1 of isolate D47 and an uninoculated MSM control (); degradation of diuron in MSM (W) by the same inoculum is shown for comparison.
(1 g lÿ1; replacing NaCl in the original MSM), MSM + isoproturon + glucose + NH4Cl. For comparison, bottles were also prepared with 20 ml MSM + diuron (20 mg lÿ1), but with no additional carbon or nitrogen source. Bottles not receiving 0.1 ml glucose solution were amended with 0.1 ml sterile deionized water. Duplicate bottles of each medium were inoculated with 0.5 ml bacterial suspension, giving a ®nal density of 7.7 106 cfu mlÿ1. A control bottle containing MSM + isoproturon received 0.5 ml MSM in place of the bacterial inoculum. Degradation of isoproturon was again slow when the pesticide provided the sole source of carbon and nitrogen, with over 50% remaining after 29 d incubation (Fig. 7). A similar rate was also seen with treatments receiving glucose or NH4Cl. However, in the presence of both glucose and NH4Cl, degradation was very rapid, with almost 90% degradation after just 6 d. During incubation, the inoculum density in this treatment increased markedly, rendering the culture opaque in comparison to the other, almost clear, cultures. Diuron degradation was again rapid, with only 12% remaining after just 2 d incubation. In the isoproturon treatment amended with glucose and NH4Cl, a single metabolite accumulated, with the same retention time as 4-isopropylaniline. Only trace amounts of this metabolite were detected in the other slow-degrading isoproturon treatments.
3. Discussion No previous reports of rapid biodegradation of diuron in ®eld soils have been reported; degradation is usually slow and follows ®rst order kinetics (Hill et al., 1955; Madhun and Freed, 1987). In our study, degradation in one soil sample out of the 10 examined was
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characteristically slow until some time between 29 and 43 d incubation, when a dramatic increase in rate occurred. It is dicult to attribute this rapid degradation of diuron to peculiar physical or chemical characteristics of this one soil sample. Soils from adjacent sites on transect 1, showing slow rates of diuron degradation, were of similar pH and, separated by only 5 m, were unlikely to have diered appreciably in other, unmeasured, properties. The ®eld site had, to all knowledge, never been treated with diuron, thus precluding the development of an adapted micro¯ora as a result of repeated exposure to the compound. One could perhaps speculate on whether the annual application of (structurally similar) isoproturon to this ®eld in¯uenced the diuron-degrading potential of the soil micro¯ora. However, failure to detect rapid diuron degradation in nearby, physically and chemically similar, soil samples suggests that this is unlikely. The rapid degradation that occurred in this soil sample was subsequently enhanced by retreatment with diuron, resulting in a DT50 of less than 24 h following the fourth and ®fth applications. The retreatment of a `slow-degrading' soil sample did not result in an increase in the rate of diuron degradation, over the 20 d duration of the experiment. It cannot be ruled out that, after more prolonged incubation, the rate of diuron degradation in this soil may have increased, as observed for the `active' soil in the initial incubation. However, there are no previous reports of acceleration of degradation in sites subjected to multiple diuron applications, indicating that eventual microbial adaptation to diuron degradation is not the norm. All ®ve diuron-degrading colonies were identical in appearance and in response to characterisation tests and would appear to be the same organism. Therefore it is likely that this bacterium constituted the major culturable, and possibly only, diuron-degrading component of the mixed culture. It was not possible to assign the bacterium to a de®nitive genus from the results of the characterisation tests alone; this would require further fatty acid- or nucleic acid-based procedures. Introduction of the higher density cell suspension of this isolate into fumigated soil containing `aged' diuron residues resulted in rapid degradation. Degradation in non-fumigated soil receiving the same inoculum load was somewhat slower. With the low density inoculum, detectable degradation occurred only in the fumigated soil. This could be attributed to a growth-promoting eect resulting from the liberation of nutrients from dead cells, or to a reduction in competition or antagonism by eradication of the indigenous soil micro¯ora. The bene®cial eect of soil fumigation on the ecacy of subsequently introduced inoculum has been shown by Gunalan and Fournier (1993) and Duquenne et al. (1997). Diuron residues had been present in the soil
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for over 12 weeks, during which time the overall rate of degradation had been very slow; yet on introduction of the diuron-degrading isolate, residue levels declined rapidly. Previous studies with a range of pesticides have demonstrated that increased residence time in soil results in stronger adsorption, with desorption to the soil solution becoming more dicult (Walker, 1987; Blair et al., 1990; Gaillardon et al., 1991; Walker et al., 1995). Working with sulphonylurea herbicides, Duy et al. (1993) provided evidence that degradation occurred primarily in the water phase of soil, with restricted rates of release from the adsorbed phase leading to reduced rates of degradation over time. These studies would suggest that `aged' residues are relatively unavailable to microbial attack. In the current experiment, aged diuron residues were degraded rapidly by the introduced bacterium. However, in the absence of parallel studies with freshly-added diuron, conclusions cannot be drawn on the possible in¯uence of increased pesticide residence time in soil on diuron degradation by this bacterium. This diuron-degrading bacterium was able to degrade both N 0 -methoxy phenylurea herbicides (linuron and monolinuron) and N 0 N 0 -dimethyl phenylureas (diuron, metoxuron), but had low activity against isoproturon. In contrast, in many previous investigations into bacterial degradation of phenylureas, activity has only been demonstrated against methoxysubstituted compounds (WallnoÈfer, 1969; Engelhardt et al., 1971; Roberts et al., 1993). An exception to this is the study of Roberts et al. (1998), where a bacterial isolate was obtained which was able to degrade both N 0 N 0 -dimethyl-substituted compounds (isoproturon, diuron) and also the methoxy-substituted compound linuron. However, in studies with phenylurea-degrading fungi, both dimethyl- and methoxy-substituted compounds have been shown to be readily metabolised by an individual isolate (Weinberger and Bollag, 1972; Tillmanns et al., 1978; Shelton et al., 1996). In the current study, degradation rate of phenylurea herbicides and derivatives would appear to be related to several structural features of the molecule. Linuron, with an N 0 -methoxy group, was degraded more rapidly than diuron, with a methyl in place of the methoxy group of linuron. However, substitution of one or two hydrogen atoms in place of the N 0 -methyl groups of diuron (DCPMU and DCPU, respectively) resulted in total inhibition of degradation. Substituents on the phenyl ring also greatly in¯uenced the rate of degradation. Removal of the 3-chlorine substituent from linuron, i.e. monolinuron, reduced the rate of degradation; substitution of a methoxy group for the 4-chlorine of diuron, i.e. Metoxuron, gave considerably slower degradation. The 4-isopropyl group of isoproturon, in place of the 3,4-dichloro substituents of diuron, caused a greater than 10-fold reduction in the rate of degra-
dation. These comparisons suggest that the size or nature of the urea N 0 -substituents is a determining factor in the occurrence of degradation and that ring chlorination, particularly at the 4-position, would appear to greatly enhance rates of degradation. In studies with several fungal isolates, Sharabi and Bordeleau (1969) observed a similar degradationenhancing in¯uence of 3,4-dichloro substitution with a range of acylanilides, although none of their isolates showed activity towards diuron. Roberts et al. (1993) also showed the apparent requirement for chlorination of the phenyl ring in studies with a mixed linurondegrading culture: chlorbromuron was degraded, but metobromuron, lacking the chlorine ring substituent, was not. In contrast, Vroumsia et al. (1996) found that isoproturon (no ring chlorination) was more readily degraded than diuron, in studies with a range of fungal isolates. In the current study, the rate of isoproturon degradation was greatly enhanced in the presence of a supplementary carbon and nitrogen source. This suggests that the degrading enzymes involved had low activity towards isoproturon, which was compensated for by the considerable increase in inoculum density (and hence enzyme production) which occurred under these conditions. Degradation of diuron and linuron by this isolate resulted in the accumulation of only one detectable break-down product, which would appear to be 3,4dichloroaniline; isoproturon degradation resulted in the apparent accumulation of 4-isopropylaniline. Detection of these metabolites would suggest that degradation proceeded by hydrolysis of the bond between the amino group of the aniline moiety and the carbonyl group of the substituted urea component. The degradation process may perhaps occur by a sequence of reactions, through a number of intermediate products, rather than by a direct hydrolysis; however, these may be short-lived (or polar) and hence were not detected by HPLC. There was no evidence of further breakdown of 3,4-DCA; in liquid culture, concentrations of this metabolite increased in line with the decrease in levels of parent compound and then remained stable. Degradation of monolinuron and metoxuron also resulted in accumulation of unidenti®ed metabolites; these were presumed to be the respective anilines. This pattern of degradation suggests that the bacterium was utilising the substituted urea component of the molecule as a carbon and nitrogen source. Studies with the carbamate insecticide carbofuran have also identi®ed bacterial isolates which, in a similar fashion, hydrolyse the methyl-carbamate side chain from the molecule, but do not metabolise the aromatic remainder, 7-carbofuran phenol (Karns et al., 1986; Chaudhry Rasul and Ali, 1988; Parekh et al., 1994). This hydrolysis yields methylcarbamic acid, which spontaneously decomposes to yield CO2 and
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methylamine (Getzin, 1973), with the latter compound believed to be used by the bacteria as a carbon or nitrogen source (Karns et al., 1986; Chaudhry Rasul and Ali, 1988). Hydrolysis of diuron at the carbonyl group would produce dimethylcarbamic acid which, as for methylcarbamic acid, would decompose to CO2 and dimethylamine. It is possible that, as with the carbofuran degrading isolates, the latter compound is being utilised by the bacterium as a carbon and nitrogen source. However, in a previous investigation into bacterial metabolism of linuron (Engelhardt et al., 1972), both 3,4-dichloroaniline and N,O-dimethylhydroxylamine (from the urea side chain) were found to accumulate as degradation products, with CO2 also detected (from the urea carbonyl group). This earlier study would therefore suggest that, in contrast to the carbofuran-degrading isolates, the amine produced after hydrolysis of linuron is not being utilised by the bacterium. Further studies are required to determine which components of the urea side chain are being utilised by the diuron-degrading bacterium in our study. In other work with diuron, monomethyl and demethylated degradation products have been detected in addition to the aniline, suggesting that degradation in soil usually proceeds by successive N-demethylation, followed by hydrolysis of the urea to give the aniline (Dalton et al., 1966; Tillmanns et al., 1978; Ellis and Camper, 1982). In our experiments, these additional metabolites were detected in the initial soil incubations, but never during degradation mediated by isolate D47, in liquid culture, or when added to soil. The apparent inability of this isolate to degrade the monomethyl and demethylated breakdown products of diuron con®rms that diuron degradation did not proceed by N-demethylation. Bacteria isolated in the study of Roberts et al. (1998) appeared to degrade isoproturon by stepwise demethylation, with one isolate also showing degradation of diuron. In their study, diuron degradation by this isolate was accompanied by production of DCPMU (but not 3,4-dichloroaniline), suggesting occurrence of degradation by the usually observed demethylation pathway. In conclusion, this would appear to be the ®rst report of accelerated biodegradation of diuron in soil and only the second of a single bacterium able to degrade this herbicide. The apparent con®nement of rapid diuron-degrading activity to a single sub-site within a previously untreated ®eld is curious. It would be interesting to determine whether this was a fortuitous, chance occurrence, or if this isolate (or other diuron-degrading microorganisms) is in fact of wider distribution, albeit perhaps at very low population densities. The rapid diuron degradation observed on addition of this isolate to non-sterile soil suggests that, if introduced to sites treated with this or the other phenylureas against which it was active, equally rapid her-
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bicide degradation would occur in the ®eld. If the bacterium was suciently competitive to survive in soil after the phenylureas had disappeared, then it is also possible that future applications of these herbicides would undergo rapid degradation. The apparent con®nement of rapid diuron-degrading activity to a single sub-site in the ®eld, however, suggests that prior to laboratory treatments with diuron, the bacterium was occupying a very isolated niche. In this situation, it would be unlikely to have a signi®cant eect on herbicide ecacy.
Acknowledgements We are grateful to the UK Biotechnology and Biological Sciences Research Council for funding this work and also to Alun Morgan, Horticulture Research International, Wellesbourne, for characterisation of isolates.
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