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Dynamics of carbofuran-degrading microbial communities in soil during

three successive annual applications of carbofuran

S.L. Trabue, A.V. Ogram, L.-T. Ou*

Soil and Water Science Department, P.O. Box 110290, University of Florida, Gainesville, FL 32611-0290, USA

Received 4 October 1999; received in revised form 3 May 2000; accepted 29 May 2000

Abstract

The in¯uence of repeated annual ®eld applications of the insecticide±nematicide carbofuran on carbofuran-degrading microbial commu-nities was studied at a site in Florida that exhibited enhanced degradation toward the chemical. Three successive annual applications of carbofuran at 4.5 kg ha21y21did not result in an increase in the size of the microbial community capable of mineralizing uniformly ring-labeled 14C-carbofuran (carbofuran-ring degraders) in surface soil (0±15 cm depth). After the second annual application, however, the community capable of mineralizing carbonyl-labeled14C-carbofuran (carbofuran hydrolyzers) in the treated surface soil was signi®cantly larger than that after the ®rst annual application. Communities of methylamine utilizers in treated and untreated soils were much larger than the communities of carbofuran phenol degraders, but not statistically different from the sizes of carbofuran hydrolyzers. In addition to no increase in the number of carbofuran ring degraders in the treated site during three consecutive annual applications of carbofuran, laboratory addition of 10mg carbofuran g21to treated and untreated soils collected in 1995 did not result in an increase in the number of carbofuran ring

degraders. This suggests that degradation of the ring structure of carbofuran in soil is a cometabolic process.q_{2001 Elsevier Science Ltd.}

Keywords: Carbofuran; Most-probable-number; Carbofuran ring degraders; Carbofuran hydrolyzers; Enhanced soil

1. Introduction

It has been known since 1981 that repeated ®eld applica-tions of the insecticide±nematicide carbofuran (2,3-dihy-dro-2,2-dimethyl-7-benzofuranyl methylcarbamate) cause enhanced degradation of the chemical in the soil (Felsot et al., 1981; Read, 1983; Camper et al., 1987; Turco and Konopka, 1990). As a consequence of enhanced degrada-tion, there may be a loss in the ef®cacy of the pesticide to control target pests, resulting in crop failure (Felsot et al., 1981; Racke and Coats, 1990). Enhanced degradation of pesticides in soils is a microbial process (Racke and Coats, 1990). There are two schools of thought on the mechanism by which microorganisms in soil develop enhanced degradation. One is as a consequence of repeated applications of a pesticide, where an increase in the number of microorganisms capable of degrading the chemical results in more rapid degradation of the chemical compared to untreated soil. Evidence supporting this hypothesis is based on the observations that after addition of the phenoxy herbicide 2,4-D (Ou, 1984; Holben et al., 1992; Ka et al.,

1994) or the organophosphate insecticide isofenphos (Racke and Coats, 1987), the numbers of soil microorganisms capable of degrading the chemicals increased substantially. The other hypothesis is that repeated applications of a pesti-cide result in an increase in the enzyme activity, but not community size, speci®cally toward the degradation of the pesticide. Evidence for the second hypothesis is supported by the ®ndings that the sizes of EPTC-degrading commu-nities in soils with or without a history of ®eld applications of the herbicide were similar (Moorman, 1988).

At present, published information on the changes of carbofuran-degrading community sizes in soils after devel-opment of enhanced degradation toward carbofuran is mixed. Hendry and Richardson (1988) and Dzantor and Felsot (1989, 1990) pointed to an increase in the number of carbofuran degraders in soils with enhanced degradation compared to the number of the indigenous carbofuran degraders. Racke and Coats (1988), Merica and Alexander (1990), Scow et al. (1990) and Robertson and Alexander (1994) failed to link an increase in the number of carbofuran degraders with the number of carbofuran applications in enhanced soils.

There are likely to be three groups of microorganisms that may be involved in the degradation of carbofuran in soils

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(Chaudhry and Ali, 1988). Groups 1 and 2 are capable of hydrolyzing carbofuran and utilizing methylamine, one of the hydrolysis products, as a sole source of N, and as sole source of C and N for growth, respectively. Group 3 utilizes the aromatic ring as a sole source of carbon. It is likely that group 3 also has the capacity to hydrolyze carbofuran and then utilize the aromatic ring of carbofuran phenol, one of the hydrolysis products, for growth. Thus, all three groups are likely involved in hydrolysis of the carbamate linkage. The majority of carbofuran-degrading bacteria that have been isolated from soils belong to group 2. Topp et al. (1993) isolated a methylotrophic bacterium from an agricul-tural soil that hydrolyzed carbofuran. If some carbofuran hydrolyzers are methylotrophic bacteria, the number of the microorganisms should increase after carbofuran hydro-lysis. Hendry and Richardson (1988) reported a signi®cant increase in the size of carbofuran hydrolyzers from 1:6£

103 to 3:1£10 5

cells g21after one laboratory treatment of soil with carbofuran. However, Merica and Alexander (1990) and Robertson and Alexander (1994) failed to link carbofuran degradation with the growth of carbofuran hydrolyzers as well as ring degraders.

We located a ®eld site in Florida that had been treated with carbofuran annually two consecutive times, enhanced degradation of carbofuran in soil at this site was observed (Ou, unpublished observation). This site provided an unique opportunity to ®nd out whether enhanced degradation of carbofuran was due to an increase in microbial communities capable of degrading carbofuran or due to an increase in enzyme activity per cell (cometabolism). We determined changes of microbial communities capable of degrading carbofuran (carbofuran ring degraders and carbofuran hydro-lyzers) in soil from this site after two to three successive annual ®eld applications of carbofuran. In addition, microbial community sizes capable of degrading carbofuran phenol or methylamine were determined as part of this study.

2. Materials and methods

2.1. Chemicals

Technical grade carbofuran (99% purity), analytical grade carbofuran phenol (99.3% purity), carbonyl-labeled (CBL)14C-carbofuran (speci®c activity, 131 MBq mmol21), and uniformly ring-labeled (URL) 14C-carbofuran (speci®c activity, 229 MBq mmol21) were provided by FMC Corpora-tion (Princeton, NJ). Prior to use, the two 14C-labeled compounds were puri®ed to.98% radiopurity by thin-layer chromatography (TLC) using preparative silica gel G TLC plates (Ou et al., 1982, 1985). URL 14C-carbofuran phenol was obtained by alkaline hydrolysis of URL14C-carbofuran overnight at 708C, and was puri®ed by preparative TLC to .98% radiopurity. Analytical grade methylamine hydro-chloride (.99% purity) was purchased from Aldrich (Milwaukee, WI).

2.2. Field plots, carbofuran treatments and soil sampling

Field plots at an experimental site located in northeast of Florida (60 km northeast of Gainesville, FL) were treated with carbofuran annually in March or April from 1992 to 1996 at a rate of 4.5 kg ha21. This site had been under continuous potato cultivation for more than 10 y. Control (untreated) plots at this site had never been treated with carbofuran or any structurally similar pesticides, but received fertilizers at rates identical to the treated plots. Soil samples were collected from three plots at two depths (0±15 and 45±60 cm) 4 weeks after annual applications of carbofuran in three consecutive years (third, fourth and ®fth annual applications in 1994, 1995 and 1996, respectively). Soil samples from each depth were combined and mixed. Soil samples were stored in plastic bags in the dark at 4₈C and used within 3 months after collection. All soil samples were classi®ed as Ellzey ®ne sand (sandy, siliceous, hyperthermic Arenic Ochraqualfs). Key soil properties of the samples have been reported by Trabue et al. (1997).

2.3. 14C-most-probable-number (MPN) assay

A14C-MPN technique similar to that used by Ou (1984) to determine 2,4-D-degrading microbial community sizes in soils was employed to determine the sizes of microbial communities capable of degrading carbofuran or carbofuran phenol. URL or CBL 14C-carbofuran were used to deter-mine community sizes capable of deter-mineralizing the aromatic ring structure or hydrolyzing the carbamate linkage of carbofuran. URL 14C-carbofuran phenol was used to esti-mate the numbers of microorganisms capable of mineraliz-ing the phenolic structure of carbofuran phenol.

One gram of soil (oven-dry weight basis) was transferred to a sterile capped MPN tube containing 9 ml of a minimal mineral medium, including 10mg tryptone ml21 (Ou, 1984). The medium also contained 10mg ml21of technical grade carbofuran and 16.7 Bq ml21of URL14C-carbofuran, CBL 14C-carbofuran or URL 14C-carbofuran phenol. Five replicates of successive 5- to 10-fold dilutions were made. All tubes were incubated in the dark at 288C for 28 d. Control MPN tubes were treated as described above, except that soil was not added to the tubes. At the end of the incubation, 0.1 ml of concentrated HCl was added to each tube, and after mixing, they were kept in a fume hood for 4± 6 h. Afterwards, 0.5 ml of the MPN media was assayed for

14

C-activity by liquid scintillation counting (LSC). Tubes were scored as positive when #70% of the initial 14 C-activity remained in solution. 14C-activity in all control

14

C-MPN tubes was unchanged after 28 d.

To determine community size changes after laboratory treatment of carbofuran, 100 g of soil (oven-dry weight basis) were added to a 250 ml Erlenmeyer glass ¯ask with a cotton plug and mixed with 1.0 mg of technical grade carbofuran to give a concentration of 10mg g21. The ¯asks were incubated in the dark at ambient temperature

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(23^28C), and once a week their weights were checked, sterile deionized water was added to compensate for any water loss. Periodically, 1 g of soil was removed and placed in a sterile MPN tube containing 9 ml of the MPN medium (including 10mg tryptone ml21), technical grade carbofuran (10mg ml21), and either URL 14C-carbofuran, CBL 14 C-carbofuran or URL 14C-carbofuran phenol. Community sizes were estimated as above.

2.4. MPN for methylamine utilizers

An MPN technique that determined the community size capable of utilizing an organic substrate for growth was used for determination of the number of microorganisms capable of utilizing methylamine for growth (Alexander, 1982). The MPN method was similar to the 14C-MPN assay, except that non-labeled methylamine hydrochloride equivalent to 100mg methylamine ml21 was added to the MPN tubes as a sole source of carbon and no tryptone was added. Five replicates of successive 5-fold dilutions were made. Tubes were scored positive if turbidity developed in the MPN solutions in the tubes after 28 d at 288C. Control tubes, which contained the MPN medium and methylamine, were all devoid of turbidity after 28 d.

2.5. Statistical analysis

Numbers of microorganisms per gram soil based on the MPN assays were obtained using the Eureka software (Borland International, Scotts Valley, CA). The MPN and the 95% con®dence limits were estimated by the software.

3. Results

3.1. Carbofuran degrading communities in ®eld soils

The numbers of microorganisms capable of mineralizing the aromatic ring of carbofuran (ring degraders) in treated surface soils (0±15 cm depth) collected 4 weeks after three annual ®eld applications of carbofuran were not statistically different (Table 1). The MPN in treated subsurface soils (45±60 cm depth) were also not statistically indistinguish-able, with the exception of the subsurface soil collected in 1995. The MPN in this soil was signi®cantly larger than the MPN in the other two subsurface soils. The MPN in the treated surface soils were statistically larger than the MPN in the corresponding subsurface soils, with the exception of the surface and subsurface soils collected in 1995. The MPN in the two soils were not statistically different. The MPN in untreated surface soils collected in 1994 and 1995 were not signi®cantly different from each other, nor were they signif-icantly different from the treated surface soils, with the exception of the untreated surface soil collected in 1995. The MPN in this soil was statistically smaller than the MPN in the treated surface soil collected in the same year. The MPN in untreated subsurface soils collected in three consecutive years as well as in untreated surface soils collected in 1996 were zero.

The MPN of microorganisms capable of hydrolyzing the carbamate linkage of carbofuran (carbofuran hydrolyzers) in the treated and untreated soils collected in 1994 and 1995 (Table 2) were three to ®ve orders of magnitude larger than the MPN of carbofuran ring degraders. The MPN of carbo-furan hydrolyzers in the treated surface soil collected in 1995 was signi®cantly larger than the MPN in the treated

S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81

Table 1

MPN (cells g21_{) of carbofuran ring degraders in treated and untreated} surface soils collected in three consecutive years (1994±1996); CI Ð con®dence interval

Depth (cm) MPN Lower 95% CI Upper 95% CI

Treated soil (1994)

0±15 21.6abca _6.5 _71.4

45±60 2.0d 0.6 6.2

a _{Values followed by the same letter are not signi®cantly different}

P,0:05:

Table 2

MPN (cells g21_{) of carbofuran hydrolyzers in treated and untreated surface} soils collected in 1994 and 1995; CI Ð con®dence interval

Depth (cm) MPN Lower 95% CI Upper 95% CI

Treated soil (1994)

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and untreated surface and subsurface soils collected in 1994, but not signi®cantly different from the MPN in the untreated surface soil collected in 1995. However, the MPN in the untreated surface soil collected in 1995 was not statistically different from the MPN in the treated surface soil, and untreated surface and subsurface soils collected in 1994, but signi®cantly larger than the MPN in the treated subsur-face soil collected in 1994. Due to larger than usual preci-pitation in late winter and early spring in 1995, the water table at the site at the time of sampling was fairly shallow; occurring at 50±55 cm from the soil surface.

3.2. Carbofuran phenol- and methylamine-degrading communities in ®eld soils

The MPN of microorganisms capable of utilizing methy-lamine in the treated and untreated surface soils collected in 1995 were much larger 5£105 times) than the MPN of microorganisms capable of mineralizing the aromatic ring of carbofuran phenol (Table 3). The MPN of methylamine utilizers in the treated and untreated surface soils were not statistically different from each other. It is interesting to point out that the MPN of methylamine utilizers and carbo-furan hydrolyzers in the two soils were not statistically different (Tables 2 and 3).

The numbers of carbofuran phenol ring degraders in the treated and untreated soils averaged 16 and 9 cells g21

soil, respectively, and were statistically indistinguishable (Table 3). The MPN of carbofuran ring degraders and carbofuran phenol ring degraders in treated and untreated soils were all small (Tables 1 and 3). They were not statistically different in the untreated soil (Tables 1 and 3). But the MPN of carbofuran ring degraders in the treated soil was statistically larger than the MPN of carbofuran phenol ring degraders.

3.3. Growth of carbofuran degraders after a laboratory treatment of carbofuran

Laboratory treatment of carbofuran at 10mg g21 to the ®eld treated surface and subsurface soils collected in 1995 did not result in a statistically signi®cant increase in the MPN of carbofuran ring degraders after 28 d of incubation (Fig. 1A). After 14 d, the MPN in the ®eld treated surface soil was signi®cantly smaller than before the laboratory treatment. The MPN of carbofuran ring degraders in the ®eld untreated surface soil did not change after laboratory treatment with carbofuran, with the exception that 14 d after the treatment, the MPN was signi®cantly smaller than before the treatment. In the untreated subsurface soil, carbo-furan ring degraders were not detected until 28 d after the laboratory treatment of carbofuran, when 4.5 cells g21were detected in this soil.

The MPN of carbofuran-hydrolyzers in the ®eld treated surface soil collected in 1995 was initially very large1:23£

107cells g21soil), and after laboratory treatment with carbo-furan at a rate of 10mg g21soil, the MPN remained stable during the ®rst 7 d of incubation (Fig. 1B). After 14 d, the MPN declined signi®cantly. The MPN of carbofuran hydro-lyzers in the ®eld untreated surface soil did not change during the ®rst 3 d after the laboratory application of carbofuran; however, between days 3 and 7, the MPN increased signi®-cantly. After 14 d, it declined to the level statistically similar to that before the treatment.

3.4. Growth of carbofuran phenol degraders and methylamine utilizers after laboratory treatment with carbofuran phenol and methylamine

After laboratory treatment of URL14C-carbofuran phenol

Table 3

MPN (cells g21_{) of methylamine utilizers and carbofuran phenol ring} degraders in treated and untreated surface soils collected in 1995; CI Ð con®dence interval

Degraders MPN 95% CI Upper 95% CI

Treated soil

Methylamine 7:93£10

6_aa ₂ :40£10

6 ₂

:62£10

7

Carbofuran phenol 16.0b 6.2 41.2

Untreated soil

Methylamine 4:93£10

6_a ₁

:49£10

6 ₁

:62£10

7

Carbofuran phenol 8.5b 3.3 21.9

P,0:05:

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at 10mg g21 to ®eld treated and untreated surface soils collected in 1995, the MPN of carbofuran phenol ring degra-ders in these two soils were statistically indistinguishable during the entire 28 d of incubation (Fig. 2A). Even though the average MPN of carbofuran phenol degraders in the surface soil after 28 d was 6.8 times larger than before the laboratory treatment, due to large errors associated with the MPN determination, the two numbers were not statisti-cally different. Results similar to the MPN of carbofuran phenol degraders were obtained for the MPN of methyla-mine utilizers in treated and untreated surface soils (Fig. 2B). The average MPN in the treated soil 7 d after the laboratory treatment was 10 times larger than before the

laboratory treatment, although they were not signi®cantly different.

4. Discussion

In this study, we chose MPN assays over other enumera-tion techniques. Diluenumera-tion-plate count methods were inade-quate. Although small semi-transparent watery colonies developed on basal mineral-carbofuran agar plates after 5±7 d of incubation, no carbofuran degradation and no growth were observed when biomass of individual colonies was inoculated into the liquid basal mineral-carbofuran medium (Ou, unpublished observations). Methods such as gene probing are not available for those strains. Only one gene involved in carbofuran degradation has been cloned. This gene, the methyl carbamate degrading (mcd) gene, was cloned fromAchromobactersp. WM111 and encodes for a carbofuran hydrolase (Tomasek and Karns, 1989). mcd

appears to be not widely distributed among carbofuran degraders in the US. DNA isolated from many soils that have been exposed to carbofuran did not hybridize with this gene (Derk et al., 1993). 14C-MPN assays at present appear to be the only means for determination of carbo-furan-degrading communities in soils.

The sequence of carbofuran degradation in soil is ®rst via hydrolysis to carbofuran phenol and methylamine. Carbo-furan phenol is degraded further through aromatic ring clea-vage, and eventually to CO2 and H2O (Ou et al., 1982;

Trabue et al., 1997). Therefore, the same microorganisms that mineralized the aromatic ring of carbofuran were likely also to be responsible for carrying out mineralization of the aromatic ring of carbofuran phenol. It was not clear why the MPN for carbofuran ring degraders was statistically larger than the MPN for carbofuran phenol ring degraders in trea-ted soil collectrea-ted in 1995 where there was enhanced degra-dation of carbofuran (Trabue et al., 1997). They were all small in numbers, however.

Carbonyl-labeled 14C-carbofuran was used to determine the number of carbofuran hydrolyzers in soil based on the

14

C-MPN technique. When the carbamate linkage of carbo-furan is broken by hydrolysis, 14C-carbonyl is immediately converted to 14CO2, carbofuran phenol and methylamine

(Fig. 3). Due to unavailability of furanyl ring-labeled

Fig. 2. Most probable numbers (MPN) of carbofuran phenol ring degraders (A) and methylamine utilizers (B) in ®eld treated and untreated surface soils after receiving 10mg carbofuran phenol g21and 10mg methylamine g21, respectively, in the laboratory during 14 or 28 d of incubation. Soil samples were collected from treated and untreated plots in 1995.

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14

C-carbofuran, the number of microorganisms capable of degrading the furanyl ring could not be determined. However, it is conceivable that carbofuran ring degraders may also have the capacity to mineralize the furanyl ring. We found that URL14C-carbofuran in treated and untreated surface and subsurface soils collected in 1995 from the same site was rapidly mineralized to 14CO2(Trabue et al., 1997).

Prior to this study, the treated site had been treated with carbofuran annually for two consecutive years and soil of this site was found to exhibit enhanced degradation toward carbofuran after the second annual treatment (1993) (Ou, unpublished observation). Enhanced degradation was conti-nually observed from 1994 to 1996 (Trabue, Unpublished PhD thesis, University of Florida, 1997; Trabue et al., 1997). Despite the fact that carbofuran was applied to the treated site annually at a rate of 4.5 kg ha21for ®ve conse-cutive years, the MPN of carbofuran ring degraders in the surface soils during the last three annual applications were small and not statistically different. This suggests that mineralization of the ring structure of carbofuran was mainly by cometabolism.

The fact that the MPN of carbofuran hydrolyzers and methylamine utilizers were not statistically different may be fortuitous. However, some carbofuran-degrading bacteria are methylotrophic organisms (Chaudhry and Ali, 1988; Singh et al., 1993; Topp et al., 1993). They were capable of hydrolyzing carbofuran and utilizing methyla-mine as a sole source of C for growth. It is known that methylotrophic bacteria utilize 1 C compounds, including methane and methylamine, as a sole source of C for growth (Hanson and Hanson, 1996). Large communities of metha-notrophs ranging from 106to 108cells g21soil exists in oxic soils (Hanson and Hanson, 1996). Since methanotrophs are a subgroup of methylotrophs, the sizes of methylotrophic communities in soils should be at least equal or larger than the sizes of methanotrophic communities. In our study, community sizes of methylamine utilizers in the two soils were similar to the reported values for methanotrophs.

Our ®ndings suggest that some carbofuran hydrolyzers may also be methylamine utilizers. Hydrolysis of 4.5 kg carbofuran ha21(equivalent to 2mg g21) will result in the formation of 280 ng methylamine g21soil. Thus, the methy-lamine will provide 110 ng C and 130 ng N g21for serving as C and N sources for methylotrophic bacteria. A typical bacterial cell has a dry mass of 280 fg and consists of 50% C and 14% N (Neidhardt et al., 1990). Thus, 280 ng methyla-mine g21 could support from 7:9£10

5

to 3:3£

106cells g21soil, if N and C are not limiting factors, respec-tively. Due to the large difference between the lower and upper con®dence intervals (Table 3), even an increase of 3:3£10

6

cells g21would not result in a statistically signif-icant increase in the community size of methylamine utili-zers. In this study, ®ve replicates for each dilution were employed for all MPN experiments, an increase to 10 repli-cates may provide smaller difference between lower and upper con®dence intervals. As a consequence, release of

280 ng methylamine g21 from hydrolysis of carbofuran may result in a statistical increase in MPN for methylamine utilizers.

Communities of carbofuran ring degraders in the ®eld treated and untreated soils after receiving 10mg carbofur-an g21

in laboratory did not increase numerically, suggest-ing that the rsuggest-ing structure of carbofuran did not provide a carbon source for the growth of microorganisms, even though the structure was microbially degraded. This ®nding was supported by the results of MPN estimations of carbo-furan ring degraders in soil from the treated plots during three consecutive annual ®eld applications of carbofuran (Table 1). Similarly, the MPN of carbofuran phenol ring degraders in the treated and untreated soils after receiving 10mg carbofuran phenol g21did not increase numerically as well. Therefore, enhanced degradation of carbofuran in the treated soil (Trabue et al., 1997) may have been due to an increase in enzyme activity responsible for carbofuran ring degradation. Merica and Alexander (1990) also found that the MPN of carbofuran ring degraders did not increase in enhanced soil in 2 and 12 d after receiving 10mg URL

14

C-carbofuran g21.

In conclusion, our results indicated that communities of microorganisms capable of degrading the ring structure of carbofuran during three consecutive years of carbofuran treatments remained small and stable, and hence the degra-dation is a cometabolic process.

Acknowledgements

We thank John E. Thomas for technical assistance. Sincere appreciation is extended to David P. Weingartner for ®eld treatments of carbofuran. This study was partially supported by a grant from USDA. Florida Agricultural Experimental Station journal series No. R-06805.

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