Pestic. Sci. 1993, 39, 55-60

Evidence for the Enhanced Biodegradation of Napropamide in Soil Allan Walker, Nisha R. Parekh, Steven J. Roberts & Sarah J. Welch Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK (Revised manuscript received 29 March 1993; accepted 21 May 1993)

Abstract: In laboratory incubations, the time to 50 % loss of napropamide was approximately 60, 21 and 8 days in soil treated for the first, second and third time respectively. In a survey of soils from commercial fields, there was evidence that enhanced biodegradation of the compound had been induced by normal field applications-in some soils by a single previous treatment. Confirmation of the observations of rapid rates of loss in pre-treated soil was obtained in experiments with three formulations of napropamide. The rate of degradation in enhanced soils was unaffected by treatment of the soils with the antifungal antibiotic cycloheximide, but was inhibited by the antibacterial antibiotic chloramphenicol. Mixed bacterial cultures able to degrade the herbicide were obtained from three rapid-degrading soils by enrichment culture. Isolates from two of them were able to degrade the herbicide in pure culture. These bacteria have, as yet, not been characterised.

1 INTRODUCTION

The herbicide napropamide shows selectivity in certain brassica crops, but its main use in the United Kingdom is for weed control in perennial crops such as straw- berries, pome fruit, and hardy ornamental nursery stock. Early research indicated that napropamide was relatively stable to degradation in the and that half-lives under ideal warm, moist soil conditions could exceed 50-60 days. The compound is photochemically unstable3 and rapid losses may occur when it is surface-applied in spring or early summer; the herbicide must be in- corporated into soil following application at these times of year. Application to the soil surface in late autumn or winter is not followed by such rapid l o ~ s e s , ~ and current recommendations for use in perennial crops permit application to the soil surface between 1 November and 1 March. The results of recent laboratory and field experiments have indicated that napropamide may be susceptible to enhanced degradation in soil, the process by which the rate of degradation of a pesticide is increased significantly in soil treated previously with the same c o m p o ~ n d . ~ In some fruit-growing systems, applications of napropamide may be made at rates up to 2.5 kg a.i. ha-' in successive seasons, and the likelihood of enhanced degradation may consequently be quite high. The objectives of the present experiments were to

confirm that enhanced degradation of napropamide can be induced in previously untreated soil, to establish the extent to which enhanced degradation is apparent in commercial field soils, and to make preliminary studies of some microbiological aspects of the problem.

2 EXPERIMENTAL METHODS AND RESULTS

2.1 Enhanced degradation in laboratory incubations

A sample of soil (3 kg) from Rush Pits field at Horticulture Research International, Wellesbourne was treated with a commercial wettable powder formulation (500 g a.i. kg-') of napropamide to give a concentration of 8 mg kg-l dry soil using the methods described in detail previously.6 The soil was a sandy loam with 2.1 YO organic matter and a pH of 7.2 (1 : 1 ratio, soil : distilled water) and it had a water content at an applied pressure of 33 kPa of 12.0 YO (w/w). The treated soil was divided into six replicate amounts (500 g) which were incubated in 1-litre polypropylene containers at 25°C with moisture adjusted to 12.0%. The soil in two of the six containers was sampled at intervals over the subsequent 105 days when 45 g soil was removed and frozen until analysis. After this 105-day period, the soil from the containers which had not been sampled was removed, spread in

55 Pestic. Sci. 0031-613X/93/$06.00 0 1993 SCI. Printed in Great Britain

56 A . Walker et al.

separate plastic trays on the laboratory bench and dried until 20 g water had been lost. This was replaced by 20 ml of a suspension of the formulated napropamide in water sufficient to add a further 8 mg a.i. kg-' to the soil. After thorough mixing, the soils were returned to their containers and incubated again. The soil in two of the containers was sub-sampled as before over a period of 10 weeks, and then the soil in the final two containers that had not been sub-sampled was treated with a third dose of the herbicide. These soils were again sub-sampled as before over the subsequent five weeks. Napropamide residues were measured by HPLC following extraction of the herbicide with methanol. Aliquots of soil (40 g) were extracted by shaking with methanol (50 ml) for 1 h on a wrist-action shaker. The samples were allowed to stand until the soil had settled, and sub-samples of the clear supernatant were then injected into the chromatograph. A stainless steel column (25 cm x 5 mm i.d.) packed with Lichrosorb-RP18 was used and the solvent system used was methanol + water (90 + 10 by volume) at a flow rate of 1 ml min-l. Detection was by UV absorbence at 220 nm, and the retention time was 3.0 min.

50 100 250

Time after initial treatment (days)

Fig. 1. Degradation in soil of successive applications of napropamide. Vertical lines indicate times of retreatment.

The results are shown in Fig. 1, in which the residual concentration of napropamide is plotted against time after initial treatment of the soil. They indicate that the time to 50% initial loss was approximately 60 days for the first application, 21 days for the second application and 8 days for the third application.

TABLE 1 Sites, Soil Properties, Napropamide Pre-treatment Histories and Times for 50 % Initial

Loss of Herbicide (DT,,)

Site

A

B

C

D

E

F

G

H

I

J

K

L

Location Soil Napropamide Organic p H DT,, treatments matter (days)

(% wfw)

Hereford A0 A3

Worcester BO B1 B2

Winchester CO c2

Maidstone DO D1

Worcester EO E l E2

Stafford FO F l F2

Colchester GO G2

Upminster HO H1 H2

Brentwood I0 I3

Gt Baddow JO J2

Enfield KO K3

L1 Jersey LO

None 1984, 1985, 1986 None 1990 1988, 1989 None 1990, 1991 None 1989 None 1990 1990, 1992 None 1990 1989, 1990 None 1988, 1989 None 1989 1989, 1991 None 1986, 1988, 1990 None 1989, 1990 None 1988, 1990, 1991 None 1991

2.45 2.40 3.83 4.8 5 2.76 3.64 3.42 2.6 1 2.55 2.70 4.2 1 3.66 4.33 3.37 4.92 3.46 3.26 5.78 5.92 3.27 2-22 2.64 268 2.75 4.87 6.00 2.56 282

6.2 1 22 6.35 2.5 5.97 9 5 6.76 2.6 6.77 3.0 6.28 4.6 6.09 4.6 6.07 > 45 6.15 2.8 5.24 36 6.83 2.1 6-43 1.8 6.8 1 31 5.77 5.8 6.16 4.4 6.68 > 45 7.17 2.1 743 42 7.22 6.1 7.03 4.1 7.10 3.2 7.43 3.1 6.71 > 45 6.43 1.8 6.36 > 45 6.76 2.2 5.77 > 45 6.40 12

Enhanced biodegradation of napropamide in soil

120 I I 10 o l (a) Site

80

60

- 40 $!

c 20

3

p!

1

.- c

'0 10 20 30 40 1 50

(b) Site G

80

60

G2

'0 A 10 20 30 40 !

(d) Site I 10

'0 10 20 30 40 !

57

0

3

Days

Fig. 2. Degradation of napropamide in soils from commercial farms. Notation of soils is listed in Table 1.

2.2 Enhanced degradation in soils from commercial fields

Soil samples were collected from a number of field sites with known histories of napropamide use. At each site, soil was collected from the appropriate napropamide pre-treated area or areas, and from an adjacent site with a similar soil but with no history of napropamide use. Details of the sites and pretreatment histories of the soils are listed in Table 1. In total there were 28 soils, of which three had been treated three times previously with the herbicide, seven had been treated twice before, and six had been treated once before. The soils varied in texture from clay loams to sandy loams, pH varied from 5.2 to 7.4 and organic matter from 2.2 to 6.0%. The moisture content of the soils at an applied pressure of 33 kPa was measured using a pressure membrane apparatus7 and the soils were treated with napropamide (Section 2.1) and incubated at 25°C for a period of 45 days. Sub-samples were taken at intervals and napropamide residues were measured as before. To avoid cross-contamination, all equipment used was either disposable or had been autoclaved, and aseptic techniques were used as far as possible.

In all previously treated soils, the rate of napropamide degradation was relatively rapid, and the time to 50% loss was always less than 15 days (DT,,, Table 1). The DT,, was derived by linear interpolation between appropriate residue measurements. The rate of degra- dation in the previously untreated soils was variable,

almost always slower than in the corresponding pre- treated soils, but occasionally unexpectedly rapid. Some examples to illustrate the main features of the results are shown in Fig. 2. Residues are expressed as percentages of those measured at time zero. The data are for a series of soils, pretreated one, two or three times before, and for their respective previously untreated controls. The data for the control soils illustrate the variability in degradation rate observed in apparently untreated samples.

2.3 Studies with different formulations of napropamide

In order to confirm the validity of the conclusion that enhanced degradation was occurring in soils from commercial fruit farms, a further experiment was made with soils from sites F, G and I (Table 1) which had received two or three previous applications of the herbicide and with their respective controls which had no previous history of napropamide use. Twelve sub- samples (40 g) of the six soils were weighed into sterile conical flasks (100 ml) and napropamide in 5 ml water was added to give a concentration of 7.5 mg a.i. kg-l soil. A further 12 sub-samples of Rush Pits soil (section 2.1) were treated in the same way. The herbicide was added to four replicate samples of each soil as a suspension of the wettable powder formulation, a dispersion of a suspension concentrate formulation, or as an aqueous solution of pure, analytical grade napropamide. All flasks were capped with aluminium foil, allowed to stand at room temperature for 4 h, shaken vigorously to mix

58 A . Walker et al.

l4 I 'O1 -7 12 9 p 10

8 4

Y

a 8

$ 6 0

Q

m 2 2

control soil F2 soil G2 soil K3 soil LO n z f j

4

2

n " control soil FO soil GO sol1 10 soil F2 soil G2 soil 13

Fig. 3. Napropamide remaining in different soils five days after application of the herbicide as (m) wettable powder or (a) suspension concentrate formulations, or (0) analytical grade

herbicide. Analysis by (a) HPLC or (b) GLC.

the herbicide into the soil and incubated at 25°C for five days. The flasks containing the Rush Pits soil were frozen at -15°C after the initial mixing to serve as non- degrading controls. After the five-day incubation period, two samples of each treatment, including the frozen controls, were extracted with methanol and napropamide was measured by HPLC as before. The two remaining samples were extracted by shaking with acetone (50 ml) and napropamide residues were measured by GLC. The GLC conditions were described previously by Walker et aL4

The results of the analyses are shown in Fig. 3. They indicate similar results, irrespective of formulation or analytical method, and confirm extensive degradation within five days in the soils that had received three previous doses of the herbicide, and little degradation in the three previously untreated control soils.

2.4 Studies with antibiotics

Further sub-samples (6 x 30 g) of soils F2, G2 and K3 (Table 1) together with similar sub-samples of soil LO and Rush Pits soil were weighed into sterile conical flasks (100 ml). A solution of cycloheximide (2 ml, 750 mg litre-') or chloramphenicol(2 ml, 750 mg litre-'), or sterile distilled water (2 ml) was added to duplicate sub-samples of each soil. The samples were shaken vigorously and incubated at 25°C for 24 h. After this time an aqueous suspension of the wettable powder formulation of napropamide (2 ml; 150 mg a.i. litre-') was added to all samples, followed by thorough shaking. All of the samples were incubated for a further six days

Fig. 4. Napropamide remaining after six days in different soils pre-treated with aqueous solutions of (a) cycloheximide, (a)

chloramphenicol, or (m) with distilled water.

with the exception of those with the Rush Pits soil which were frozen at - 15°C to serve as controls. At the end of the six-day period, residual napropamide was extracted by shaking with methanol (50 ml) as before, followed by HPLC determination of the herbicide.

The results (Fig. 4) indicated rapid loss of naprop- amide in the pre-treated soils when amended with distilled water or cycloheximide, but an apparent reduction in the rate of loss when these soils were amended with chloramphenicol. There was little dif- ference between treatments in the incubated control (soil LO), although the concentrations in this soil were somewhat lower than those recovered from the frozen controls.

2.5 Microbiological studies

2.5.1 Enrichment culture The enrichment culture technique used to isolate de- grading bacteria from napropamide-degrading soil has been described previously by Roberts et ~ 1 . ~ 3 ' The enrichment medium (referred to as MB) was a mineral base with no added carbon or nitrogen.*.' Napropamide was added to the medium before autoclaving as a solution in ethanol (4000 mg litre-') to give a final concentration of 10 mg litre-'. Samples (25 g) of soils G2, I3 and K3 (Table l), together with soil LO as a previously untreated control, were treated with naprop- amide in conical flasks (50 ml) to give an initial herbicide concentration of 8 mg kg-l as described in Section 2.3 above and incubated for three days at 25°C. Sub-samples of soil (500 mg) were then used to inoculate 20 ml of MB+napropamide medium in 100-ml conical flasks which were incubated on a shaking platform in a controlled temperature room (23°C). Cultures were sampled at intervals of one, two or three days and the napropamide concentration was measured by HPLC, as before, following dilution of 0.1 ml liquid culture with 0.9 ml methanol. When the napropamide concentration had fallen to approximately 50% of its initial value, 0.5 ml of culture was transferred to a similar flask of the same medium. At each transfer stage, samples of the

Enhanced biodegradation of napropamide in soil 59

soil LO Control

10

. . . . . . . . . . . . . . . .

I I & I - 0 5 10 15 20 0'

Days

Fig. 5. Napropamide degradation in shake flask culture in a mineral salts medium during repeated sub-culturing using soil K3 (pre-treated three times with napropamide) and soil LO

(previously untreated) as the initial inoculum.

liquid cultures were preserved by freezing at c. - 76°C following the addition of sterile glycerol to give a final concentration of 150 g k g l .

As the results for all three pre-treated soils were similar, only the results for soil K3 are presented (Fig. 5). Bacterial cultures were obtained from all the soils and their ability to degrade napropamide remained stable through five transfers in liquid medium over a period of 17 days. In an identical experiment with the LO control soil, no degradation of napropamide was recorded during the full 19-day period of the experiment (Fig. 5).

2.5.2 At the second enrichment sub-culture, 0.1 ml aliquots of serial ten-fold dilutions in sterile tap water were spread onto plates of MB + napropamide + YE agar (mineral base medium; napropamide, 10 mg litre-l; Bacto yeast extract, 0.1 g litre-l; Difco Bacto Agar, 15 g litre-'). Napropamide in ethanol (4000 mg litre-') was added to the medium immediately after autoclaving to obviate the possibility of reaction with the agar during autoclaving. Following incubation for up to six days at 25"C, 20 well- separated colonies were selected at random from the dilution plates for each soil and purified by streaking on MB + napropamide + YE agar plates.

Each isolate was tested for its ability to degrade napropamide in pure culture. Cells grown on MB+ napropamide+YE agar plates (up to six days, 25°C) were suspended in a small volume of sterile tap water to give a concentration of c.10' cells ml-' (based on turbidity). These suspensions (0.5 ml) were then used to inoculate 20 ml of MB + napropamide liquid medium in 100-ml flasks which were incubated as before, with regular sampling for HPLC analysis. When the con-

Degrading ability of pure cultures

*-. 'A . h - . 0 2 4 6 8 10

0

Days Fig. 6. Napropamide degradation by bacterial isolate number

0300 obtained from soil K3.

centration of napropamide had declined to less than 50% of its initial value, sub-cultures in further shake- flasks (0.5 ml to 20 ml) were made.

Observation of the dilution plates indicated that the liquid enrichment cultures contained a mixture of bacteria. Three out of twenty isolates from soil G2 and four out of twenty isolates from soil K3 degraded napropamide in pure culture in MB + napropamide liquid medium, but no napropamide-degrading isolates were obtained from the other soil tested (13). The ability to degrade napropamide remained stable on repeated sub-culturing in either liquid medium or on agar plates. Degradation of napropamide by one of the isolates (number 0300) through a series of transfers in MB+ napropamide liquid medium is shown in Fig. 6.

3 DISCUSSION

In recent years, there has been increasing research interest in the enhanced degradation of soil-applied pesticides, the phenomenon by which they are degraded more rapidly following repeated application to the same soil. The subject has been reviewed in detail by Racke and Coats." Walker and Welch,', demonstrated that enhanced degradation could occur with a wide range of soil-applied herbicides if selection pressures were suf- ficiently intensive. With many compounds, they also demonstrated that, although significant effects could be induced in laboratory incubations, these were unlikely to occur under practical field conditions. However, the results indicated that enhanced degradation of the amide herbicides propyzamide and napropamide could be induced by application of commercial rates of herbicide in the field, and that the enhancement was relatively stable. The related herbicide diphenamid is also sus- ceptible to enhanced degradation, suggesting that sub- stituted amides may be particularly prone to this phenomenon." The present results confirm these earlier observations with napropamide. The data presented in

60 A . Walker et al.

Fig. 1 demonstrate clearly that enhanced degradation is induced readily by repeated treatment at relatively high doses in laboratory incubations, and the results in Table 1 and Fig. 2 demonstrate that enhanced degradation has been induced in a number of soils following commercial applications of the herbicide under field conditions. The data indicate that even a single pre-treatment with a commercial rate of herbicide can induce consider- able enhancement (Fig. 2(a)). The data in Fig. 2(d) are presented to illustrate an anomalous result that was observed with two paired soil samples; the rate of loss of napropamide from the previously untreated sample was as rapid as that from the pre-treated soil. At both of these sites (CO and 10, Table l), the untreated sample was taken from an area immediately adjacent to that from which the pre-treated sample was obtained, whereas in all of the other comparisons, untreated soils were taken from a different field from that which had been treated with napropamide. Previous experiments have demon- strated that rapid-degrading ability is easily transferred from one soil to another, with just 1 g of rapid-degrading soil transferring enhanced degrading ability to 1 kg of previously untreated soil.12, l3 It is possible, thereafter, that some contamination of the control sample had occurred, either under natural conditions in the field by movement of soil from the treated to the untreated area, or during sampling by using the same soil sampler to remove first the treated soil and second the untreated soil. In retrospect, it was unfortunate that no specific instructions with respect to methods of handling the samples were given to the different individuals collecting the soils.

The results in Fig. 3 further confirm that in several soil samples enhanced degradation of napropamide has been induced by normal agronomic use of the herbicide. Up to 90 % degradation was observed following incubation of napropamide in soils that had received three previous doses of the herbicide, compared with less than 10% degradation in the three previously untreated soils. A consistent effect was observed with three different formulations of the herbicide when residues were determined following extraction with two different solvents and using two analytical procedures.

The data in Fig. 4 provide evidence that soil bacteria are the main micro-organisms responsible for degra- dation of napropamide ; the anti-fungal antibiotic cyclo- heximide14 had little effect on the rate of loss compared with the significant inhibition of degradation by chlor- amphenicol, which is primarily an anti-bacterial anti- biotic.'* The results in Figs 5 and 6 confirm the likely involvement of soil bacteria, since both mixed and pure bacterial cultures able to degrade napropamide were obtained from rapid-degrading soils by enrichment in a liquid mineral salts medium with the herbicide as sole

carbon and nitrogen source. The results in Figs 5 and 6 are for one of the three soils examined using enrichment culture and appropriate isolation techniques. All three soils gave stable, mixed cultures able to degrade the herbicide, but pure cultures were obtained from only two of them. It is possible that the random colony selection process used in these experiments 'missed ' the active- degrader(s) in the third soil, although more compre- hensive selection procedures have been similarly un- successful with several other soil-applied pesticide^.^. l5

Experiments are currently in progress to characterise the bacterial isolates in detail and to investigate the primary route by which they degrade napropamide.

ACKNOWLEDGEMENTS

We are grateful to The Agricultural and Food Research Council and The Ministry of Agriculture, Fisheries and Food for financing this work, and to Zeneca Agro- chemicals for the sample of analytical grade naprop- amide and their interest in this work. The assistance given by Mr Andrew Greenfield, ADAS, Oxford, with identification of sites and collection of soils is also gratefully acknowledged.

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