Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1987 Enhanced microbial degradation of insecticides in soil Kenneth David Racke Iowa State University

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Enhanced microbial degradation of insecticides in soil

Racke, Kenneth David, Ph.D.

Iowa State University, 1987

UMI 300N.ZeebRd. Ann Arbor, MI 48106

Enhanced microbial degradation of insecticides in soil

by

Kenneth David Racke

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department: Entomology Major; Entomology (insecticide Toxicology)

Approved:

Members of the Committee; Signature was redacted for privacy.

Charge of Major Work

Signature was redacted for privacy.

For the Ma^r Department Signature was redacted for privacy.

Signature was redacted for privacy. For the Graduate College

Iowa State University Ames, Iowa

1987 TABLE OF CONTENTS Page

GENERAL INTRODUCTION 1

CHAPTER I. ENHANCED DEGRADATION OF ISOFENPHOS BY SOIL MICROORGANISMS 10

ABSTRACT 12

INTRODUCTION 13

MATERIALS AND METHODS 15

EXPERIMENTAL PROCEDURES 18

RESULTS AND DISCUSSION 22

ACKNOWLEDGMENTS 36

LITERATURE CITED 37

CHAPTER II. COMPARATIVE DEGRADATION OF ORGANOPHOSPHORUS INSECTICIDES IN SOIL: SPECIFICITY OF ENHANCED MICROBIAL DEGRADATION 41

ABSTRACT 43

INTRODUCTION 44

MATERIALS AND METHODS 46

EXPERIMENTAL PROCEDURES 51

RESULTS 53

DISCUSSION 64

ACKNOWLEDGMENTS 69

LITERATURE CITED 70 ill

Page CHAPTER III. ENHANCED DEGRADATION AND THE COMPARATIVE FATE OF CARBAMATE INSECTICIDES IN SOIL 75

ABSTRACT 77

INTRODUCTION 78

MATERIALS AND METHODS 80

RESULTS 85

DISCUSSION 94

ACKNOWLEDGMENTS 98

LITERATURE CITED 99

SUMMARY 103

LITERATURE CITED 105

ACKNOWLEDGMENTS 111 1

GENERAL INTRODUCTION

Literature review

Pesticides in soil Pesticides are an integral component of modern

agriculture. Since the introduction of the synthetic, organic pesticides

in the post-World War II era, they have come to be relied upon for the

control of a multitude of pests in a variety of cropping systems. This is

especially true for corn agriculture, wherein soil-applied pesticides are the key component of the farmer's arsenal against an array of noxious weeds and the key insect pest, the corn rootworm complex (Piabrotica spp.). In

Iowa alone in 1985, around 6 million acres of corn were treated with soil insecticides, and over 12 million acres with soil herbicides (Wintersteen and Hartzler, 1987).

Once applied to the soil, pesticides are inextricably caught up in the cycles and processes operating within this system. I have defined soil as a "complex ecosystem composed of populations of micro- and macro-organisms in an abiotic matrix of mineral and organic matter". Delwiche (1967) viewed the soil as a processing depot, continually receiving reduced organic compounds and continually oxidizing them to CO^ and HgO. Thus, pesticide dissipation follows the introduction of pesticides into the soil system. Pesticides or their products may move out of the soil system with water (leaching, runoff) or air (volatilization, wind erosion), or may be reversibly sorbed to soil mineral or organic matter. While chemical or photochemical degradation may also play a role in pesticide dissipation, biodégradation represents one of the most important mechanisms for pesticide dissipation in soil (Kaufman, 1974? Alexander, 1981). 2

Biodégradation was recognized early as a major factor governing pesticide fate in soil (Audus, 1951)• The interaction between pesticides and microorganisms has been the subject of considerable interest ever since, and a number of reviews and books have dealt with the topic (Bollen,

1961; Bollag, 1974» Tu and Miles, 1976; Laveglia and Dahm, 1977; Hill and

Wright, 1978; Lai, 1984). Matsumura (1985) outlined a useful scheme for classifying pesticide/microbe interactions. The first type of interaction he characterized as incidental metabolism. In this case, the pesticide is merely incidentally degraded by general enzymes produced by the microbe during the course of its normal metabolic activities, and the microbe gains no benefit from degradation of the pesticide. This type of interaction has been termed 'cometabolism' (Horvath and Alexander, 1970; Alexander, 1981), and has been touted as the major type of interaction occurring between pesticides and microbes in soil (Alexander, 1981; Matsumura, 1985).

However, recent developments in soil microbiology have increasingly drawn attention to a second type of interaction. This second type of interaction is simply termed 'catabolism', and signifies that the pesticide or a product is beneficially metabolized by the microbe as an energy (Clark and

Wright, 1970; Sethunathan and Yoshida, 1973) or nutrient (Cook et al.,

1978; Karns et al., 1986) source. A third type of interaction which would be microbially beneficial is detoxification metabolism. In this case, a microbial toxicant is metabolically altered by the microbe to an inactive or less active form.

It would appear that judged strictly by the aims of soil-pest control, pesticide dissipation would run counter to the objectives of the 3

agriculturalist, for a pesticide applied to soil must persist long enough

to provide effective control of the target pest. This is especially true for the corn rootworm, which displays a prolonged hatching period during late spring and early summer (Krysan, 1986), long after the planting date

at which time soil insecticides are applied. In fact, the earliest soil insecticides applied for corn rootworm control, aldrin, heptachlor, and

BHC, were quite recalcitrant, and in some cases one application provided two years of control (Lilly, 1956). However, due to environmental toxicological considerations, the prolonged persistence of these early compounds came to be regarded as a negative phenomenon (Lichtenstein, 1957;

Carson, 1962; Korschgen, 1970). It was in this context that the

'biodegradable' organophosphorus and carbamate insecticides were introduced for soil use. These compounds were valued specifically because of their high activity against soil pests and their eaise of biodégradation in soil.

It can be seen that a somewhat paradoxical situation has come to exist as regards what is expected of soil pesticides. On the one hand, we wish to employ compounds that will persist long enough in the soil to provide effective pest control, and on the other hand we desire compounds not to persist for unreasonable lengths of time in environmentally significant quantities. The tense nature of this paradox is revealed in the dilemma posed by the phenomenon of enhanced microbial degradation of soil pesticides.

Enhanced microbial degradation of soil pesticides Enhanced microbial degradation is a special form of pesticide biodégradation in which an increased rate of pesticide degradation in soil is associated with 4

repeated use of that compound. Application of a susceptible pesticide to soil apparently induces a microbial adaptation, so that a subsequent dose of pesticide is much more rapidly degraded than the first. This adaptation involves the induction of a population of specific pesticide-degrading microorganisms capable of catabolically utilizing the pesticide as an energy or nutrient source (Kaufman and Edwards, 1983»

Kaufman et al., 1985). It is not known whether the observed increase in numbers of pesticide-degrading microbes results from multiplication of preadapted microbes, or from the selection of new metabolic activities produced by genetic changes (Clarke, 1974; Senior et al,, 1976; Spain et al., 1980). As a microevolutionary event, enhanced biodégradation is functionally equivalent to the appearence of antibiotic resistance in microorganisms (Chakrabarty, 1976; Williams, 1978). Enhanced microbial degradation of organic compounds has also been documented in aquatic ecosystems (Spain et al., 1980; Pfaender et al., 1985), and has been exploited for some time in waste treatment technology (McKinney et al.,

1956; Malaney, 1960; Haller, 1978).

Enhanced degradation is not a new phenomenon in soil. Early work on the biodégradation of the herbicide 2,4-Dichlorophenoxyacetic acid in soil clearly provided evidence of reduced persistence following repeated applications (Newman and Thomas, 1949; Audus, 1951). Enhanced degradation of several other phenoxyacetic acid herbicides was also noted (Fryer and

Kirkland, 1970; Kirkland and Fryer, 1972), and this enhanced degradation was subsequently linked to increased populations of herbicide-degrading microorganisms in soils previously exposed to these herbicides (Fournier, 5

1980; Fournie? et al., 1981). Enhanced degradation of phenoxyacetic acid

herbicides has been well studied, and both the enzymatic degradation

pathways and genetic control of adapted bacterial catabolism have been elucidated (Tiedje et al., 1969; Fisher et al., 1978; Don and Pemberton,

1981; Ghosal et al., 1985). Since the discovery of 2,4-D enhanced degradation, a number of other reports of enhanced degradation have surfaced (Raghu and MaCrae,- 1966; McClure, 1972; Sethunathan and Pathak,

1972; Rodriguez and Borough, 1977; Forrest et al., 1981). But because most of these instances were not associated with widespread pest control failures, enhanced degradation was relegated to the status of academic curiosity.

The subject of enhanced microbial degradation was recently brought into the limelight when it was identified as the underlying cause of pest control failures in the Midwest corn-growing region. Reduced efficacy of the insecticide carbofuran for corn rootworm control was first noted in the mid to late 1970s (Tollefson, 1986). The first report of reduced persistence of carbofuran following repeated use (Greenhalgh and Belanger,

1981) coincided with evidence that enhanced microbial degradation was the cause (Felsot et al., 1981). Subsequent reports confirmed the occurrence of enhanced carbofuran degradation over a wide geographic area (Read, 1986;

Kaufman and Edwards, 1983; Harris et al., 1984). Several carbofuran-degrading bacteria, isolated from soils displaying enhanced carbofuran degradation, are capable of utilizing carbofuran as a nitrogen source in pure culture (Karns et al., 1986; Chaudhry et al., 1986). The key enzyme involved in carbofuran catabolism, carbofuran hydrolase, has 6

been isolated recently (Derbyshire et al., 1987), and work is now in

progress to unravel the genetic basis of bacterial carbofuran metabolism

(Karns, personal communication; U.S. Dept. of Agriculture, Beltsville, MD).

Soon after the discovery of enhanced carbofuran degradation, another

event occurred that generated interest in enhanced degradation. This was

the discovery that EPTC, a commonly used corn herbicide, failed to provide

adequate weed control following several consecutive years of use (Doersch

and Harvey, 1979; Harvey and Schuman, 1981). Not long after enhanced

microbial degradation of EPTC was convincingly demonstrated (Rahman and

James, 1983; Wilson, 1984; Kaufman et al., 1985), the same phenomenon was

shown to affect the fate of several other thiocarbamate herbicides in soil

(Gray and Joo, 1985; Skipper et al., 1986; Harvey et al., 1986). A

bacterium has recently been isolated from EPTC soils which carries a

degradative plasmid encoding the catabolism of EPTC as an energy source

(Tarn et al., 1987).

The occurrence of enhanced degradation of both carbofuran and EPTC in connection with pest control failures has ignited much interest in enhanced

microbial degradation (Fox, 1983). Responses from several segments of society have occurred. Corn growers who have experienced pest control failures due to enhanced degradation have expressed consternation at this unexpected event (Cannell, 1983; Anonymous, 1984). Considerable confusion also seems to exist among growers regarding what enhanced degradation is, which pesticides have been affected, and what remedies are available.

The agricultural chemical industry has also responded to enhanced degradation. There has been a considerable effort to minimize corporate 7-

damage due to enhanced degradation. Companies marketing pesticides which

have failed due to enhanced degradation have sought to implement measures

designed to ameliorate the situation, such as the recommendation of

pesticide rotations or the incorporation of microbial inhibitors into formulations (Prochnow, 1981; Miaullis et al., 1982; Brady, 1986). Several

pesticides have apparently been removed from the market or stopped in development due to problems with enhanced degradation (Murphy and Cohick,

1985). Companies not yet directly experiencing product failure have sought to 'prove' that their pesticide is not susceptible to enhanced degradation in an effort to placate growers' concerns (Belcher et al., 1986; Hamm and

Thomas, 1986; Powell and Herman, 1987). A few companies have, in addition, sought to exploit the situation provided by enhanced degradation to call into question the reliability of competitors' products (Staetz, 1986; 1987;

Powell and Herman, 1987). Unfortunately, this has contributed substantially to the confusion existing among growers.

The federal government, under the auspices of the U.S. Department of

Agriculture, also has displayed interest in enhanced degradation. The USDA sponsored a workshop on enhanced microbial degradation during October of

1984, and has subsequently provided over a quarter of a million dollars in research funds for university research on enhanced degradation through the

Pesticide Impact Assessment Program.

Interest in enhanced degradation also has been generated within the academic and research community. The Entomological Society of America sponsored a special symposium on enhanced degradation during March of 1984, as did the American Chemical Society during April of 1984» International 8

interest in enhanced degradation is evidenced by the 10 papers and 1

plenary lecture devoted to this topic at the Sixth International Congress

of Pesticide Chemistry (lUPAC) held during August of 1986.

Although enhanced microbial degradation has been recognized to occur

with pesticides in soil for some years, and in spite of current research

activity, much remains unclear. Currently, there is no universally

accepted definition of exactly what is enhanced microbial degradation. As

a result, no standard criteria for confirmation of enhanced degradation are

recognized. This has led to an unfortunate situation in which one

publication may invoke enhanced degradation based solely on observations of

reduced field efficacy, while another may invoke enhanced degradation based

solely on decreased persistence following numerous lab-administered soil

pesticide treatments.

Dissertation objectives

The overall goal of this dissertation was to generate accurate

research information on enhanced microbial degradation that would

contribute to its understanding and to the formulation of informed

responses to it. The first specific objective was to develop a

standardized methodology for the study of enhanced microbial degradation in

soil. This was needed both to expound a definition of enhanced degradation

and to develop criteria for its confirmation. The apparent failure of the insecticide isofenphos (Amaze) following repeated use provided an ideal situation in which to accomplish this. The second specific objective was to determine the specificity of enhanced microbial degradation in soil.

The question to be answered was whether the induction of enhanced 9

degradation of an insecticide in soil due to repeated use also resulted in

the enhanced degradation of similar insecticides not previously applied to

that soil. Investigations on the specificity of enhanced degradation would

provide basic information on the breadth of the adaptation involved. The

specificity of enhanced microbial degradation was examined both for the

organophosphorus and carbamate insecticide classes.

Explanation of dissertation format

This dissertation has been completed following an alternate format,

with each chapter comprising a paper submitted for publication to the

Journal of Agricultural and Food Chemistry. Chapter I addresses the enhanced degradation of isofenphos, and has recently been published.

Chapters II and III address the question of enhanced degradative specificity, and have been submitted for publication. LITERATURE CITED sections are included at the end of each chapter. An additional LITERATURE

CITED section at the end of the dissertation lists sources cited in the

GENERAL INTRODUCTION and SUMMARY sections. 10

CHAPTER I. ENHANCED DEGRADATION OF ISOFENPHOS

BY SOIL MICROORGANISMS 11

ENHANCED DEGRADATION OF ISOFENPHOS

BY SOIL MICROORGANISMS

Kenneth D. Racke and Joel R. Coats

Department of Entomology, Iowa State University, Ames, Iowa 50011 12

ABSTRACT

Laboratory experiments were conducted to investigate the enhanced

degradation of isofenphos (l-methylethyl-2-[[ethoxy[(l-methylethyl)amino]-

phosphinothioyl]-oxy] benzoate) in soil and to elucidate the microbiology

of this phenomenon. [U ring-''^C]Isofenphos was most rapidly degraded in

Iowa cornfield soils that had a history of isofenphos insecticide use.

Between 13 and 42^ of an applied dose of 5 ppm remained as isofenphos after

4 weeks in soil with isofenphos use history, whereas between 63 and 13%

remained in comparable nonhistory soils. Soils with enhanced isofenphos

degradation contained an adapted population of soil microorganisms

responsible for the degradation observed. Degradation products of

isofenphos detected in cultures of adapted soil microorganisms included

isopropyl salicylate, ''^COg, and polar products. A bacterial strain

(Pseudomonas sp.) isolated from soil with enhanced isofenphos degradation

proved capable of utilizing isofenphos as a sole carbon source. 13

INTRODUCTION

Enhanced degradation is the phenomenon whereby a soil-applied

pesticide is rapidly degraded by a population of microorganisms that has

adapted due to previous exposure to the pesticide. This phenomenon has

been observed with several herbicides (Newman and Thomas, 1949; Audus,

1951; Fryer and Kirkland, 1970; Harvey and Schuman, 1981), insecticides

(Sethunathan and Pathak, 1972; Felsot et al., 1981; Read, 1983), and

fungicides (Walker et al., 1985; Yarden et al., 1986). The type of

pesticide metabolism involved is microbially beneficial and is accompanied

by an increase in the numbers of specific pesticide-degrading soil microorganisms (Kaufman and Edwards, 1983). The practical implication of enhanced degradation is that the pesticide involved may be so rapidly degraded as to reduce its efficacy against the target pest (Felsot et al.,

1982).

Isofenphos (1-methylethy1-2-[[ethoxy[(1-methylethy1)amino]- phosphinothioyl] -oxyjbenzoate) is an organophosphorus insecticide that was registered for use against soil-dwelling pests of corn (Amaze). Failure of isofenphos to control damage caused by larval corn rootworms (Piabrotica spp.) resulted in its withdrawal from that market after the 1983 growing season. Although initial reports indicated that isofenphos and its oxon metabolite were rather persistent in soil (Chapman and Harris, 1982;

Felsot, 1984), recent evidence indicates that isofenphos is much less persistent after a second consecutive year of its application (Chapman et al., 1986).

The current research was initiated to determine if enhanced microbial 14

degradation of Isofenphos had occurred. These laboratory studies were designed to investigate both the fate and degradation of [^^C]isofenphos in soil as well as the microbiology of its degradation. 15

MATERIALS AND METHODS

Chemicals

Uniformly ring-labelled isofenphos, technical and analytical grade isofenphos, and several of its metabolites were obtained from Mobay

Chemical Corporation, Kansas City, MO. [^^cjiaofenphos was purified by using thin-layer chromatography with development in (4:3)hexane-acetone (R^

0.77). The obtained radiopurity was greater than 98%, The fungicide cycloheximide (3-[2(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]- glutarimide) and the bactericide chloramphenicol (D(-)-threo-2,2-dichloro-

N-[b-hydroxy-a-(hydroxymethy1)-p-nitrophenethy1]acetamide) were purchased from Sigma Chemical Company, St. Louis, MO.

Soils and treatments

Surface samples of Iowa soils were collected from cornfields at the end of the growing season, sieved to remove debris, and stored in a moist condition at 4°C. Soils chosen for study included those with isofenphos history, in which isofenphos had not performed well after a second year of use, and comparable soils with no recent insecticide history. Additional soils had received prior applications of other organophosphorus or carbamate insecticides. Properties of soils in which isofenphos degradation was extensively studied (soils I-IV) are listed in Table I.

Soil samples were treated with ["*^0]isofenphos in acetone at 5 ppm as described by Lichtenstein and Schulz (1959). Aliquots of treated soil were taken for incubation and also for analysis of initial insecticide 16

Table I, Soils Used to Evaluate the Enhanced Degradation of Isofenphos

Texture Soil Isofenphos % H_0 No. Series History PH O.M. Sand Silt Clay 1/3 bar

I Tama No 7.3 3.9 8.2 58.6 33.2 32.9

II Readlyn No 6.9 2.9 34.8 36.0 29.2 23.6

III Canisteo No 7.4 4.8 30.2 34.0 35.8 31.5

IV Tama Yes 6.7 3.2 5.8 63.8 30.4 28.7

V Canisteo Yes 7.2 4.8 25.3 38.3 36.4 31.2

VI Canisteo Yes 7.4 4.9 27.0 38.9 34.2 32.3

^Soils I and IV (Tama), and soils III, V, and VI (Canisteo) were companion soils taken from adjacent fields.

^For isofenphos soils, includes last two years; other soils had no insecticide applications for at least ten years. 17

concentration. Soils that received antimicrobial treatments were treated

with aqueous solutions of either chloramphenicol or cycloheximide at 100

ppm. Soil sterilization was by autoclaving at 121°C for 1 h.

Extraction and analyses

At the end of the incubation period, [''^C]isofenphos residues in soil

were extracted twice with acetone-methanol (l:l), once with

acetone-methanol-dichloromethane (1:1:1), and partitioned into

dichloromethane as described by Lichtenstein et al. (1973). Aqueous

samples of [''^Cjisofenphos-treated microbial cultures were acidified with

concentrated HCl before partitioning.

Isofenphos and its metabolites were characterized by thin-layer

chromatography (TLC) and autoradiography, as described by Hsin and Coats

(1986). The values for these compounds with development of the

chromatogram in hexane-acetone (4:3) were; isopropyl salicylate (0.86), isofenphos (0.77), isofenphos oxon (0.47), and salicylic acid (0.06).

Confirmatory analysis was by high-pressure liquid chromatography using a

Waters model 6000A system with 254-nm UV detector. The extracts were dissolved in mobile phase (10# H^O in methanol) and injected onto a

Bondapak C^g column (0.6 x 30 cm) with a flow rate of 0.3 ml/min.

Retention times for isopropyl salicylate, isofenphos, and isofenphos oxon were 16.2, 14.8, and 13.3 min, respectively. Unextractable, soil-bound

['''^C]insecticide residues were recovered by combustion to ^^COg in a

Packard sample oxidizer. Quantification of "*^0, including that present in vapor traps and in alkaline CO^ traps, was by liquid scintillation counting. 18

EXPERIMENTAL PROCEDURES

Assay of enhanced microbial degradation of iaofenphos

To determine whether the degradation of iaofenphos was affected by-

soil insecticide history, a soil degradation assay similar to that

described by Bartha and Pramer (1965) was used. A 25-g portion of each

soil, in duplicate, was treated with 5 ppm of [^^C]isofenphos (0.1 uCi) and then placed within an 8-oz French square bottle along with an equal volume of sterile silica sand (75 g). The soil-sand mixtures were moistened so that the total water content of each was 15 mL. The presence of the sand in this ratio ensured that all soil-sand mixtures were maintained at approximately field moisture capacity (1/3 bar soil moisture tension) and thus allowed comparison of isofenphoa degradation rates in texturally diverse soils (Keeney and Bremner, 1967). Glass vials containing 0.1N NaOH were placed in each jar and served aa COg traps. The jars were closed tightly and incubated at 25°C in the dark for 1 week, during which time the 14 COg traps were replaced and analyzed for COg daily. The evolution of

^^'COg was used as an indicator of isofenphoa degradation.

To determine whether soil microorganisms were involved in the degradation of isofenphoa obaerved, the effect of varioua antimicrobial treatmenta on the evolution of ^^COg from [''^Cjiaofenphoa-treated soil was investigated. A soil with isofenphoa hiatory and in which [''^C]iaofenphos had been rapidly degraded to ''^COg was chosen for study (soil IV). The

[^^Cjisofenphos degradation assay waa used with eight jars of soil. Two aoila were autoclaved before iaofenphos treatment to determine the effect of aterilization on the observed degradation. Two aoila were treated with 19

the fungicide cycloheximide at 100 ppm, and two soils were treated at 100

ppm with the bactericide chloramphenicol. Two soils then remained

untreated. The soil degradation assay was conducted as described

previously.

Effect of enhanced degradation on the fate of isofenphos in soil

This study determined the fate of [^^C]isofenphos as affected by

enhanced degradation. Six soils, three with a history of enhanced

degradation and three with no recent insecticide history, were treated with

5 ppm of [''^C]isofenphos (0.5 uCi) in duplicated 100-g samples and added to

250-mL glass jars (5 cm diam. x 11 cm ht.). Soils were moistened to field capacity (1/3 bar soil moisture tension) with distilled water and incubated at 25°C in the dark by using a flow-through incubation system (Kearney and

Kontson, 1976; Ferris and Lichtenstein, 1980). Air was periodically purged from the tightly sealed soil jars through both vapor and CO^ traps, and this allowed monitoring of ["*^0]isofenphos degradation to Distilled water was added as necessary to replace that lost due to evaporation.

After either 1 or 4 weeks, the soil in each jar was analyzed for the presence of [^^C]isofenphos and its metabolites as previously described.

Because soil-bound residues represented a major end product of the applied [^^C]isofenphos in several soils, a study was also conducted to investigate the potential for further metabolism of these soil-bound residues. Soils that had been incubated for either 1 or 4 weeks with

[^^C]isofenphos and extracted to remove soluble residues were used.

These soils, which contained only bound residues, were then reincubated for 1 week to monitor further bound residue degradation by using the 20

methodology described by Racke and Lichtenstein (1985). The role of soil

microorganisms was assessed by reincubating bound residue soil that had

been sterilized by autoclaving. The criterion for further degradation of 14. these soil-bound residues was the evolution of COg.

The metabolism of isofenphos by soil microorganisms

The metabolism of [''^C]isofenphos was studied in soil-free cultures of adapted soil microorganisms. An aqueous extract of soil microorganisms was prepared by shaking a 50-g sample of soil with 200 mL of basal salts medium

(Spain et al., 1980), following which the soil was removed by centrifugation. The soil used as the source of inoculum was one in which enhanced degradation of isofenphos had been confirmed (Soil IV) and had been treated with 100 ppm of isofenphos 1 week before use to increase the numbers of isofenphos-degrading microorganisms present in the supernatant.

A 75-mL aliquot of this supernatant was measured into each of two glass culture jars (5 cm diam. x 11 cm ht.) and then treated with 10 ppm of

[''^C]isofenphos (1.0 uCi) dissolved in 10 uL of acetone. The cultures were aerobically incubated at 25°C by using a flow-through incubation system

(Gorder and Lichtenstein, 1980). During the 36-h incubation period, samples of culture medium were periodically withdrawn and analyzed for the A A presence of [ C]isofenphos and its metabolites. Vapor traps and COg traps were also monitored periodically for the presence of evolved

Additional metabolism studies were also conducted as described, but with either 50 ppm of salicylic acid added to the culture in addition to the [^^C]isofenphos or with the aqueous soil extract sterilized before insecticide treatement. 21

Enumeration and identification of isofenphos-degrading soil microorganisms

This study examined the specific soil microbial population involved in

enhanced degradation of isofenphos. A most-probable-number method was used to enumerate isofenphos-degrading microorganisms in soils I through VI

(Lehmicke et al., 1979; Somerville et al., 1985). Soil was serially diluted and placed in media containing [''^C]isofenphos at 10 ppm as the sole carbon source. The presence of isofenphos-degrading microorganisms was confirmed by the production of considerable quantities of (>20# of applied by the end of a 3-week incubation period.

For comparison, the total numbers of aerobic bacteria, fungi, and actinomycetes present in these same six soils were determined by standard plate count (Wollum, 1982). An agar was employed for culturlng soil bacteria and actinomycetes (Ridge and Rovira, 1971) and a rose bengal agar was used for culturlng soil fungi (Martin, 1950). Final microbial counts were performed after 1 week, and each determination was performed in duplicate.

To identify the specific soil microorganisms involved in enhanced isofenphos degradation, isolates obtained from the most-probable-number study were streaked on basal salts agar with Isofenphos provided as the sole carbon source. Colonies growing on this medium were characterized by utilizing standard microbial taxonomlc techniques (Skerman, 1967; Buchanan and Gibbons, 1974). 22

RESULTS AND DISCUSSION

Assay of enhanced mlorobial degradation of Isofenphos

The soil degradation assay provided an estimate of the rate of

[''^C]isofenphos degradation in soils with a variety of insecticide

histories. Results indicate that less than 3^ of the applied insecticide

was degraded to ''^COg in 1 week in soils with no recent insecticide history

(Table II). In contrast, soils with a history of isofenphos use degraded

32% of the applied [^^C]isofenphos to ''^COg. Quantities of ''^COg evolved from individual isofenphos-history soils were quite variable and ranged 14. from 5 to 56^ of applied C. This increased degradation rate also occurred in soil treated with 100 ppm of isofenphos 1 week before the assay, with 69% of the subsequently applied isofenphos degraded to

''^COg. Isofenphos degradation in soil was not affected by previous use of any other organophosphorus or carbamate insecticide and was not accelerated in soil in which enhanced degradation of carbofuran had occurred.

The effects of various antimicrobial treatments on the degradation of

[''^C]isofenphos to in isofenphos-history soil (soil IV) were investigated. With no treatment applied, there was a rapid, accelerating production of ^^COg from [''^Cjisofenphos-treated soil during the incubation period (Figure 1). This accelerating degradation is indicative of microbial involvement in the degradation process. The involvement of microorganisms was confirmed by the negligible ^^COg production from soil that had been sterilized by autoclaving. Although the addition of the fungicide cycloheximide did not affect the degradation of isofenphos, application of the bactericide chloramphenicol had virtually the same 23

Table II. The Evolution of CO- from [ Cjisofenphos-Treated Soils During a 1-Week Incubation as Influenced by- Soil Insecticide History

Soil treated with [''^Cjisofenphos at 5 ppm^

Soil insecticide^ ^. history Number of soils (?S of applied C)

None 5 2.98 0.99

Isofenphos 4 31.52 19.28

Organophosphorus 8 2.07 0.58

Carbamate 6 2.36 + 1.61

A 25-g aliquot of each soil, in duplicate, was treated with 5 ppm of [ ^Cjisofenphos (O.IOuCi), mixed with 75 g of sand, and incubated for 1 week.

^Indicates past 2 years of soil insecticide use. Organo- phosphorus includes terbufos, phorate, ethoprop, and fonofos. Carbamate includes only carbofuran.

^Represents mean cumulative ''^C0„ evolved and trapped in 0.1N NaOH during a 1-week incubation + S.E. 24

SOIL TREATED WITH (I^C) ISOFENPHOS ANTIMICROBIAL TREATMENT 25

NONE

P CYCLOHEXIMIDE 20

IS

(M 10

CHLORAMPHENICOL -V AUTOCLAVING

1 3 S 7

DAYS OF INCUBATION

Figure 1. The effect of.antimicrobial treatments on the evolution of COg from [ ^Cjisofenphos-treated soil 25

effect as autoclaving. This suggests that soil bacteria are mainly

responsible for the rapid degradation of isofenphos.

Data indicate that isofenphos is rapidly degraded by microorganisms in soils previously exposed to this insecticide. The observed enhanced degradation of isofenphos is consistent with reports of decreased isofenphos persistence in soils receiving repeated isofenphos applications

(Chapman et al., 1986).

Effect of enhanced degradation on the fate of isofenphos in soil

This study was a comparison of the fate and degradation of

[^^cjisofenphos in control soils (no recent insecticide history) and in isofenphos-history soils with enhanced isofenphos degradation. Isofenphos was quite persistent in the control soils (l, II, and III), in which between 63 and 75^ of the applied [^'^C]isofenphos remained after 4 weeks

(Table III),. This is consistent with earlier observations that determined the half-life of isofenphos in soil to be between 4*5 and 12 weeks (Felsot,

1984; Chapman and Harris, 1982). In soils with enhanced isofenphos degradation (IV, V, and VI), however, only 13^ (0.6 ppm) to 42^ (2.1 ppm) of the initially applied dose of [''^C]isofenphos remained after 4 weeks.

This decreased persistence could indeed reduce the extended efficacy of isofenphos in soil for a key soil pest of corn, the corn rootworm, against which it is reported to have an LC^^ in soil of 1.2 ppm (Sutter, 1982). 14 The distribution of the remaining C was quite different in comparing the control soils with the isofenphos-history soils. In control soils (I, 14 II, and III), [ C]isofenphos oxon was the major metabolite detected, accounting for up to 15^ of the initially applied [^^C]isofenphos. In Table III. Effect of Insecticide History on the Fate and Degradation of [^^Cjlsofenphos in Soil, During a 4-Week Incubation

* Soil Insecticide History^

Fraction None None None Isofenphos Isofenphos Isofenphos I II III IV V VI

recovered in % of applied [^^C]isofenphos Soil Isofenphos 62.8° 74.2* 75.2* 12.9® 24.9^ 41.9®

Isof. Oxon 15.2° 11.3* 8.1® 2.8^ 4.lS 5.6^

Other^ 0.9° 0.2° 0.8° 0.8° 0.7° 0.6°

Bound 9.2° 8.6°* 7.9* 23.6® 25.8^ 15.7® 8 M C 10.0° 5.6* 5.2* 52.4® 34.3^ 31.0®

Volatiles 0.2° 0.2° ND^ 0.4^ ND ND

Total 98.2°* 100.1* 97.1° 92.8°® 89.6® 94.8°®

^[^^C]lsofenphos was uniformly applied to 100-g portions of soil at 5 ppm (0.5 uCi).

Includes previous 2 growing seasons.

^ ^eans followed by the same letter in each horizontal row are not significantly different at the 3% level (Student-Newman-Keuls Test).

^Includes polar, water-soluble products and traces of other metabolites.

^ND = not determined. 27

these soils, there was a steady accumulation of this oxidative metabolite,

constituting, for example, 6.9 and 15.2# of the applied after 1 and 4

weeks of incubation, respectively (Soil I). The accumulation of this

persistent metabolite has been reported to occur in soil mainly through nonbiological means (Felsot, 1984). Soil-bound residues and were also produced in control soils, but generally represented a minor fate of the applied [^^C]isofenphos.

The same degradation products of [''^C]isofenphos were also detected in isofenphos-history soils (IV, V, and VI), but the rate of formation and final distribution of these products were strikingly different. Only small quantities of [''^C]isofenphos oxon were detected, representing, for example, 2.8 and 4«1# of applied after 1 and 4 weeks of incubation, respectively (Soil V). A large percentage of the applied [^^Cjisofenphos was converted to and soil-bound residues in these soils. Degradation to "''^COg accounted for 1/3 to 1/2 of initially applied [''^C]isofenphos during the 4-week incubation period. The most rapid evolution of occurred during the first week of incubation (Figure 2).

Large quantities of soil-bound residues were produced in soils in which isofenphos was rapidly degraded, and these residues tended to accumulate largely during the first week of incubation. In general, between 53 and 71# of the total bound residues found after 4 weeks were already present after only 1 week. An experiment conducted to determine if these residues were susceptible to further degradation revealed that small amounts (<5#) of the soil-bound residue could be released upon reincubation. This further degradation, as evidenced by the production of 28

SOIL TREATED WITH (I^C) ISOFENPHOS SOIL INSECTICIDE HISTORY

ISOFENPHOS IV

,ci ISOFENPHOS V -

ISOFENPHOS VI _

NONE I

"ONE

12 3 4

WEEKS OF INCUBATION

Figure 2. E#eot of insecticide history on the evolution of ''^COp from [ C]isofenphos-treated soils 29

14 COg* proved to be mediated by soil microorganisms. It is possible that

some of the ''^COg detected during the incubation of [''^C]isofenphos in soil •

was formed as the result of degradation of soil-bound residues formed. It

has been reported that, in general, an increase in insecticide degradation

in soil results in an increase in the quantity of soil-bound residues formed (Lichtenstein et al., 1977).

Inasmuch as no other isofenphos metabolites were detected in soils, it is unclear exactly what degradative reactions of isofenphos precede the formation of COg and soil-bound residues. It is likely that the intermediate metabolites formed were rapidly metabolized to other products, which would be consistent with earlier metabolic investigations of enhanced degradation in soil that also failed to detect intermediate metabolites of other insecticides (Rodriguez and Dorough, 1977; Felsot et al., 1981).

The metabolism of isofenphos by soil microorganisms

[^^cjisofenphos was rapidly degraded by a culture of microorganisms from soil with enhanced isofenphos degradation (Table IV). No degradation was noted in cultures that had been sterilized. As the quantity of

["*^0]isofenphos in nonsterile systems decreased, a temporary buildup of

[ Cjisopropyl salicylate was observed, with this hydrolysis product constituting 6% of the recovered after 6 h of incubation. Later in the incubation period, there was also a temporary buildup of polar, water-soluble products. The final product of isofenphos metabolism was the same as that in soil, with evolution substantially increasing as the quantities of isofenphos, isopropyl salicylate, and polar products declined. Trace quantities of salicylic acid and an unknown nonpolar 30

Table IV. The Metabolism of [^^Cjlsofenphos by a Mixed Culture of Soil Microorganisms

Hours of incubation

1 6 12 24 36

Distribution of in % of recovered

Isofenphos 89.0 82.0 69.4 36.3 11.6

Isopropyl salicylate 2.4 6.2 4.8 0.4 0.4

Polar 5.4 5.5 14.5 30.5 20.5 8 M C 0.0 0.9 2.4 23.9 62.1

Other^ 3.1 5.5 8.8 8.9 5.5

75-mL aqueous soil extract, with associated microorganisms, was treated with 5 ppm of [ ^C]isofenphos (1.0 uCi), in duplicate.

^"Other" includes that collected in organic vapor traps, as well as traces of salicylic acid and an unknown metabolite. 31

metabolite were also detected in cultures, but no isofenphos oxon.

The appearance of isopropyl salicylate in isofenphos-treated cultures

of soil microorganisms implicates a hydrolytic step in the metabolism of

isofenphos, which is common with the microbial metabolism of many

organophosphorus and carbamate insecticides (Sethunathan and Pathak, 1972;

Siddaramappa et al., 1973; Rodriguez and Dorough, 1977; Barik et al.,

1979). Because isopropyl salicylate was not detected as a metabolic

product during the degradation of isofenphos in soil, it is not certain

that this product is formed as a hydrolysis product by soil microorganisms

in situ. Insecticide hydrolysis products, however, are difficult to detect

in soil (Felsot et al., 1981; Chapman et al., 1985).

Large quantities of polar, water-soluble products of [^^C]isofenphos

degradation were formed in cultures of microorganisms, but were not similarly observed in soil. An experiment therefore was conducted to see whether these polar residues once applied to soil could again be extracted.

Within 1 h of application to soil, approximately 90$ of applied '''^C-polar residues were present as unextractable, soil-bound residues. If these same polar residues had been produced in soil, they may have constituted the soil-bound residues detected in the soil degradation study.

Because traces of [''^C]salicylic acid, a secondary hydrolysis product of isofenphos, were detected in isofenphos-treated microbial cultures, a second investigation of isofenphos metabolism was conducted. In this study, 50 ppm of salicylic acid was added to microbial cultures along with the [''^C]isofenphos in an attempt to force the temporary accumulation of any [^^cjgalicylic acid produced. Although the disappearance rate of 32

[^'^C]isofenphos was not affected by this addition, the production of

was delayed as compared with the previous study. The eventual evolution of

was preceded by a temporary accumulation of [''^C]salicylic acid in the medium, which accounted for 9.6% of recovered after 12 h of incubation. The implication is that salicylic acid is a probable secondary hydrolysis product of isofenphos. The lack of its accumulation in the previous experiment may be due to its extremely rapid metabolism once formed. Given that some soil bacteria are known to carry degradative plasmids for salicylic acid metabolism (Chakrabarty, 1972), it is possible that its formation may represent a key factor in the susceptibility of isofenphos to enhanced microbial degradation.

Enumeration and identification of isofenphos-degrading soil microorganisms

Results of the microbial most-probable-number determination (Figure 3) indicate that soils in which enhanced isofenphos degradation has occurred contain a population of microorganisms capable of metabolizing this insecticide. The three soils in which isofenphos was rapidly degraded (IV,

V, and VI) each contained, per gram, several thousand microorganisms capable of using isofenphos as a sole carbon source. Control soils (l, II, and III) contained no such microbial population. In comparison with the total numbers of microorganisms in these soils (Figure 3), isofenphos-degrading microorganisms represent only a small fraction of the total microflora present. The presence of an isofenphos-degrading population of soil microorganisms seems to provide a satisfactory explanation for the occurrence of enhanced degradation in these soils. A single application of isofenphos at 100 ppm to control soils (l, II, and 33

^ BACTERIA (10^)

FUNQI (10®)

I I ACTIN0MVCETB8 (10*)

I I Ê li M I- m ISOFENPHOS DEORAOERS (lO^)

NONE NONE NONE ISOFENPHOS ISOFENPHOSLu ISOFENPHOS I II III IV V VI

SOIL INSECTICIDE HISTORY

Figure 3. Numbers of microorganisms and isofenphos-degrading micro­ organisms in soil 34

III) resulted in the appearance of a similar degradatory population after a

1-week incubation. This suggests that enhanced degradation of isofenphos is an inducible phenomenon. Similarly, Fournier et al. (1981) reported an inducible population of 2,4-D-degrading microorganisms in soils in which enhanced degradation of this herbicide had occurred.

A bacterial strain was isolated from isofenphos-treated culture medium, and it proved capable of using isofenphos as a carbon source. This gram-negative, short rod (length; 1.3 uM) metabolized ["*^0]isofenphos to

and was identified as a species of Pseudomonas. This strain produced a light yellow, nonfluorescent pigment and was both motile and highly aerobic. When given a choice of either isofenphos or glucose as an energy source, the bacteria preferentially chose glucose. Pseudoraonads are common soil bacteria capable of metabolizing a wide variety of aromatic compounds

(Wheelis, 1975). Several Pseudomonas spp. have been isolated from soil that metabolize organophosphorus or carbamate insecticides (Kazano et al.,

1972; Siddaramappa et al., 1973; Kilbane et al., 1982), and it is a pseudomonad that has been implicated in the enhanced degradation of carbofuran (Felsot et al., 1981). Also, with salicylic acid in the microbial pathway of isofenphos degradation, it is noteworthy that some pseudoraonads carry the salicylic acid degradative plasmid (Chakrabarty,

1972). Several other bacterial isolates are being evaluated for isofenphos degrading ability.

In conclusion, isofenphos has undergone enhanced degradation in soil due to the induction of an isofenphos-degrading population of microorganisms. Further research is needed to provide a comprehensive 35

understanding of this increasingly important phenomenon if the effective use of biodegradable soil pesticides is to be continued. 36

ACKNOWLEDGMENTS

Special thanks are expressed to D. D. Michael for invaluable help provided with the microbial aspects of this project. Thanks are also expressed to Dr. Jon Tollefson and the corn insects research group for help in locating candidate soils, and to Dr. Tom Loynachan for manuscript ideas.

Results of this study were presented at the 190th National Meeting of the

American Chemical Society, Chicago, IL, Sept 1985; AGRO 38. Funding for this project was provided by grants from the USDA North Central Region

Pesticide Impact Assessment Program and the Iowa Corn Promotion Board. 37

LITERATURE CITED

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Barik, S.; Wahid, P. A.; Ramakrishna, C.; Sethunathan, N. "A Change in the Degradation Pathway of Parathion After Repeated Applications to Flooded Soil". J. Agric. Food Chem. 1979, 1391-1392.

Bartha, R.; Pramer, D. "Features of a Flask and Method for Measuring the Persistence and Biological Effects of Pesticides in Soil". Soil Soi. 1965, 100, 68-70.

Buchanan, R. E.; Gibbons, N. E., Eds. Bergey's Manual of Determinative Bacteriology, 8th ed.; The Williams and Wilkins Co.: Baltimore, MD, 1974*

Chakrabarty, A. M. "Genetic Basis of the Biodégradation of Salicylate in Pseudomonas". J. Bacterid. 1972, 112, 815-823.

Chapman, R. A.; Harris, C. R. "Persistence of Isofenphos and laazophos in a Mineral and an Organic Soil". J. Environ. Soi. Health, Part B 1982, B17, 355-361.

Chapman, R. A.; Harris, C. R.; Moy, P.; Henning, K. "Biodégradation of Pesticides in Soil; Rapid Degradation of Isofenphos in a Clay Loam After a Previous Treatment". J. Environ. Sci. Health, Part B 1986, B21, 269-276.

Chapman, R. A.; Moy, P.; Henning, K. "A Simple Test for Adaptation of Soil Microorganisms to the Degradation of the Insecticide Carbofuran". J. Environ. Soi. Health, Part B 1985, B20, 313-319.

Felsot, A. S. "Persistence of Isofenphos (Amaze) Soil Insecticide Under Laboratory and Field Conditions and Tentative Identification of a Stable Oxygen Analog Metabolite by Gas Chromatography". J. Environ. Sci. Health, Part B 1984, 13-27.

Felsot, A. S.; Maddox, J. V.; Bruce, W. "Enhanced Microbial Degradation of Carbofuran in Soils with Histories of Furadan Use". Bull. Environ. Contam. Toxicol. 1981, 781-788.

Felsot, A. S.; Wilson, J. G.; Kuhlman, D. E.; Steffey, K. L. "Rapid Dissipation of Carbofuran as a Limiting Factor in Corn Rootworm (Coleoptera; Chrysomelidae) Control in Fields with Histories of Continuous Carbofuran Use". J. Econ. Entomol. 1982, 7^, 1098-1103.

Ferris, I. G.; Lichtenstein, E. P. "Interactions Between Agricultural Chemicals and Soil Microflora and Their Effects on the Degradation of [ ^CjParathion in a Cranberry Soil". J. Agric. Food Chem. 1980, 28, 1011-1019. 38

Fournier, J. C.; Codaccioni, P.; Soulas, G. "Soil Adaptation to 2,4-D Degradation in Relation to the Application Rates and the Metabolic Behaviour of the Degrading Microflora". Chemosphere 1981, 10, 977-984.

Fryer, J. D.; Kirkland, K. "Field Experiments to Investigate Long-Term Effects of Repeated Applictions of MCPA, Tri-Allate, Simazine and Linuron: Report After 6 Years". Weed Res. 1970, 133-158.

Gorder, G. W.; Lichtenstein, E. P. "Degradation of Parathion by Cranberry Soil Microorganisms". Can. J. Microbiol. 1980, 475-481.

Harvey, R. G.; Schuman, D. B. "Accelerated Degradation of Thiocarbamate Hebicides with Repeated Use". Abstr. Weed Sci. Soc. Am. 1981, 124.

Hsin, C. Y.; Coats, J. R. "Metabolism of Isofenphos in Southern Corn Rootworm". Pestic. Biochem. Physiol. 1986, 336-345.

Kaufman, D. D.; Edwards, D. F. "Pesticide/Microbe Interaction Effects on Persistence of Pesticides in Soil". In Pesticide Chemistry; Human Welfare and the Environment, Vol. 4; Miyamoto J., Kearney, P. C., Eds.; Pergamon Press: Oxford, England, 1983.

Kazano, H.; Kearney, P. C.; Kaufman, D. D. "Metabolism of Methylcarbamate Insecticides in Soils". J. Agric. Food Chem. 1972, 975-979.

Kearney, P. C.; Kontson, A. "A Simple System to Simultaneously Measure Volatilization and Metabolism of Pesticides from Soils". J. Agric. Food Chem. 1976, 424-4266.

Keeney, D. R.; Bremner, J. M. "Determination and Isotope-Ratio Analysis of Different Forms of Nitrogen in Soils: 6. Mineralizable Nitrogen". Soil Sci. Soc. Am. Proc. 1967, 31_* 34-39.

Kilbane, J. J.; Chatterjee, D. K.; Karns, J. S.; Kellogg, S. T.; Chakrabarty, A. M. "Biodégradation of 2,4,5-Trichlorophenoxyacetic Acid and Chlorophenols". Appl. Environ. Microbiol. 1982, 72-78.

.Lehmicke, L. G.; Williams, R. T.; Crawford, R. L. " C-Most-Probable-Number Method for Enumeration of Active Heterotrophic Microorganisms in Natural Waters". Appl. Environ. Microbiol. 1979, 38, 644-649.

Lichtenstein, E. P.; Fuhremann, T. W.; Schulz, K. R.; Liang, T. T. "Effects of Field Application Methods on the Persistence and Metabolism of Phorate in Soils and its Translocation into Crops". J. Econ. Entomol. 1973, 66, 863-866.

Lichtenstein, E.^P.; Katan, J.; Anderegg, B. N. "Binding of Persistent and Nonpersistent C-Labelled-Insecticides in an Agricultural Soil". J. Agric. Food Chem. 1977, 25, 43-47. 39

Lichtenstein, E. P.; Schulz, K. R. "Breakdown of Lindane and Aldrin in Soils". J. Boon. Entomol. 1959» 118-124.

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Racke, K. D.; Lichtenstein, E. P. "Effects of Soil Microorganisms on the Release of Bound C Residues from Soils Previously Treated with [ C]Parathion". J. Agric. Food Chem. 1985, 33, 938-943.

Read, D. C. "Enhanced Microbial Degradation of Carbofuran and Fensulfothion After Repeated Applications to Acid Mineral Soil". Agric. Ecosyst. Environ. 1983, 10^, 37-46.

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Rodriguez, L. D.; Dorough, H. W. "Degradation of Carbaryl by Soil Microorganisms". Arch. Environ. Contam. Toxicol. 1977, 47-56.

Sethunathan, N.; Pathak, M. D. "Increased Biological Hydrolysis of Diazinon after Repeated Application in Rice Paddies". J. Agric. Food Chem. 1972, 586-589.

Siddaramappa, R.; Rajaram, K. P.; Sethunathan, N. "Degradation of Parathion by Bacteria Isolated from Flooded Soil". Appl. Microbiol. 1973, 846-849.

Skerraan, V. B. D. A Guide to the Identification of the Genera of Bacteria, 2nd ed.; The Williams and Wilkins Co.: Baltimore, MD, 1967.

Somerville, C. C.; Monti, C. A.; Spain, J. C. "Modification of the ^^C Most-Probable-Number Method for Use with Nonpolar and Volatile Substrates". Appl. Environ. Microbiol. 1985, 49, 711-713.

Spain, J. C.; Pritchard, P. H.; Bourquin, A. W. "Effects of Adaptation on Biodégradation Rates in Sediment/Water Cores from Estuarine and Freshwater Environments". Appl. Environ. Microbiol. 1980, 4^, 726-734.

Sutter, G. R. "Comparative Toxicity of Insecticides for Corn Rootworm (Coleoptera; Chrysomelidae) Larvae in a Soil Bioassay". J. Econ. Entomol. 1982, 75, 489-491.

Walker, A.; Brown, P. A.; Entwistle, A. R. "Enhanced Degradation of Iprodione and Vinclozolin in Soil". Pestic. Soi. 1986, T7, 183-193. 40

Wheelis, M. L. "The Genetics of Dissimilarity Pathways in Pseudomonas". Annu. Rev. Microbiol. 1975, 505-524.

Wollum, A.G. In Methods of Soil Analysis; Page. A. L., Miller, R. H., Keeney, D. R., Eds.; American Society of Agronomy; Madison, WI, 1982.

Yarden, 0.; Aharonson, N.; Katan, J. Abstracts of Papers, Sixth International Congress of Pesticide Chemistry, Ottawa, Canada; International Union of Pure and Applied Chemistry; Oxford, England, 1986; paper 6C-24. 41

CHAPTER II. COMPARATIVE DEGRADATION OF ORGANOPHOSPHORUS

INSECTICIDES IN SOIL: SPECIFICITY OF

ENHANCED MICROBIAL DEGRADATION 42

COMPARATIVE DEGRADATION OF ORGANOPHOSPHORUS

INSECTICIDES IN SOIL: SPECIFICITY OF

ENHANCED MICROBIAL DEGRADATION

Kenneth D. Racke and Joel R. Coats

Department of Entomology, Iowa State University, Ames, Iowa 50011 43

ABSTRACT

Laboratory experiments investigated the comparative degradation of 6

organophosphorus insecticides in soil as affected by enhanced microbial

degradation. The degradation rates and product distributions of

chlorpyrifos, fonofos, ethoprop, terbufos, and phorate were not

dramatically altered in soils containing microbial populations adapted to rapidly degrade isofenphos. An Arthrobacter sp. isolated from soils with a

history of isofenphos use rapidly metabolized isofenphos in pure culture,

but did not metabolize or ooraetabolize any of the other 5 organophosphorus insecticides. Likewise, only fonofos was rapidly degraded in soil with a long history of fonofos use. None of the organophosphorus insecticides was rapidly degraded in soil containing oarbofuran-degrading microbial populations. Results indicate that the phenomenon of enhanced microbial degradation of soil insecticides may exhibit some degree of specificity. 44

INTRODUCTION

Organophosphorus and carbamate Insecticides are widely used in the

Midwest to control such soil-dwelling pests of corn as larval corn

rootworms (Piabrotica spp.) and cutworms (Agrotis spp). Corn is now the

crop receiving the most intensive use of soil-applied insecticides, and in

Iowa alone, approximately 6 million acres are treated annually with soil

insecticides (Wintersteen and Hartzler, 1987). Although the degradation of

individual insecticides in soil has been investigated, few comparative

studies of their degradation have been conducted (Fuhremann and

Lichtenstein, 1980).

Enhanced microbial degradation is an increasingly important phenomenon

affecting the degradation of pesticides in soil (Tollefson, 1986).

Enhanced degradation occurs when a population of soil microorganisms, which

has adapted due to previous exposure to a pesticide, rapidly degrades a

subsequent application of the pesticide. The result of this enhanced

degradation is a failure of the pesticide to adequately control the target

pests due to dramatically decreased persistence (Felsot et al., 1982).

Enhanced degradation of several commonly used soil pesticides has been reported, including carbofuran (Felsot et al., 1981), EPTC (Wilson, 1984), and isofenphos (Racke and Coats, 1987a).

Enhanced degradation is, at its core, a pesticide/microbe interaction.

The decreased persistence of the pesticide involved results from rapid catabolism by populations of pesticide-adapted soil microorganisms

(Fournier et al., 1981; Kaufman and Edwards, 1983; Racke and Coats, 1987a).

Pesticide catabolism by adapted soil microorganisms usually involves a 45

hydrolysis, with further metabolism and utilization of one or more hydrolysis products as carbon or nutrient sources (Karns et al., 1986; Tarn et al., 1987; Racke and Coats, 1987a).

The current research was initiated to study the comparative degradation of 6 organophosphorus insecticides in soil as affected by enhanced microbial degradation. The study focused on determining the specificity of the enhanced degradative phenomenon, with special emphasis on the specificity of enhanced isofenphos degradation.

>. 46

MATERIALS AND METHODS

Chemicals

The following radiolabelled insecticides, along with model

metabolites, were obtained from the respective manufacturers: [^^C]-[U ring]-isofenphos (Mobay Chemical Corporation, Kansas City, MO),

[^^C]-[2,6-phenyl]-chlorpyrifos (Dow Chemical Company, Midland, Ml),

ring]-fonofos (Stauffer Chemical Company, Mountain View, CA),

[^^C]-[ethyl-1]-ethoprop (Rhone-Poulenc Inc., Monmouth Junction, NJ),

[^^C]-[methylene]-terbufos and [''^C]-[methylene]-phorate (American Cyanamid

Corporation, Princeton, NJ). All compounds were of greater than 98% radiopurity. Chemical structures of these insecticides are shown in Figure

1, and selected properties are shown in Table I.

Soils

Surface samples of Iowa soils were collected from cornfields at the end of the growing season, sieved to remove debris, and stored in a moist condition at 4°C. Two soils (ll and IV - 'isofenphos-history•) had histories of isofenphos use and performance failures and contained populations of isofenphos-degrading microorganisms (Racke and Coats,

1987a). A companion soil for each isofenphos soil (I and III) was taken from an adjacent field with no recent insecticide exposure. A soil with 5 consecutive years of fonofos use was also used (V). A final soil (VI -

'mixed-insecticide-history') had been exposed to a variety of organophosphorus and carbamate insecticides over the last 10 years and contained a microbial population that had adapted to rapidly degrade several carbamate insecticides, including carbofuran, carbaryl, bendiocarb. 47

CpoCH(CH,)j (CH,),CHNJ /= ISOFENPHOS _)p-o

C.H.O CHLORPYRIFOS CI C,

C,H.O, FONOFOS P—S

o C.H.S. II ETHOPROP )p-oc,H,

s C.H.O. II TERBUFOS /P—s—CHj—s—cccHjjj C,H,0

PHORATE )p-S-CHj-S-C,H5 C,H.O

Figure 1. Structures of organophosphorus insecticides used in the comparative degradation study 48 •

Table I. Selected Properties of Organophosphorus Insecticides Studied

Water ^ Soil Compound Solubility Half-Life Location Reference (ppm) (weeks)

12 Field Chapman and Harris (1962) laofenphos 22.1 5 - 8 Field Abou-Assaf et al. (1967) >14 Lab Felsot (1984)

2 - 6 Field Chapman and Harris (1980a) Chlorpyrifos 0.7 2 - 7 Field Pike and Getzin (1961) 4 - 12 Lab Getzin (1961)

6 Field Kiigemagi and Terriere (1971) Fonofos 15.7 6 Field Ahmad et al. (1979) 3 - 5 Lab Miles et al. (1979)

Ethoprop 750 2 - 12 Field Smelt et al. (1977) 14 Lab Jordan et al. (1966)

<2 Field Ahmad et al. (1979) Terbufos 5.5 2 Field Szeto et al. (1986) 1 Lab Chapman et al. (I982b)

<1 Field Suett (1975) Phorate 17.9 <1 Lab Getzin and Chapman (i960) <2 Lab Fuhremann and Lichtenstein (1980)

®Bowman and Sans (1979, 1983). 49

and cloethocarb (Racke and Coats, 1987b). Properties of soils used in the

comparative degradation study are listed in Table II.

Extraction and analyses

Both insecticide-treated soils and microbial cultures were analyzed

for insecticide and metabolites at the end of the respective incubation

period. [''^C]Insecticide residues in soil were extracted twice with

acetone-methanol (1:1), once with acetone-methanol-dichloromethane (1:1:1),

and partitioned into dichloromethane as described by Fuhremann and

Lichtenstein (1980). Aqueous samples of [''^C]insecticide-treated microbial

cultures were acidified with concentrated HCl before partitioning.

Thin-layer chromatographic and autoradiographic systems were used to

characterize isofenphos (Racke and Coats, 1987a), chlorpyrifos (Getzin,

1981), fonofos (Fuhremann and Lichtenstein, 1980), ethoprop (Menzer et al.,

1971), terbufos (Laveglia and Dahm, 1975), phorate (Fuhremann and

Lichtenstein, 1980), and their respective metabolites. Qualitative

confirmation of the identity of parent insecticides was by gas-liquid

chromatograpy, using a Varian 3700 chromatograph equipped with a 10#

DC-200/2# OV-225 on 80/100 mesh chromosorb column (2mm x 90cm) and a TSD

(N-P) detector. Unextractable, soil-bound [''^C]insecticide residues were

recovered by combustion to ^^COg in a Packard sample oxidizer.

Quantification of ^^C, including that present in vapor traps, on TLC

plates, and in alkaline COg traps, was by liquid scintillation counting. 50

Table II. Soils Used in the Comparative Insecticide Degradation Study

Texture Soil Insecticide % HpO No. Series History pH O.M. Sand Silt Clay 1/3 bar

I Tama None 7.3 3.9 8.2 58.6 33.2 32.9

II Tama Isofenphos 6.7 3.2 5.8 63.8 30.4 28.7

III Canisteo None 7.4 4.8 30.2 34.0 35.8 31.5

IV Canisteo Isofenphos 7.2 4.8 25.3 38.3 36.4 31.2

V Festina Fonofos 7.3 2.5 17.1 60.1 22.9 26.6

VI Readlyn Carbofuran 6.9 2.8 41.3 32.0 26.7 22.4

®Soils I and II (Tama), and soils III and IV (Canisteo) were companion soils taken from adjacent fields.

^For isofenphos soils, includes last two years; for fonofos soil includes last 5 years; carbofuran soil had carbofuran previous year, and mixed carbamate and organophosporus insecticide exposure every other year for 10 years, with alternate years untreated. 51

EXPERIMENTAL PROCEDURES

Comparative degradation of organophosphorus insecticides In soil

The 6 soils utilized in the study (Table II) were separately treated

with each of the 6 [''^C]insecticides at 5 ppm (0.5 or 1.0 uCi) in acetone

(Fuhremann and Lichtenstein, 1980) in duplicate 100-g samples and added to

250-mL glass jars (5 cm diam. x 11 cm ht.). The effect of laboratory

induction of enhanced degradation was also investigated by pretreating an

insecticide-free soil (soil I) with 10 ppm of isofenphos at 2-week

intervals for a total of 4 pretreatments. Two weeks after the last

pretreatment, this soil was then treated with [^^C]insecticides in the same

manner as the other 6 soils. Soils were moistened to field capacity (l/3

bar soil moisture tension) with distilled water and incubated at 25°C in the dark by using a flow-through incubation system (Ferris and

Lichtenstein, 1980). Air was periodically purged from the tightly sealed soil jars through both vapor and CO^ traps, and this allowed maintenance of aerobic conditions and monitoring of [''^C]insecticide degradation to ''^COg.

Distilled water was added as necessary to replace that lost due to evaporation. After 4 weeks, the soil in each jar was analyzed for the 1/1 presence of the respective [ C]insecticide and its metabolites as previously described.

Comparative metabolism of organophosphorus insecticides by an isofenphos-degrading microorganism

To isolate microorganisms involved in the enhanced degradation of isofenphos, aqueous suspensions of soils II and IV were incubated on basal-salts agar plates containing formulated isofenphos insecticide (Amaze 52

20G) as the sole carbon source. Colonies appearing on the plates were

tested for their ability to mineralize [''^C]isofenphos to (Racke and

Coats, 1987a) and were characterized by using standard microbial taxonomic

methodology (Gerhardt, 1981).

The comparative metabolism of the 6 organophosphorus insecticides was

determined in a pure culture of the most active degrader, an Arthrobacter

sp. Late exponential phase cultures of this Arthrobacter sp., grown on

basal-salts medium with salicylate as the sole carbon source, were

harvested by centrifugation (SOOjg x 30 rain) and washed twice by suspension

of the pelleted cells in basal-salts medium. One-milliliter aliquots of

1.0 Abs (Beckman DB spectrophotometer, 540 nm) bacterial suspension were

added to 25-mL Erlenmeyer flasks containing 4 mL of basal-salts medium,

following which one set was sterilized by autoclaving. Glass-fiber discs

impregnated with 250 ug of [''^C]insecticide (0.1 uCi) were then added

separately to the cultures, with half of the cultures receiving both

[^^C]insecticide and 250 ug of isofenphos to test for cometabolism. The flasks were incubated on a throw-shaker at 120 RPM for 12 h at 25°C, after which the cultures were analyzed for the presence of [''''''C] insecticides and

metabolites as described earlier. 53

RESULTS

Comparative degradation of organophoaphorua insecticides in soil

Results of the comparative degradation study will be summarized for

each Insecticide.

Isofenphos (Table III) and its oxon metabolite were most rapidly

degraded in soils with previous exposure to isofenphos (ll and IV). This

rapid degradation resulted in the production of significantly more COg and

soil-bound residues than in companion soils (I and III). The overall

degradation of Isofenphos was not significantly altered in soil with

fonofos history (V) or mixed insecticide history (Vl). A relatively

constant 62-76# of the applied isofenphos persisted after the 4-week

incubation in soils with a variety of textural properties and no prior

isofenphos exposure. Isofenphos degradation was extremely rapid in the

isofenphos-pretreated soil (10 ppm x 4 pretreatments), with more than 30%

of the applied insecticide being mineralized to COg within 2 days (results

not shown in Table III).

Chlorpyrifos (Table IV) was as persistent or more persistent in the isofenphos-history soils (II and IV) than in the companion soils (l and

III). However, a significantly different distribution of degradation

products was evident in comparing the Tama soils (I and II) with the

Canlsteo soils (III and IV). Chlorpyrifos was more persistent, yet gave rise to greater COg in the Tama soils, whereas greater production of soil-bound residues and 3,5,6-trlchloropyridinol occurred in the Canlsteo soils. The degradation of chlorpyrifos was not distinctly greater in either the fonofos-history soil (V) or the mixed-Insecticide-history soil Table III. Effect of Insecticide History on the Fate and Degradation of [^^C]lsofenphos in Soil, During a 4-Week Incubation

Soil Insecticide History^

Fraction None Isofenphos None Isofenphos Fonofos Mixed I II III IV V VI

recovered in % of applied C]isofenphos Soil Isofenphos 52.8^ 12.9® 75.2^ 24.9® 76.3^ 74.9^

Isof. Oxon 15.2* 2.8® 8.1^ 4.1® 12.1^ 11.6^

Bound 9.2* 23.6® 7.9^ 25.8® 8.0^ 7.3^

^^COg 10.0* 52.4® 5.2^ 34.5® 4.7^ 4.5^

Other° 0.9* 0.8* 0.8* 0.7* 1.4* 0.7*

Total 98.2* 92.8® 97.1* 89.6® 102.4^ 99.1*^

^[^^C]lsofenphos was uniformly applied to duplicate 100-g portions of soil at 5 ppm (0.5 uCi). Results from soils I-IV previously published (Racke and Coats, 1987a).

^Soil insecticide history includes last 2 years for II and IV, last 10 years for I and III, and last 5 years for V. Soil VI had carbofuran last year and mixed carbamate and organo- phosphorus insecticide exposure previously.

'^Includes volatile products other than CO^ as well as polar, water-soluble products.

^ ^eans followed by the same letter in each horizontal row are not significantly different at the 5% level (Student-Hewman-Keuls Test). Table IV. Effect of Insecticide History on the Fate and Degradation of [^^C]Chlorpyrifos in Soil, During a 4-Week Incubation

Soil Insecticide History^

Fraction None Isofenphos None Isofenphos Fonofos Mixed I II III IV V VI

recovered in % of applied [^^C]chïorpyrifos Soil Chlorpyrifos 23.6* 50.3® 11.9^ 14.1^ 33.9® 55.8^

T.C. Pyridinol 16.3* 5.6® 34.4^ 36.0^ 28.7® 11.8^

Chior. Oxon 1.9* 0.6® 0.9® 1.0® 1.8* 0.6®

Bound 20.8* 21.5* 39.1® 35.2^ 18.6® 15.4^

25.0* 16.4® 6.5^ 7.6^ 9.1® 10.9^

Other° 3.6* 2.2® 2.5® 3.7* 2.1® 1.0^

Total 91.1* 96.6* 95.2* 97.4^ 94.1^ 95.5*

^[^^C]Chlorpyrifos was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

Soil insecticide history includes last 2 years for II and IV, last 10 years for I and III, and last 5 years for V. Soil VI had carbofuran last year and mixed carbamate and organo- phosphorus insecticide exposure previously.

'^Includes volatile products other than CO^ as well as polar, water-soluble products.

* ^eans followed by the same letter in each horizontal row are not significantly different at the 3% level (Student-Newman-Keuls Test). 56

(VI). Chlorpyrifos was most persistent in the soils with pH < 7 (II and

VI).

The degradation of fonofos (Table V) was not dramatically affected by

soil isofenphos history. A decreased persistence of fonofos and

methyl-phenyl-sulfone and an increased production of COg were evident in

Tama soils (I and II) as compared with Canisteo soils (ill and IV). The

degradation of fonofos was most rapid in the fonofos-history soil (V), with

less than of the applied fonofos remaining in the soil after 4 weeks.

This increased degradation resulted in much greater production of

with 41^ of the applied [^^Cjfonofos cumulatively mineralized to ''^COg

during the first 11 days of incubation. Fonofos was most persistent in the

soil with mixed insecticide history.

Ethoprop (Table VI) was equally or more persistent in the isofenphos-history soils (II and IV) than in the companion soils (ill and

V). As was the case with fonofos, greater degradation of ethoprop, especially to COg, was noted in the Tama soils (l and II) compared with the

Canisteo soils (ill and IV). The persistence of ethoprop was not reduced in the fonofos-history soil (V), and it was the greatest in the mixed-insecticide-history soil (Vl). Significant amounts (>0.5#) of extractable ethoprop metabolites were not detected.

Neither terbufos (Table VII) nor phorate (Table VIII) was very persistent in any of the 6 soils, and both were nearly completely metabolized after 4 week's to their primary and secondary oxidative products, sulfoxides and sulfones. In all soils, greater quantities of terbufos sulfoxide were present than terbufos sulfone, whereas phorate Table V. Effect of Insecticide History on the Fate and Degradation of [^^CjFonofos in Soil, During a 4-Week Incubation

Soil Insecticide History^

Fraction None Isofenphos Hone Isofenphos Fonofos Mixed I . II III IV V VI

recovered in ^ of applied [^^C]fonofos Soil Fonofos 34.5^ 35.3* 51.2® 55.2® 7.8^ 60.4®

Meth. Ph. Sulfone 1.1* 1.4* 6.2® 5.3^ 0.2® 0.4^ de „ d de e de _(ie Fono. Oxon 0.4 0.7 0.5 0.2® 0.4 0.6

Bound 28.9^ 37.3® 27.7^ 21.3® 33.1® 20.8®

29.6^ 25.6® 15.5^ 12.6® 53.7^ 15.1^

Other^ 1.4^ 0.9®f 0.9®f 1.0^ 2.1® 0.7®

Total 96.0^ 101.2® 101.8* 95.6* 97.3* 97.9*

^[^^C]Fonofos was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

^Soil insecticide history includes last 2 years for II and IV, last 10 years for I and HI, and last 5 years for V. Soil VI had carbofuran last year and mixed carbamate and organo- phosphorus insecticide exposure previously.

"^Includes volatile products other than CO^ as well as polar, water-soluble products.

^ ^eans followed by the same letter in each horizontal row are not significantly different at the 3% level (Student-Newman-Keuls Test). Table VI. Effect of Insecticide History on the Fate and Degradation of [^^C]Ethoprop in Soil, During a 4-Week Incubation

b Soil Insecticide History

Fraction None Isofenphos None Isofenphos Fonofos Mixed I II III IV V VI

recovered in % of applied [^^C]ethoprop Soil Ethoprop 6.9 14.6'= 24.4 25.3 24.1 56.8* de de g Bound 31.7 29.Sf 33.7' 31.8 25.3^ 19.2 g 50.9 50.6^ 39.4 38.Gf 45.7 23.4 de de de de Other° 0.2 0.2 0.1 0.0 0.1 0.1

Total 89.8 95.3 97.7 95.7 95.3 99.5

^[^^C]Ethoprop was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

^Soil insecticide history includes last 2 years for II and IV, last 10 years for I and HI, and last 5 years for V. Soil VI had carbofuran last year and mixed carbamate and organo- phosphorus insecticide exposure previously.

^Includes volatile products other than CO^ as well as polar, water-soluble products.

^ ®Means followed by the same letter in each horizontal row are not significantly different at the 5% level (Student-Newman-Keuls Test). Table VII. Effect of Insecticide History on^the Fate and Degradation of [^^C]Terbufos in Soil, During a 4-Week Incubation

Soil Insecticide History^

Fraction None Isofenphos None Isofenphos Fonofos Mixed I II III IV V VI

recovered in % of applied [^^C]terbufos Soil Terbufos 1.4'= 1.0*^® 2.4®^ 3.5^ 0.3^ 3.8^

Terb. Sulfoxide 31.3^ 37.0® 53.4^ 52.3^ 42.6® 59.1^

Terb. Sulfone 22.9^ 20.6* 11.7® 9.5® 17.6^ 11.6®

Bound 17.6^ 22.9® 17.0^ 16.3^ 15.1® 11.2^

18.5*^ 16.3® 13.4^ 13.lf 14.5® 12.2^

Other° 9.2* 6.6® 4.0^ 5.9® 8.1* 4.9®f ef Total 100. 104.4® 101.8®^ 100.6*^ 98.2* 102.8

^[^^CjTerbufos was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

Soil insecticide history includes last 2 years for II and IV, last 10 years for I and HI, and last 5 years for V. Soil VI had carbofuran last year and mixed carbamate and organo- phosphorus insecticide exposure previously.

°Includes volatile products other than CO^ as well as polar, water-soluble products.

^ Cleans followed by the same letter in each horizontal row are not significantly different at the 3% level (Student-Newman-Keuls Test). 60

suifone was the major phorate metabolite detected. The persistence of

these oxidative metabolites was as great or greater in the

isofenphos-history soils (II and IV) than in the companion soils (I and

III). Differences also were not evident in the fonofos soil (V) nor in the

mixed insecticide-history soil (Vl). Greater quantities of terbufos

sulfone, in relation to quantities of terbufos sulfoxide, were present in

the Tama soils (l and II) than in the Canisteo soils (ill and IV).

Insecticide degradation results for the isofenphos-pretreated soil

(soil I: 10 ppm x 4 pretreatments) are not shown because, with the

exception of isofenphos, results were not significantly different from

those obtained in soil I with no pretreatments.

Comparative metabolism of organophosphorus insecticides by an

isofenphos-degrading microorganism

Several bacterial isolates from isofenphos-history soils (II and IV)

were capable of degrading isofenphos to "'^CO^. One bacterium isolated

from soil II was identified as a Pseudomonas sp. and was capable of

utilizing isofenphos as a sole carbon source (Racke and Coats, 1987a).

However, this isofenphos-degrading trait was unstable and was lost after

successive culturings on a rich medium, perhaps indicating plasmid

involvement. In contrast, an Arthrobacter sp. isolated from soils II and

IV exhibited a very stable isofenphos-degrading trait, and this isolate

proved to be the most efficient isofenphos degrader. A pure culture of

this bacterium completely degraded 10, 50, or 100 ppm of [''^C]isofenphos in

less than 6 h, with the production of nearly equal quantities of 14 COg and

[^^C]polar, water-soluble products. Although preliminary evidence suggests Table VIII. Effect of Insecticide History on the Fate and Degradation of [^^CjPhorate in Soil, During a 4-Week Incubation

Soil Insecticide History

Fraction None Isofenphos None Isofenphos Fonofos Mixed I II III IV V VI

recovered in % of applied [^^C]phorate Soil Phorate 0.4^ 0.4* 0.2* 1.1® 0.5* 0.2*

Phor. Sulfoxide 3.8* 2.7® 8.2^ 8.9^ 8.1^ 18.2®

Phor. Sulfone 35.0* 46.0® 46.8® 49.5^ 49.6^ 57.7®

Bound . 22.7*® 23.6® 20.1^ 19.5^ 21.6* 13.8®

^^COg 19.3* 26.9® 12.0^ 11.3® 16.4^ 10.8®

Other® 18.3* 1.0® 13.6^ 11.8® 9.4^ 2.8^

Total 99.5* 100.5* 100.8* 102.2*® 105.7^ 103.5*

^[^^CjPhorate was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

^Soil insecticide history includes last 2 years for II and IV, last 10 years for I and HI, and last 5 years for V. Soil VI had carbofuran last year and mixed carbamate and organo- phosphorus insecticide exposure previously.

'^Includes volatile products other than CO^ as well as polar, water-soluble products.

^ ^Means followed by the same letter in each horizontal row are not significantly different at the 5% level (Student-Newman-Keuls Test). 62

that this Arthrobacter may be able to utilize isofenphos as a sole carbon

source, growth on isofenphos in pure culture was not conclusively

quantified. The strain exhibited typical Arthrobacter characteristics,

including snapping division and a rod to coccus, gram-negative to

gram-positive life cycle. This isolate utilized a variety of simple

organic substrates as sole carbon sources (e.g., benzoate, salicylate,

phenyl acetate), tolerated up to 4*5# NaCl, was highly aerobic and

nonmotlle, sensitive to penicillin, carbeniclllln, and bacitracin, and

insensitive to colistln.

A pure culture of the isofenphos-degrading Arthrobacter sp. was tested

for the ability to metabolize or cometabolize chlorpyrlfos, fonofos,

ethoprop, terbufos, or phorate. After a 12-h exposure to each of these

Insecticides individually, or In combination with isofenphos, no evidence

of metabolism or cometabolism of any of the insecticides other than

isofenphos was noted (Figure 2). In the case of isofenphos, 45^ of the

applied Isofenphos was recovered as polar, water-soluble products

after 12 h. When this incubation was repeated in a flow-through system

(Racke and Coats, 1987a), 50% of the applied [''^C]isofenphos was mineralized to ''^COg. 63

PARENT INSECTICIDE METABOLITES iF U 100 (/)

TfS f 80H S

i 60 0 aR

5 40

20

1o S N S N CHLO FONO ETHO TERB PHOR INSECTICIDE

Figure 2. Metabolism of organophosphorus insecticides in a pure culture of Arthrobacter sp. (S = sterile, N = nonsterile) 64

DISCUSSION

Each of the organophosphorus insecticides exhibited different

persistence and a different product distribution in soil. On average, the

persistence of parent insecticide after 4 weeks, in order of decreasing

persistence, was: isofenphos > fonofos > chlorpyrifos > ethoprop »

terbufos > phorate. This is consistent with earlier reports of the

persistent nature of isofenphos (Chapman and Harris, 1982; Felsot, 1984)

and of the relatively greater persistence of fonofos than of both

chlorpyrifos (Miles et al., 1979) and phorate (Ahmed et al., 1979}

Fuhremann and Lichtenstein, 1980). The ephemeral nature of terbufos and

phorate in soil has been well documented (Getzin and Chapman, I960; Suett,

1975; Sellers et al., 1976; Chapman et al., 1982a,b), and it is terbufos sulfoxide and phorate sulfone that are most persistent in soil and may contribute to target-pest toxicity (Laveglia and Dahm, 1975; Chapman and

Harris, 1980b; Harris and Chapman, 1980).

The importance of soil properties in modulating the degradation of insecticides has long been recognized (Edwards, 1966). In the current study, all the organophosphorus insecticides were most persistent in the

Readlyn soil, which had the least organic matter content, greatest sand content, and a near-neutral pH. Isofenphos, fonofos, ethoprop, terbufos, and phorate were all more persistent in the higher-organic-matter Canisteo soils than in the Tama soils. Increased organic matter in soil is often associated with increased adsorption of pesticides (Stevenson, 1972), which can render pesticides less susceptible to microbial degradation (Ogram et al., 1985). In the present study, only chlorpyrifos was less persistent in 65

the Canisteo soils than in the Tama soils, being represented by greater

quantities of 3,5»6-trichloropyridinol in the former soils. Chemical

degradation is an important factor modulating chlorpyrifos persistence in

soil (Miles et al., 1979), and increased production of its hydrolysis

product in soil has been correlated with increased soil organic matter

content (Chapman and Harris, 1980a; Getzin, 1981).

Clearly, the degradation of isofenphos was profoundly affected by its

prior application to soil. Decreased persistence of isofenphos in the

field after several years of use has been documented (Chapman et al., 1986;

Abou-Assaf et al., 1986). Racke and Coats (1987a) subsequently

demonstrated that isofenphos had undergone enhanced degradation by

inducible populations of soil microorganisms.

The involvement of an Arthrobacter in the enhanced degradation of

isofenphos is not surprising inasmuch as Arthrobacter spp. are

metabolically diverse degraders in soil (Hagedorn and Holt, 1975) and have

been implicated in the enhanced degradation of both 2,4-D and BPTC (Tiedje et al., 1969; Tara et al., 1987). Several soil bacteria have been isolated that could rapidly metabolize isofenphos, including a Corynebacterium sp.

(Murphy and Cohick, 1985), a Pseudomonas sp. (Racke and Coats, 1987a), and a Streptomyces sp. (Gauger et al., 1986). Thus, it is possible that the enhanced degradation of isofenphos in soil may be due to the activities of more than one bacterial strain. Indeed, several genera of bacteria have been implicated in the enhanced degradation of carbofuran (Felsot et al.,

1981; Karns et al., 1986) and diazinon (Siddaramappa et al., 1973; Forrest et al., 1981). 66

Although fonofos was rapidly degraded in the soil with fonofos

history, more research is needed to determine if it too has undergone

enhanced degradation. Unlike the rapid degradation of isofenphos and

carbofuran, preliminary results in several soils indicate that the ability

of a soil to rapidly degrade fonofos does not persist through the winter.

It may be important to distinguish between temporary soil adaptation to

rapidly degrade a pesticide and the persistence of that adaptive trait to

affect a subsequent field application of the chemical.

None of the other organophosphorus insecticides was rapidly degraded

in soil in which enhanced degradation of isofenphos was evident. Likewise,

only fonofos was rapidly degraded in the fonofos-history soil. It was in

the soil with mixed insecticide history that the organophosphorus

insecticides were most persistent, although several carbamates had

undergone enhanced degradation in this soil (Racke and Coats, 1987b).

In several instances, the enhanced degradation of a specific pesticide

in a given soil has evidently involved a rather broad adaptation, with

other members of the same pesticide class also subject to rapid

degradation. This has been true to some extent with the carbamate

insecticides (Harris et al., 1984; Felsot, 1986), thiocarbamate herbicides

(Wilson, 1984; Harvey et al., 1986), and phenoxyacetic acid herbicides

(Kirkland and Fryer, 1972). However, the enhanced degradation of

organophosphorus insecticides in soil has usually been associated with a

lesser degree of cross adaptation. Thus, the enhanced degradation of

diazinon has displayed some degree of specificity within the

organophosphorus class (Sethunathan and Pathak, 1972; Forrest et al.. 67

1981). Likewise, the enhanced degradation of chlorfenvinphos is specific,

and the degradation of other organophosphorus insecticides is not

accelerated in soil capable of rapidly degrading chlorfenvinphos (Read,

1987). Horng and Kaufman (1987) did find some evidence that cross

adaptations may be associated with the enhanced degradation of some

organophosphorus insecticides in soil. Cross-adaptations involving members

of different classes of pesticides have not generally been noted (Forrest

et al., 1981; Harris et al., 1984).

The isofenphos-degrading Arthrobacter sp. isolated in the current

investigation was able to rapidly metabolize this compound, yet did not

metabolize or cometabolize the other organophosphorus insecticides tested.

Because hydrolysis has been shown to be an important step in the enhanced

microbial degradation of isofenphos (Racke and Coats, 1987a), it is

possible that the enzymes involved in isofenphos hydrolysis may be somewhat

specific. Of the 6 organophosphorus insecticides studied, isofenphos is

unique in possessing a phosphoramide bond (Figure I), and this may confer

properties requiring the action of a different type of hydrolytic enzyme.

Although the hydrolase isolated from diazinon-metabolizing Flavobacterium

and Pseudomonas strains is able to hydrolyze some of the other

organophosphorus insecticides (Kearney et al., 1986), only the hydrolysis

product of diazinon is utilized as a carbon source by these bacteria.

Consequently, only diazinon is rapidly degraded by these bacteria in soil

(Sethunathan and Pathak, 1972; Forrest et al., 1981). It is possible that the metabolic specificity exhibited by organophosphorus-degrading bacteria may relate to their ability to beneficially metabolize the hydrolysis 68

products formed.

In conclusion, results indicate that the enhanced degradative phenomenon may exhibit a high degree of specificity in soil and at the microbial level. Further research is needed to shed light on the importance of this phenomenon in soil, and provide a basic understanding of the microbial ecology and genetics of its occurrence. 69

ACKNOWLEDGMENTS

Special thanks are expressed to D. D. Michael for help with the microbial aspects of this project. Thanks are also expressed to J. J.

Tollefson for help in locating candidate soils, to F. D. Williams for suggestions on experimental design, and to T. E. Loynachan for manuscript suggestions. Results of this study were presented at the 191st National

Meeting of the American Chemical Society, New York City, April 1986; AGRO

86. Funding for this project was provided by grants from the Iowa Corn

Promotion Board and the USDA North Central Region Pesticide Impact

Assessment Program. 70

LITERATURE CITED

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Ahmad, N.; Walgenbach, D. D.; Sutter, G. R. "Comparative Disappearance of Fonofos, Phorate and Terbufos Soil Residues Under Similar South Dakota Field Conditions". Bull. Environ. Contam. Toxicol. 1979, 423-429.

Bowman, B. T.; Sans, W. W. "The Aqueous Solubility of Twenty-Seven Insecticides and Related Compounds". J. Environ. Soi. Health, Part B 1979, B14. 625-634.

Bowman, B. T.; Sans, W. W. "Further Water Solubility Determinations of Insecticidal Compounds". J. Environ. Soi. Health, Part B 1983, B18, 221-227.

Chapman, R. A.; Harris, C. R. "Persistence of Chlorpyrlfos in a Mineral and an Organic Soil". J. Environ. Sol. Health, Part B 1980a, B15, 39-46.

Chapman, R. A.; Harris, C. R. "Insecticidal Activity and Persistence of Terbufos, Terbufos Sulfoxide and Terbufos Sulfone in Soil". J. Econ. Entomol. 1980b, 536-543.

Chapman, R. A.; Harris, C. R. "Persistence of Isofenphos and Isazophos in a Mineral and an Organic Soil". J. Environ. Soi. Health, Part B 1982, B17, 355-361.

Chapman, R. A.; Tu, C. M.; Harris, C. R.; Harris, C. R. "Biochemical and Chemical Transformations of Phorate, Phorate Sulfoxide, and Phorate Sulfone in Natural and Sterile, Mineral and Organic soil". J. Econ. Entomol. 1982a, 75, 112-117.

Chapman, R. A.; Tu, C. M.; Harris, C. R.; Dubois, D. "Biochemical and Chemical Transformations of Terbufos, Terbufos Sulfoxide, and Terbufos Sulfone in Natural and Sterile, Mineral and Organic Soil". J. Econ. Entomol. 1982b, 75, 955-960.

Chapman, R. A.; Harris, C. R.; Moy, P.; Hennlng, K. "Biodégradation of Pesticides in Soil: Rapid Degradation of Isofenphos in a Clay Loam After a Previous Treatment". J. Environ. Sci. Health, Part B 1986, B21, 269-276.

Edwards, C. A. "Insecticide Residues in Soils". Residue Rev. 1966, 13, 83-132. 71

Felaot, A. S. "Persistence of Isofenphos (Amaze) Soil Insecticide Under Laboratory and Field Conditions and Tentative Identification of a Stable Oxygen Analog Metabolite by Gas Chromatography". J. Environ. Soi. Health, Part B 1984, 13-27.

Felsot, A. S. "Cross-Adaptations for Biodégradation of Methylcarbamate Insecticides in Soil". Abstracts of Papers, 192nd National Meeting of the American Chemical Society, New York City, NY; American Chemical Society: Washington, DC, 1986; AGRO 87.

Felsot, A. S.; Maddox, J. V.; Bruce, W. "Enhanced Microbial Degradation of Carbofuran in Soils with Histories of Furadan Use". Bull. Environ. Contam. Toxicol. 1981, 781-788.

Felsot, A. S.; Wilson, J. G.; Kuhlman, D. E.; Steffey, K. L. "Rapid Dissipation of Carbofuran as a Limiting Factor in Corn Rootworm (Coleoptera: Chrysomelidae) Control in Fields with Histories of Continuous Carbofuran Use". J. Econ. Entomol. 1982, 75, 1098-1103.

Ferris, I. G.; Lichtenstein, E. P. "Interactions Between Agricultural Chemicals and Soil Microflora and Their Effects on the Degradation of [ C]Parathion in a Cranberry Soil". J. Agric. Food Chem. 1980, 28, 1011-1019.

Forrest, M.; Lord, K. A.; Walker, N.; Woodville, H. C. "The Influence of Soil Treatments on the Bacterial Degradation of Diazinon and Other Organophosphorus Insecticides". Environ. Pollut., Series A 1981, A24, 93-104.

Foumier, J, C.; Codaccioni, P.; Soulas, G. "Soil Adaptation to 2,4-D Degradation in Relation to the Application Rates and the Metabolic Behaviour of the Degrading Microflora". Chemosphere 1981, _1£, 977-984.

Fuhremann, T. W.; Lichtenstein, E. P. "A Comparative Study of the Persistence, Movement, and Metabolism of Six Carbon-14 Insecticides in Soils and Plants". J. Agric. Food Chem. 1980, 446-452.

Gauger, W. K.; MacDonald, J. M.; Adrian, N. R.; Mathees, D. P. "Characterization of a Streptomycete Growing on Organophosphate and Carbamate Insecticides". Arch. Environ. Contam. Toxicol. 1986, 137-141.

Gerhardt, P., Ed. Manual of Methods for General Bacteriology; American Society for Microbiology: Washington, DC, 1981.

Getzin, L. W. "Degradation of Chlorpyrifos in Soil; Influence of Autoclaving, Soil Moisture, and Temperature". J. Econ. Entomol. 1981, 74» 158-162.

Getzin, L. W.; Chapman, R. K. "The Fate of Phorate in Soils". J. Econ. Entomol. I960, 53, 47-51. 72

Hagedorn, C.; Holt, J. G. "A Nutritional and Taxonomic Survey of Arthobacter Soil Isolates". Can. J. Microbiol. 1975, 353-361.

Harris, C. R.; Chapman, R. A. "Insecticidal Activity and Persistence of Phorate, Phorate Sulfoxide, and Phorate Sulfone in Soils". Can. Entomol. 1980, 112, 641-653.

Harris, C. R.; Chapman, R. A.; Harris, C.; Tu, C. M. "Biodégradation of Pesticides in Soil: Rapid Induction of Carbamate Degrading Factors After Carbofuran Treatment". J. Environ. Soi. Health, Part B 1984, B19, 1-11.

Harvey, R. G.; McNevin, G. R.; Albright, J. W.; Kozak, M. E. "Wild Proso Millet (Panicum miliaceum) Control with Thiocarbamate Herbicides on Previously Treated Soils". Weed Soi. 1986, 773-780.

Horng, L. C.; Kaufman, D. D. "Accelerated Biodégradation of Several Organophosphate Insecticides". Abstracts of Papers, 193rd National Meeting of the American Chemical Society, Denver, CO; American Chemical Society: Washington, DC, 1987; AGRO 44.

Jordan, E. G.; Montecalvo, D. M.; Norris, F. A. "Metabolism of Ethoprop in Soil". Abstracts of Papers, 192nd National Meeting of the American Chemical Society, Anaheim, CA; American Chemical Society; Washington, DC, 1986; AGRO 36.

Karns, J. S.; Mulbry, W. W.; Nelson, J. 0.; Kearney, P. C. "Metabolism of Carbofuran by a Pure Bacterial Culture". Pestic. Biochem. Physiol. 1986,' 211-217.

Kaufman, D. D.; Edwards, D. F. "Pesticide/Microbe Interaction Effects on Persistence of Pesticides in Soil". In Pesticide Chemistry; Human Welfare and the Environment, Vol. 4; Miyamoto, J., Kearney, P.C., Eds.; Pergamon Press; Oxford, England, 1983.

Kearney, P. C.; Karns, J. S.; Mulbry, W. W. "Engineering Soil Microorganisms for Pesticide Degradation". Abstracts of Papers, Sixth International Congress of Pesticide Chemistry, Ottawa, Canada; International Union of Pure and Applied Chemistry: Oxford, England, 1986; paper SS 2-01.

Kiigemagi, U.; Terriere, L. C. "The Persistence of Zinophos and Dyfonate in Soil". Bull. Environ. Contam. Toxicol. 1971, 355-359.

Kirkland, K.; Fryer, J. D. "Degradation of Several Herbicides in a Soil Previously Treated with MCPA". Weed Res. 1972, _12, 90-95.

Laveglia, J.; Dahm, P. A. "Oxidation of Tebufos (Counter) in Iowa Corn and Soil". Environ. Entomol. 1975 , 4.» 715-718. 73

Menzer, R. E.; Iqbal, Z. M.; Boyd, G. R. "Metabolism of D-Ethyl ^,^,Dipropyl Phosphorodithioate (Mocap) in Bean and Corn Plants". J. Agric. Food Chem. 1971, _19, 351-356.

Miles, J. R. W.; Tu, C. M.; Harris, C. R. "Persistence of Eight Organophosphorus Insecticides in Sterile and Non-Sterile Mineral and Organic Soils". Bull. Environ. Contam. Toxicol. 1979, 22^, 312-318.

Murphy, J. J.; Cohick, A. D. "Behavior of Amaze in Midwestern Soils". Presented at the 40th meeting of the North Central Branch of the Entomological Society of America, Lexington, KY; Entomological Society of America: Hyattsville, MD, 1985; paper 121.

Ogram, A. V.; Jessup, R. E.; Ou, L. T.; Rao, P. S. C. "Effects of Sorption on Biological Degradation of 2,4-Diohlorophenoxy Acetic Acid in Soils". Appl. Environ. Microbiol. 1985, 582-587.

Pike, K. S.; Getzin, L. W. "Persistence and Movement of Chlorpyrifos in Sprinkler-Irrigated Soil". J. Econ. Entomol. 1981, 74, 385-388.

Racke, K. D.; Coats, J. R. "Enhanced Degradation of Isofenphos by Soil Microorganisms". J. Agric. Food Chem. 1987a, 94-99.

Racke, K. D.; Coats, J. R. "Enhanced Degradation and the Comparative Fate of Carbamate Insecticides in Soil"., submitted for publication in J. Agric. Food Chem., 1987b.

Read, D. C. "Greatly Accelerated Microbial Breakdown of Chlorfenvinphos and Fensulfothion, and Influence of Microbes on Other Insecticides, in Acid Mineral Soil Treated with Chlorfenvinphos Three Years in Succession"., submitted for publication in J. Econ. Entomol., 1987.

Sellers, L. G.; Owens, J. C.; Tollefson, J. J.; Dahm, P. A. "Residues of Terbufos (Counter) in Iowa Corn and Soil". J. Econ. Entomol. 1976, 69, 133-135.

Sethunathan, N.; Pathak, M. D. "Increased Biological Hydrolysis of Diazinon after Repeated Application in Rice Paddies". J. Agric. Food Chem. 1972, 586-589.

Siddaramappa, R.; Rajaram, K. P.; Sethunathan, N. "Degradation of Parathion by Bacteria Isolated from Flooded Soil". Appl. Microbiol. 1973, 846-849.

Smelt, J. H.; Leistra, M.; Voerman, S. "Movement and Rate of Decomposition of Ethoprophos in Soil Columns Under Field Conditions". Pestic. Soi. 1977, 8, 147-151.

Stevenson, F. J. "Organic Matter Interactions Involving Herbicides in Soils". J. Environ. Qual. 1972, 1, 333-343. 74

Suett, D.L. "Persistence and Degradation of Chlorfenvinphos, Chlormephos, Disulfoton, Phorate and Pirimiphos-Ethyl Following Spring and Late Summer Soil Application". Pestic. Soi. 1975» 385-393.

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Tollefson, J. J. "Why Soil Insecticidal Immunity"? Solutions 1986, 30, 48-55.

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Wintersteen, W.; Hartzler, R. "Pesticides Used in Iowa Crop Producton in 1985" Iowa State Cooperative Extension Service Bulletin Pm-1288, Jan 1987. 75

CHAPTER III. ENHANCED DEGRADATION AND THE COMPARATIVE FATE

OF CARBAMATE INSECTICIDES IN SOIL 76

ENHANCED DEGRADATION AND THE COMPARATIVE FATE

OF CARBAMATE INSECTICIDES IN SOIL

Kenneth D. Racke and Joel R. Coats

Department of Entomology, Iowa State University, Ames, Iowa 50011 77

ABSTRACT

Laboratory experiments investigated the comparative degradation of 5 carbamate insecticides in soil as affected by enhanced microbial degradation. Soils with prior field exposure to carbofuran, cloethocarb, or several carbamates contained adapted microbial populations capable of rapidly degrading carbofuran. Bendiocarb was rapidly degraded in all soils displaying enhanced carbofuran degradation, but carbaryl and cloethocarb were most rapidly degraded only in soil with prior exposure to several carbamates or to cloethocarb. The persistence of aldicarb and its oxidative metabolites, aldicarb sulfoxide and aldicarb sulfone, was not dramatically altered in soils with enhanced carbofuran degradation.

Results indicate that, although cross-adaptations for enhanced degradation exist within the carbamate insecticide class, structural similarity may play a role in modifying the expression of enhanced degradation in soil. 78

INTRODUCTION

Enhanced degradation is the phenomenon whereby a soil-applied

pesticide is rapidly degraded by a population of microorganisms that has

adapted because of previous exposure to it or a similar pesticide. The

result of this enhanced degradation may be a failure of the pesticide to

adequately control the target pest due to dramatically decreased

persistence (Felsot et al., 1982; Harvey et al., 1986). Pesticide

catabolism by adapted soil microorganisms is microbially beneficial in that

the pesticide or a hydrolysis product serves as a microbial carbon, energy,

or nutrient source (Fournier et al., 1981; Karns et al., 1986; Racke and

Coats, 1987a; Tam et al., 1987). Enhanced degradation has been reported

for a number of insecticides (Sethunathan and Pathak, 1972; Felsot et al.,

1981; Read, 1983; Racke and Coats, 1987a; Read, 1987a), herbicides (Newman

and Thomas, 1949; Audus, 1951; Kirkland and Fryer, 1972; Wilson, 1984; Gray

and Joo, 1985; Skipper et al., 1986), and fungicides (Walker et al., 1986;

Yarden et al., 1986).

Carbofuran is a carbamate insecticide that has been widely applied to

soil to control insect pests of corn, most notably larval corn rootworms

(Piabrotica spp.). Decreased persistence of carbofuran in fields with

prior carbofuran exposure (Greenhalgh and Belanger, 1981) led to the

discovery that enhanced microbial degradation was responsible for the decreased persistence and associated control failures observed (Felsot et al., 1981, 1982, 1985). Application of carbofuran to soil results in the appearance of a bacterial population capable of rapidly metabolizing subsequent applications of carbofuran (Felsot et al., 1981; Karns et al., 79

1986; Chaudhry et al., 1986). In addition to carbofuran, two other

carbamate insecticides, carbaryl (Rodriguez and Dorough, 1977) and aldicarb

(Read, 1987a), are also susceptible to enhanced degradation in soil.

There has been much interest in determining the specificity of the

microbial adaptation involved in enhanced degradation. In some cases,

microbial adaptation to degrade a specific pesticide rapidly in soil has resulted in the ability to degrade a variety of related pesticides (Audus,

1951; McClure, 1972; Kaufman and Edwards, 1983; Skipper et al., 1986), whereas in other cases, the adaptation has been more narrowly confined to a single pesticide (Sethunathan and Pathak, 1972; Forrest et al., 1981; Racke and Coat's, 1987b; Read, 1987b). Repeated laboratory treatments of soil with carbofuran were reported to induce rapid degradation of carbofuran and a number of other carbamate insecticides (Harris et al., 1984). A carbofuran-degrading bacterium isolated from a soil column percolated with

200 ppm of carbofuran has been shown to be capable of catabolizing several other carbamate insecticides (Karns et al., 1986).

The purpose of the present study was to investigate the specificity of enhanced carbamate degradation in soil as induced by normal field applications, A comparative approach examined the metabolism of a series of carbamate insecticides in the laboratory in soils with confirmed enhanced carbofuran degradation and in companion soils taken from adjacent fields with no recent insecticide history. Insecticides chosen for study were carbofuran, bendiocarb, carbaryl, cloethocarb, and aldicarb (Figure

1). 80

MATERIALS AND METHODS

Chemicals

The following radiolabelled insecticides, along with model

metabolites, were obtained from the respective sources: [''^C]-[U

ring]-carbofuran and [^^C]-[oarbonyl]-carbofuran (FMC Corporation,

Princeton, NJ), [^^^0-[2-benzodioxole]-bendiocarb (Fisons Corporation,

Bedford, MA), [''^C]-[l-naphthyl]-carbaryl (Nuclear Chicago, Chicago, IL),

[^^C]-[4-phenyi]-cloethocarb (BASF Corporation, Parsippany, NJ),

[^^C]-[methylthio]-aldicarb (California Bionuclear Corporation, Sun Valley,

CA). All compounds were of greater than SB% radiopurity. Chemical

structures of these insecticides are shown in Figure 1.

Soils

Surface samples of soils (Table l) were collected from cornfields at

the end of the growing season, sieved to remove debris, and stored in a

moist condition at 4°C. Two soils (II and III) had received a carbofuran application the previous 2 growing seasons. A companion soil (l) came from an adjacent field with no recent insecticide history. Two soils were from fields on an experimental farm, one soil (IV) with no recent insecticide history and the other soil (V) with extensive insecticide history. The soil with extensive insecticide history had carbofuran applied the previous year and mixed carbamate and organophosphorus insecticide treatments every other year for 10 years with alternate years untreated. Carbamate insecticides previously used on soil V included carbofuran, bendiocarb, and cloethocarb. The final pair of soils came from within one field, one soil

(VI) from a plot with no recent insecticide history and the other soil 81

0-C-NHCH CARBOFURAN

0-C-NHCH3 BENDIOCARB 'VCHj

0-C-NHCH, CARBARYL

O-C-NHCH,

O-CH-CHjCI CLOETHOCARB OCH,

0-C-NHCH] ALDICARB il CH-Ç-SCHj CH,

Figure 1. Structure of carbamate insecticides used in the comparative degradation study 82

(VII) from a plot that had received an annual treatment of cloethocarb for

each of the last 4 years. The last field application of carbofuran and

cloethocarb failed to provide adequate control of corn rootworms

(Piabrotica spp.) in soils II, III, and V, and in soil VII, respectively.

Characteristics of soils used in the comparative degradation study are

listed in Table I.

Confirmation that the adaptation resulting in enhanced carbofuran

degradation had occurred in the carbamate-history soils (II, III, V, VII)

was made by using an assay of [^^C]-[carbonyl]-carbofuran degradation to

^^COg, demonstrating microbial involvement, and enumerating adapted,

carbofuran-degrading microorganisms according to the methodology of Racke

and Coats (1987a).

Soil insecticide treatment

The 7 soils used in the study (Table I) were treated separately with

each of the 5 [^^C]insecticides (1.0 uCi) in acetone (Lichtenstein and

Schulz, 1959) in duplicate 100-g samples and added to 250-mL glass jars (5

cm diam. x 11 cm ht.). [^^^]-[U ring]-carbofuran was used for this study.

* Soils were moistened to field capacity (l/3 bar soil moisture tension) with

distilled water and incubated at 25°C in the dark by using a flow-through

incubation system (Ferris and Lichtenstein, 1980). Air was periodically

purged from the tightly sealed jars through both vapor and CO^ traps, which

allowed maintenance of aerobic conditions and monitoring of

[^^C]insecticide degradation to ^^COg. 83

Table I. Soils Used in the Comparative Insecticide Degradation Study-

Percent Soil Insecticide Texture Carbofuran No, Series^ History pH O.M. Degraded Sand Silt Clay 1-W Assay®

I Bourbon None 6.4 8.1 56.6 26.0 17.4 17.8

II Bourbon Carbofuran 6.7 3.7 63.8 20.5 15.7 91.1

III Tama Carbofuran 7.0 4.4 8.6 58.7 32.7 85.2

IV Readlyn None 7.0 3.9 41.3 34.7 24.0 19.7

V Readlyn Carbamates 6.7 4.3 29.3 41.3 29.4 81.5

VI Webster None 6.6 4.9 34.6 36.0 29.4 24.5

VII Webster Cloethocarb 7.2 5.1 37.8 34.8 27.4 86.6

®Soils I and II (Bourbon), soils IV and V (Readlyn), and soils VI and VII (Webster) were companion soils taken from adjacent fields.

^For soils with no insecticide history (I,IV,VI), includes last 10 years; soils II and III had carbofuran applied last 2 years; soil VII had cloethocarb applied last 4 years ; soil V had received carbofuran previous year, and mixed carbamate and organophosphorus insecticides every other year for 10 years, with alternate years untreated.

.?A 25-g aliquot of each soil, in duplicate, was treated with 5 ppm of [ ^C]-[carbonyl]-carbofuran (0.1 uCi), mixed with 75 g of sand, and incubated for.1 week. 'Percent Carbofuran Degraded' is based on the quantity of COg evolved. Results are means of duplicate tests. 84

Extraction and analyses 1 After a 4-week incubation, [ AC]insecticide residues in soil were extracted twice with acetone:methanol (1:1), once with acetone:methanolrdichloromethane (1:1:1), and partitioned into dichloromethane as described by Lichtenstein et al. (1973). Thin-layer chromatographic (TLC) and autoradiographic systems were used to characterize carbofuran (Metcalf et al., 1968), bendiocarb (Hsin and Coats,

1987), carbaryl (Leeling and Casida, 1966), aldicarb (Bull et al., 1967), and their respective metabolites. Organic-soluble extracts of cloethocarb-treated soils were spotted on silica gel TLC plates and developed in chloroform:ethyl acetate:N-hexane (5:2:1) (cloethocarb

R^=0.53). Qualitative confirmation of the identity of parent insecticides was by gas-liquid chromatograpy, using a Varian 3700 chromatograph equipped with a 10^ DC-200/2# OV-225 on 80/l00-mesh chromosorb column (2mm x 90cm) and a TSD (N-P) detector. Unextractable, soil-bound [^''''C]Insecticide residues were recovered by combustion to ^^COg in a Packard sample oxidizer. 85

RESULTS

Confirmation of carbofuran enhanced degradation

The [^^C]-[carbonyl]-carbofuran degradation assay (Table l) revealed

that carbofuran was rapidly degraded in soils with prior carbofuran

exposure This rapid degradation was also evident in the soil

with cloethocarb history (VII). Results clearly divided the soils into two

categories, those with prior carbamate exposure that degraded 82 to 91^ of

the applied carbofuran (II,III,V,VII) in 4 weeks, and those with no recent

insecticide exposure that degraded 18 to Z5% of the applied carbofuran

(I,IV,VI). The evolution rate of ''^CO^ from the carbamate-history soils

accelerated during the first few days of incubation (e.g., soil II, Figure

2), which is indicative of enhanced microbial degradation (Kaufman and

Edwards, 1983; Racke and Coats, 1987a). Sterilization or application of

bactericide (chloramphenicol) inhibited the degradation of carbofuran,

implicating microbial involvement. A most-probable-number assay indicated 5 5 that between 1.5 % 10 and 7.4 % 10 carbofuran-degrading microorganisms

were present per gram of carbamate-history soil (II,III,V,VII). In 5 contrast, an average of 0.5 x 10 carbofuran-degraders was detected in insecticide-free soils (I,IV,VI). Thus, all lines of evidence confirmed the occurrence of enhanced carbofuran degradation in the carbamate-history soils.

Comparative degradation of carbamate insecticides in soil

The comparative degradation of 5 carbamate insecticides was evaluated in each of the 7 soils to determine which of the other carbamate insecticides would also be rapidly degraded in soil displaying enhanced 86

SOIL TREATED WITH (^^C) CARBOFURAN + ANTIMICROBIAL TREATMENT g 100

NONE CYCLOHEXIMIDE 80

60

40

CHLORAMPHENICOL

-V AUTOCLAVING 1 3 5 7 DAYS OF INCUBATION

Figure 2. Effect oJJ'.antimicrobial treatments on the evolution of ''^COp from a [ C]-[carbonyl]-carbofuran-treated soil (ll) 87

carbofuran degradation. Results of the comparative degradation study will

be summarized for each insecticide.

As anticipated by the [^4c]-[carbonyl]-carbofuran degradation assay,

very little [^^Cj-Eu ring]-carbofuran (Table II) persisted after the 4-week

incubation in soils with prior carbamate exposure (II,III,V,VII). In

contrast, between 29 and 31% of the initially applied carbofuran remained

in the companion soils (l,ÏV,Vl). The rapid degradation of carbofuran

resulted in the production of large quantities of soil-bound residues.

Bendiocarb (Table III) persistence in soil was similarly affected by

prior carbamate exposure. In all soils in which carbofuran had been

rapidly degraded, bendiocarb was likewise rapidly degraded, and only about

1% of the initially applied bendiocarb persisted in these soils

(II,III,V,VII) after 4 weeks. In contrast, between 30 and 47^ of the

applied bendiocarb persisted in the companion soils (I,IV,VI). Rapid

degradation of bendiocarb resulted in increased production of soil-bound residues and also in increased production of in comparison with companion soils.

Carbaryl (Table IV) degradation results were mixed in the carbamate-history soils. In one carbofuran-history soil (ll), carbaryl was slightly less persistent and, in another soil (ill), markedly more persistent, than in the companion soil (l). However, carbaryl degradation was much accelerated in soil with mixed carbamate history (v) and in soil with cloethocarb history (VIl). The increased degradation of carbaryl in these 2 soils (V,VII) resulted in the production of large quantities of

which by the second week of incubation accounted for Table II. Effect of Insecticide History on the Fate and Degradation of [^^C]Carbofuran in Soil, During a 4-Week Incubation

Soil Insecticide History^

Fraction None Carbofuran Carbofuran None Carbamates None Cloethocarb I II III IV V VI VII

^^C recovered in 56 of applied [^^C]carbofuran

Carbofuran 29.4^ 0.9® 1.2® 56.0^ 0.8® 56.6^ 1.6®

3-Keto Carbo. 3.4 0.3 0.3 4.5 0.2 1.4 0.4

Other^ 3.1 1.6 1.1 2.0 1.7 1.2 1.8

Soil-Bound 41.9^ 69.2® 60.3^ 29.5® 71.8^ 27.2® 76.9^

^^co^ 13.8^ 30.4® 36.4^ 5.8® 14.0^ 8.6^ 11.0^

Total 91.5^* 102.3^ 99.3*^® 97.8^^ 88.5® 94.9^® 91.7^^®

^[^^C]Carbofuran was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

Soil insecticide history includes last 10 years for I, IV, and VI; includes last 2 years for soils II and HI; includes last 4 years for soil VII; soil V had received carbofuran previous year, and mixed carbamate and organophosphorus insecticides every other year for 10 years, with alternate years untreated.

^Primarily water-soluble metabolites with traces of organic-soluble metabolites.

^ Means followed by the same letter in each horizontal row are not significantly different at the IjS level (Student-Newman-Keuls Test). Table III. Effect of Insecticide History on the Fate and Degradation of [^^C]Bendiocarb in Soil, During a 4-Week Incubation

Soil Insecticide History^

Fraction None Carbofuran Carbofuran None Carbamates None Cloethocarb I II III IV V VI VII

14c recovered in % of applied [^^cjbendiocarb

Bendiocarb 34.6* 0.9® 1.6® 30.4^ 1.3® 47.2® 0.9®

Other° 3.5 2.7 2.1 2.0 1.9 2.3 2.1

Soil-Bound 38.5^ 43.4® 51.9^ 38.4^ 42.4® 28.3® 51.8^

I^COg 19.5^ 42.4® 36.2^ 23.3® 43.2® 18.7^ 36.2^

Total 96.0 89.4 91.9 94.1 88.8 96.5 91.1

^[^^C]Bendiocarb was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

Soil insecticide history includes last 10 years for I, IV, and VI; includes last 2 years for soils II and HI; includes last 4 years for soil VII; soil V had received carbofuran previous year, and mixed carbamate and organophosphorus insecticides every other year for 10 years, with alternate years untreated.

"^Primarily water-soluble metabolites with traces of organic-soluble metabolites.

®Means followed by the same letter in each horizontal row are not significantly different at the 1% level (Student-Newman-Keuls Test). Table IV. Effect of Insecticide History on^the Fate and Degradation of [^^C]Carbaryl in Soil, During a 4-Week Incubation

Soil Insecticide History^

Fraction None Carbofuran Carbofuran None Carbamates None Cloethocarb I II III IV V VI VII

recovered in % of applied [^^Cjcarbaryl

Carbaryl 26.8^ 17.7® 42.6^ 38.6® 5.8^ 54.1^ 9.3»

Other° 0.2 0.1 2.7 0.1 0.0 0.2 0.1

Soil-Bound 8.6^ 6.8® 11.3^ 7.9^® 4.8® 10.6^ 7.4*®

55.3^ 68.2® 42.2^ 46.9® 80.6^ 22.9^ 80.0»

Total 90.9^ 92.8^® 97.2® 93.5*^® 91.2"^ 87.8^ 96.8®

^[^^C]Carbaryl was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

^Soil insecticide history includes last 10 years for I, IV, and VI; includes last 2 years for soils II and III; includes last 4 years for soil VII; soil V had received carbofuran previous year, and mixed carbamate and organophosphorus insecticides every other year for 10 years, with alternate years untreated.

"^Primarily water-soluble metabolites with traces of organic-soluble metabolites.

^Means followed by the same letter in each horizontal row are not significantly different at the 1% level (Student-Newman-Keuls Test). 91

72 and 63% of the applied [^^C]carbaryl, respectively.

The degradation of cloethocarb (Table V) was also only moderately altered in soils with oarbofuran history. Although significantly less cloethocarb persisted in both carbofuran-history soils (II,III) than in the companion soil (l), the difference observed was much less than that observed with carbofuran or bendiocarb. As with carbofuran, bendiocarb, and carbaryl, cloethocarb was rapidly degraded in the carbamate-history soil (V). Rapid degradation of cloethocarb was also observed in soil with prior exposure to cloethocarb (VIl). The rapid degradation of cloethocarb in soils V and VII resulted in greater formation of soil-bound residues and 14 slightly greater COg production than that observed in companion soils

(IV,VI).

Aldicarb (Table VI) was not very persistent in any of the soils tested, with less than 2% of applied aldicarb remaining after the 4-week incubation in each soil. Aldicarb was instead represented by its insecticidal metabolites, aldicarb sulfoxide and aldicarb sulfone. The quantities of aldicarb sulfoxide recovered from the carbamate-history soils were not significantly less than those recoverd from the corresponding companion soils. This was not true for aldicarb sulfone, of which significantly less was present in the carbamate-history soil (V) and the cloethocarb-history soil (VIl) than in the companion soils (IV,VI). The decrease in aldicarb sulfone present was accompanied by corresponding increases in and soil-bound residue production. Table V. Effect of Insecticide History on the Fate and Degradation of [^^C]Cloethocarb in Soil, During a 4-Week Incubation

Soil. Insecticide History^

Fraction None Carbofuran Carbofuran None Carbamates None Cloethocarb I II III IV V VI VII

14c recovered in % of applied [^^cjcloethocarb

Cloethocarb 48.3* 24.0® 41.1^ 50.0* 1.2® 67.0^ 2.6® c ^ d _ _d d _ d _ „d Other 1.3 2.1 1.1 1.2 1.0 2.0 0.9

Soil-Bound 31.3* 43.9® 41.3® 32.1* 67.0^ 22.8® 67.7^

I^COg 14.1* 15.3* 12.4® 9.3^ 21.7® 8.1^ 18.2^

Total 95.0*® 85.2® 95.9*® 92.8*® 90.9*® 99.9* 89.3*^

^[^^CjCloethocarb was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

Soil insecticide history includes last 10 years for I, IV, and VI; includes last 2 years for soils II and III; includes last 4 years for soil VII; soil V had received carbofuran previous year, and mixed carbamate and organophosphorus insecticides every other year for 10 years, with alternate years untreated.

'^Primarily water-soluble metabolites with traces of organic-soluble metabolites.

^ ^eans followed by the same letter in each horizontal row are not significantly different at the level (Student-Newman-Keuls Test). Table VI. Effect of Insecticide History on the Fate and Degradation of [^^C]Aldicarb in Soil, During a 4-Week Incubation

Soil Insecticide History^ Fraction None Carbofuran Carbofuran None Carbamates None Cloethocarb I II III IV V VI VII

14c recovered in % 5 of applied [^^Qjaidicarb

Aldicarb 1.1 1.6 0.6 0.6 0.5 0.5 1.4

Aid. Sulfoxide 3.1^ 16.4® 11.5^® 10.7® 11.9^® 13.6^ 12.0^2

Aid. Sulfone 17.6^ 17.8* 18.1* 18.4* 7.3® 20.3^ 12.9®

Other^ 7.5" 16.0® 11.7^ 13.7®^ 12.of 11.1^ 13.3®^

Soil-Bound 19.8^ 15.3® 19.9* 20.6* 24.3^ 19.9* 21.7* - r 8 J \ C 38.2^ 29.6® 33.7*^^ 31.8®f 37.7* 30.7® 35.2*f

Total 87.3* 96.7® 95.5® 95.8® 93.7® 96.1® 96.6®

^[^^C]Aldicarb was uniformly applied to duplicate 100-g portions of soil at 5 ppm (1 uCi).

Soil insecticide history includes last 10 years for I, IV, and VI; includes last 2 years for soils II and III; includes last 4 years for soil VII; soil V had received carbofuran previous year, and mixed carbamate and organophosphorus insecticides every other year for 10 years, with alternate years untreated.

^Primarily water-soluble metabolites with traces of organic-soluble metabolites.

®Means followed by the same letter in each horizontal row are not significantly different at the level (Student-Newman-Keuls Test). 94

DISCUSSION

Evidence from the current investigation clearly suggests that

cross-adaptations of enhanced degradation occur within the carbamate insecticide class. Previous exposure of soil to carbofuran, cloethocarb, or several carbamates induced the rapid degradation of carbamate compounds that had not been previously applied to a given soil. Evidence for induction of relatively nonspecific enhanced carbamate degradation in soil has been reported for soils exposed to carbofuran in the field (Kaufman and

Edwards, 1983; Felsot, 1986) and soils subject to repeated carbofuran applications in the lab (Harris et al., 1984» Chapman et al., 1986b;

Felsot, 1986). However, most of the previous laboratory studies in which enhanced degradation was induced by repeated laboratory treatments did not distinguish between cometabolism and true cross-adaptation of the subsequently applied carbamates. Crosa-adaptation would imply the ability of a compound to induce the appropriate microbial response (e.g., enzyme production) in the absence of large quantities of previous substrate.

Cross-adaptations for enhanced degradation have also been reported within the phenoxyacetic acid herbicide class (Newman and Thomas, 1949; Audus,

1951; Kirkland and Fryer, 1972) and within the thiocarbamate herbicide class (Wilson, 1984; Skipper et al., 1986; Harvey et al., 1986). This is in contrast to the lack of cross-adaptations associated with enhanced degradation within the organophosphorus insecticide class as expressed in soil (Sethunathan and Pathak, 1972; Forrest et al., 1981; Racke and Coats,

1987b; Read, 1987b). In addition, enhanced carbofuran degradation does not affect the degradation of organophosphorus insecticides (Racke and Coats, 95

1987b), thiocarbamate herbicides (Kaufman and Edwards, 1983), or

dithiocarbaraate herbicides (Harris et al., 1984) in soil.

Also evident from the current investigation is that not all the

carbamates were as rapidly degraded as carbofuran in soil displaying

enhanced carbofuran degradation. It is possible that similarity in

chemical structure may play a role in moderating the cross adaptations

expressed. Bendiocarb, a carbamate that is extremely similar in structure

to carbofuran (Figure 1), was rapidly degraded in each soil that rapidly

degraded carbofuran. With carbaryl and cloethocarb, this was not the case,

however, because in soils exposed previously only to carbofuran, the

increase in carbaryl or cloethocarb degradation was slight or absent.

Felsot (1986) reported that, although bendiocarb, trimethacarb, and

isoprocarb were all rapidly degraded in soil with enhanced carbofuran

degradation, bufencarb was not. Similarly, Read (1987a) found that soil repeatedly treated with carbofuran in the laboratory developed the capacity to degrade carbofuran, propoxur, and cloethocarb rapidly, but not oxamyl or aldicarb. Chapman et al. (1986b) determined that, although one laboratory pretreatment of soil with carbofuran induced rapid degradation of both carbofuran and carbaryl, 4 pretreatments were needed to induce rapid cloethocarb degradation, and oxamyl was not rapidly degraded even after 9 carbofuran pretreatments. A range of selectivity of cross-adaptions involved in enhanced degradation has also been reported within the phenoxyacetic acid herbicide class (Kirkland and Fryer, 1972) and the thiocarbamate herbicide class (Wilson, 1984; Skipper et al., 1986; Harvey et al., 1986). 96

The idea that a range of intensity of cross-adaptations exists within

the carbamate insecticide class is supported by work with a

carbofuran-degrading bacterium isolated from soil. Karns et al. (1986)

reported that an Achromobacter sp. isolated from carbofuran-treated soil

could hydrolyze not only carbofuran, but also several other carbamates.

However, the rate of hydrolysis depended on similarity of structure to

carbofuran, with hydrolysis rates for the following compounds arranged in

decreasing order; carbofuran > carbaryl > propoxur > aldicarb >

0-nitrophenyl dimethyl carbamate. Work conducted with carbofuran

hydrolase, the carbofuran- hydrolyzing enzyme isolated from this

Achromobacter sp., revealed that, although this enzyme was very efficient

in hydrolyzing carbofuran and carbaryl, it was much less efficient in

hydrolyzing aldicarb in vitro (Derbyshire et al., 1987).

It is possible that both inherent and induced soil properties may play

a role in modulating the extent to which carbamate cross-adaptations are expressed. Factors such as soil pH, moisture, organic-matter content, and temperature have been shown to affect the development and expression of enhanced carbofuran degradation (Read, 1983; Chapman et al., 1986a,b).

These same factors may also affect the magnitude of the cross-adaptations observed. Chapman et al. (1986b) reported that soils collected from the same plots but during different seasons displayed different degrees of carbamate cross-adaptations in response to repeated laboratory carbofuran treatments. Although the ecology of carbofuran-degrading microorganisms in soil has not been examined, members of 3 bacterial genera have been implicated in enhanced carbofuran degradation (Felsot et al., 1981; Karns 97

et al., 1986; Chaudhry et al., 1986). It is not known whether specific

bacterial species or species complexes display similar selectivity or even

genetic bases for carbamate metabolism. In the current investigation,

carbofuran, bendiocarb, carbaryl, and cloethocarb were rapidly degraded in

soil with prior exposure to various carbamates, yet only carbofuran and

bendiocarb were unequivocally subject to enhanced degradation in soils

previously exposed only to carbofuran. It is possible that different

populations of carbamate-degrading microorganisms may have been induced,

depending on the range of substrates to which the soil had previously been

exposed. Prior use of cloethocarb induced rapid degradation of carbofuran

in the current investigation, but prior use of carbofuran only slightly

accelerated cloethocarb degradation. Cloethocarb persistence in soil has

previously been reported to be unaffected by enhanced carbofuran

degradation (Hamm and Thomas, 1986).

The susceptibility of carbamate insecticides to enhanced degradation

is a significant development that may limit the long-term usefulness of

these compounds in soil. Recognition of enhanced carbofuran degradation as

the cause of pest control failures may be partly resonsible for the substantial decrease in its use against corn rootworms in Iowa (Becker and

Stockdale, 1980; Wintersteen and Hartzler, 1987). Enhanced degradation may also have played a substantial role in recent decisions to suspend development of bendiocarb and cloethocarb for this same market. Additional research regarding the microbial ecology and genetics of enhanced degradation, as well as factors that render particular pesticides or groups of pesticides susceptible to enhanced degradation, is needed. The authors thank K. R. Titus and D. D. Michael for assistance with chemical and microbial analyses associated with this project. Thanks are also expressed to L. W. Bledsoe, J. D. Oleson, and J. J, Tollefson for help in locating candidate soils and to T. E. Loynachan and L. Somasundaram for manuscript suggestions. Results of this study were presented at the 194th

National Meeting of the American Chemical Society, New Orleans, LA, Aug

1987; AGRO 23. Funding for this project was provided by grants from the

Iowa Corn Promotion Board, the USDA North Central Region Pesticide Impact

Assessment Program, and BASF Corporation. 99

LITERATURE CITED

Audus, L. J. "The Biological Detoxification of Hormone Herbicides in Soil". Plant Soil 1951, 3, 170-192.

Becker, R.; Stockdale, H. J. "Pesticides Used in Iowa Crop Production in 1978 and 1979" Iowa State Cooperative Extension Service Bulletin Pm-964, Jan 1980.

Bull, D. L.; Lindquist, D. A.; Coppedge, J. R. "Metabolism of 2-Methyl-2- (Methylthio)Propionaldehyde 0;-(Methylcarbamoyl)0xime (Temik, UC-21149) in Insects". J. Agric. Food Chem. 1967, 15^, 610-616.

Chapman, R. A.; Harris, C. R.; Harris, C. "The Effect of Formulation and Moisture Level on the Persistence of Carbofuran in a Soil Containing Biological Systems Adapted to its Degradation". J. Environ. Sci. Health, Part B 1986a, 57-66.

Chapman, R. A.; Harris, C. R.; Harris, C. "Observations on the Effect of Soil Type, Treatment Intensity, Insecticide Formulation, Temperature and Moisture on the Adaptation and Subsequent Activity of Biological Agents Associated With Carbofuran Degradation in Soil". J. Environ. Sci. Health, Part B 1986b, 125-141.

Chaudhry, G. R.; Ou, L. T.; Wheeler, W. B. "Degradation of Carbofuran by Soil Microorganisms". Abstracts of Papers, Sixth International Congress of Pesticide Chemistry, Ottawa, Canada; International Union of Pure and Applied Chemistry; Oxford, England, 1986; paper 6C-12.

Derbyshire, M. K.; Karns, J. S.; Kearney, P. C.; Nelson, J. 0. "Purification and Characterization of a N-Methylcarbamate Pesticide Hydrolyzing Enzyme"., submitted for publication in J. Agric. Food Chem., 1987.

Felsot, A. S. "Effect of Conditioning Soil for Enhanced Biodégradation of Carbofuran on Dissipation of Other Methyl Carbamate Insecticides". Abstracts of Papers, Sixth International Congress of Pesticide Chemistry, Ottawa, Canada; International Union of Pure and Applied Chemistry; Oxford, England, 1986; paper 6B-08.

Felsot, A. S.; Maddox, J. V.; Bruce, W. "Enhanced Microbial Degradation of Carbofuran in Soils with Histories of Furadan Use". Bull. Environ. Contam. Toxicol. 1981, 781-788.

Felsot, A. S.; Steffey, K. L.; Levine, E.; Wilson, J. G. "Carbofuran Persistence in Soil and Adult Corn Rootworra (Coleoptera; Chrysoraelidae) Susceptibility; Relationship to the Control of Damage by Larvae". J. Econ. Entomol. 1985, 78, 45-52. 100

Felsot, A. S.; Wilson, J. G.; Kuhlman, D. E.; Steffey, K. L. "Rapid Dissipation of Carbofuran as a Limiting Factor in Corn Rootworm (Coleoptera: Chrysomelidae) Control in Fields with Histories of Continuous Carbofuran Use". J. Econ. Entomol. 1982, 1098-1103.

Ferris, I. G.; Lichtenstein, E. P. "Interactions Between Agricultural Chemicals and Soil Microflora and Their Effects on the Degradation of [ C]Parathion in a Cranberry Soil". J. Agric. Food Chem. 1980, 28, 1011-1019.

Forrest, M.; Lord, K. A.; Walker, N.; Woodville, H. C. "The Influence of Soil Treatments on the Bacterial Degradation of Diazinon and Other Organophosphorus Insecticides". Environ. Pollut., Series A 1981, A24, 93-104.

Fournier, J. C.; Codaccioni, P.; Soulas, G. "Soil Adaptation to 2,4-D Degradation in Relation to the Application Rates and the Metabolic Behaviour of the Degrading Microfora". Chemosphere 1981, _1£, 977-984•

Gray, R. A.; Joo, G. K. "Reduction in Weed Control After Repeat Applications of Thiocarbamate and Other Herbicides". Weed Soi. 1985, 33, 698-702.

Greenhalgh, R.; Belanger, A. "Persistence and Uptake of Carbofuran in a Humic Mesisol and the Effects of Drying and Storing Soil Samples on Residue Levels". J. Agric. Food Chem. 1981, 231-235.

Hamm, R. T.; Thomas, J. H. "Microbial Degradation of Cloethocarb and Carbofuran in 2 Different Soils". Abstracts of Papers, Sixth International Congress of Pesticide Chemistry, Ottawa, Canada; International Union of Pure and Applied Chemistry: Oxford, England, 1986; paper 6C-14-

Harris, C. R.; Chapman, R. A.; Harris, C.; Tu, C. M. "Biodégradation of Pesticides in Soil: Rapid Induction of Carbamate Degrading Factors After Carbofuran Treatment". J. Environ. Sci. Health, Part B 1984, B19, 1-11.

Harvey, R. G.; McNevin, G. R.; Albright, J. W.; Kozak, M. E. "Wild Proso Millet (Panicum miliaceum) Control with Thiocarbamate Herbicides on Previously Treated Soils". Weed Sci. 1986, 773-780.

Hsin, C. Y.; Coats, J. R. "Bendiocarb Metabolism in Adults and Larvae of the Southern Corn Rootworm Diabrotica undecimpunctata Howardi Barber". J. Pestic. Sci. 1987, 12, 301-309.

Karns, J. S.; Mulbry, W. W.; Nelson, J. 0.; Kearney, P. C. "Metabolism of Carbofuran by a Pure Bacterial Culture". Pestic. Biochem. Physiol. 1986, 25, 211-217. 101

Kaufman, D. D.; Edwards, D. F. "Pesticide/Microbe Interaction Effects on Persistence of Pesticides in Soil". In Pesticide Chemistry; Human Welfare and the Environment, Vol. 4; Miyamoto, J., Kearney, P. C., Eds.; Pergamon Press: Oxford, England, 1983.

Kirkland, K.; Fryer, J. D. "Degradation of Several Herbicides in a Soil Previously Treated with MCPA". Weed Res. 1972, _12, 90-95.

Leeling, N. C.; Casida, J. E. "Metabolites of Carbaryl (1-Naphthyl Methylcarbamate) in Mammals and Enzymatic Systems for Their Formation". J. Agric. Food Chem. 1966, 281-290.

Lichtenstein, E. P.; Fuhremann, T. W.; Schulz, K. R.; Liang, T. T. "Effects of Field Application Methods on the Persistence and Metabolism of Phorate in Soils and its Translocation into Crops". J. Econ. Entomol. 1973, 66, 863-866.

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Metcalf, R. L.; Fukuto, T. R.; Collins, C.; Borck, K.; El-Aziz, S. A.; Munoz, R.; Cassil, C. C. "Metabolism of 2,2-Dimethyl-2,3-Dihydrobenzofuranyl-7-N-Methyl- carbamate (Furadan) in Plants, Insects, and Mammals". J. Agric. Food Chem. 1968, _1^, 300-311.

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Racke, K. D.; Coats, J. R. "Enhanced Degradation of Isofenphos by Soil Microorganisms". J. Agric. Food Chem. 1987a, 35, 94-99.

Racke, K. D.; Coats, J. R. "Comparative Degradation of Organophosphorus Insecticides in Soil: Specificity of Enhanced Microbial Degradation". J. Agric. Food Chem. 1987b, in press.

Read, D. C. "Enhanced Microbial Degradation of Carbofuran and Fensulfothion After Repeated Applications to Acid Mineral Soil". Agric. Ecosyst. Environ. 1983, 22.» 37-46.

Read, D. C. "Greatly Accelerated Microbial Degradation of Aldicarb in Re-Treated Field Soil, in Flooded Soil, and in Water". J. Econ. Entomol. 1987a, 80, 156-163. 102

Read, D. C. "Greatly Accelerated Microbial Breakdown of Chlorfenvinphos and Fensulfothion, and Influence of Microbes on Other Insecticides, in Acid Mineral Soil Treated with Chlorfenvinphos Three Years in Succession"., submitted for publication in J. Econ. Entomol., 1987b.

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Sethunathan, N.; Pathak, M. D. "Increased Biological Hydrolysis of Diazinon after Repeated Application in Rice Paddies". J. Agric. Food Chem. 1972, 586-589.

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Walker, A.; Brown, P. A.; Entwistle, A. R. "Enhanced Degradation of Iprodione and Vinclozolin in Soil". Pestic. Sci. 1986, 183-193.

Wilson, R. G. "Accelerated Degradation of Thiocarbamate Herbicides in Soil with Prior Thiocarbamate Herbicide Exposure". Weed Sci. 1984, 32, 264-268.

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SUMMARY

This dissertation has addressed several aspects of enhanced microbial

degradation of pesticides in soil.

Research on the enhanced degradation of isofenphos and carbofuran has

provided important information regarding the essential nature of enhanced

degradation. Enhanced microbial degradation may now be defined as "the

phenomenon whereby a pesticide is rapidly catabolized by a population of

soil microorganisms that has adapted due to previous exposure to it or a

similar pesticide". Several criteria for confirmation of enhanced

microbial degradation have also been developed. Minimum criteria for

confirming the enhanced microbial degradation of a pesticide include;

1. Demonstration of reduced pesticide persistence in soils previously exposed to the pesticide or a similar compound.

2. Implication of microbial involvement in the rapid degradation process.

3. Identification of an adapted population of pesticide-degrading soil microorganisms.

These criteria deal strictly with enhanced microbial degradation as a pesticide-microbe interaction, and the effect of this occurrence on actual pest control efficacy must be ascertained from controlled field experiments. The results of this study indicate that by these criteria, both isofenphos and carbofuran have undergone enhanced microbial degradation. The evidence also indicates that cloethocarb and fonofos may 104

also have undergone enhanced microbial degradation. No evidence pointing

to enhanced microbial degradation of chlorpyrifos, terbufos, phorate, or

ethoprop was produced.

Research on the specificity of enhanced microbial degradation has demonstrated that the degree of specificity associated with this phenomenon varies depending on the pesticide involved. The enhanced degradation of isofenphos was very specific, indicating that a rather narrow microbial catabolic adaptation was involved. On the other hand, enhanced degradation of carbofuran was much less specific, and cross-adaptations for enhanced degradation of other carbamates were evident. No interactions between organophosphorus and carbamate enhanced degradation were discovered.

The use of biodegradable soil pesticides will continue to be a major agricultural pest management strategy. Enhanced microbial degradation is a problem which looms as a threat to the continued viability of this approach. However, research information generated by this dissertation and other studies in progress should lay the foundation for informed responses to this problem. It would seem that much of the burden of responsibility for ensuring that new soil pesticides are biodegradable yet not extremely susceptible to enhanced degradation falls on the agricultural chemical industry. However, extensive investigations by the academic and research community dealing with the microbial ecology, biochemistry, and genetics of enhanced degradation are needed. This basic information will not only prove useful as it relates to soil pesticide study, but also should facilitate the design of biodegradative technologies for the disposal of pesticides and for environmental decontamination. 105

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ACKNOWLEDGMENTS

Upon completing this dissertation, I am mindful once again of how

thankful I am for all the help and encouragement I've received throughout

my graduate student career from many individuals. I owe a special debt of gratitude to Dr. J. R. Coats, who has overseen my tenure here at Iowa State as my major professor. I am very appreciative of his support during my

Ph.D. program, for his friendship, and for the freedom he-allowed me in my investigations. I am also indebted to ray former major professor at the

University of Wisconsin, Dr. E. P. Lichtenstein, who started me on my way in pesticide science and turned me into a researcher. I express my thanks to my Ph.D. committee members. Dr. J. M. Bremner, Dr. T. E. Loynachan, Dr.

J. J. Tollefson, and Dr. F. D. Williams, for their support, advice, patience, and ideas freely shared. I'd also like to thank my faithful laboratory assistants, D. D. Michael and K. R. Titus, for their valuable and cheerful assistance.

"So then it does not depend on the man who wills or the man who runs, but on God who has mercy." Romans 9:16