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Enhanced biodegradation of carbofuran in soil with a history of repeated applications of carbofuran and characterization of bacterial degraders isolated from the soil
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ENHANCED BIODEGRADATION OF CARBOFURAN IN SOIL WITH A HISTORY OF
REPEATED APPLICATIONS OF CARBOFURAN AND CHARACTERIZATION OF
BACTERIAL DEGRADERS ISOLATED FROM THE SOIL












By

STEVEN LEE TRABUE

















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1997















ACKNOWLEDGMENTS


I thank Dr. Li Tse Ou for allowing me the opportunity to pursue a Ph.D. degree

under his guidance and for all his support he gave me throughout my studies. I also thank

the members of my supervisory committee, Drs. George O'Connor, Andrew Ogram,

Suresh Rao, David Sylvia, and Lonnie Ingram, for their inputs. I would especially like to

thank Drs. Ogram and Sylvia for answering many of my questions. Appreciation is

extended to the USDA Special Water Quality Research Program for partially funding this

research, and to Dr. Randal Brown for giving me a generous assistantship when my

source of funding ended.

I would like to thank those who assisted me in my endeavors. The opportunities

to use the laboratory facilities of Drs. Mary Collins, Art Hornsby, Andrew Ogram, David

Sylvia, William Stall, and Henry Aldrich are greatly acknowledged. Dr. Sylvia Coleman,

Dave Cantlin, Wei Jing, Bill Reeve, Cheryl Hodge, and Larry Schwandes for their help in

the lab were appreciated. I would especially like to thank John Thomas who provided me

help in time of need and was always willing to share a joke or two when I needed it most;

he will be missed. I think Joyce Taylor and Celia Earl for their encouragements. I thank

my fellow graduate students who have left or still remain: thanks for keeping a smile on

my face. Specifically I wish to thank Dr. Chris Pedersen, Dave Farmer, Jose Escamilla,

and Patrick Mulroy for all the late night conversations. To Dongping Dai, Hector Castor,

ii









Randy Sillian, Chris Bliss, Gerco Hoogeweg, and Elisa D'Angelo, I enjoyed the many

laughs and conversation we shared. I thank Keun-Yook Chung for sitting with me in

many of our classes, and I wish you well.

I would like to express my gratitude to those outside the University of Florida

who helped me through. I wish to thank Ken and Kathy French and Gil and Marian Prost

for being the examples of how couples should live their lives. I am ever grateful to my

parents and family and thank them for their love and encouragement, for without them I

know I would not have finished my studies. I missed them tremendously and cherished

the time we spent together.

I would like to thank Dr. Ou for working with me these past four years. He

helped me find my way when I was floundering and encouraged me when I was down,

not always with words but with his life. I am truly a better scientist and person for

knowing him.





















iii















TABLE OF CONTENTS






ACKNOWLEDGMENTS ............................................ ii

ABSTRACT ...................................................... .. vi

CHAPTERS

1 INTRODUCTION ................................................. 1

2 LITERATURE REVIEW ........................................ 3

History of Carbamate Pesticides ...................................... 3
Toxicology of Carbofuran ................. ........................ 5
Characteristic Properties of Carbofuran ................................. 9
Degradation of Carbofuran in Nonenhanced Soils ....................... 12
Enhanced Degradation of Carbofuran ................................. 18
Carbofuran Degradation by Soil Microorganisms Involved in Enhanced
Degradation ................ ............................ 30

3 EXPERIMENTAL PROCEDURE .................................. 37

Field Work ................ ................................... 37
Characterization of Carbofuran-Degrading Soil Isolates ................... 48

4 DEGRADATION IN SURFACE AND SUBSURFACE SOILS ............ 60

Introduction ................ .................................. 60
Results ......................................................... 61
Discussion ................................................ 82





iv









5 INFLUENCE OF MICROBIAL POPULATIONS ON ENHANCED
DEGRADATION OF CARBOFURAN IN SOIL ........................ 93

Introduction ..................................................... 93
Results ...................................................... 94
Discussion ................ .................................. 106

6 CHARACTERIZATION OF CARBOFURAN-DEGRADING BACTERIA.. 115

Introduction . . ....................................... . 115
Results and Discussion ........................................... 116

7 CONCLUSIONS ................... ............................ 138

GLOSSARY ...................................................... 143

LIST OF REFERENCES ................ ............................. 144

BIOGRAPHICAL SKETCH ................ .......................... 158




























v















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ENHANCED BIODEGRADATION OF CARBOFURAN IN SOIL WITH A HISTORY
OF REPEATED APPLICATIONS OF CARBOFURAN AND CHARACTERIZATION
OF BACTERIAL DEGRADERS ISOLATED FROM THE SOIL


By

Steven Lee Trabue

December 1997


Chairperson: Dr. Li Tse Ou
Major Department: Soil and Water Science

Carbofuran is a broad-spectrum N-methylcarbamate insecticide used to control

certain soil borne insects and nematodes. Enhanced biodegradation of carbofuran has

been attributed to the loss of pesticidal efficacy in soil with a history of carbofuran use.

Microorganisms are responsible for enhanced degradation of carbofuran in soil. There is

little information linking enhanced degradation with carbofuran-degrading microbial

populations in soil profiles. One of the objectives of this research was to measure the

degradation rate of carbofuran in soil related to carbofuran use in soil. Soil samples were

collected from a site in Florida that had either a previous history or no history of exposure

to carbofuran. Mineralization rates of carbofuran in soils collected from different soil

depths were measured using ["'C-CAL (carbonyl-labeled)] or ['4C-URL (uniformly ring-

vi









labeled] carbofuran. Metabolites in soils were measured using ['4-URL] carbofuran.

Carbofuran degraded more rapidly in soils previously treated with the pesticide. Surface

soils degraded carbofuran more rapidly than subsurface soils, and cultivated soils

degraded carbofuran more rapidly than noncultivated soils. Hydrolysis of carbofuran was

the major route for enhanced biodegradation of carbofuran in soils which had received

prior applications.

The second objective was to measure changes in the carbofuran-degrading

microbial populations in soils as a function of carbofuran treatment history and soil

depth. Numbers of microorganisms capable of mineralizing ['4C-CAL] and ['4C-URL]

carbofuran, and ["4C-URL] carbofuran phenol, as well as methylamine degraders were

determined using MPN techniques. Repeated field applications of carbofuran increased

the number of carbofuran-hydrolyzing microorganisms, but other types of degraders

remained unchanged.

The final objective was to isolate and characterize carbofuran-ring-degrading

bacteria. Three bacteria were isolated from soil and identified as Sphingomonas sp.

according to their fatty acid methyl ester profiles. Growth of these isolates was

determined using the Petroff-Hausser bacteria counter technique. Metabolites of

carbofuran were determine using ["'C-URL] carbofuran. Mineralization rates were

determined using ['4C-CAL] and ['4C-URL] carbofuran. The bacteria mineralized

between 84-91% of the ["4C-CAL] carbofuran in 72 hours, while during the same period

only 40-47% of the ["4C-URL] carbofuran was mineralized. Incorporation of "C into the

biomass was found to be between 20-33% of the applied ['4C-URL] carbofuran.

vii















CHAPTER 1
INTRODUCTION


The broad spectrum insecticide and nematicide carbofuran (2,3-dihydro-2,2-

dimethyl-7-benzofuran-7yl methylcarbamate) came into use in agriculture in the late

1960s. Carbofuran originally exhibited excellent control over the corn rootworm

(Diabrotica spp.), but repeated use of carbofuran in the same fields resulted in the failure

to control target pests. This loss of efficacy was attributed to enhanced biodegradation of

carbofuran, and microorganisms were the culprits responsible for enhanced degradation.

Currently, there is a substantial body of information on enhanced degradation of

carbofuran in soils. However, there is little information linking enhanced degradation of

carbofuran with the ecology of carbofuran-degrading microorganisms in soils.

Information is needed on the effect of repeated field applications of carbofuran on the

degradation rates of carbofuran in soil profiles, and the ecology of the microorganisms

involving in the degradation of carbofuran in soils.

In the first half of Chapter 2, I provide background information on carbofuran,

including its toxicity, physical and chemical properties, and its degradation in

nonenhanced soils. The second half of this chapter, I review enhanced degradation of

carbofuran in soils, and present information on soil bacteria that have various capacities




1









2

of degrading carbofuran. In Chapter 3, the experimental procedures used in my research

are discussed.

In Chapter 4, I investigated the development of enhanced degradation in a Florida

sandy soil repeatedly field treated with carbofuran. The purpose of the study was to

determine the effect of repeated field applications of carbofuran on the degradation

potential of carbofuran in enhanced and nonenhanced soils. In addition, metabolite

formation was used to determine the disappearance and degradation pathways of

carbofuran in enhanced and nonenhanced soils. In Chapter 5, I1 attempted to link the

degradation of carbofuran with changes in the carbofuran-degrading populations in

enhanced and nonenhanced soils. The fluctuations were monitored as a function of the

soils carbofuran field application histories, and as a function of time after a laboratory

treatment of carbofuran. The characterization of three carbofuran-degrading bacteria

isolated from soils is addressed in Chapter 6. The goals of the characterization were to:

1) measure the growth of the soil isolates on carbofuran; 2) measure the degradation rates

of carbofuran by the soil isolates; 3) measure the mineralization rates of carbofuran by the

soil isolates in different growth media; 4) attempt to elucidate the degradation pathways

of carbofuran by the soil isolates; and 5) attempt to determine if the genes for carbofuran

degradation are located on chromosomal or plasmid DNA.















CHAPTER 2
LITERATURE REVIEW



History of Carbamate Pesticides



The history of carbamate chemicals can be traced back several hundred years to

Africa (Holmstedt, 1972). Carbamate compounds were derived from calabar beans used

in witchcraft trials. The calabar beans were used to pronounce guilt or innocence upon an

accused individual. If village leaders believed there was validity to the charges against

the accused, the individual was forced to drink a concoction of mature calabar bean seeds

and water. If the accused were fortunate, he or she would quickly regurgitate the mixture,

but the less fortunate were doomed to uncontrollable shaking and frothing at the mouth

that eventually led to death. Those who regurgitated the calabar bean were pronounced

innocent, and those who died were pronounced guilty and justice was served.

In the mid 1800s, Europeans brought calabar bean seeds back to Scotland giving

the legume plant the name Physostigma venconosum. The natural toxin from the plant

was identified as a vegetable alkaloid in 1863, with the main toxin, physostigmine, being

purified one year later (Holmstedt, 1972). The structure of physostigmine was elucidated

in 1925 (Figure 2-1) and successfully synthesized in 1935 (Julian and Piki, 1935).



3










4











CH3


N N





Physostigmine









CHCH,



CH3



Carbofuran





Figure 2-1. Chemical stucture of physostigimine and carbofuran.









5

In the mid to late 1940s, Geigy Chemical Company attempted to develop an

insect repellent. They tested a series of carbamate compounds, discovering that these

compounds were poor repellents but that they were toxic to horseflies, aphids, and other

small insects. At this point, Geigy decided to pursue developing carbamates insecticides

rather than carbamate repellents. All of the insecticides developed by Geigy were

dimethyl carbamates. In 1953, Union Carbide Corporation synthesized another class of

carbamate compounds in which the dimethyl carbamoyl moiety was replaced with a

monomethyl moiety. These aryl N-methylcarbamates were shown to have superior

insecticidal activity compared to the dimethyl carbamic acids (Kolbezen et al., 1954). It

is from these aryl N-methylcarbamate insecticides that carbofuran is derived (Figure 2-1).

Carbofuran is exclusively a field applied insecticide and nematicide used for various

crops some of the more important ones include corn and rice.


Toxicology of Carbofuran



Mode of Action

Carbamate compounds are fairly potent inhibitors of cholinesterase similar to that

of the organophosphate insecticides. Acetylcholinesterase hydrolyze acetylcholine, and

the inhibition of this enzyme results in the accumulation of acetylcholine in the central

nervous system (CNS) synapses. This accumulation results in an over-stimulation of the

acetylcholine receptors causing the interruption of nerve impulse transmission for insects,

that eventually leading to death (Kuhr and Dorough, 1976). In vertebrates, the









6

accumulation of acetylcholine in the synapses of neuromuscular junction results in an

over-stimulation of the acetylcholine receptors that results in death due to respiratory

failure (Gupta, 1994). Carbamate compounds react with the OH group of serine in the

active site of the enzyme (Gupta, 1994). In addition to inhibition of acetylcholinestase, it

is speculated that carbofuran inhibits the activities of other enzymes that use serine in the

active sites of the molecule (Gupta, 1994).



Toxicity of Carbofuran

Animals

Concern over the use of carbofuran in fields is due to the potential toxicity toward

humans based on animal studies. The most sensitive mammal to carbofuran poisoning

appears to be mice with LD50 of 2.0 mg kgl' by oral ingestion (Fahmy et al., 1970). Other

studies have shown that carbofuran is acutely toxic to a host of other mammals with LD50

for oral ingestion ranging from 2.5 mg kg"' for rats to 19 mg kg-' for dogs (Gupta, 1994).

Carbofuran is most toxic through oral and inhalation routes of exposure, but is less toxic

through the dermal route (LDS0 885 mg kg-' for rabbits).

Birds

Birds in the wild are particularly susceptible to carbofuran poisoning, and because

of large numbers of bird kills, the formulation and method of application of carbofuran

has been regulated. Granular formations of carbofuran are reportedly highly toxic to

birds (Balcomb, 1983). The LD50 values for oral ingestion of carbofuran in ducks and

quail were 0.42 mg kg-' (Hudson et al., 1972) to 5 mg kg-' (Osweiler et al., 1985),









7

respectively. Elliot et al. (1996) reported a secondary killing of nine birds of prey with

symptoms of anticholinesterase poisoning. Their crop contents contained duck parts that

were contaminated with carbofuran. Dietrich et al. (1995) reported the deaths of eight

buzzards (Buteo buteo) five of which had crops contents of earthworms that were

contaminated with carbofuran.

Fish

Aquatic organisms are also susceptible to carbofuran from agriculture runoff and

accidental spraying. The 24 hour LC50 values for bluegill ( Lepomis macrochirus) and

fathead minnow (Pimephelas promelas) were 0.1 mg L-' and 2.24 mg L-', respectively.

The 96 hour LC50 values were 0.088 mg L-' and 1.99 mg L"' for bluegill and fathead

minnow, respectively (Trotter et al., 1991). Application of carbofuran to flooded rice

plots resulted in killing less than 10 % of the green sunfish (Lepomis Cyanellus), and

mosquito fish (Gambusia affinis) (Davey et al., 1976).

Soil microorganisms

Many pesticides have been shown to have deleterious effects on the microbial

communities, but when applying carbofuran to a soil, its impact on the general microbial

community varies depending on the individual soil. Tu (1972a; 1972b) reported that

carbofuran was inhibitory to bacteria and fungi in a sandy loam (pH 8.2), whereas in

neutral to acidic sandy loam soils, carbofuran had little effect on the size of the microbial

biomass (Tu, 1978). Das et al. (1995) reported that carbofuran had little effect on the

fungal population size in the rice rhizosphere, but bacterial population sizes were

stimulated by the addition of carbofuran. In organic soils, the bacterial and fungal









8

populations were stimulated by carbofuran application to the soil (Tu, 1978; Mathur et

al.,1976). The management practice used in applying carbofuran to a soil influenced the

effect of carbofuran on the microbial community. Soils that had carbofuran applied

banded exhibited a faster increase in the microbial biomass than soils that receiving

carbofuran through broadcast (Mathur et al., 1976).

Nitrification is considered to be the most sensitive biological function in soil that

can be negatively impacted by pesticide applications (Rajagopal et al.,1984). Several

researchers reported carbofuran applied to soil at rates between 5 and 500 utg g-' was not

inhibitory to nitrification in terms of nitrite formation (Lin et al., 1972; Mathur et al.,

1976; Ramakrishna and Sethunathan, 1982). Rather, it has been reported that carbofuran

stimulated nitrification in soils previously exposed to the pesticide ( Ramakrishna and

Sethunathan, 1982). Thus, it appears that carbofuran has little if any negative impact on

the general microbial size after a single agriculture application of carbofuran.

Soils with a previous history of carbofuran exposure exhibited no effect on the

size of the bacterial and fungal populations upon repeated exposure to the pesticide

(Duah-Yentumi and Johnson, 1986). A 4-fold increase in bacterial population size was

reported within a rice rhizosphere after repeated treatment with carbofuran

(Venkateswarlu and Sethunathan, 1978). A single application of carbofuran reportedly

stimulated the nitrogenase activity associated with the rhizosphere of rice (Kanungo et

al.,1995). This stimulation in nitrogenase activity continued for two additional

applications, but after the fourth application the nitrogenase activity did not increase

significantly. The results of these studies suggest that carbofuran has no adverse effects









9

on the general microbial community even after repeated treatments. It appears that

carbofuran may stimulate the activities of soil bacteria when repeatedly applied to the

soil. This conclusion is supported by a recent finding on the N-methylcarbamate aldicarb

that after 19 years of continuous application tosoil has resulted in an increase in the total

microbial biomass (Hart and Brooks, 1996).


Characteristic Properties of Carbofuran



Physical and Chemical Properties

The physical and chemical properties of carbofuran are presented in Table 2.1

There is a little controversy over the solubility of carbofuran at 250 C. Bowman and Sans

(1979) measured carbofuran solubility to be 700 jlg ml-', but FMC, the maker of the

pesticide, reported carbofuran solubility to be 351 lag ml-'. Other data presented by

Bowman and Sans (1979) support the 351 ulg ml"' value; they measured the solubility of

carbofuran to be 320 gLg ml-' at 190 C. It appears that the hydrolysis products of

carbofuran may have been inadvertently measured and included within the 700 gg ml-'.

Carbofuran has a low vapor pressure which suggests that there will be a negligible loss

of carbofuran in soil due to volatilization.



Carbofuran Sorption

Pussemier et al. (1989) investigated the molecular parameters of a pesticide that

determine its sorption characteristics. For arylcarbamates, they determined that the









10

Table 2-1. Physical and chemical properties of carbofuran.a

Chemical formula C12 H15 NO3

Molecular weight 221.6 g mol"'

Physical state White crystalline soild

Flammability Not flammable

Melting point 15 to 1540C

Density 1.180 (20C)

Vapor pressure 2 x 10-5 mm Hg (330C)
1.1 x 104 mmHg (500C)

Octanol/water partition coefficient 42.5

Water solubility 351 glg ml' (250C)

K. 9-36 ml g-'
a data from Trotter et al. (1991) and Wauchope et al. (1992)











hydrophobicity of the compound was strongly correlated with sorption of the compound

on various sorbates (r = 0.6-0.91). For a range of soils, carbofuran sorption was strongly

correlated with the soils organic carbon content (r2 = 0.81), with a K. value of 30 ml g-'

for carbofuran (Sukop and Cogger, 1992). This value corresponded closely to the K,.

value of 26 ml g-' determined by Felsot and Wilson (1980). The average K.. value for

carbofuran in soil is 22 ml g-' with values ranging from 9-36 ml g'1 (Wauchope et al.,

1992). Felsot and Lew (1989) found that the bioavailability of carbofuran was controlled

by the organic carbon content of a soil. They reported that organic carbon accounted for

the greatest proportion of variability in the LC50 (r2 = 0.89) and LC95 (r2 = 0.88) of the

pesticide in soil.



Carbofuran Transport in Soils.

Based on the low potential for sorption (low Kc), carbofuran should be expected

to be fairly mobile in soils. In a microcosm study, Lichtenstien and Liang (1987) found

that over a period of 36 days, > 29% of the applied 14C-activity was measured in runoff

and percolation water. While in a field experiment, carbofuran was found to move after a

rain fall event during which the surface transport of carbofuran was not associated with

any sediment but rather with the run-off water (Caro et al., 1973). Williams et al. (1995)

reported that carbofuran was more mobile than models predicted and suggested that the

physical soil properties influenced the movement of the pesticide more than the chemical

properties of the pesticide. Williams et al. (1995) stressed that improved management

practice is the key to reducing potential contamination of local environments. This was









12

supported by Caro et al. (1973) who detected more carbofuran in runoff from fields that

had received carbofuran by broadcast application than by band application.



Degradation of Carbofuran in Nonenhanced Soils



Aerobic Soils

Degradation mode in aerobic soil

The predominate mechanism of carbofuran degradation under field conditions

was reportedly to be highly site specific. Both biological and chemical degradation

contribute to carbofuran degradation in soils. Chemical hydrolysis of carbofuran is

higher in alkaline soils than in neutral to lower pH soils (Getzin, 1973). In sterile neutral

or acidic soils, degradation rates of carbofuran were lower than in corresponding non-

sterile soils (Getzin, 1973). In alkaline soil, the degradation rates of carbofuran were

similar whether they were sterile or nonsterile.

In field studies, the areas where carbofuran was rapidly degraded were higher in

clay and water contents (Caro et al., 1973). This is contradicted by the finding that the

clay content of a soil had a negative impact on the degradation of carbofuran (Abdellate

et al., 1967). In other studies, the clay content was not linked at all to the degradation

rates of carbofuran (Ou et al, 1982; Charnay and Fournier 1994). Soil properties ( pH,

organic matter, cation-exchange capacity (CEC)), and the types of microbial populations

also can not be linked directly to the degradation rates of carbofuran (Ou et al., 1982).

The soil properties that have been shown to be linked to the degradation rate of









13

carbofuran are temperature and water contents (Caro et al., 1973; Mathur et al., 1976;

Telekar et al., 1977; Ou et al., 1982).

The biotic influence on carbofuran degradation is evident in that higher

temperatures and water contents which increase microbial activity also influence the

degradation rates of carbofuran in soil (Kieft et al., 1995). The main mechanism of

carbofuran degradation in organic soils has been shown to be a microbially mediated

process (Greenhalgh and Belanger, 1981; Mathur et al.,1976). The higher microbial

activity in surface soils than in subsurface soils has been attributed to the increased rates

of carbofuran degradation in the surface soils (Buyanovsky et al., 1993; Mallawatantri et

al., 1996). Thus, it appears that in neutral to acidic soils biological degradation is the

dominate mechanism for carbofuran degradation.

Metabolism of carbofuran in aerobic soil

The metabolites detected in field and microcosm studies revealed that only small

amounts (2-10%) of carbofuran were converted to its oxidation products 3-hydroxyl-

carbofuran and 3-ketocarbofuran (Caro et al., 1973; Lichtenstien and Liang, 1987).

Lichtenstien and Liang (1987) using ['4C-CAL (carbonyl labeled)] carbofuran determined

that most of the extractable '4C-activity was associated with carbofuran.

In batch studies using ['4C-URL (uniformly ring labeled)] carbofuran, there were

low levels (< 4.0%) of the oxidation products 3-hydroxylcarbofuran and 3-ketocarbofuran

detected, and in addition the hydrolysis products carbofuran phenol and 3-ketocarbofuran

phenol were also detected in small amounts (< 4.0%) (Getzin, 1973; Ou et al., 1982).

Carbofuran was the major extractable compound, yet the major portion of "'C-activity









14

was actually associated with soil-bound residues (50-95%) (Getzin, 1973; Ou et al.,

1982). The precursor compounds of the soil-bound residues appear to be the carbofuran

phenolic compounds, since incubation of ['4C-URL] carbofuran phenol resulted in similar

'4C distribution patterns as ['4C-URL] carbofuran (Getzin, 1973). The batch carbofuran

degradation studies using ['4C-URL] carbofuran, reveal that hydrolysis of the carbamate

moiety from carbofuran was the main pathway of degradation in soils (Figure 2-2). This

is typical of the metabolism of other N-methylcarbamates in soils (Kazano, et al., 1972).

Half-lives of carbofuran in aerobic soil

Half-life values of carbofuran in soil under field conditions ranged from 15-117

days (Caro et al., 1973, Greenhalgh and Belanger, 1981; Williams et al., 1995). Half-life

values in mineral soils under field conditions were dependent on the method of

carbofuran application, with banded application having half-life values almost twice as

long as broadcast values (Caro et al., 1973). In organic soils under field conditions, half-

life values were similar whether applied by broadcast or banded (Mathur et al., 1976;

Greenhalgh and Belanger, 1981). Half-life values of carbofuran under laboratory

conditions ranged from 14 days to more than lyear (Getzin, 1973; Ou et al., 1982 and

Short and Enfield, 1988). It is interesting to note the degradation rate of carbofuran is

three times faster in soils with a history of continuous monoculture cultivation and

organophosphate insecticide use than in soils not in cultivation. (Rouchaud et al., 1989).











0 0 0
CH Co C C-o o HCH,

0 CH3 H 0 3 CH
CH3 CH3 CH3
OH 0
Carbofuran 3-Hydroxylcarbofuran 3-Ketocarbofuran







OH OH OH
O CH3 0 CH3 0 CH3
CH3 I CH,3 CH3
OH 0
Carbofuran phenol 3-Hydroxylcarbofuran phenol 3-Ketocarbofuran phenol










C02+ H20 + Bound Residues + Biomass

Figure 2-2. Carbofuran degradation in nonenhanced aerodic soils.









16

Anaerobic Soils

Degradation mode in anaerobic soil

Carbofuran was reported to be susceptible to photodecomposition with close to

25% of the applied carbofuran being degraded after exposure to direct sunlight during a

96 hour incubation period (Deuel et al.,1979), but only 10% of the applied carbofuran

was degraded after exposure to laboratory light over a similar time period. Loss of

carbofuran through volatilization was found to be negligible (Deuel et al.,1979), as to be

expected based on carbofuran vapor pressure for carbofuran is 2.5 x 10'5 mM Hg at 33 C.

The redox potential of a given soil was found not to affect the persistence of carbofuran

in flooded soils (Panda et al., 1988). The predominate abiotic mechanism for carbofuran

degradation in flooded ecosystems is believed to be alkaline hydrolysis (Venkateswarlu et

al., 1977; Venkateswarlu and Sethunathan, 1978; Panda et al., 1988; Morra et al., 1996).

In a rice paddy, the pH of flood water in immediate contact with the soil surface was 8.5

while surface soil pH was 7.3 and the subsurface soil pH was 6.9 (Panda et al., 1988).

Thus, the abiotic degradation of carbofuran appears to be related to the pH of the soil

solution, and exposure of carbofuran to direct sunlight.

Based on the degradation rates of carbofuran in autoclaved soils and non-auto-

claved soils, biological degradation of carbofuran is a major factor in anaerobic soils

(Venkateswarlu et al., 1977; Arunachalam and Lakshman, 1990). A number of bacteria

capable of degrading carbofuran have been isolated from anaerobic soils including

bacteria capable of utilizing carbofuran as a sole source of C for growth and energy

(Venkateswarlu et al., 1977; Venkateswarlu and Sethunathan, 1984; Rajagopal et al.,









17

1984; Ramanand et al., 1991). Despite the presence of carbofuran-degrading

microorganisms in anaerobic soils, the link between the decreased efficacy of carbofuran

and a buildup of carbofuran degrading microorganisms has yet to be proven.

Metabolism of carbofuran in anaerobic soil

The only compound detected in paddy water or soils is the parent compound

carbofuran ( Deuel et al., 1978; Johnson and Lavy, 1995). Only trace amounts (< 1%) of

3-ketocarbofuran were occasionally detected in paddy soils, while 3-hydroxylcarbofuran

was detected at trace amounts in the overlying water and the flooded soil (Johnson and

Lavy, 1995). Due to the rapid dissipation in a Texas rice paddy, neither carbofuran nor

its metabolites were detected in paddy soils, but carbofuran and its metabolite 3-

ketocarbofuran were detected in the overlying water (Deuel et al., 1978).

The major metabolite of ['4C-URL] carbofuran in flooded ecosystems was the

hydrolysis product carbofuran phenol (Venkateswarlu and Sethunathan, 1979, Panda et

al., 1988; Lalah et al., 1996). In these reduced oxygen environments, 3-

hydroxylcarbofuran and 3-ketocarbofuran phenol never accumulated to more than 1 % of

the applied '4C activity (Venkateswarlu and Sethunathan, 1978; Lalah et al.,1996). The

phenyl ring of carbofuran appears to be recalcitrant to degradation in flooded ecosystems,

with cumulative "'CO2 production never exceeding 7 % of the applied ["4C-URL]

carbofuran (Venkateswarlu and Sethunathan, 1978; Lalah et al., 1996). Soil bound

residues of ['4C-URL] carbofuran in anaerobic soils never exceeded 10 % after one month

of incubation (Venkateswarlu and Sethunathan, 1978, Lalah et al., 1996). These studies

suggest that the degradation of carbofuran in flooded ecosystem is mainly via hydrolysis









18

to carbofuran phenol, with the aromatic ring remaining largely intact in the anaerobic

environments (Figure 2-3).

Half-lives of carbofuran in anaerobic soil

The half-life values for carbofuran in field paddy soils range from 10 to 58 days

(Nicosia et al., 1991; Johnson and Lavy, 1995). In laboratory studies, the half-life values

of carbofuran in a bulk soil ranged from 11.9 to 15.1 days (Panda et al., 1988), while the

half-life value in an anaerobic rhizosphere was 10.8 days (Das et al., 1995).



Enhanced Degradation of Carbofuran



Background Information

Evidence for enhanced degradation

The concern over the fate and persistence of chlorinated pesticides coupled with a

growing environmental conscience in our society resulted in an inclination for the

development of environmentally benign (less toxic and biodegradable) pesticides. Classes

of chemicals such as organophosphates and carbamates that are less persistent than highly

halogenated pesticides such as DDT have proven to be effective in controlling target

pests. One of these pesticides was bufencarb, a N-methylcarbamate insecticide used for

the control of corn pests, most notably the corn rootworm larval (Diabrotica spp.). In the

early 70's, farmers complained to state extension agencies in Iowa about poor control of

target insects in fields treated with bufencarb, but the poor control of bufencarb could not









19


0


H CH3
O51 y CH3


Carbofuran







OH
0 CH,
I cf, + NH2CH3

Carbofuran phenol Methylamine






Figure 2-3. Carbofuran degradation in nonenhanced anaerobic soil.









20

be reproduced in research plots (Tollefson, 1986). Due to extensive complaints about

poor performance, state extension services recommended not using bufencarb for corn

rootworm larval control (Tollefson, 1986). During this time, the carbamate insecticide

trimethacarb also exhibited poor pesticidal efficacy, and similar to bufencarb, it was

determined that insect resistance could not sufficiently explain the observed failures

(Chou et al., 1978). Other carbamate and organophosphate pesticides also exhibited

similar pest control failures during the 1970's (Sethunathan and Pathak, 1972; Chou et al.,

1978; Felsot et al., 1985). The lost of pest control from these pesticides has now been

attributed to enhanced degradation in soils and microorganisms are responsible for

enhanced degradation.

Carbofuran was introduced in 1967, and gave outstanding protection from attack

by corn rootworm larvae (Tollefson, 1986). Carbofuran was also found to be effective in

control of brown planthopper (Nilaparvata lugens) and green leafhopper (Nephotettix

virescens) in rice paddies (IRRI, 1975). From the mid to late 1970's, news on the

ineffective control of target pests in fields previous treated with carbofuran started to

surface. Tollefson (1986) found in 1975 that soils previously treated with carbofuran

exhibited greater root damage than soils with no history of exposure. He repeated the

study in 1976 and again soils previously treated with carbofuran resulted in excessive

root damage as opposed to soils with no history of exposure. The evidence linking loss

of control of target pest and accelerated degradation of carbofuran began to accumulate.

Williams et al. (1976) reported that in Canada pest control problems were found

in vineyards that had been previously treated with carbofuran, and that the levels of









21

carbofuran residues were lower than what was expected. They were able to show that in

organic soils a significant portion of carbofuran degradation was biological, and from

these soils they even isolated a few soil organisms which possessed the capacity to

degrade carbofuran. However, Williams et al. (1976) failed to supply any evidence for

enhanced rates of carbofuran degradation in the soil. Greenhalgh and Belanger (1981)

found that organic soils that were retreated with carbofuran had lower residue levels of

carbofuran after 30 days compared to plots treated for the first time.

In the Philippines, the Internation Rice Research Insitute (1977) reported that rice

fields treated with carbofuran suffered worse hopperburn than untreated fields.

Hopperbum results from infestation of brown planthopper attacks on rice plants. It was

thought that the loss of efficacy for carbofuran in the treated paddy soil was due to a rapid

buildup of carbofuran-degrading bacteria (Venkateswarlu and Sethunath, 1978). This

was due in part to the isolation of a bacterium that was capable of degrading carbofuran

from a rice field (Venkateswarlu et al.,1977). However, after repeatedly treating flooded

soils with carbofuran, there were no evidence linking carbofuran treatments with loss of

pest control via rapid degradation of carbofuran (Venkateswarlu and Sethunath, 1978).

Confirmation of enhanced degradation

Felsot et al. (1981) produced one of the first studies linking the loss of pest

control to enhanced degradation that was, in turn, associated with microbial degradation.

The study was conducted due to concern by corn producers over the poor control of

northern and western corn rootworms by carbofuran. Resistance to carbofuran by

rootworm could not adequately be related to this failure (Felsot et al., 1981). Felsot and









22

coworkers collected soils from corn fields in Illinois with various histories of carbofuran

treatment. Soil samples were collected from four corn fields with a history of carbofuran

applications and poor pest control, two corn fields with a history of phorate applications,

a field that had never been treated with any pesticides, and a crawl space under a house

treated with chlordane. All the soils that were previously treated with carbofuran

degraded the pesticide more rapidly when compared to soils with no history of carbofuran

use. Sterile treated soils lost the ability to rapidly degrade the pesticide indicating that

enhanced degradation was a microbial process. In addition, Felsot and coworker were

able to isolate a two of soil bacteria that degraded carbofuran.

Repeated applications of carbofuran to a soil under laboratory conditions (500 tig

carbofuran g-' soil) resulted in carbofuran degradation rates 600-1000 times faster than in

a soil with no history of exposure (Read, 1983). This rapid carbofuran degradation

coincided with a reduction in the lag period prior to initiation of degradation. In addition,

Read (1983) was able to link enhanced degradation to the loss of control of target

organisms by showing that the reduced residue levels in soils previously exposed to

carbofuran resulted in a decrease in the percentage of cabbage maggot egg mortality.

The work of Read (1983) and Felsot et al. (1981) demonstrated that the inability

of carbofuran to control target pests in soils was associated with the rapid breakdown of

the compound. Furthermore, microorganisms were shown to be responsible for this rapid

breakdown of carbofuran. However, the question remained were we seeing an

enhancement of carbofuran degradation or were we witnessing only soils that had the

capacity to degrade carbofuran at rapid rates? Felsot et al., (1981) noted that soils









23

collected along the fence rows next to carbofuran treated soils also exhibited accelerated

degradation rates of carbofuran.

To demonstrate that enhanced degradation of carbofuran resulted from the

repeated applications of carbofuran to the same field, Suett (1986) determined carbofuran

degradation rates in eight U.K. soils with histories of carbofuran applications, and in soils

collected from adjoining fields that had no prior history of exposure to carbofuran. These

soils covered a wide range of physical and chemical properties. Suett showed that the

degradation of carbofuran was more rapid in soils previously treated with the pesticide,

and that the "initial" lag phase for each soil was shorter in previously exposed soils as

compared to soils with no history of carbofuran application. One of the soils fumigated

with dazomet did not degrade carbofuran despite repeated carbofuran applications. The

reduced microbial activity may reflect a general reduction in the microbial biomass due to

the fumigant. Subsequently, Camper et al. (1987) and Turco and Konopka (1990) also

found in South Carolina and Indiana soils with a previous history of carbofuran

application, a more rapid degradation than adjoining soils not previously treated with

cabofuran.



Factors that Influence Enhanced Degradation

Key soil physical, chemical and biological factors.

Extensive efforts have been made to understand the basic mechanisms involved in

the development of enhanced degradation of pesticides in soils. One question that has

often been asked is: what are the characteristics of a soil that develops enhanced









24

degradation from repeated applications of carbofuran? Researchers have attempted to

determine the key soil factors that affect the mineralization and disappearance rates of

carbofuran in soil. The key soil factors that had been studied included soil pH, organic

matter content, clay content, CEC, C:N ratio, organic N content and CaCO3 content.

None of these soil factors were found to correlate with the mineralization or

disappearance of carbofuran in enhanced and nonenhanced soils (Ou et al., 1982;

Charnay and Fournier, 1994). Only water content and temperature were correlated to the

mineralization and disappearance of carbofuran in soil (Ou et al., 1982; Chapman et al.,

1986; Chapman and Harris, 1990; Parkin and Shelton, 1994). The soil water content was

found to be the most dominate factor in determining the spatial and temporal variations of

carbofuran degradation rates in enhanced soils (Parkin and Shelton, 1994).

Total soil aerobic microorganisms and aerobic bacterial populations, along with

amidase and urease activity, were poorly correlated with carbofuran degradation rates in

enhanced and nonenhanced soils (Ou et al., 1982; Dzantor and Felsot, 1990). In addition,

initial carbofuran-hydrolyzing population size was also poorly correlated with the

development of enhanced degradation of carbofuran in soil (Dzantor and Felsot, 1990;

Charnay and Fournier, 1994). Thus, Charnay and Fournier (1994) concluded that only a

soil's exposure history to carbofuran can be correlated with the development of enhanced

degradation.

Enhanced degradation and chemical treatment

It appears that carbofuran must be applied to soils at a certain threshold level prior

for enhanced degradation to occur (Chapman et al.,1986; Hendry and Richardson, 1988;









25

Chapman et al., 1990). Mineral soils were found to develop enhanced degradation at

application rates of 1.0 jlg carbofuran g-' soil (Chapman et al.,1986; Chapman and Harris,

1990), but enhanced degradation did not occur at application rates below 0.1 uLg

carbofuran g-' soil (Chapman et al.,1986; Hendry and Richardson, 1988). In organic

soils, the application rate of carbofuran required to induce enhanced degradation was 10.0

utg carbofuran g'- soil, and at application rates below 1.0 ug carbofuran g-' soil, enhanced

degradation did not develop (Chapman and Harris, 1990). The higher loading rates for

carbofuran to develop enhanced degradation in organic soils is attributed to the higher

sorption capacity of the organic soils and, thus, the decreased bioavailability of

carbofuran to the microorganisms. Further evidence supporting a threshold concentration

required for triggering enhanced degradation is found in the increased spatial variability

of enhanced degradation activity in no-till plots compared to conventional tillage plots

(Parkin and Shelton, 1992). In no-till plots, enhanced degradation of carbofuran

corresponded to the placement of carbofuran, while in conventional tillage plots the

homogenizing of the soil resulted in a lack of any observed spatial distribution of

enhanced degradation activities.

Enhanced degradation of carbofuran is not only induced by a threshold level of

carbofuran treatment, but also by structurally similar carbamate pesticides (Harris et al.,

1984; Racke and Coats, 1988; Dzantor and Felsot, 1989; Morel-Chevillet et al., 1996).

This phenomenon is known as "cross-conditioning." However, not all carbamate

compounds induced enhanced degradation of carbofuran. Recently, a soil in France was

shown to exhibit enhanced degradation toward carbofuran after pretreatment with 15









26

different aryl methylcarbamate pesticides, but soils that were incubated with primicarb

(2-dimethylamino-5,6-dimethylpyrimidin-4-yl dimethylcarbamate) and formetanate ( 3-

dimethylaminomethyleneiminophenyl methylcarbamate) did not develop enhanced

degradation toward carbofuran (Morel-Chevillet et al., 1996).

Knowing that structurally similar compounds have the potential to induce

enhanced degradation of carbofuran, one could ask what is the response of soils with

enhanced degradation toward other unrelated agricultural chemicals? The N fertilizer

urea, when applied to enhanced soils, exhibited an inhibitory response to the

mineralization of ['4C-CAL] carbofuran (Merica and Alexander, 1990), while other N

fertilizers (sodium nitrate and ammonium nitrate) had no effect on the mineralization of

['4C-CAL] carbofuran in enhanced soils (Hendry and Richardson, 1988; Racke and Coats,

1990; Merica and Alexander, 1990). The addition of organophosphate pesticides to soils

has been shown to have inhibitory effects on enhanced degradation of carbofuran (Racke

and Coats, 1990; Talebi and Walker, 1994). In particular, the organophosphates ethoprop

and paraoxon have been reported to markedly reduce carbofuran degradation in enhanced

soils (Racke and Coats, 1990; Talebi and Walker, 1994). The reduction of carbofuran

degradation may result from inhibition of the enzymes involved in the hydrolysis of the

carbamate linkage to carbofuran (Talebi and Walker, 1994). Inhibition of the enzymes is

not unexpected since it has been shown that N-methylcarbamates do inhibit

phenylcarbamate hydrolyzing enzymes (Kaufman et al., 1970), and possibly some

organophosphates as well.









27

Duration of enhance degradation of carbofuran.

The number of field applications of carbofuran to a soil before enhanced

degradation occurs and the duration of enhanced degradation has been investigated.

Several researchers reported that after one field application of carbofuran resulted in an

increase in the degradation of the chemical (Getzin and Shanks, 1990; Harris et al., 1988).

Racke and Coats (1990) reported that enhanced degradation may persist for different

periods of time depending upon soil type and possibly soil depth. Recently, Suett et al.

(1993) reported that several soils in the U.K. sustained enhanced degradation of

carbofuran without being treated with the pesticide for over 5 years. Eagle (1986) found

that some soils that had received a single carbofuran treatment were still able to degrade

carbofuran at an enhanced rate even after 4 years without a treatment of carbofuran.

Role of biomass and enzyme activity on enhanced degradation

The capacity of a soil to exhibit enhanced degradation of a pesticide has been

directly attributed to the ability of the soil microbial community to degrade the pesticide

at increasing rates until the pesticide is no longer of any efficacious value. The following

questions are often debated by scientists as to the main mechanism responsible for a soil

to develop enhanced degradation: 1) does repeated exposure of the pesticide result in

increased microbial biomass capable of degrading the pesticide; or 2) does repeated

exposure of the pesticide result in an increase in the enzyme activity specifically toward

the degradation of the pesticide?

Evidence supporting the first hypothesis is based on the degradation of the

herbicide 2,4-D (Ou, 1984; Holben et al., 1992; Ka et al., 1994) and the organophosphate









28

isofenphos (Racke and Coats, 1987). Ou (1984) reported that soils treated with 2,4-D

exhibited an increased in the degrading population with time after treatment with the

pesticide. Racke and Coats (1987) found the numbers of isofenphos degraders in soils

progressively increased as the number of field applications increased resulting in a

progressive increase in the degradation rate of the chemical.

Evidence for the second hypothesis is based on the numbers of EPTC-degrading

microorganisms in soils with and without a history of EPTC field application. Moorman

(1988) found that the numbers of EPTC-degraders in soils with a history of EPTC

applications were not significantly different from the numbers of EPTC-degraders in soils

with no history of EPTC application. In addition, the number of EPTC-degraders in

soils did not significantly increase 25 days after the application of EPTC despite the

increased degradation of EPTC in those soils.

What do these studies on carbofuran-degraders reveal about the nature of

enhanced degradation of carbofuran? The answer is enhanced degradation may include

both. Several studies point to an increase in the number of carbofuran-degraders in soils

with enhanced degradation compared to the indigenous carbofuran-degrading

microorganisms (Hendry and Richardson, 1988; Dzantor and Felsot, 1989; Dzantor and

Felsot, 1990). While other studies fail to link an increase in the number of carbofuran

degraders with the number of carbofuran applications (Racke and Coats, 1988; Merica

and Alexander, 1990; Scow et al. 1990; Robertson and Alexander, 1994).

Hendry and Richardson (1988) reported a significant increase in the number of

carbofuran-hydrolyzers from 1.6 x 103 to 3.1 x 105 cells g-' after one treatment with









29

carbofuran. Subsequent treatments of carbofuran did not result in a significant increase in

the number of carbofuran-hyrolyzers in the laboratory treated soil.

Racke and Coats (1988) reported that the higher number of carbofuran-

hydrolyzers in carbofuran treated soil was not significant to the number in the control (no

carbofuran history) soil, and they concluded that the enhanced degradation of carbofuran

in the treated soil could not be explained by differences in the number of carbofuran-

hydrolyzers in the different soils. In similar findings, Scow et al. (1990) and Robertson

and Alexander (1994) concluded that the microorganisms capable of mineralizing '4C-

carbofuran did not grow at the expense of either the methylamine or the phenyl moiety of

carbofuran, rather, carbofuran was degraded by a cometabolic process.

Microorganisms involved in carbofuran degradation

Since enhanced degradation of carbofuran is biological, the next question to ask is

what are the major groups of microorganisms involved in the degradation of carbofuran.

The use of selective antibiotics allows understanding of the roles the major groups of

microorganisms capable of degrading a pesticide. The use of selective antibiotics in

soils with enhanced degradation toward EPTC revealed that bacteria were the

predominate factor of enhanced degradation in soils ( Dick et al., 1990).

The bactericides chloramphenicol and streptomycin and a fungicide

cycloheximide have been used to determine whether or not bacteria or fungi are the main

group responsible for the degradation of carbofuran in soils. Studies that employed the

bactericides chloramphenicol demonstrated that the mineralization of ['4C-CAL]

carbofuran was predominantly bacteria in nature (Lenvanon, 1994; Racke and Coats,









30

1987). Wootton et al. (1993) investigated the role of fungi in the degradation of

carbofuran and concluded that fungi did not play a major role in the degradation of

carbofuran. These studies implicated bacteria as the major biological source of carbofuran

degradation in enhanced soils.



Carbofuran Degradation by Soil Microorganisms Involved in Enhanced Degradation



The ability to isolate cultures of pesticide degrading microorganisms is evidence

that pesticides are being metabolized in the field. Mixed cultures allow for greater

understanding of the biological mechanisms involved in the degradation of pesticides in

the environment. However, it is through the isolation of axenic cultures that detailed

genetic and physiological mechanisms of pesticide degradation are obtained. The

isolation and characterization of 2,4-D degrading bacteria (Don and Pemberton, 1981)

that has allowed researchers to obtain information on the evolution (Fulthorpe et al.,

1995) and the general ecology of the pesticide degrading microbial communities (Xia et

al., 1995; Ka et al., 1995).

The first carbofuran-degrading bacteria (actinomycete) isolated from soil

degraded carbofuran in the presence of a second C source (Williams et al., 1976). The

isolation of these bacteria resulted in the warning of high numbers of actinomycetes in

soil may result in rapid degradation of carbofuran. The early carbofuran-degrading

isolates degraded carbofuran slowly taking over 20 days for the complete degradation of

carbofuran (Venkateswarlu et al., 1977; Felsot et al., 1981; Venkateswarlu and









31

Sethunathan, 1985). Achromobacter sp. strain WM111 (Karns et al., 1986) was the first

soil bacterial isolate that exhibited the degradation kinetics that were comparable to the

rapid degradation of carbofuran observed in enhanced field soils.

Phenotypic classification based on the metabolism of carbofuran

Chaudhry and Ali (1988) classified carbofuran degrading bacteria into three

different groups (I, II, III) based on their phenotypic metabolism of carbofuran (Figure 2-

4) : Group I utilized carbofuran as the sole source of nitrogen and converted carbofuran

into carbofuran phenol; Group II utilized carbofuran as a sole source of carbon and

nitrogen and converted carbofuran to carbofuran phenol; and Group III utilized

carbofuran as a sole source carbon and nitrogen and converted carbofuran to carbofuran

phenol and further mineralized the carbofuran phenol to CO2 and H2 0. The majority of

soil bacteria isolated from soil thus far belong to Groups I and II. Little is known about

the physiology of soil isolates, the enzymes involved in the degradation and the genetics

of these organisms.

Group I

Carbofuran-degrading bacteria belonging to Group I are capable of hydrolyzing

carbofuran to carbofuran phenol and methylamine and utilizing the methylamine as a N

source (Figure 2.4). Achromobacter sp. strain WM 11 belongs to this group. This

isolate degraded not only carbofuran but other N-methylcarbamate pesticides, including

carbaryl, baygon, and aldicarb (Karns et al., 1986). WM111 degraded > 99% of the

applied carbofuran within 42 hours with a doubling time of 3 hours in a N free mineral

medium supplemented with glucose (7,200 mg ml-') and carbofuran (200 mg ml-'). The









32


0
CH Jo Group III

0 CH3
CH, -; CO2 + H20

Carbofuran







CO2 + H20




OH /Group II
OJ 0 CH3
cI + NHI-CH
Methylamine

Carbofuran phenol + Group I
Second Carbon
Source





CO2 + H20





Figure 2-4. Metabolic classification of carbofuran-degrading bacteria.









33

enzymes for carbofuran degradation are induced in the presence of carbofuran and

methylamine, but the expression of the hydrolase enzyme was inhibited when a rich

source of N was introduced into the growth medium. The gene encoding for carbofuran

hydrolase, mcd, was located on a plasmid and has been cloned (Tomasek and Karns,

1989). The enzyme properties have been characterized (Derbyshire et al., 1987; Karns

and Tomasek, 1991). Chaudhry and Ali (1988) also reported the isolation of Group I

type soil bacteria, but did not report any additional information on these bacteria.

Group II

The Group II carbofuran degrading bacteria are capable of hydrolyzing carbofuran

to carbofuran phenol and methylamine, and utilizing the methylamine as a C and N

source (Figure 2.4). The majority of the soil isolates capable of degrading carbofuran

belong to this classification. One of these isolates exhibited synergistic carbofuran

degradation behavior with another soil bacterium not capable of hydrolyzing carbofuran

(Singh et al., 1993). Some of the isolates expressed carbofuran hydrolase activity

constitutively with increased activity in the presence of carbofuran (Topp et al., 1993).

Group II isolates have been found to exhibit greater hydrolase activity than Group I

isolates (Chaudhry and Ali, 1988).

Some of the soil bacteria in this group were found to hybridize with the mcd

gene probe (Topp et al., 1993; Parekh et al., 1995). The majority of the soil bacteria in

Group II failed to hybridize to probes from the mcd clone (Chapalamadugu and

Chaudhry, 1992; Parekh et al., 1995). All the soil bacteria that hybridized with the mcd









34

probe harbored plasmids greater than the size of 100 kbp (Topp et al., 1993; Parekh et al.,

1994).

Group III

The isolates that belong to Group III are capable of hydrolyzing carbofuran to

carbofuran phenol and methylamine, and further degrading carbofuran phenol to carbon

dioxide, cellular components, and soluble products (Figure 2.4). More than 100 bacteria

that have the capacity to degrade carbofuran, but to date only five of these soil bacteria

are capable of degrading carbofuran phenol (Chaudry and Ali, 1988; Ramanand et al.,

1988; Head et al., 1992; Feng et al., 1997a). Group III isolates generally contain

multiple plasmids (Chaudry and Wheeler, 1988; Head et al., 1992; and Feng et al.,

1997a). None of the soil bacteria from Group III have hybridized to the mcd probe

(Chapalamadugu and Chaudhry, 1992; Feng et al., 1997a). Plasmids of the Group III

isolates may harbored the genes responsible for degradation of carbofuran. However, the

degradation of carbofuran by Sphingomonas sp. CF06 was the only known case involving

plasmids (Feng et al., 1997a).

Carbofuran Hydrolase Enzyme.

The only carbofuran hydrolase enzyme characterized to date was isolated from the

Achromobacter sp. WM111, and the enzyme existed as a homodimer. (Derbyshire et al.,

1987; Karns and Tomasek, 1991). Three other N-methylcarbamate hydrolase enzymes

have been isolated from three different soil bacteria (Mulbry and Eaton, 1991;

Chapalamadugu and Chaudhry, 1993; Hayatsu and Nagata, 1993). Two of these

hydrolases (Mulbry and Eaton, 1991; Hayatsu and Nagata, 1993) were very similar to









35

that of the carbofuran hydrolase reported by Kams and Tomasek (1991). The two

hydrolase enzymes were homodimers with a broad range of N-methylcarbamate activity

(Mulbry and Eaton, 1991; Hayatsu and Nagata, 1993). The hydrolase enzyme isolated by

Mulbry and Eaton (1991) has also been found to have enzymatic activity toward

carbofuran, but the hydrolase enzyme isolated by Hayatsu and Nagata was not tested for

its ability enzyme to hydrolyze carbofuran. One of the hydrolase enzyme was found to be

an esterase (Hayatsu and Nagata, 1993). The third carbaryl hydrolase enzyme exhibited

no enzyme activity toward carbofuran and did not have broad specificities toward other

N-methylcarbamates (Chapalamadugu and Chaudhry, 1993).

Genetics of Carbofuran Degradation

The genetics of carbofuran degradation by soil microorganisms is largely

unknown. The mcd gene from the Achromobacter sp. strain WMI 11 is harbored on a

large plasmid (greater than 100 kbp), and is the only gene coding for carbofuran

degradation that has been cloned (Tomasek and Kams, 1989). The soil bacteria that

hybridized to the mcd probe were all found to harbor the gene on large plasmids as was

the case for the original strain WMI11 (Topp et al., 1993; Parekh et al., 1995). A

comparison of carbofuran degrading microorganisms that had sequence homology with

the mcd gene probe were found to breakdown into five plasmid restriction length

polymorphism patterns (RFLP) (Parekh et al., 1996). Two of the soil isolates that are

from different RFLP groups and geographical areas, exhibited different chromosomal

and plasmid backgrounds, indicating that the carbofuran degrading genes did not

independently evolve, but rather was acquired from a plasmid already containing the set









36

of genes from the degradation of carbofuran (Karns, 1990). Thus, it appears that

enhanced degradation of carbofuran may result from the transfer of catabolic plasmids

harboring the genes for carbofuran degradation.

The involvement of plasmids in the mineralization of the aromatic ring of

carbofuran has largely been speculative with only plasmids present in the strains capable

of completely mineralizing carbofuran as evidence (Chaudry and Wheeler, 1988; Head et

al., 1992). Sphingomonas sp. strain CF06 harbors five plasmids some of which were

required for mineralizing the aromatic ring of carbofuran (Feng et al., 1997a). The

plasmids of CF06 contained at least six putative insertion sequence (IS) elements that

were cloned (Feng et al., 1997a). They found three of the five plasmids hybridized with

the IS elements. Since the acquisition of catabolic genes has been attributed to IS

elements (Tomasek et al., 1989), IS elements may play a role in the evolution of

carbofuran metabolism (Feng et al., 1997a). Understanding how soil bacteria acquire and

express the genes responsible for enhance degradation may provide researchers potential

tools to minimize the impact of enhanced degradation in agriculture fields.















CHAPTER 3
EXPERIMENTAL PROCEDURE



Field Work



Site Selection and Soil Characterization

Soil samples

Soil samples were collected from experimental plots at the University of Florida

Agricultural Experiment Station near Hastings, FL. The experimental site was located 6

miles SE of the city of Hastings off County Road 13. Soil at this site is classified as

Ellzey fine sand (sandy, siliceous, hyperthermic, Arenic Ochraqualfs). The majority of

this site was under potato cultivation for more than 20 years. Some experimental plots

under potato cultivation had been treated with carbofuran annually from 1993 to 1996,

and others had never been treated with carbofuran or any other structurally similar

pesticides. Sample designations were according to cultivation and histories of carbofuran

exposure. Soil samples from plots under potato cultivation and treated with carbofuran

were designated Cultivated Treated (CT). Samples from cultivated plots not receiving

carbofuran treatment were designated Cultivated Nontreated (CN). Soil samples from

plots not in cultivation but previously exposed to carbofuran were designated

Noncultivated Treated (NT), and samples from plots with no exposure to carbofuran and

37









38

not under cultivation were designated Noncultivated Nontreated (NN). Table 3-1 shows

the histories of carbofuran applications and cultivation, and application rates of

carbofuran to the plots where soil samples were collected. A schematic of the field plots

layout is shown in Figure 3-1.

The CT plots have been under potato cultivation for the past 10 years prior to the

beginning of this project, and CN plots were in potato production for only five years.

The NT soil samples came from a strip of grass along the drainage ditch adjacent to the

CT plots that was assumed to have been exposed to carbofuran due to its close proximity

to the CT plots. NN soil was an undeveloped grass field next to a woodland area where

groundwater flowed away from the grass field toward the experimental plots of CN; the

risk of exposure to carbofuran was considered negligible.

In July 1994, one month after annual field application of carbofuran, surface and

subsurface CT and CN soil samples were collected to a depth of 60 cm (top of the water

table) at 15 cm increments. A composite of three soil cores were taken from various

locations of the plot using a 10 cm diameter bucket auger. Soils with no carbofuran

exposure were collected first, while those soils previously exposed to carbofuran were

collected afterward.

In March 1995, one month after annual field application of carbofuran surface and

subsurface soils were collected as previously described for CT and NT soil samples,

except this time soil cores were not composited as done previously. Also in March 1996,

soil samples CT and NN, were collected exactly the same way as in March 1995. All soil

samples were stored in the dark at 40C and were used within four months.









39

Table 3-1. Description of the field plots where soil samples were collected and their
histories of carbofuran exposure.

Year Application rate Cultivation

kg ha-'

Cultivated Treated Plotsa (CT)

1994 4.5 Potato

1995 4.5 Potato

1996 4.5 Potato

Cultivated Nontreated Plotsb (CN)

1994 0 Potato/Onions

Noncultivated Treated Plots (NT)

1995 0 None

Noncultivated Nontreated Plots (NN)

1996 0 None
aThese plots were treated with carbofuran the first time in 1992 at a rate of 4.5 kg ha'
bAlthough these plots were not treated with carbofuran, they received fertilizers at rates
similar to the CT plots.


















Cultivated Untreated


Cultivated Treated



















Figure 3-1. Layout of the University of Florida Agriculture Experimental Station near Hastings, FL.



0









41

Soil characterization

The soil properties that were determined included: water content, pH, particle

size, and organic carbon content. Methods for particle size analysis, water content, pH,

and organic carbon content were as those described in Methods of Soil Analysis, Parts I

and II (American Society of Agronomy, 1986). Particle size analysis was performed

following the pipette method (Gee and Bauder, 1986). Soil water content was obtained

by gravimetric determination (Gardner, 1986). Soil pH was measured in a 1:1 soil extract

using distilled water (McLean, 1982). Soil organic carbon content was determined

following the Walkey-Black procedure (Nelson and Sommers, 1982).



Carbofuran Biodegradation Potential

Chemicals

Technical grade carbofuran (99% purity) and uniformly ring-labeled ['4C-URL]

and carbonyl-labeled [14C-CAL] carbofuran were provided as gifts from FMC Corp.

(Princeton, NJ). Radio-purity of the chemicals was verified by thin-layer

chromatography (TLC). Chemicals were not used unless radio-purity was greater than 98

%. If needed, the labeled chemicals were purified by preparative TLC to greater than

98%. All other chemicals were either HPLC grade, or the highest grade commercially

available.

Carbofuran mineralization

Mineralization of ['4C-CAL] and ["4C-URL] carbofuran was used to determine

the potential of the Ellzey soils to biodegradation carbofuran. One hundred grams of soil









42

(oven dry weight basis) were added to 250 ml Erlenmeyer glass flasks and mixed with

radio-labeled and technical grade carbofuran at a rate of 1.7 KBq and 1 mg, respectively.

After hand-mixing with a sterile spatula for 5 minutes, each flask was tightly closed with

a rubber stopper under which a stainless steel vial containing 0.5 ml of KOH (5.3 mol L"')

was hung (Ou, 1991). For the soil samples that were treated with ['4C-CAL] carbofuran,

KOH traps were replaced with new traps on days 1, 3, 7, 14 and 28. The KOH traps used

in trapping the evolved '4CO2 from the ['4C-URL] carbofuran treated samples were

replaced on days 3, 7, 14, 21, and 28. At the same time, the flasks were weighed, and

deionized water was added to compensate for any water loss.

KOH in the trap vials was diluted (1:10) with distilled water to 5 ml. The '4C in

the diluted KOH solution (0.5 ml) was quantified by liquid scintillation counting (LSC)

using a Beckman liquid scintillation counter model LS 5801 (Palo Alto, CA).

Extraction of soil

After 28 days of incubation, 10 g of soil was removed from the flasks and placed

in 50 ml glass culture tubes with Teflon liner screw caps along with 20 ml of methanol.

These tubes were shaken for one hour on a reciprocal shaker. After one hour of shaking,

methanol from the soil extracts was separated from the soil by vacuum filtration using a

Whatman no. 41 filter paper. Volumes of soil extracts were measured, and a small

aliquot (0.5 ml) was removed for the determination of '4C-activity.

Combustion of extracted soil

The extracted soil was placed in a mortar and homogenized by mixing and

grinding with a pestle for 3-5 minutes. Two-tenths of a gram of the soil was used for the









43

determination of the '4C-activity in the extracted soil. The '4C-activity in the extracted

soil was combusted to '4CO2 in a Packard Tri-Carb sample oxidizer. The '4CO2 was

trapped in a scintillation solution containing an organic amine, and quantified by LSC.

Statistical analysis

The initial amounts of '4C02 evolved from soils treated with '4C-carbofuran will

be compared for different soils, soil depth and number of carbofuran treatments using a

Student t-test. Carbofuran loses its pesticide efficacy upon the hydrolysis of the

carbamate moiety that results in the evolution of 14CO2 from ['4C-CAL] carbofuran.

Therefore, the monitoring the evolution of '4CO2 from ['4C-CAL] carbofuran treated soils

is probably the best indicator for evaluating enhanced degradation of carbofuran in soil.



Carbofuran Disappearance and its Metabolite Formation

Extraction of soil samples collected in 1994

One hundred fifty grams of soil (oven-dry weight basis) collected in 1994 were

placed in a 500 ml glass Erlenmeyer flask along with 1.5 mg of technical grade

carbofuran, 17 KBq of ['4C-URL] carbofuran, and 1.5 ml of deionized water and mixed

for five minutes. After mixing by hand for five minutes with spatulas, each flask was

tightly closed with a rubber stopper under which a stainless steel KOH trap containing 0.5

ml KOH solution (5.3 mol L-') was hung (Ou, 1991).

After removing the KOH traps, 10 g of soil were removed from the 500 ml flask

and placed in a 50 ml glass culture tubes with a Teflon liner screw caps. Fresh KOH

vials were hung in the flasks. Methanol (20 ml) was used to extract the ['4C-URL]









44

carbofuran and its '4C-metabolites from the soil in the tubes. Tubes were shaken for one

hour on a reciprocal shaker. The extracts were filtered under vacuum through Whatman

no. 41 filter papers. The volumes of the soil extracts (25-30 ml) were measured and 0.5

ml of the methanol extract was removed to determine the '4C-activity in the methanol

extracts by LSC. Water in the extracts was removed by anhydrous sodium sulfate, and

the extracts were concentrated under a gentle stream of N2 to approximately 0.3 ml.

Extracted soils were combustion following the procedure described previously.

Thin-layer chromatography (TLC) analysis

Carbofuran and its metabolites in the concentrated extracts were separated on

silica gel G TLC glass plates (E. Merk, Demstdt, Germany). The plates were developed

to a distance of 15 cm using a solvent system of diethyl ether and hexane (3:1). The TLC

plates were exposed for 3 to 4 weeks to Kodak x-ray films (SB-5) for autoradiographical

analysis (Ou et. al., 1982). Radioactive area of the TLC gels corresponding to

carbofuran, metabolite standards, and unknown were scrapped, transferred to scintillation

vials and quantified by LSC.

Extraction of soil samples collected in 1996

A similar procedure used to extract soil samples collected in 1994, with minor

modifications, was also used to extract soil samples collected in 1996. In this study, 10 g

of soil were removed from each flask and placed in a culture tube and extracted with 20

ml of methanol as previously mentioned. Soil suspensions were first filtered under

vacuum through Whatman no. 41 filter papers, and then washed three times in succession

with 20 ml of methanol. After the volumes of the methanol extracts were determined, the









45

'4C-activity in the extracts was quantified by LSC. Water in the extracts was removed by

anhydrous sodium sulfate, and the extracts were concentrated to approximately 5 ml on a

roto-evaporator (Brinkman Instruments, Westbury, NY). The extracts were transferred to

small glass vials and concentrated under a gentle stream of N2 to approximately 0.3 ml.

The procedures for the determination of '4C-activity in the extracted soil samples and the

determination of metabolites by a TLC-autoradiographical procedure were as described

previously.



Growth of Carbofuran-Degrading Populations

Most-Probable-Number (MPN) assay

A '4C-MPN technique was used to estimate the microbial population size in soil

that was capable of degrading the carbamate and aromatic ring moieties of carbofuran

(Ou, 1984). ['4C-CAL] and ['4C-URL] carbofuran were used to determine the population

size in soil capable of hydrolyzing carbofuran and capable of mineralizing the aromatic

ring structure of carbofuran, respectively. A MPN assay that determined the population

size capable of utilizing a substrate for growth was used for determination of the

population size capable of utilizing methylamine (Alexander, 1982). A description of

soil samples, depth, incubation time, and specific degrading population is shown in Table

2-3.

One hundred grams of soil (oven-dry basis) were added to a 250 ml Erlenmeyer

glass flask and mixed with 1.0 mg technical grade carbofuran. The flasks were incubated









46

Table 3-2. Substrates, soil depths and sampling time for the MPN experiments.

Year Soil Substrate Soil Depth Sampling time

cm days

1994 CT, CN ['4C-CAL]a 0-15, 45-60 0

CT, CN [14C-URL]b 0-15, 45-60 0

1995 CT, NT ['4C-CAL] 0-15, 45-60 0, 1, 3, 7,14,28

CT, NT ['4C-URL] 0-15, 45-60 0, 1, 3,7, 14,28

CT, NT '4Carbofuran 0-15 0,1, 3, 7, 14,28
phenol

CT, NT methylamine 0-15 0,1, 3, 7, 14,28

1996 CT, NN ['4C-URL] 0-15,45-60 0, 1, 3,5, 7,14
a [l4C-CAL] = [14C-CAL] carbofuran
b [14C-URL] = [14C-URL] carbofuran









47

in the dark at ambient temperature (23+ 2 C), and once a week they were checked for

water loss and deionized water was added to compensate for any water loss. At

predetermined time intervals (Table 2.2), 1 g of soil was removed and placed in a sterile

capped MPN tube that contained 9 ml of a sterile

minimal mineral medium (MMA) consisting of the following ingredients per liter:

K2HPO4, 4.8 g; KH2PO4, 1.2 g; NH4NO3, 1.0 g; MgSO4, 0.25 g; CaC12 2H20, 40 mg;

Fe2(S04)3, 1 mg. In addition, 10 mg of tryptone was added to the MMA prior to

sterilization. Technical grade carbofuran (10 mg) and '4C-carbofuran or '4C-carbofuran

phenol were added after autoclaving of the MMA. The MPN medium used to enumerate

carbofuran degraders contained approximately 400 dpm ml-' of ['4C-CAL] carbofuran or

800 dpm mll' of ['4C-URL] carbofuran, and the MPN medium used to enumerate

carbofuran phenol degraders it contained approximately 500 ml"' dpm of ['4C-URL]

carbofuran phenol. When enumerating methylamine degraders in soil, only unlabeled

methylamine hydrochloride was added to the tubes as the sole source of carbon at a rate

of 100 mg L' of methylamine.

After adding 1 g of soil to the MPN medium, five replicates of successive 5 to 10

fold dilution were made. All tubes were incubated in the dark at 28 "C for four weeks.

Control MPN tubes were treated identically as above except that soil was not added to the

tubes. At the end of the incubation period, 100 giL of concentrated HC1 was added to

each tube mixed and let stand for 4 hours. After standing for 4 hours, 0.5 ml of the MPN

media was assayed for '4C-activity remaining in solution as determined by LSC. Tubes

were scored as positive when less than 60 % of the initial '4C-activity remained in









48

solution. '4C in all the control '4C-MPN tubes were unchanged after 28 days of

incubation. For methylamine degraders, tubes were scored positive if turbidity was

observed in the MPN solutions in the tubes. Controls for the methylamine MPN tubes

were all devoid of any turbidity.

Statistical analysis

MPN numbers were calculated using the Eureka (Borland International, Scotts

Valley, CA). The equations calculate the MPN number and the 95% confidence limits of

the MPN numbers. Data processed into the program include dilution factors, replicates at

each dilution, number of positives at each dilution, and the weight of soil (g) initially

added to MPN tubes. The mean value for the various degrading microbial populations

were compared for differences between soils and differences between soil depth. Growth

in the various degrading microbial populations were also compared with initial

degrading populations. The results were used to determine if enhanced degradation of

carbofuran is a function of increased growth or increased activity of the microorganism

capable of degrading carbofuran.



Characterization of Carbofuran-Degrading Soil Isolates



Isolation and Metabolism of Carbofuran by Soil Isolates.

Isolation procedures

A batch enrichment technique was used to isolate carbofuran-degrading bacteria

from soil. Technical grade carbofuran was applied to 100 g CT soil at a concentration of









49

10 mg kg-'. The flasks were incubated in the dark at ambient temperature (232 C), and

the flasks were checked for water loss once a week and deionized water was added to

compensate for any water loss. Carbofuran was reapplied every 4 weeks, and after 4

months of incubating 10 g of the soil was removed and inoculated into a 250 ml

Erlenmeyer flask that contained 100 ml sterile MMA and carbofuran at a rate of 400 .Lg

ml-'. In addition to the batch enrichment technique, soil was also inoculated directly into

sterile MMA that contained 400 u.g of carbofuran ml-'. The flasks were incubated at

room temperature on a rotary shaker at 100 rpm.

Once every two weeks, 10 ml of sample was transferred to a fresh MMA

containing carbofuran (400 gg ml-'). After 4 successive 1-to-10 transfers, liquid cultures

were free of soil particles, and a red pigmented color developed along with slight

turbidity in some flasks. Small amounts of the culture fluids that developed the red color

were streaked onto carbofuran-MMA (400 jtg carbofuran ml-'1 MMA) agar plates.

Colonies that developed on carbofuran-MMA agar plates were restreaked onto fresh

carbofuran-MMA agar plates. Colonies that developed on new carbofuran-MMA plates

were then streaked on Luria-Bertani (LB) agar plates for checking the purity of the

isolated culture. Once colonies developed on the LB agar plates and determined to be a

pure culture, they were restreaked again on carbofuran-MMA agar plates. For confirming

that the isolate was capable of degrading carbofuran, a small amount of biomass from a

colony was inoculated into liquid carbofuran-MMA containing a small amount of ['4C-

CAL] or ['4C-URL] carbofuran (30 Bq ml-') (Ou and Sharma, 1989). Those bacterial









50

isolates were considered to have the capacity to degrade carbofuran if they mineralized

['4C-CAL] and/or ['4C-URL] carbofuran.

Identification of soil bacteria isolated

Unknown bacterial isolates capable of utilizing carbofuran as a sole source C for

growth were sent to the Plant Pathology Department at the University of Florida for

analysis of their fatty acid methyl-ester signature profiles using the MIDI system. In

addition, each bacterial isolate was also identified by gram stain (Difco Laboratory,

Detroit, MI), motility, and biochemical properties (Oxi/Ferm Tubes II, Becton Dickinson

Cockeysville, MD).

Mineralization of carbofuran as sole source of carbon

The purpose of subsection was to determine the mineralization rate of carbofuran

by the soil bacterial isolates as related to their growth. A small amount of biomass from a

colony was inoculated to a 250 ml flask that contained 50 ml of carbofuran-MMA (50 plg

carbofuran ml-' MMA). After 24-48 hours of incubation, 5 ml of the culture fluid were

transferred into a fresh carbofuran-MMA. After the second transfer, one-day old cultures

were inoculated into 250 ml Biometer flasks (Bellco, Vineland, NJ) containing 50 ml of

MMA, and technical grade carbofuran, and ['4C-CAL] or ['4C-URL] carbofuran at rates

of 50 ug ml' and 4 KBq ml-', respectively. The side arms of the flasks contained 5 ml of

0.5 M KOH solution for trapping '4CO2. At predetermined time intervals, KOH was

removed from the side arms and replaced with fresh KOH. Trapped '4CO2 in the KOH

was quantified by LSC. After 72 hours, 10 ml of culture solution was removed and

vacuum filtered through 0.2 ulm Nylon filters (Micron Separations, Inc., Westboro, MA).









51

The filters were washed three times with 5 ml of MMA. '4C-activity in filtered extracts

and washed filters were quantified by LSC. All samples were done in triplicate. The '4C-

activity detected in the KOH solution represented the portion of 4C-carbofuran being

mineralized, while '4C retained on the filters represented the '4C being incorporated into

the biomass and '4C in the filtered extracts represented the '4C-activity in the cell free

medium.

Growth of soil bacteria

In conjunction with the sampling of the KOH traps, growth of the soil bacteria in

the carbofuran-MMA was measured using a Petroff-Hasuer bacteria counter (Becke et al.,

1990). During the replacement of KOH traps, 100 p.1 of culture solution was also

removed and diluted with an equal volume of a phosphate buffer (0.1 mol L"' K2HPO4

and 0.1 mol L"' KH2PO4 pH 7.2). After mixing, two drops of the diluted culture solution

were deposited on the surface of the counting chamber along with a glass slip cover.

After allowing 15 minutes for bacterial cells to settle down, cells were counted according

to the procedure of Becke et al. (1990). All samples were done in duplicate.

Mineralization of carbofuran without supplement of an extra N source.

Metabolism of carbofuran as a sole source of N was studied in an identical

experimental set-up with the study on the metabolism of carbofuran as a sole source of

carbon. In this experiment the mineral media (MMB) consisted of the following

ingredients L-' H20: K2HPO4, 0.48 g; KH2PO4, 0.12 g; MgSO4, 0.25 g; CaC12 2H20, 40

mg; Fe2(S04)3, 1.0 mg. In addition, carbofuran (200 pgg ml-') and glucose (325 ptg ml-')

were added to the MMB prior to sterilization. The radio-labeled carbofuran was applied









52

at a rate of 6 Bq ml-' for ['4C-CAL] carbofuran and 80 Bq ml-' for ['4C-URL] carbofuran.

All glassware was acid washed in a 1% HC1 solution (3 hours) and triple rinsed with

distilled water prior to use. The mineralization and mass balance of the 4C-activity

followed similar procedure described in the section of "Mineralization of carbofuran as a

sole source of C."

Mineralization of carbofuran in soil extract

The metabolism of carbofuran in soil extract was also studied in a similar

procedure used for determination of mineralization of 14C-carbofuran in MMA as a sole

source of C. The soil extract was prepared by autoclaving a mixture of 500 grams of CT

surface soil and 500 ml of deionized water at 121 C for 1 hour (Ou, 1991). The soil

suspension was carefully transferred to centrifuged tubes, and they were centrifuged for

20 minutes at 10000 rpm. The supernatant fluid was vacuum filtered through 0.22 mm

cellulose acetate filters (Coming, Inc., Coming, NY). Technical grade and radio-labeled

carbofuran were added to the soil extract at 50 l.g ml-' and 80 Bq ml"', respectfully.



Carbofuran Degradation Pathways

Degradation and metabolite formation

In this experiment, the degradation of carbofuran and the formation of its

metabolites were monitored by means of TLC-autoradiographical analysis. Ten ml of

one-day-old cultures grown in carbofuran-MMA was inoculated into a 1000 ml

Erlenmeyer flask containing MMA (500 ml), carbofuran (50 plg ml-'), and ['4C-URL]

carbofuran (80 Bq ml-'). After inoculation, each flask was tightly closed with a rubber









53

stopper under which a stainless steel vial containing 0.5 ml of 5.3 mol L-' KOH was hung

(Ou, 1991).

At predetermined time intervals, the stainless steekltraps were replaced with new

traps containing fresh KOH. The removed KOH solutions were diluted with deionized

water to 5 ml, and the trapped '4CO2 in the KOH was quantified by LSC. While changing

the traps, 10 ml of culture solutions was removed from the flasks, and they were vacuum

filtered through 0.2 mm Nylon filters. The filters were washed three times with 5 ml of

MMA. After the volumes of the filtered fluids was determined, the '4C-activity in the

fluids were determined by LSC. '4C-activity retained on the Nylon filters were quantified

by LCS.

Prior to extraction, the filtered extracts were acidified with 60 uil of concentrated

HC1. They were then extracted twice with 25 ml of ethyl acetate. Moisture in ethyl

acetate extracts were removed by anhydrous sodium sulfate and evaporated to dryness

using a roto-evaporator. The residue in each flask was redissolved in 5 ml of anhydrous

methanol and the methanol solutions were transferred to glass vials. The volume of the

methanol solutions volume to 0.3 ml by gently passing N2 gas over the extracts.

Carbofuran and its metabolites in the concentrated extracts were separated and quantified

by TLC-autoradiographical analysis and LSC as described previously in the soil

metabolite section.

Red-colored metabolite(s),

The purpose of these experiments was to isolate, purify, and identify the red-

colored metabolite associated with the metabolism of carbofuran. Three bacterial isolates









54

capable of degrading carbofuran produced a water soluble red-colored pigment(s) when

grown in carbofuran-MMA or carbofuran-MMB. One of these isolates was designated to

be HPL strain and was used for this study. The HPL strain was inoculated into a

carbofuran-MMA solution with a carbofuran concentration of 400 p.g ml-', and incubated

on a shaker (100 rpm) for 1 week at 28 C. After one week, the culture solution

developed a dark red color, and the culture fluid was centrifuged at 10,000 rpm for 20

minutes. The supernatant was collected and acidified to pH 3. The acidified supernatant

was vacuum filtrated through a C,8 reverse phase column (SepPack, Waters, Milford,

MA). The retained pigment in the column was eluted with anhydrous methanol into glass

vials.

Methanol extract was spotted on a silica gel G preparative TLC glass plate (E.

Merk, Darnstadt, Germany). The plate was developed to a distance of 15 cm using the

same developing solvent used for the separation of carbofuran and its metabolites as

described previously. A red-colored band traveled 2.0 cm (Rf = 0.12) from where the

extract was originally spotted. The gel associated with the red-colored band on the plate

was scrapped and placed in a glass beaker. The gel was extracted 3 times with deionized

water. The water extract was acidified to a pH of 3 and then vacuum filtered through a

solid phase C,8 column. Methanol was used to remove the red-colored pigment from the

column. The methanol extract was spotted on a preparative TLC plate for a second

purification of the metabolite.

The purified methanol extract was further purified by loading the extract into a

silica gel (particle size 63-200 mm) column 2.5 x 20 cm (Spectrum Chromatography,









55

Houston, TX). The methanol extract was eluted from the column under gravity. The

mobile phase was a mixture of ethyl acetate:acetic acid (100:1), and fractions collected

every 10 minutes. Fractions were combined, and the ethyl acetate:acetic acid was

removed by a roto-evaporator as previously described. Residues were redissolved in

anhydrous methanol (dried over sodium sulfate). Samples were sent to the Stine-Haskell

Research Center (DuPont Agriculture Products, Newark, DE) for liquid chromatography/

mass spectrometry analysis (HPLC/MS).

Cell-free culture extracts

The purpose of these experiments was to determined the carbofuran degradation

pathway by the carbofuran-degrading bacterial strain TA05. The TA05 strain was chosen

for the isolation of unknown metabolite(s) because it degraded carbofuran more rapidly

than the strain HPL. The procedures for the isolation of the unknown metabolites in one-

day-old cell-free culture extracts were similar to the previously section. In this isolation

procedure, the one-day-old cell-free culture extract was not further purified on preparative

TLC plates or silica gel columns. Rather, the methanol extract from the solid phase C18

column was dried by flushing with N2 gas and redissolved into methylene chloride. The

methylene chloride samples were sent to Charles Schmindt of the Environmental

Engineering Science Department at the University of Florida for gas chromatography/

mass spectrometer analysis (GC/MS).









56

Genetics of Carbofuran Degradation

The purpose of this study was to determine whether the genes responsible for the

degradation of carbofuran reside on the chromosome or on plasmids. One of the isolates

(TA strain) capable of degrading carbofuran harbored a single plasmid. The plasmid in

the TA strain has been shown to have regions of homology with the plasmids of another

carbofuran degrading isolate (Sphingomonas sp. strain CF06). The CF06 strain harbored

five plasmids and these plasmids were shown to be responsible for the mineralization of

['4C-URL] carbofuran (Feng et al., 1997a).

Plasmid curing

A small amount of biomass from a colony of the TA strain was inoculated into

liquid LB medium and grown at 42 C with shaking for two days. Curing and isolation of

the cured TA strain was done by Drs. X. Feng and A. Ogram at Washington State

University in the Crop and Soil Science Department. The cured TA05 strain was provided

by Drs. Feng and Ogram. This cured strain was obtained by growing the parent strain in

LB broth at high temperature (42 C). This strain lost the capacity to mineralize both the

['4C-CAL] and ['4C-URL] carbofuran.

Isolation of plasmids

Plasmid DNA was isolated from the TA strain using a modified procedure

developed by Feng et al. (1996a). Table 3-3 shows the procedures used to isolate plasmid

from the strain in a large scale (500 ml) and a small scale (5-10 ml). Purified plasmid

(chromosomal free) DNA was obtained by the CsCI density gradient ultracentrifugation









57

Table 3-3. Procedures used to isolate plasmid DNA (Feng et al., 1996a).

Procedure Large scale Small scale

1. Collect cells from culture by centrifugation (10,000 500 ml 5-10 ml
rpm for 15 min.).

2. Resuspend cells in 6.7% sucrose-50 mM Tris-lmM 5 ml 200 jp1
EDTA pH 8.0. Warm to 37C for 5-10 min.

3. Add lysozyme (100 mg ml in 25 mM Tris pH 8.0). 100 p1 10 pl1
Mix well and incubate at 370C for 30 min.

4. Add alkaline SDS solution (3% SDS, 0.2 M NaOH). 8 ml 400 plI
Mix immediately with gentle shaking and incubate
on ice for 10 min.

5. Add ice cold sodium acetate (pH 4.8). Mix gently 6 ml 300 l.1
and incubate on ice for 30-60 min.

6. Centrifuge (10,000 rpm for 15 min.) At 4C and
recover the supernatant.

7. Add 0.6 volumes 2-propanol and mix well. Spin at 12 ml 600 ml
12,000 rpm for 15 min. and pour off supernatant

8. Resuspend DNA pellet in 0.5 ml TE buffer (10 mM 500 ml 500 u.1
Tris-HCl, ImM EDTA; pH 8.0)

9. Add phenol:chloroform:isoamy alcohol (25:24:1). 750 pi1 500 pll
Mix and spin for 2 min. at max. speed (15,000 rpm)

10. Add chloroform:isoamy alcohol (24:1). Mix and spin 500 l.1 500 p.1
at max. speed for 2 min.

11. Transfer the aqueous phase to another tube and add 3 50 l.1 50 .1
M sodium acetate.

12. Add two volumes of ethanol (100%). Mix well and 1000 p1I 1000 pl
spin at max. speed for 2 min.

13. Wash pellets by adding 75% ethanol and invert tube 1000 pll 500 p.1
and let tube air dry for 30 min.

14. Resuspend pellet in TE buffer with RNAase (0.1 mg 200 pI1 30 pll
ml-'). Store at 40C.









58

procedure (Sambrook et al., 1989). The plasmid DNA was stored in sterile HPLC grade

water at 40C until used.

Introduction of TA05 plasmid into Pseudomonas fluorescens M480R strain

Competent cells Competent cells of P. fluorescens M480R strain were the host to

receive the plasmid DNA isolated from the TA strain by electroporation. A single colony

of M480R strain was inoculated into 10 ml of LB broth and incubated overnight at 28 C

on a shaker. One ml of the overnight culture was inoculated into 100 ml of LB broth and

incubated as before. Cells were grown to 0.4 to 0.5 absorbance (600 nm) (exponential

growth) and immediately chilled in an ice bath for 1-2 minutes. Cells were then

centrifuged for 10 minutes at 6,000 rpm. After the supernatant was discarded, the cells

were resuspended into a cold 10 % glycerol solution and centrifuged again. This washing

with glycerol was repeated a total of 5 times. After the final washing, cells were

resuspended in 400 1l of the glycerol solution and subdivided into several micro-

centrifuge tubes (40 ll per tube) and stored at -70 C.

Electroporation The plasmid DNA isolated from the TA05 strain was introduced

into the competent cells of P. fluorescens M480R strain by electroporation. The

competent cells (40 g.1) were mixed with 1 gg of the plasmid DNA from the TA strain

and electroporated with a Gene Pulser apparatus II (Bio-Rad, Richmond, CA).

Electroporated cells were initially incubated in cold LB broth for 8 to 10 minutes

followed by incubation at room temperature for 3 hours. Transformants were selected for

their ability to grow on carbofuran-MMA agar plates with carbofuran as a sole source of

C or N. The positive control in the electroporation was the introduction of the plasmid









59

pRK415 into the M480R strain. pRK415 is the plasmid that carries the antibiotic

resistance to tetracycline. The negative control was the substitution of water for the TA

plasmid.















CHAPTER 4
CARBOFURAN DEGRADATION IN SURFACE AND SUBSURFACE SOILS


Introduction



Carbofuran is a broad-spectrum N-methyl carbamate insecticide used to control

certain soil borne insects and nematodes. In 1993, carbofuran was listed as the fifth most

heavily used field insecticide in the US (Gianessi and Anderson, 1995). Although

carbofuran is moderately persistent in soil, exhibiting an average field half-life of 50 days

(Wauchope et al., 1992), its relatively high mobility (K. = 30) (Sukop and Cogger,

1992), and toxicity toward mammals (Fahmy et al., 1970) and aquatic organisms (Trotter

et al., 1991) raises concerns over its use in agriculture. These concerns are heightened by

the fact that carbofuran residues have been detected in groundwater and in surface water

(Erickson and Norton, 1990; Shahane, 1994).

Measurements of the biodegradation potential of most pesticides are typically

done on the top 15-20 cm of soil. This may over estimate the biodegradation potential in

the subsurface horizons. The biodegradation potential of carbofuran was reportedly

lower in the subsurface soil (> 50 cm depth) compared to surface soil (Buyanovsky et al.,

1993). In addition, Mallawatantri et al. (1996) reported that the mineralization of

carbofuran in the A horizon was significantly greater than in lower horizons for soils with


60









61

no prior exposure to carbofuran. This reduction in the biodegradation potential of

carbofuran in subsurface soils has been attributed to a reduction in microbial activity of

the subsurface environments (Buyanovsky et al., 1993).

The repeated use of carbofuran in agricultural fields has resulted in its accelerated

degradation in soil (Suett, 1986). A consequence of the shortened half-life of carbofuran

is a reduction in its insecticidal efficacy against target pests (Read, 1983). Enhanced

degradation of carbofuran has been attributed to carbofuran-degrading microorganisms

that develop after repeated exposure to the pesticide (Hendry and Richardson, 1988).

The purpose of this study was to characterize the degradation of carbofuran in

surface and subsurface soils under various carbofuran treatment histories and cultivation

practices. This study characterizes the degradation of carbofuran in surface and

subsurface soils according to its biodegradation potential and solvent extractable of

carbofuran using ['4C-CAL] and ['4C-URL] carbofuran. The biodegradation potential is

accessed by measuring the evolution of 4CO2 from soil applied with ['4C-CAL] and [14C-

URL] carbofuran, and the disappearance of solvent extractable ['4C-URL] carbofuran

from soil. In addition, '4C-metabolites and "4C recovery in soil are also determined.


Results



Soils

The selected properties of the soils used for this study are given in Table 4-1. The

annual application of carbofuran in the cultivated treated (CT) soils began in the March,









62

Table 4-1. Selected characteristics of the Ellzey soil samples used in this study.

Depth pH Moisture Organic Sand Silt Clay
content Carbon

cm ml kg-' g kg-' %

CT soil

0-15 5.9 99 4.2 97 1 2

45-60 5.1 159 6.2 92 5 3

CN soil

0-15 6.9 151 15.5 92 5 3

45-60 5.2 181 5.7 95 2 3

NT soil

0-15 5.7 80 5.3 97 1 2

45-60 6.2 175 6.1 92 5 3

NN soil

0-15 6.0 208 19.5 90 8 2

45-60 5.6 172 4.5 95 3 2









63

1992, and continued through March, 1996. The low organic carbon content in the surface

(0-15 cm) CT soils (4.2 g kg-') reflects the continuous cultivation in this plot for over ten

years, while the higher organic carbon content in the cultivated nontreated (CN) surface

soil (15.5 g kg-') reflects the shorter period of time this field has been in cultivation (five

years). The noncultivated nontreated (NN) surface soil has never been under cultivation

which is evident by the high organic carbon content (19.5 g kg-'). The typical organic

carbon content in the surface horizon of the Ellzey soil series is between 10-30 g kg-'. It

is interesting to note that the noncultivated treated (NT) soil has not been under

cultivation, but its organic carbon content in the surface soil is similar to that of the

treated soil. This may result from traffic (next to a dirt road) that could result in soil

compaction and reduced biological activity. The lower organic carbon content (less than

10 g kg-') in subsurface soils (45-60 cm) for all soils samples is a characteristic of Ellzey

soil series.



Mineralization of ['4C-CAL] in Surface and Subsurface Soils

Surface soil (0-15 cm depth).

The mineralization of ['4C-CAL] carbofuran was rapid in the surface CT soils

collected in 1994 and 1996 (Figure 4-1 A) with none exhibiting any lag periods. The CT-

94 mineralized 12 % of the applied '4C-activity in the first 24 hours and over 90% after

three days. The CT-96 exhibited an even greater rate of mineralization than the CT-94

with over 70 % of the ['4C-CAL] carbofuran applied being mineralized within 24 hours

(Figure 4-1A). This initial mineralization rate was significantly higher (a < 0.05) for the








64

100 -


80 / CT soil '94'
A -- CT soil '96'
S60 ---- CTsoil-fum '94'


40 ~


20
I 20


^ o
w B
4 80 -

60-
O CU soil '94'
T -i- NU soil '96'
S 40


20 --- -- -



0 5 10 15 20 25 30

Time (days)



Figure 4-1. Mineralization of ['4C-CAL] carbofuran in surface (0-15 cm) Ellzey soil: A)
treated soil; B) control soils. Error bars represent the standard deviations of analysis.









65

CT-96 compared to that collected in 1994. The fumigated CT-94 mineralized ['4C-CAL]

carbofuran slowly with less than 11% of the '4C-applied mineralized after 28 days of

incubation. This is in sharp contrast to the nonfumigated CT soil samples collected in

1994 and 1996 (Figure 4-1 A).

The mineralization pattern for [14C-CAL] carbofuran in the control soils was

somewhat different from the treated soils. In the CN soil, there was a three day lag

period prior to rapid mineralization of ['4C-CAL] carbofuran with greater than 80 % of

the applied '4C being mineralized after 14 days (Figure 4-1B). The NN soil initially did

not mineralize ['4C-CAL] carbofuran rapidly with less than 20 % of the applied '4C being

mineralization after 28 days of incubation (Figure 4-1B). When comparing cumulative

14CO2 production, there was a significantly greater amount of '4CO2 evolved in the CT

soils than in the CN soil after 7 days of incubation (a < 0.05). There was a significant

higher mineralization rate in the cultivated soils than in the nonculitvated soil (a < 0.05).



Subsurface soil (45-60 cm depth).

There was a day lag period of 3 days prior to the onset of rapid mineralization in

the CT subsoil collected in 1994 (Figure 4-2A), while in the CT subsoil collected in 1996

the lag period was not observed. The CT-96 also mineralized a significantly higher (a <

0.05) amount of ["4C-CAL] carbofuran after 3 days of incubation compared to the CT-94.

The CT-96 mineralized more than 10% and 80 % of the applied "'C in 1 and 7 days,

respectively. The CT sample collected in 1994 mineralized greater than 80 % of the








66

100
100 -,- ----------------------------------


80- A


/- 60- /


4 -
]- /' I -0- CT soil '94'
S20 / CT soil '96'
S_ / -- CT soil-fum
o 0 __,_____.-A,
0

B
80-


S60-
O -0- CU soil '94'
U -A-- NU soil '96'
40-


20-

= "= I-IU

0 5 10 15 20 25 30

Time (days)

Figure 4-2. Mineralization of ['4C-CAL] carbofuran in subsurface (45-60 cm) Ellzey soil:
A) treated soil; B) control soils. Error bars represent the standard deviations of analysis.









67

applied ["C-CAL] carbofuran within 7 days. After 3 days of incubation, the surface CT

soils mineralized significantly (a < 0.05) more ["C-CAL] carbofuran than the

corresponding subsurface CT soil (Figures 4-1A and 4-2A). In the fumigated CT soil, the

lag period lasted seven days prior to the onset of rapid mineralization of [14C-CAL]

carbofuran (Figure 4-2A).

Mineralization of [14C-CAL] carbofuran was very low in the subsurface control

soils (CN and NN). The mineralization in these soils was slightly more than 10% of the

applied '4C during the 28 days of incubation. The subsurface control soils (CN and NN)

mineralized a significantly (a < 0.05) smaller amount of ["C-CAL] carbofuran than the

CT soils.



Mineralization of ['4C-URL] Carbofuran in Surface and Subsurface soils

Surface soils (0-15 cm depth).

The mineralization of [14C-URL] carbofuran in all CT soils was rapid with more

than 30% of the "'C applied being mineralized in the first three days of incubation (Figure

4-3A). Mineralization for the surface soils peaked after 14 days of incubation. There

were no differences in the initial degradation rates or total CO2 evolution from any of

the surface CT-94, CT-95 and CT-96. There were differences between the mineralization

rates of ["C-CAL] carbofuran and ['4C-URL] carbofuran, with the ['4C-CAL] carbofuran

exhibiting a faster mineralization than ['4C-URL] carbofuran.








68

100 -

A
80


60-


-40

a --0- CT soil '94'
S20 -U- CT soil '95'
o* -/- CT soil '96'
0

a, ]B
| 80-


60


40 / -0- CN soil '94'
/ -- NT soil '95'
20 --- NN soil '96'

0

0 5 10 15 20 25 30

Time (day)



Figure 4-3. Mineralization of ['4C-URL] carbofuran in surface (0-15 cm) Ellzey soil: A)
treated soil; B) control soils. Error bars represent the standard deviations of analysis.









69

The control soil samples (CN, NT and NN) exhibited various patterns of ['4C-URL]

carbofuran mineralization (Figure 4-3B). In the CN soil, there was a lag period of 3 days

prior to the onset of rapid mineralization of ['4C-URL] carbofuran. This mineralization

reached a plateau after 14 days with 56 % of the applied "4C being mineralized at the end

of 28 days of incubation. The NT soil exhibited no lag period (note: based on the

mineralization curve, a short lag period of less than 3 days might exit), and 11 % of the

applied ['4C-URL] carbofuran was mineralized after three days, and it also peaked on day

14, with 67% of the applied 14C being mineralized after 28 days of incubation (Figure 4-

3B). The NN soil mineralized ['4C-URL] carbofuran slowly but steadily with less than

10% of the applied 14C being mineralized after 28 days of incubation (Figure 4-3B).

There was a significantly higher mineralization of ['4C-URL] carbofuran in the

CT soils than in the CN soil during the first 3 days of incubation. After 3 days, there was

no difference between CT, CN and NT soils for cumulative mineralization of ['4C-URL]

carbofuran. There was a significantly faster (ca < 0.05) mineralization of ['4C-URL]

carbofuran in the CT soil than in the NN soil, however.

Subsurface soils (45-60 cm depth).

The mineralization of the CT soil collected in 1994 exhibited a lag period of 3

days prior to the onset of rapid mineralization of ['4C-URL] carbofuran (Figure 4-4A).

The CT soil samples collected in 1995 and 1996 rapidly mineralized ['4C-URL]

carbofuran without a lag period (Figure 4-4A). Total cumulative "4CO2 evolution in CT-

94, CT-95, and CT-96 was not significantly different despite CT-94 and CT-96








70

100 -

A

80-




S/ -4- CT soil '94'
S-U- CT soil '95'
S20- / -/A-- CT soil '96'
40
o' 0. -^ l------------


S80- B


60-
0
UU
Z 40- /
/ -0- CU soil '94'
/ -- NT soil '95'
20 / NU soil'96'



0 5 10 15 20 25 30

Time (day)


Figure 4-4. Mineralization of ['4C-URL] carbofuran in subsurface (45-60 cm) Ellzey soil:
A) treated soil; B) control soils. Error bars represent the standard deviations of analysis.









71

mineralizing on average 22% more of the ['4C-URL] carbofuran. For the CT-96, the total

amount of ['4C-URL] carbofuran mineralized throughout the 28 days of incubation was

significantly larger (a < 0.05) in the subsurface soil than in the surface soil, but not

significant in 1994 or 1995.

The CN soil samples exhibited a seven day lag period prior to the onset of

mineralization of ['4C-URL] carbofuran. After the lag period, the mineralization was

linear from days 7 to 21, with total cumulative '4CO2 production being 58% of the applied

'4C at the end of 28 days of incubation. In the NT soil, a lag period of 3 days was

observed prior to the onset of rapid mineralization of ['4C-URL] carbofuran with 64% of

the total applied '4C being mineralized in 28 days. There was little mineralization of ['4C-

URL] carbofuran in the NN soil, with about 1.5 % of the applied 14C being mineralized

after 28 days of incubation (Figure 4-4B).

Once rapid mineralization occurred in the CN and NT soils, there was not a

significant differences in the total cumulative '4CO2 production in the CT soils treated

with ['4C-URL] carbofuran than in the CN and NT soils. During the 28 days of

incubation, there was a significantly (a < 0.05) faster mineralization of [14C-URL]

carbofuran in the CT soils than in the NN soil. The CN and NT soils also exhibited a

greater mineralization of ['4C-URL] carbofuran than the NN soil after the onset of rapid

mineralization.

'4C Recovery in soil

The recovery of the '4C-applied in the Ellzey soils treated with ['4C-URL]

carbofuran ranged from 67.2 to 98.3 % at the end of 28 days of incubation (Table 4-2).









72

Table 4-2. Distribution of "4C activity in Ellzey soil treated with ['4C-URL] carbofuran
(10 mg kg-') at the end of 28 days of incubation.

Depth Evolved Extractable Nonextractable Recovery
14CO2 14C 14C

cm % of applied '4C

CT soil '96'

0-15 46.7( 1.0)a 10.9(0.6) 36.4(2.7) 92.3(4.2)

45-60 77.2 ( 1.7) 4.2(0.3) 16.9(0.5) 95.4(3.0)

CT soil '95'

0-15 56.3( 4.2) 5.2(0.4) 27.3(2.6) 88.8(3.2)

45-60 57.6(10.5) 6.9(0.8) 32.2(7.8) 98.5(2.0)

CT soil '96'

0-15 46.1(4.1) 4.0(0.6) 31.1(4.9) 85.7(2.2)

45-60 74.0(0.9) 3.8(0.8) 20.3(0.2) 98.1(1.3)

CN soil '94'

0-15 56.2(1.2) 2.8(2.1) 32.5(8.3) 89.3(13.3)

45-60 59.2 (3.2) 9.8(4.6) 28.4(3.7) 92.6( 7.1)

NT soil '95'

0-15 67.6(1.6) 5.2(1.4) 19.9(4.5) 92.8(5.8)

45-60 64.0(5.7) 5.7(1.0) 28.6(1.1) 98.3(2.1)

NN soil '96'

0-15 8.6(1.6) 56.9(4.3) 1.6(0.7) 67.2(2.9)

45-60 1.7(0.2) 86.2(3.4) 10.1(1.0) 98.0(2.2)
a = values within parentheses are the standard deviations of the measurements









73

Excluding the NN surface soil, "C recoveries ranged from 85.7 to 98.3%. The CT soils

collected in 1994, 1995 and 1996 were the only Ellzey soils to exhibit a different pattern

of '4C distribution between its surface and subsurface soils. The CT subsurface soils

mineralized between 1 to 31% (on average 20 %) more of the applied '4C activity than the

surface soils, while the CT surface soils averaged about 10% more nonextractable '4C

bound residues. The CT subsurface soils exhibited greater percentage of carbofuran

mineralized than all other Ellzey soils.

The CT, CN, and NT soils were capable of mineralizing the aromatic ring of

carbofuran and exhibited similar trends of "C-activity distribution. The trends being that

these soils had low recovery of extractable '4C-activity (2.8-10.9%) with nonextractable

'4C-activity ranging from 16.9-36.4%. The NN soils were the only soils that did not

mineralize the aromatic ring structure extensively. These soils had larger quantities of

extractable '4C-activity (56.9-86.2), but smaller quantities of nonextractable '4C (1.6-

10.1%).

Carbofuran metabolism in the Ellzey soils

In this study, ["4C-URL] carbofuran was employed to monitor the disappearance

of carbofuran and its toxic metabolites 3-hydroxylcarbofuran and 3-ketocarbofuran in the

Ellzey soils, as well as less toxic phenolic products. The total toxic residues (TTR)

include carbofuran, 3-hydroxylcarbofuran and 3-ketocarbofuran. Determination of

metabolites led to the estimation of the half-lives for TTR in the Ellzey soils and possible

elucidation of the degradation pathway of carbofuran. The TLC Rf values for carbofuran

and its toxic metabolites 3-hydroxylcarbofuran and 3-ketocarbofuran were 0.42, 0.19 and









74

0.37, respectively. The Rf values for the phenolic metabolites carbofuran phenol, 3-

hydroxyl-carbofuran phenol, and 3-ketocarbofuran phenol were 0.73, 0.53, and 0.59,

respectively.

Degradation pathway of carbofuran in Ellzey soils

The distribution of extractable '4C-carbofuran and its '4C metabolites varied for

Ellzey soil samples collected in 1994 and 1996. In the CT soil samples collected in 1994

and 1996, carbofuran phenol was the only metabolite detected in both surface and

subsurface soils (Tables 4-3 and 4-4). There was also an unknown polar metabolite(s) (Rf

= 0.0) detected in all soil samples except in the CN subsurface soil. Carbofuran phenol,

3-ketocarbofuran, and 3-ketocarbofuran phenol were occasionally detected in CN surface

and subsurface soil samples. 3-Hydroxylcarbofuran and 3-ketocarbofuran were detected

after 3 days of incubation in the NN surface soil samples, while carbofuran phenol and 3-

ketocarbofuran phenol were detected only once, and that was after a week of incubation.

Carbofuran phenol and 3-ketocarbofuran were occasionally detected in the NN subsurface

sample (Table 4-4). The proposed degradation pathway for carbofuran in the CT, CN,

and NN of the Ellzey soils are depicted on Figure 4-5.

Disappearance of carbofuran in Ellzey soils.

The disappearance of the carbofuran in the CT soils followed similar pattern for

the mineralization of [4C-CAL] carbofuran in the CT soils. Initial rapid disappearance of

carbofuran was observed in the CT surface soils collected in 1994 and 1996 with less

than 1% of the applied '4C remaining after 7 and 3 days, respectively (Tables 4-3 and 4-

4). The subsurface CT soils sampled in 1994 exhibited a lag period of 3 days prior to









75

Table 4-3. Carbofuran and carbofuran metabolites detected from solvent extracts in
cultivated treated soils and cultivated nontreated soils in 1994. Soils were treated with
['4C-URL] carbofuran.


Days Carbofuran 3-Keto TTRa Carbofuran 3-Keto Unknown
carbofuran phenol carbofuran Rf = 0.0
phenol

% of applied '4C

CT soil (0-15 cm)

1 65.8( 0.1)b 0.0 65.8( 0.1) 0.9(1.2) 0.0 2.5(1.7)

3 19.7( 3.6) 0.0 19.7(3.6) 2.3(2.3) 0.0 9.3(8.5)

7 0.8( 0.2) 0.0 0.8(0.2) 0.0 0.0 6.8(2.4)

CT soil (45-60)

1 83.8( 3.8) 0.0 83.8( 3.8) 0.0 0.0 0.0

3 78.4( 2.2) 0.0 78.4(2.2) 2.0(0.5) 0.0 0.8(0.54)

7 0.6( 0.1) 0.0 0.6(0.1) 0.0 0.0 7.0(1.3)

CN soil (0-15 cm)

3 37.5(14.0) 0.0 37.5(14.0) 0.0 0.0 0.0

7 3.8( 5.1) 3.1(4.7) 6.9(0.4) 0.0 0.0 0.7(1.0)

CN soil (45-60 cm)

3 72.7( 5.0) 0.0 72.7( 5.0) 0.0 0.0 0.0

7 71.7(22.6) 15.6(9.1) 87.2(31.7) 0.0 0.0 0.0

14 33.5(17.0) 0.0 33.5(17.0) 2.5(0.8) 3.4(0.3) 0.0

21 10.1( 5.2) 0.0 10.1(5.2) 0.0 7.2(0.6) 0.0
aTTR = total toxic residues
b = values within parentheses are the standard deviations of the measurements









Table 4-4. Carbofuran and carbofuran metabolites detected from solvent extracts in cultivated treated and noncultivated untreated soil
samples in 1996.
Days Carbofuran 3-Hydroxyl 3-Keto Carbofuran 3-Ketocarbofuran Unknown
carbofuran carbofuran phenol phenol Rf = 0.0

% applied "4C

Cultivated Treated (0-15 cm)

3 0.9(0.2)- 0.0 0.0 0.1(0.1) 0.0 5.0(1.0)

7 0.8(0.4) 0.0 0.0 0.0 0.0 3.2(0.2)

Cultivated Treated (45-60 cm)

3 8.8(0.0) 0.0 0.0 3.0(0.2) 0.0 11.9(0.1)

7 0.8(0.4) 0.0 0.0 0.0 0.0 0.0

Noncultivated Untreated (0-15 cm)

3 79.6(0.4) 0.0 3.0(0.5) 0.0 0.0 0.8(0.1)

7 64.9(7.1) 3.6(1.6) 4.8(1.6) 0.0 0.0 1.4(0.0)

21 72.6(2.9) 0.6(0.6) 2.8(2.8) 1.1(0.5) 3.4(2.2) 0.9(0.1)

Noncultivated Untreated (45-60 cm)

3 97.5(4.9) 0.0 0.0 0.0 0.0 1.2(0.1)

7 94.6(1.2) 0.0 0.0 0.0 0.0 0.0

21 88.0(0.0) 0.0 0.0 3.7(2.0) 0.5(0.0) 0.2(0.2)
a values within parentheses are the standard deviations of the measurements









0 0
CH 'N CH3 N )L-


H3 H3
OH 0e
3-Hydroxylcarbofuran 3-Ketocarbofuran





OH OH













CO2 + H20 + Bound Residues + Biomass
Figure 4-5. Degradation pathways of carbofuran in soil. Highlighted area represents enhanced soils









78

rapid disappearance of carbofuran with less than 1% of the applied '4C remaining after 7

days. The CT subsurface soil collected in 1996 degraded carbofuran significantly more

rapidly after 3 days than the CT soil collected in 1994. In both of the years the CT soils

were sampled, the surface soils degraded carbofuran significantly faster than the

subsurface soils.

The disappearance of carbofuran in the control CN soil (Tables 4-3) did not

follow the same pattern as those of the mineralization of ['4C-CAL] carbofuran (Figure 4-

1). The lag period prior to rapid disappearance of carbofuran in the surface soil appeared

to be shorter than for the mineralization of ['"C-CAL] carbofuran, less than 3 days. The

lag period prior to rapid disappearance of carbofuran in the CN subsurface soil was

shorter than for the mineralization of [14C-CAL] carbofuran as well. In the NN soil, the

pattern was similar for the disappearance of carbofuran and mineralization of ["C-CAL]

carbofuran for both the surface and subsurface soils with little degradation of carbofuran

occurring. The CT surface and subsurface soils collected in 1996 exhibited a

significantly more rapid loss of carbofuran than any of the control soils. The CN surface

soil degraded carbofuran more rapidly than the NN surface soil.

Half-life values of carbofuran based on disappearance of carbofuran in Ellzey soils.

The half-life values for carbofuran in Ellzey soils were estimated based on the

disappearance of carbofuran using first-order kinetics. The disappearance rate constants

(k,) and half-lives (t,,2) are listed on Table 4-5. Most of the rates approximate first-order

kinetics as indicated by correlation coefficients (r2). The half-life value for carbofuran in









79

Table 4-5. The estimated half-life values for carbofuran disappearance, total toxic
residues (TTR) disappearance and the hydrolysis rates carbofuran in Ellzey soils.

TTR and carbofuran Hydrolysis of
disappearance carbofuran

Depth k, t1/2 r2 k, t,, r2

cm days-' days days-' days
xl02 xl02

CT soil '94'

0-15 71.1 1.0 0.990 82.1 1 0.919

45-60 74.7 1.0 0.863 25.0 3 0.885

CT soil '96'

0-15 80.0 1.0 0.829 57.9 1 0.884

45-60 83.1 1.0 0.999 25.6 3 0.965

CT soil 'fum'

0-15 NDa ND ND 0.4 170 0.989

45-60 ND ND ND 4.1 17 0.931

CN soil '94'

0-15 45.7(47.2)b 2(2) 0.971(0.979) 13.1 5 0.946

45-60 7.8(10.5) 9(7) 0.842(0.930) 1.0 68 0.891

NN soil '96'

0-15 1.0( 1.0) 70(62) 0.637(0.445) 1.0 71 0.995

45-60 0.6(0.6) 117(117) 0.987(0.988) 0.4 176 0.983
aND = not determined
b value within the parentheses represent the half-life values for carbofuran disappearance
if they are different from the TTR disappearance









80

the NN surface soil collected in 1996 was not followed well by first order rate kinetics, as

evidenced by the poor r2 (0.445). Due to the rapid degradation of carbofuran in the CT

surface soil collected in 1996, it was not possible to obtain good data for the half-life

estimation. The half-lives of carbofuran in the CT surface soil samples ranged one to less

than one day and from 1 to 3 days for the subsurface soils (Table 4-5).

The CN soils that had not been previously exposed to carbofuran exhibited half-

life values that are not characteristic of soils with no history of exposure to carbofuran,

but rather similar to a soil with a prior exposure history. The calculated half-life values

for carbofuran were 2 and 7 days in the surface and subsurface CN soil, respectively. The

NN surface and subsurface soils exhibited little mineralization of ['4C-CAL] carbofuran

(Figures 4-1B and 4-2B), and this was evident in the half-life values for carbofuran being

62 and 117 days for surface and subsurface soils, respectively.

Half-life values of TTR disappearance and carbofuran hydrolysis in Ellzey soils.

The half-lives of TTR disappearance were estimated based on the disappearance

of TTR in the Ellzey soils, and the half-lives of the hydrolysis of ['4C-CAL] carbofuran in

the soils were estimated based on the evolution of '4CO2. The hydrolysis of carbofuran

and the mineralization of ['4C-CAL] carbofuran should coincide since both are measures

the hydrolysis of the carbamate group from carbofuran. The half-life values were

estimated based on the first-order kinetics for biodegradation (Table 4-5). Most of the

rates approximate first-order kinetics as indicated by their r2 values. In the CT soils, the

disappearance of carbofuran and TTR were similar since none of the two toxic

metabolites 3-hydroxylcarbofuran and 3-ketocarbofuran were detected. The half-lives of









81

TTR disappearance in the CT soils was one day for surface soils, and 1 to 3 days for

subsurface soils. However, for the fumigated CT soils collected in 1994, the half-life

values were much larger than for the CT soils collected in 1994 and 1996. The calculated

half-lives for the fumigated surface and subsurface soils were 170 and 17 days,

respectively.

The half-life values for the hydrolysis of carbofuran in the CN soil ranged from 2

to 5 days in surface soil and from 9 to 68 days in subsurface samples. The large

variations of the half-life values in the CN samples reflects the differences in the

observed lag periods between the TTR disappearance and mineralization of ['4C-CAL]

carbofuran in the CN soil samples. The lag periods for TTR disappearance were shorter

than the lag periods for the mineralization of ['4C-CAL] carbofuran. The NN soil

samples exhibited little degradation of carbofuran and this was evident in their calculated

half-lives. The half-lives of carbofuran in surface NN soils samples ranged from 70 to

71 days and from 117 to 175 days in the subsurface soils.



Discussion



Metabolism of ['4C-CAL and '4C-URL] Carbofuran in Ellzey Soil.

Mineralization of [14C-CAL] vs. ['4C-URL1 carbofuran.

As mentioned above, the mineralization of [14C-CAL] carbofuran was more rapid

than that of [14C-URL] carbofuran in all samples of the Ellzey soil. This reflects that the

linkage of the carbamate group is readily subject to biological hydrolysis resulting in the









82

release of 4CO2. In addition, the aromatic ring structure is more resistant to microbial

attack than is the carbamate group. The carbonyl carbon from the carbamate group of

carbofuran is already at its highest oxidation state and thus can not be assimilated by

aerobic heterotrophic organisms. At present, the mechanisms have not been identified

for the enzymatic cleavage of the ring structure of carbofuran and the resulting

product(s), as well as subsequent degradation pathways that lead to the formation of the

final oxidation products, CO2 and H20.

Lag period for carbofuran degradation.

The CT soils that had been continuously treated with carbofuran exhibited more

rapid mineralization of ['4C-CAL] carbofuran and loss of TTR than the soils CN and NN

that did not receive carbofuran. The differences in the mineralization rates of the freshly

applied ['4C-CAL] carbofuran were evident in the duration of the initial lag periods in the

CT soils and those of the nontreated soils (Figure 4.1). Suett (1986) reported that the

most notable losses of carbofuran from degradation were in the initial stages of the

incubation for soils exhibiting enhanced degradation. Other researchers observed that the

lag periods were greatly reduced in soils exhibiting enhanced degradation of carbofuran

(Harris et al., 1984; Camper et al., 1987; Turco and Konopka, 1990). This lag period was

correlated with the duration between the last application of carbofuran, with the most

recent application exhibiting shorter lag periods (Suett et al., 1993). Reduction in the lag

periods can be achieved by pre-treating soils with carbofuran (Read, 1983; Chapman et

al., 1986; Dzantor and Felsot, 1989), and other aryl N-methylcarbamates (Dzantor and

Felsot, 1989; Dzantor and Felsot, 1990; Morel-Chevillet, 1996).









83

In this study, the initial lag periods prior to the onset of rapid carbofuran

degradation were similar in the CT surface soils taken in 1994 and 1996, yet the

degradation rates were significantly different. A lag period of 3 days was observed for

the CN soils prior to the onset of the rapid degradation of carbofuran. The CT soil

collected in 1996 exhibited a significantly greater capacity for degrading carbofuran than

soil collected in 1994. This greater capacity suggests that a larger population of

carbofuran-hydrolyzing microorganisms were present in the CT soil collected in 1996

than in 1994. This degrader population probably developed from the two additional

annual field applications of carbofuran received by the CT surface soil. Hendry and

Richardson (1988) observed similar trends in soils repeatedly treated with carbofuran in a

laboratory study. They reported that on a soil with no previous history of carbofuran

treatment, only 7% of the applied ['4C-CAL] carbofuran was mineralized in 3 days, but in

subsequent treatments, the mineralization rate progressively increased. After the third

treatment, more than 90 % of the applied ['4C-CAL] carbofuran was mineralized in 19

hours. The increase in the mineralization of ['4C-CAL] carbofuran was linked to an

increase in the number of carbofuran-hydrolyzing microorganisms after each additional

treatments (Hendry and Richardson, 1988).

The NN soil samples never exhibited any lag period, and carbofuran was

mineralized at a slow but constant rate (Figure 4.1B). The constant rate of carbofuran

degradation in the NN soils may suggest that: 1) the hydrolysis of the carbamate group

from carbofuran was not linked to any growth in the microbial population (Alexander and









84

Scow, 1989); or 2) the initial microbial population capable of hydrolyzing carbofuran was

too small to be capable of significantly degrading the pesticide (Alexander, 1994).



Half-life Values for Carbofuran Disappearance and Hydrolysis.

Surface soil (0-15 cm).

Half-life values for carbofuran disappearance or hydrolysis were smaller than the

published values (Felsot et al., 1981; Suett, 1986; Turco and Konopka, 1990; Parkin and

Shelton, 1992). Published half-life values ranged form 3 to 10 days and only accounted

for the disappearance of carbofuran, and did not include the two toxic oxidation products

of carbofuran. The rapid degradation in the CT soils may reflect the number of annual

field applications to the CT soils (3 to 5 times); other enhanced soils in the literature

generally ranged from 1 to 3 applications of carbofuran.

It is interesting to note that the half-life values (2 to 5 days) of carbofuran and

TTR disappearance in the CN soil were smaller than those in the NN soils. The CN soils

were not treated with carbofuran, yet rapid degradation of carbofuran occurred. This

suggests that the CN soils might have been previously exposed to carbofuran or

structurally similar compounds. This assumption is based on the observation that the

half-lives for carbofuran in soils not previously exposed to the pesticide were much

larger, ranging from 18 to 90 days (Wauchope et al., 1992). Furthermore, half-life

values for carbofuran degradation in the NN surface soil were much larger (62 days).

Similar rates of carbofuran degradation have been reported to occur in the fence rows

near treated field plots (Felsot et al., 1981; Racke and Coats, 1990) and adjoining fields









85

(Suett, 1986). The contamination by carbofuran residues in the CN soils may have

resulted from inaccurate record keeping of pesticide application, pesticide drift during

application, or contamination via farm machinery that was used in both fields. For the

pesticides MBC (benzimidazol-2-yl carbamate) and EPTC (s-ethyl N,N-dipropyl

carbamothioate), it has also been demonstrated in the laboratory that a small amount of

soil (2% of total) from an enhanced field when inoculated into a soil not previously

exposed to the pesticide can trigger a rapid increase in the degradation of the pesticide

(Yarden et al., 1987; Dick et al., 1990). Thus, another possibility is that small amounts of

inoculum from the CT soil could have been inadvertently transferred into the CN soils via

farm equipment.

Subsurface soils (45-60 cm)

The CT surface soils degraded carbofuran more rapidly than the corresponding

subsurface soils, yet the half-life values for the degradation of carbofuran in the CT

subsurface soils (1 to 3 days) were comparable to those of other surface soils with

problems of enhanced degradation (Felsot et al., 1981; Parkin and Shelton, 1992). This is

the first report of enhanced degradation extending down into a shallow subsurface

horizon. Previous reports on pesticide degradation in subsurface soils found no link

between pesticide degradation and growth of the microbial biomass (Muller et al., 1992).

Thus, degradation of pesticides in subsurface soils was considered to be a cometabolic

processes (Moorman, 1990). Recent studies on the degradation of carbofuran in

subsurface soils have dealt exclusively with soils having no previous history of exposure









86

to the pesticide (Buyanovsky et al., 1993; Mallawatantri et al., 1996). These studies

found that the degradation of carbofuran in subsurface soils decreased with depth.



Source of Carbofuran Degraders in Subsurface Soils.

The question arises as to the source of the microorganisms in the subsurface soil

that are capable of degrading a xenobiotic chemical such as carbofuran. Do the

microorganisms capable of degrading carbofuran develop in subsoils as a result of

transport from the surface soil? Or, do these microbes develop independently in the

subsurface soil through natural adaptation? In an aquifer, Aelion et al. (1987) linked the

adaptation of p-nitrophenol (PNP) degrading microorganisms with an increase in the

number of PNP degraders in the sediment. This adaptation was linked to the

concentration of PNP, with the concentrations < 14.0 gg kg-' soil being unable to sustain

any mineralization of PNP, although adaptation developed at higher concentrations >

31.0 lg kg-' soil. Similar findings for carbofuran degradation in soils were observed, and

at concentrations below 100.0 jlg kg-' soil enhanced degradation was not induced

(Hendry and Richardson, 1988; Chapman et al., 1988).

If a threshold concentration of carbofuran is needed for the development of

enhanced degradation in soils, what is the concentration of carbofuran leaching into the

shallow subsurface soil (45-60 cm) that triggers enhanced degradation in the soil? The

computer model "Chemical Movement in Layered Soils" (CMLS) version 4.0 (Nofziger

and Homsby, 1987) was used to simulated the mass fraction of carbofuran leaching into

the subsurface soil (45-60 cm). The organic carbon contents from the CT soils coupled









87

with the estimated half-lives from the CT soils samples collected in 1994 along with

water content at field capacity for the Ellzey fine sand were entered into the program. In

addition, a carbofuran application rate of 4500 iag kg-' of soil was assumed based on the

field application rates of carbofuran. Rainfall and evapotranspiration data were obtain

from local Gainesville data collected in 1985. The computer model simulated a

carbofuran concentration of 22 jLg kg-' soil would reach the 45-60 cm depth in 8 days.

From the CMLS model simulation, there is a very low probability of carbofuran

concentration in the subsurface soil exceeding the threshold values needed for the

development of enhanced degradation to occur. It appears that subsurface

microorganisms capable of degrading carbofuran may have originated from the surface

soils. Since local Gainesville rainfall and evapotranspiration data was used in the

simulation, confidence in the estimated carbofuran concentration values should be taken

with some skepticism.

If carbofuran-degrading microorganisms in surface soil were transported through

the soil profile, by what mechanisms would this movement be accomplished? The

transport of microorganisms in soil has been shown to be greatly limited by the structure

of the soil (Smith et al., 1985), with most of the transported occurring through

macropores (Smith et al., 1985; Fontes et al., 1991). Transport through an agricultural

field has been shown to be minimal since cultivation causes the soil to be structureless

and devoid of large pores, thus retarding the movement of bacteria (Smith et al., 1985).

In sandy soils, Wollum and Cassel (1978) and Fontes et al. (1991) measured only a small

amount (< 14%) of bacteria recovered from in the effluents for a fine sand (Ellzey soil is









88

a fine sand). While in course sand columns recoveries from the effluents was greater

than 90% (Fontes et al., 1991; Gannon et al., 1991). In other packed columns, PNP

degrading bacteria were found to be mostly retained in the top 2.5 cm, while below the

top 7.0 cm, little of the PNP degrading bacteria were detected (Kelsey and Alexander,

1995). Degradation ofphenanthrene was also found to be limited to the top portion of

packed columns with little degradation of phenanthrene occurring in lower portions of the

columns (Devare and Alexander, 1995).

Another possible mechanism for the transport of carbofuran degrading organisms

from surface to subsurface soils would be through the cultivation practices used in

growing potatoes. It has been shown that soils under conventional tillage exhibit little

spatial variability in enhanced degradation of carbofuran (Parkin and Shelton, 1992). The

lack of spatial variability is a result of homogenization of soil during tillage. In the

potato fields at the Hastings site, mounds were cultivated down to a 50 cm depth (D.P.

Winegarten, personal communication) and the subsurface soils were collected from 45 to

60 cm depth. Thus, it would appear that the transport of surface carbofuran-degrading

microorganisms to subsurface soil would probably be achieved through conventional

tillage practices and movement with percolation waters.



Distribution of ['4C-URL] Carbofuran

It was noted earlier that the CT soil was the only sample of the Ellzey soils that

exhibited a different pattern of "'C distribution between its surface and subsurface soil.

These soils exhibited a greater percentage of ['4C-URL] carbofuran mineralized in the









89

subsurface soil than in surface soil, but the CT surface soil had a greater percentage of

soil bound residues than the CT subsurface soil. Soil bound residues of carbofuran are a

result of the carbofuran phenolic metabolites undergoing oxidative coupling to organic

matter and thus becoming covalently bound to the organic matter (Getzin, 1973; Willems

et al., 1996). Willems et al. (1996) found that carbofuran phenol was susceptible to

oxidative coupling in the presence of horseradish peroxidase and hydrogen peroxide.

Other phenolic compounds have also been shown to bind readily to organic matter in soil

via abiotic and biotic means (Bollag et al., 1983; Pal et al., 1994). In laboratory

incubation studies, ['4C-URL] carbofuran degradation in surface soils has been shown to

result in the formation of large amounts (59.3-94.5 %) of soil bound residue in both

enhanced (Talebi and Walker, 1993) and nonenhanced (Getzin, 1972; Ou et al., 1982)

soils, while in flooded soils, the degradation of ['4C-URL] carbofuran results in a small

amount of soil bound residue, and a large accumulation of carbofuran phenol

(Venkateswarlu and Sethunathan, 1979).

For the Ellzey soil series, soil water contents at field capacity was 156 g kg-' for

0-15 cm and 54 g kg-' for 45-60 cm soil depth. Soil water contents of CT soil samples

were determined to be 99 g kg-' at 0-15 cm and 155 g kg-' at 45-60 cm (Table 4.1). Thus,

CT soil samples collected from the 45-60 cm depth would have many of its pores filled

with water and soil samples at this depth would be more anaerobic. Reduced conditions

in the subsurface CT soils may leave carbofuran phenol less susceptible to oxidative

coupling by nonspecific abiotic and biotic reactions (Pal et al., 1994), but still susceptible

to microbial attack and mineralization.









90

The reasons are not clear as to why the CN and NT soil samples did not follow

similar '4C distribution patterns as the CT soil samples. One reason for the difference in

'4C activity distributions may be the greater organic content in the CN soil sampled.

Greater organic contents would be expected to reduce the bioavailability of carbofuran

and its metabolites to microbial attack (Ogram et al., 1985), but still leave it susceptible

to abiotic or biotic (extracelluar enzymes) attack. The result would be greater oxidative

coupling to organic matter, forming bound residues. Another possible reason may be the

reduced activity of the soil microorganisms toward the mineralization of ['4C-URL]

carbofuran in the CN and NT soil samples. Lower levels of microbial activity would

result in slower mineralization of carbofuran phenol, leaving more chance for oxidative

coupling with the organic matter to occur.

In the NT soil samples, the lack of added fertilizers to the soil may explain

reduced rates of bound residues. The NT soil samples may be nutrient limited since these

soils were not in cultivation, and the growth of fungi and other organisms that produce

peroxidases could be reduced. The reduced activity of these organisms would reduce the

oxidative coupling of the phenolic groups of carbofuran with the organic matter of soil.

This, in turn, would reduce the amount of soil bound residues of carbofuran.



Metabolites of ['4C-URL] Carbofuran

The degradation of carbofuran exhibits different patterns of degradation for each

of the Ellzey soils with various carbofuran exposure histories (Tables 4.4 and 4.5).

Degradation of carbofuran, for both ['4C-CAL] and ['4C-URL] carbofuran, in the surface









91

and subsurface soils were clearly enhanced. Because carbofuran phenol was only the

metabolite in the soils in conjunction with rapid '4CO2 production from the soils treated

with ['4C-CAL] carbofuran, hydrolysis was the only step of the initial degradation of

carbofuran in the enhanced CT soils. Oxidation of carbofuran to 3-hydroxylcarbofuran

did not appear to occur in the CT soils. As a result, carbofuran phenol was either

mineralized after hydrolysis, incorporated into the microbial biomass, or converted to

bound residues. Turco and Konopka (1990) and Talebi and Walker (1993) reported that

carbofuran in enhanced soils was mainly degraded through biological hydrolysis to

carbofuran phenol. Getzin (1973) found that the application of ["4C-URL] carbofuran

phenol resulted in a rapid incorporation of "C to soil organic matter (bound residue

formation).

Degradation of carbofuran in enhanced soils can be inhibited up to 70-80% in the

presence of the organophosphates paraoxon (Talebi and Walker, 1994) and enthoprop

(Racke and Coats, 1990), respectively. The monoxygenases that have cytochrome P450 as

their active center are inhibited by piperonyl butoxide. Application of piperonyl butoxide

to an enhanced soil in the laboratory reduced the degradation of carbofuran by 10%

(Talebi and Walker, 1994). These studies suggested that carbofuran in enhanced soils

was metabolized mainly through hydrolysis of the carbamate linkage resulting in the

formation of carbofuran phenol, methylamine and CO2. Oxidation of carbofuran to 3-

hydroxyl-carbofuran may not occur in the CT soil, or only as a minor pathway.









92

Practical Implication

Prior application of the soil fumigant telone II to the CT soil inhibited the

degradation of carbofuran in the soil and blocked enhanced degradation (Figure 4-1A).

Suett (1986) reported a similar effect of the fumigant diazomet despite repeated

applications of carbofuran (Suett, 1986). Another pesticide susceptible to enhanced

degradation is the organophosphate fenamphos (Ou et al., 1994; Ou and Thomas, 1994),

but when used in conjunction with telone II enhanced degradation of fenamphos did not

develop (L.-T. Ou, personal communication). The benefits of using soil fumigants, such

as diazomet and Telone II, with carbofuran in enhanced soils are: 1) increased efficacy of

carbofuran in the root zone; and 2) reduced risk of carbofuran contaminating the

groundwater due to rapid degradation in subsurface soil.















CHAPTER 5
INFLUENCE OF MICROBIAL POPULATIONS ON ENHANCED DEGRADATION
OF CARBOFURAN IN SOIL




Introduction



The degradation of carbofuran in soils with no previous history of exposure to the

pesticide has been shown to be both abiotic and biotic in origin (Getzin, 1973). Repeated

exposure of carbofuran to field soil may result in enhanced degradation of the pesticide

(Felsot et al., 1981; Read, 1983). Although this enhancement is of biological origin,

attempts to correlate the degree of enhanced degradation in soil with key soil physical,

chemical and biological properties have been unsuccessful (Dzantor and Felsot, 1990;

Charnay and Fournier, 1994). Turco and Konopka (1990) reported a decrease in total

biomass after applying carbofuran to a laboratory soil that exhibited enhanced

degradation of the pesticide. The inability to link the increase in the total microbial

biomass to carbofuran degradation is possibly due to a small or specialized portion of the

biomass that is responsible for degradation of the pesticide in enhanced soils.

This raises the following question regarding enhanced biodegradation: 1) does

enhanced degradation of carbofuran result from an increase in specific microbial biomass

capable of degrading the pesticide; or 2) does enhanced degradation result from an

93




Full Text

PAGE 1

ENHANCED BIODEGRADATION OF CARBOFURAN IN SOIL WITH A HISTORY OF REPEATED APPLICATIONS OF CARBOFURAN AND CHARACTERIZATION OF BACTERIAL DEGRADERS ISOLATED FROM THE SOIL By STEVEN LEE TRABUE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1997

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ACKNOWLEDGMENTS I thank Dr. Li Tse Ou for allowing me the opportunity to pursue a Ph.D. degree under his guidance and for all his support he gave me throughout my studies. I also thank the members of my supervisory committee, Drs. George O'Connor, Andrew Ogram, Suresh Rao, David Sylvia, and Lonnie Ingram, for their inputs. I would especially like to thank Drs. Ogram and Sylvia for answering many of my questions. Appreciation is extended to the USDA Special Water Quality Research Program for partially funding this research, and to Dr. Randal Brown for giving me a generous assistantship when my source of funding ended. I would like to thank those who assisted me in my endeavors. The opportunities to use the laboratory facilities of Drs. Mary Collins, Art Hornsby, Andrew Ogram, David Sylvia, William Stall, and Henry Aldrich are greatly acknowledged. Dr. Sylvia Coleman, Dave Cantlin, Wei Jing, Bill Reeve, Cheryl Hodge, and Larry Schwandes for their help in the lab were appreciated. I would especially like to thank John Thomas who provided me help in time of need and was always willing to share a joke or two when I needed it most; he will be missed. I think Joyce Taylor and Celia Earl for their encouragements. I thank my fellow graduate students who have left or still remain: thanks for keeping a smile on my face. Specifically I wish to thank Dr. Chris Pedersen, Dave Farmer, Jose Escamilla, and Patrick Mulroy for all the late night conversations. To Dongping Dai, Hector Castor, ii

PAGE 3

Randy Sillian, Chris Bliss, Gerco Hoogeweg, and Elisa D'Angelo, I enjoyed the many laughs and conversation we shared. I thank Keun-Yook Chung for sitting with me in many of our classes, and I wish you well. I would like to express my gratitude to those outside the University of Florida who helped me through. I wish to thank Ken and Kathy French and Gil and Marian Prost for being the examples of how couples should live their lives. I am ever grateful to my parents and family and thank them for their love and encouragement, for without them I know I would not have finished my studies. I missed them tremendously and cherished the time we spent together. I would like to thank Dr. Ou for working with me these past four years. He helped me find my way when I was floundering and encouraged me when I was down, not always with words but with his life. I am truly a better scientist and person for knowing him. iii

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TABLE OF CONTENTS ACKNOWLEDGMENTS ABSTRACT CHAPTERS 1 INTRODUCTION 2 LITERATURE REVIEW History of Carbamate Pesticides Toxicology of Carbofuran Characteristic Properties of Carbofuran Degradation of Carbofuran in Nonenhanced Soils Enhanced Degradation of Carbofuran 1 Carbofuran Degradation by Soil Microorganisms Involved in Enhanced Degradation 3 EXPERIMENTAL PROCEDURE Field Work Characterization of Carbofuran-Degrading Soil Isolates 4 DEGRADATION IN SURFACE AND SUBSURFACE SOILS Introduction Results Discussion iv

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5 INFLUENCE OF MICROBIAL POPULATIONS ON ENHANCED DEGRADATION OF CARBOFURAN IN SOIL 93 Introduction 93 Results 94 Discussion 106 6 CHARACTERIZATION OF CARBOFURAN-DEGRADING BACTERIA ..115 Introduction 115 Results and Discussion 116 7 CONCLUSIONS 138 GLOSSARY 143 LIST OF REFERENCES 144 BIOGRAPHICAL SKETCH 158 v

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ENHANCED BIODEGRADATION OF CARBOFURAN IN SOIL WITH A HISTORY OF REPEATED APPLICATIONS OF CARBOFURAN AND CHARACTERIZATION OF BACTERIAL DEGRADERS ISOLATED FROM THE SOIL By Steven Lee Trabue December 1997 Chairperson: Dr. Li Tse Ou Major Department: Soil and Water Science Carbofuran is a broad-spectrum N-methylcarbamate insecticide used to control certain soil borne insects and nematodes. Enhanced biodegradation of carbofuran has been attributed to the loss of pesticidal efficacy in soil with a history of carbofuran use. Microorganisms are responsible for enhanced degradation of carbofuran in soil. There is little information linking enhanced degradation with carbofuran-degrading microbial populations in soil profiles. One of the objectives of this research was to measure the degradation rate of carbofuran in soil related to carbofuran use in soil. Soil samples were collected from a site in Florida that had either a previous history or no history of exposure to carbofuran. Mineralization rates of carbofuran in soils collected from different soil depths were measured using [ 14 C-CAL (carbonyl-labeled)] or [ 14 C-URL (uniformly ringvi

PAGE 7

labeled] carbofuran. Metabolites in soils were measured using [ 14 -URL] carbofuran. Carbofuran degraded more rapidly in soils previously treated with the pesticide. Surface soils degraded carbofuran more rapidly than subsurface soils, and cultivated soils degraded carbofuran more rapidly than noncultivated soils. Hydrolysis of carbofuran was the major route for enhanced biodegradation of carbofuran in soils which had received prior applications. The second objective was to measure changes in the carbofuran-degrading microbial populations in soils as a function of carbofuran treatment history and soil depth. Numbers of microorganisms capable of mineralizing [ 14 C-CAL] and [ 14 C-URL] carbofuran, and [ 14 C-URL] carbofuran phenol, as well as methylamine degraders were determined using MPN techniques. Repeated field applications of carbofuran increased the number of carbofuran-hydrolyzing microorganisms, but other types of degraders remained unchanged. The final objective was to isolate and characterize carbofuran-ring-degrading bacteria. Three bacteria were isolated from soil and identified as Sphingomonas sp. according to their fatty acid methyl ester profiles. Growth of these isolates was determined using the Petroff-Hausser bacteria counter technique. Metabolites of carbofuran were determine using [ 14 C-URL] carbofuran. Mineralization rates were determined using [ ,4 C-CAL] and [ l4 C-URL] carbofuran. The bacteria mineralized between 84-91% of the [ 14 C-CAL] carbofuran in 72 hours, while during the same period only 40-47% of the [ 14 C-URL] carbofuran was mineralized. Incorporation of 14 C into the biomass was found to be between 20-33% of the applied [ l4 C-URL] carbofuran. vii

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CHAPTER 1 INTRODUCTION The broad spectrum insecticide and nematicide carbofuran (2,3-dihydro-2,2dimefhyl-7-benzofuran-7yl methylcarbamate) came into use in agriculture in the late 1960s. Carbofuran originally exhibited excellent control over the corn rootworm (Diabrotica spp.), but repeated use of carbofuran in the same fields resulted in the failure to control target pests. This loss of efficacy was attributed to enhanced biodegradation of carbofuran, and microorganisms were the culprits responsible for enhanced degradation. Currently, there is a substantial body of information on enhanced degradation of carbofuran in soils. However, there is little information linking enhanced degradation of carbofuran with the ecology of carbofuran-degrading microorganisms in soils. Information is needed on the effect of repeated field applications of carbofuran on the degradation rates of carbofuran in soil profiles, and the ecology of the microorganisms involving in the degradation of carbofuran in soils. In the first half of Chapter 2, 1 provide background information on carbofuran, including its toxicity, physical and chemical properties, and its degradation in nonenhanced soils. The second half of this chapter, I review enhanced degradation of carbofuran in soils, and present information on soil bacteria that have various capacities 1

PAGE 9

2 of degrading carbofuran. In Chapter 3, the experimental procedures used in my research are discussed. In Chapter 4, 1 investigated the development of enhanced degradation in a Florida sandy soil repeatedly field treated with carbofuran. The purpose of the study was to determine the effect of repeated field applications of carbofuran on the degradation potential of carbofuran in enhanced and nonenhanced soils. In addition, metabolite formation was used to determine the disappearance and degradation pathways of carbofuran in enhanced and nonenhanced soils. In Chapter 5, 1 attempted to link the degradation of carbofuran with changes in the carbofuran-degrading populations in enhanced and nonenhanced soils. The fluctuations were monitored as a function of the soils carbofuran field application histories, and as a function of time after a laboratory treatment of carbofuran. The characterization of three carbofuran-degrading bacteria isolated from soils is addressed in Chapter 6. The goals of the characterization were to: 1) measure the growth of the soil isolates on carbofuran; 2) measure the degradation rates of carbofuran by the soil isolates; 3) measure the mineralization rates of carbofuran by the soil isolates in different growth media; 4) attempt to elucidate the degradation pathways of carbofuran by the soil isolates; and 5) attempt to determine if the genes for carbofuran degradation are located on chromosomal or plasmid DNA.

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CHAPTER 2 LITERATURE REVIEW History of Carbamate Pesticides The history of carbamate chemicals can be traced back several hundred years to Africa (Holmstedt, 1972). Carbamate compounds were derived from calabar beans used in witchcraft trials. The calabar beans were used to pronounce guilt or innocence upon an accused individual. If village leaders believed there was validity to the charges against the accused, the individual was forced to drink a concoction of mature calabar bean seeds and water. If the accused were fortunate, he or she would quickly regurgitate the mixture, but the less fortunate were doomed to uncontrollable shaking and frothing at the mouth that eventually led to death. Those who regurgitated the calabar bean were pronounced innocent, and those who died were pronounced guilty and justice was served. In the mid 1 800s, Europeans brought calabar bean seeds back to Scotland giving the legume plant the name Physostigma venconosum. The natural toxin from the plant was identified as a vegetable alkaloid in 1863, with the main toxin, physostigmine, being purified one year later (Holmstedt, 1972). The structure of physostigmine was elucidated in 1925 (Figure 2-1) and successfully synthesized in 1935 (Julian and Piki, 1935). 3

PAGE 11

Physostigmine 0 1 v H CH, Carbofuran Figure 2-1. Chemical stucture of physostigimine and carbofuran.

PAGE 12

5 In the mid to late 1940s, Geigy Chemical Company attempted to develop an insect repellent. They tested a series of carbamate compounds, discovering that these compounds were poor repellents but that they were toxic to horseflies, aphids, and other small insects. At this point, Geigy decided to pursue developing carbamates insecticides rather than carbamate repellents. All of the insecticides developed by Geigy were dimethyl carbamates. In 1953, Union Carbide Corporation synthesized another class of carbamate compounds in which the dimethyl carbamoyl moiety was replaced with a monomethyl moiety. These aryl N-methylcarbamates were shown to have superior insecticidal activity compared to the dimethyl carbamic acids (Kolbezen et al., 1954). It is from these aryl N-methylcarbamate insecticides that carbofuran is derived (Figure 2-1). Carbofuran is exclusively a field applied insecticide and nematicide used for various crops some of the more important ones include corn and rice. Toxicology of Carbofuran Mode of Action Carbamate compounds are fairly potent inhibitors of cholinesterase similar to that of the organophosphate insecticides. Acetylcholinesterase hydrolyze acetylcholine, and the inhibition of this enzyme results in the accumulation of acetylcholine in the central nervous system (CNS) synapses. This accumulation results in an over-stimulation of the acetylcholine receptors causing the interruption of nerve impulse transmission for insects, that eventually leading to death (Kuhr and Dorough, 1976). In vertebrates, the

PAGE 13

6 accumulation of acetylcholine in the synapses of neuromuscular junction results in an over-stimulation of the acetylcholine receptors that results in death due to respiratory failure (Gupta, 1994). Carbamate compounds react with the OH group of serine in the active site of the enzyme (Gupta, 1994). In addition to inhibition of acetylcholinestase, it is speculated that carbofuran inhibits the activities of other enzymes that use serine in the active sites of the molecule (Gupta, 1994). Toxicity of Carbofuran Animals Concern over the use of carbofuran in fields is due to the potential toxicity toward humans based on animal studies. The most sensitive mammal to carbofuran poisoning appears to be mice with LD 50 of 2.0 mg kg" 1 by oral ingestion (Fahmy et al., 1970). Other studies have shown that carbofuran is acutely toxic to a host of other mammals with LD 50 for oral ingestion ranging from 2.5 mg kg"' for rats to 19 mg kg" 1 for dogs (Gupta, 1994). Carbofuran is most toxic through oral and inhalation routes of exposure, but is less toxic through the dermal route (LD 50 885 mg kg" 1 for rabbits). Birds Birds in the wild are particularly susceptible to carbofuran poisoning, and because of large numbers of bird kills, the formulation and method of application of carbofuran has been regulated. Granular formations of carbofuran are reportedly highly toxic to birds (Balcomb, 1983). The LD 50 values for oral ingestion of carbofuran in ducks and quail were 0.42 mg kg" 1 (Hudson et al., 1972) to 5 mg kg" 1 (Osweiler et al., 1985),

PAGE 14

7 respectively. Elliot et al. (1996) reported a secondary killing of nine birds of prey with symptoms of anticholinesterase poisoning. Their crop contents contained duck parts that were contaminated with carbofuran. Dietrich et al. (1995) reported the deaths of eight buzzards (Buteo buteo) five of which had crops contents of earthworms that were contaminated with carbofuran. Fish Aquatic organisms are also susceptible to carbofuran from agriculture runoff and accidental spraying. The 24 hour LC 50 values for bluegill ( Lepomis macrochirus) and fathead minnow (Pimephelas promelas) were 0.1 mg L" 1 and 2.24 mg L*\ respectively. The 96 hour LC 50 values were 0.088 mg L' 1 and 1 .99 mg L/ 1 for bluegill and fathead minnow, respectively (Trotter et al., 1991). Application of carbofuran to flooded rice plots resulted in killing less than 10 % of the green sunfish {Lepomis Cyanellus), and mosquito fish (Gambusia affinis) (Davey et al., 1976). Soil microorganisms Many pesticides have been shown to have deleterious effects on the microbial communities, but when applying carbofuran to a soil, its impact on the general microbial community varies depending on the individual soil. Tu (1972a; 1972b) reported that carbofuran was inhibitory to bacteria and fungi in a sandy loam (pH 8.2), whereas in neutral to acidic sandy loam soils, carbofuran had little effect on the size of the microbial biomass (Tu, 1978). Das et al. (1995) reported that carbofuran had little effect on the fungal population size in the rice rhizosphere, but bacterial population sizes were stimulated by the addition of carbofuran. In organic soils, the bacterial and fungal

PAGE 15

8 populations were stimulated by carbofuran application to the soil (Tu, 1978; Mathur et al.,1976). The management practice used in applying carbofuran to a soil influenced the effect of carbofuran on the microbial community. Soils that had carbofuran applied banded exhibited a faster increase in the microbial biomass than soils that receiving carbofuran through broadcast (Mathur et al., 1976). Nitrification is considered to be the most sensitive biological function in soil that can be negatively impacted by pesticide applications (Rajagopal et al.,1984). Several researchers reported carbofuran applied to soil at rates between 5 and 500 u£ g" 1 was not inhibitory to nitrification in terms of nitrite formation (Lin et al., 1972; Mathur et al., 1976; Ramakrishna and Sethunathan, 1982). Rather, it has been reported that carbofuran stimulated nitrification in soils previously exposed to the pesticide ( Ramakrishna and Sethunathan, 1982). Thus, it appears that carbofuran has little if any negative impact on the general microbial size after a single agriculture application of carbofuran. Soils with a previous history of carbofuran exposure exhibited no effect on the size of the bacterial and fungal populations upon repeated exposure to the pesticide (Duah-Yentumi and Johnson, 1986) A 4-fold increase in bacterial population size was reported within a rice rhizosphere after repeated treatment with carbofuran (Venkateswarlu and Sethunathan, 1978). A single application of carbofuran reportedly stimulated the nitrogenase activity associated with the rhizosphere of rice (Kanungo et al.,1995). This stimulation in nitrogenase activity continued for two additional applications, but after the fourth application the nitrogenase activity did not increase significantly. The results of these studies suggest that carbofuran has no adverse effects

PAGE 16

9 on the general microbial community even after repeated treatments. It appears that carbofuran may stimulate the activities of soil bacteria when repeatedly applied to the soil. This conclusion is supported by a recent finding on the N-methylcarbamate aldicarb that after 19 years of continuous application tosoil has resulted in an increase in the total microbial biomass (Hart and Brooks, 1996). Characteristic Properties of Carbofuran Physical and Chemical Properties The physical and chemical properties of carbofuran are presented in Table 2.1 There is a little controversy over the solubility of carbofuran at 25 C. Bowman and Sans (1979) measured carbofuran solubility to be 700 ug ml" 1 but FMC, the maker of the pesticide, reported carbofuran solubility to be 351 ug ml" 1 Other data presented by Bowman and Sans (1979) support the 351 p.g ml' 1 value; they measured the solubility of carbofuran to be 320 ug ml" 1 at 19 C. It appears that the hydrolysis products of carbofuran may have been inadvertently measured and included within the 700 p.g ml" 1 Carbofuran has a low vapor pressure which suggests that there will be a negligible loss of carbofuran in soil due to volatilization. Carbofuran Sorption Pussemier et al. (1989) investigated the molecular parameters of a pesticide that determine its sorption characteristics. For arylcarbamates, they determined that the

PAGE 17

10 Table 2-1. Physical and chemical properties of carbofuran. Chemical formula C, 2 H 15 N0 3 Molecular weight 221.6 gmol" 1 Physical state White crystalline soild Flammability Not flammable Melting point 15 to 154C Density 1.180 (20C) Vapor pressure 2x 105 mmHg (33C) 1.1 x 104 mmHg (50C) Octanol/water partition coefficient 42.5 Water solubility 351 ug mr'(25 0 C) K oc 9-36 ml g" 1 data from Trotter et al. (1991) and Wauchope et al. (1992)

PAGE 18

11 hydrophobicity of the compound was strongly correlated with sorption of the compound on various sorbates (r z = 0.6-0.91). For a range of soils, carbofuran sorption was strongly correlated with the soils organic carbon content (r 2 = 0.81), with a value of 30 ml g" 1 for carbofuran (Sukop and Cogger, 1992). This value corresponded closely to the K oc value of 26 ml g" 1 determined by Felsot and Wilson (1980). The average K,,,. value for carbofuran in soil is 22 ml g" 1 with values ranging from 9-36 ml g" 1 (Wauchope et al., 1992). Felsot and Lew (1989) found that the bioavailability of carbofuran was controlled by the organic carbon content of a soil. They reported that organic carbon accounted for the greatest proportion of variability in the LC 50 (r 2 = 0.89) and LC 95 (r 2 = 0.88) of the pesticide in soil. Carbofuran Transport in Soils. Based on the low potential for sorption (low K oc ), carbofuran should be expected to be fairly mobile in soils. In a microcosm study, Lichtenstien and Liang (1987) found that over a period of 36 days, > 29% of the applied 14 C-activity was measured in runoff and percolation water. While in a field experiment, carbofuran was found to move after a rain fall event during which the surface transport of carbofuran was not associated with any sediment but rather with the run-off water (Caro et al., 1973). Williams et al. (1995) reported that carbofuran was more mobile than models predicted and suggested that the physical soil properties influenced the movement of the pesticide more than the chemical properties of the pesticide. Williams et al. (1995) stressed that improved management practice is the key to reducing potential contamination of local environments. This was

PAGE 19

12 supported by Caro et al. (1973) who detected more carbofiiran in runoff from fields that had received carbofuran by broadcast application than by band application. Degradation of Carbofuran in Nonenhanced Soils Aerobic Soils Degradation mode in aerobic soil The predominate mechanism of carbofuran degradation under field conditions was reportedly to be highly site specific. Both biological and chemical degradation contribute to carbofuran degradation in soils. Chemical hydrolysis of carbofuran is higher in alkaline soils than in neutral to lower pH soils (Getzin, 1973). In sterile neutral or acidic soils, degradation rates of carbofuran were lower than in corresponding nonsterile soils (Getzin, 1973). In alkaline soil, the degradation rates of carbofuran were similar whether they were sterile or nonsterile. In field studies, the areas where carbofuran was rapidly degraded were higher in clay and water contents (Caro et al., 1973). This is contradicted by the finding that the clay content of a soil had a negative impact on the degradation of carbofuran (Abdellate et al., 1967). In other studies, the clay content was not linked at all to the degradation rates of carbofuran (Ou et al, 1982; Charnay and Fournier 1994). Soil properties ( pH, organic matter, cation-exchange capacity (CEC)), and the types of microbial populations also can not be linked directly to the degradation rates of carbofuran (Ou et al., 1982). The soil properties that have been shown to be linked to the degradation rate of

PAGE 20

13 carbofuran are temperature and water contents (Caro et al., 1973; Mathur et al., 1976; Telekar et al., 1977; Ou et al., 1982). The biotic influence on carbofuran degradation is evident in that higher temperatures and water contents which increase microbial activity also influence the degradation rates of carbofuran in soil (Kieft et al., 1995). The main mechanism of carbofuran degradation in organic soils has been shown to be a microbially mediated process (Greenhalgh and Belanger, 1981; Mathur et al.,1976). The higher microbial activity in surface soils than in subsurface soils has been attributed to the increased rates of carbofuran degradation in the surface soils (Buyanovsky et al., 1993; Mallawatantri et al., 1996). Thus, it appears that in neutral to acidic soils biological degradation is the dominate mechanism for carbofuran degradation. Metabolism of carbofuran in aerobic soil The metabolites detected in field and microcosm studies revealed that only small amounts (2-10%) of carbofuran were converted to its oxidation products 3-hydroxylcarbofuran and 3-ketocarbofuran (Caro et al., 1973; Lichtenstien and Liang, 1987). Lichtenstien and Liang (1987) using [ 14 C-CAL (carbonyl labeled)] carbofuran determined that most of the extractable 14 C-activity was associated with carbofuran. In batch studies using [ ,4 C-URL (uniformly ring labeled)] carbofuran, there were low levels (< 4.0%) of the oxidation products 3 -hydroxy lcarbofuran and 3-ketocarbofuran detected, and in addition the hydrolysis products carbofuran phenol and 3-ketocarbofuran phenol were also detected in small amounts (< 4.0%) (Getzin, 1973; Ou et al., 1982). Carbofuran was the major extractable compound, yet the major portion of l4 C-activity

PAGE 21

was actually associated with soil-bound residues (50-95%) (Getzin, 1973; Ou et al., 1982). The precursor compounds of the soil-bound residues appear to be the carbofuran phenolic compounds, since incubation of [ 14 C-URL] carbofuran phenol resulted in similar 14 C distribution patterns as [ 14 C-URL] carbofuran (Getzin, 1973). The batch carbofuran degradation studies using [ 14 C-URL] carbofuran, reveal that hydrolysis of the carbamate moiety from carbofuran was the main pathway of degradation in soils (Figure 2-2). This is typical of the metabolism of other N-methylcarbamates in soils (Kazano, et al., 1972). Half-lives of carbofuran in aerobic soil Half-life values of carbofuran in soil under field conditions ranged from 15-117 days (Caro et al., 1973, Greenhalgh and Belanger, 1981; Williams et al., 1995). Half-life values in mineral soils under field conditions were dependent on the method of carbofuran application, with banded application having half-life values almost twice as long as broadcast values (Caro et al., 1973). In organic soils under field conditions, halflife values were similar whether applied by broadcast or banded (Mathur et al., 1976; Greenhalgh and Belanger, 1981). Half-life values of carbofuran under laboratory conditions ranged from 14 days to more than lyear (Getzin, 1973; Ou et al., 1982 and Short and Enfield, 1988). It is interesting to note the degradation rate of carbofuran is three times faster in soils with a history of continuous monoculture cultivation and organophosphate insecticide use than in soils not in cultivation. (Rouchaud et al., 1989).

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15

PAGE 23

Anaerobic Soils Degradation mode in anaerobic soil Carbofiiran was reported to be susceptible to photodecomposition with close to 25% of the applied carbofiiran being degraded after exposure to direct sunlight during a 96 hour incubation period (Deuel et al.,1979), but only 10% of the applied carbofiiran was degraded after exposure to laboratory light over a similar time period. Loss of carbofiiran through volatilization was found to be negligible (Deuel et al.,1979), as to be expected based on carbofiiran vapor pressure for carbofiiran is 2.5 x 10' 5 mM Hg at 33 C. The redox potential of a given soil was found not to affect the persistence of carbofiiran in flooded soils (Panda et al., 1988). The predominate abiotic mechanism for carbofiiran degradation in flooded ecosystems is believed to be alkaline hydrolysis (Venkateswarlu et al., 1977; Venkateswarlu and Sethunathan, 1978; Panda et al., 1988; Morra et al., 1996). In a rice paddy, the pH of flood water in immediate contact with the soil surface was 8.5 while surface soil pH was 7.3 and the subsurface soil pH was 6.9 (Panda et al., 1988). Thus, the abiotic degradation of carbofiiran appears to be related to the pH of the soil solution, and exposure of carbofiiran to direct sunlight. Based on the degradation rates of carbofiiran in autoclaved soils and non-autoclaved soils, biological degradation of carbofiiran is a major factor in anaerobic soils (Venkateswarlu et al., 1977; Arunachalam and Lakshman, 1990). A number of bacteria capable of degrading carbofiiran have been isolated from anaerobic soils including bacteria capable of utilizing carbofiiran as a sole source of C for growth and energy (Venkateswarlu et al., 1977; Venkateswarlu and Sethunathan, 1984; Rajagopal et al.,

PAGE 24

17 1984; Ramanand et al., 1991). Despite the presence of carbofuran-degrading microorganisms in anaerobic soils, the link between the decreased efficacy of carbofuran and a buildup of carbofuran degrading microorganisms has yet to be proven. Metabolism of carbofuran in anaerobic soil The only compound detected in paddy water or soils is the parent compound carbofuran ( Deuel et al., 1978; Johnson and Lavy, 1995). Only trace amounts (< 1%) of 3-ketocarbofuran were occasionally detected in paddy soils, while 3-hydroxylcarbofuran was detected at trace amounts in the overlying water and the flooded soil (Johnson and Lavy, 1995). Due to the rapid dissipation in a Texas rice paddy, neither carbofuran nor its metabolites were detected in paddy soils, but carbofuran and its metabolite 3ketocarbofuran were detected in the overlying water (Deuel et al., 1978). The major metabolite of [ 14 C-URL] carbofuran in flooded ecosystems was the hydrolysis product carbofuran phenol (Venkateswarlu and Sethunathan, 1 979, Panda et al., 1988; Lalah et al., 1996). In these reduced oxygen environments, 3hydroxylcarbofuran and 3-ketocarbofuran phenol never accumulated to more than 1 % of the applied l4 C activity (Venkateswarlu and Sethunathan, 1978; Lalah et al.,1996). The phenyl ring of carbofuran appears to be recalcitrant to degradation in flooded ecosystems, with cumulative l4 C0 2 production never exceeding 7 % of the applied [ 14 C-URL] carbofuran (Venkateswarlu and Sethunathan, 1978; Lalah et al., 1996). Soil bound residues of [ 14 C-URL] carbofuran in anaerobic soils never exceeded 10 % after one month of incubation (Venkateswarlu and Sethunathan, 1978, Lalah et al., 1996). These studies suggest that the degradation of carbofuran in flooded ecosystem is mainly via hydrolysis

PAGE 25

18 to carbofuran phenol, with the aromatic ring remaining largely intact in the anaerobic environments (Figure 2-3). Half-lives of carbofuran in anaerobic soil The half-life values for carbofuran in field paddy soils range from 10 to 58 days (Nicosia et al., 1991; Johnson and Lavy, 1995). In laboratory studies, the half-life values of carbofuran in a bulk soil ranged from 1 1.9 to 15.1 days (Panda et al., 1988), while the half-life value in an anaerobic rhizosphere was 10.8 days (Das et al., 1995). Enhanced Degradation of Carbofuran Background Information Evidence for enhanced degradation The concern over the fate and persistence of chlorinated pesticides coupled with a growing environmental conscience in our society resulted in an inclination for the development of environmentally benign (less toxic and biodegradable) pesticides. Classes of chemicals such as organophosphates and carbamates that are less persistent than highly halogenated pesticides such as DDT have proven to be effective in controlling target pests. One of these pesticides was bufencarb, a N-methylcarbamate insecticide used for the control of corn pests, most notably the corn rootworm larval {Diabrotica spp.). In the early 70' s, farmers complained to state extension agencies in Iowa about poor control of target insects in fields treated with bufencarb, but the poor control of bufencarb could not

PAGE 26

Carbofiiran 19 CH, + NH 2 CH 3 Carbofuran phenol Methylamine Figure 2-3. Carbofuran degradation in nonenhanced anaerobic soil.

PAGE 27

20 be reproduced in research plots (Tollefson, 1986). Due to extensive complaints about poor performance, state extension services recommended not using bufencarb for corn rootworm larval control (Tollefson, 1986). During this time, the carbamate insecticide trimethacarb also exhibited poor pesticidal efficacy, and similar to bufencarb, it was determined that insect resistance could not sufficiently explain the observed failures (Chou et al., 1978). Other carbamate and organophosphate pesticides also exhibited similar pest control failures during the 1970's (Sethunathan and Pathak, 1972; Chou et al., 1978; Felsot et al., 1985). The lost of pest control from these pesticides has now been attributed to enhanced degradation in soils and microorganisms are responsible for enhanced degradation. Carbofuran was introduced in 1967, and gave outstanding protection from attack by corn rootworm larvae (Tollefson, 1986). Carbofuran was also found to be effective in control of brown planthopper (Nilaparvata lugens) and green leafhopper (Nephotettix virescens) in rice paddies (IRRI, 1975). From the mid to late 1970's, news on the ineffective control of target pests in fields previous treated with carbofuran started to surface. Tollefson (1986) found in 1975 that soils previously treated with carbofuran exhibited greater root damage than soils with no history of exposure. He repeated the study in 1976 and again soils previously treated with carbofuran resulted in excessive root damage as opposed to soils with no history of exposure. The evidence linking loss of control of target pest and accelerated degradation of carbofuran began to accumulate. Williams et al. (1976) reported that in Canada pest control problems were found in vineyards that had been previously treated with carbofuran, and that the levels of

PAGE 28

21 carbofuran residues were lower than what was expected. They were able to show that in organic soils a significant portion of carbofuran degradation was biological, and from these soils they even isolated a few soil organisms which possessed the capacity to degrade carbofuran. However, Williams et al. (1976) failed to supply any evidence for enhanced rates of carbofuran degradation in the soil. Greenhalgh and Belanger (1981) found that organic soils that were retreated with carbofuran had lower residue levels of carbofuran after 30 days compared to plots treated for the first time. In the Philippines, the Internation Rice Research Insitute (1977) reported that rice fields treated with carbofuran suffered worse hopperburn than untreated fields. Hopperburn results from infestation of brown planthopper attacks on rice plants. It was thought that the loss of efficacy for carbofuran in the treated paddy soil was due to a rapid buildup of carbofuran-degrading bacteria (Venkateswarlu and Sethunath, 1978). This was due in part to the isolation of a bacterium that was capable of degrading carbofuran from a rice field (Venkateswarlu et al.,1977). However, after repeatedly treating flooded soils with carbofuran, there were no evidence linking carbofuran treatments with loss of pest control via rapid degradation of carbofuran (Venkateswarlu and Sethunath, 1978). Confirmation of enhanced degradation Felsot et al. (1981) produced one of the first studies linking the loss of pest control to enhanced degradation that was, in turn, associated with microbial degradation. The study was conducted due to concern by corn producers over the poor control of northern and western corn rootworms by carbofuran. Resistance to carbofuran by rootworm could not adequately be related to this failure (Felsot et al., 1981). Felsot and

PAGE 29

22 coworkers collected soils from corn fields in Illinois with various histories of carbofuran treatment. Soil samples were collected from four corn fields with a history of carbofuran applications and poor pest control, two corn fields with a history of phorate applications, a field that had never been treated with any pesticides, and a crawl space under a house treated with chlordane. All the soils that were previously treated with carbofuran degraded the pesticide more rapidly when compared to soils with no history of carbofuran use. Sterile treated soils lost the ability to rapidly degrade the pesticide indicating that enhanced degradation was a microbial process. In addition, Felsot and coworker were able to isolate a two of soil bacteria that degraded carbofuran. Repeated applications of carbofuran to a soil under laboratory conditions (500 ug carbofuran g" 1 soil) resulted in carbofuran degradation rates 600-1000 times faster than in a soil with no history of exposure (Read, 1983). This rapid carbofuran degradation coincided with a reduction in the lag period prior to initiation of degradation. In addition, Read (1983) was able to link enhanced degradation to the loss of control of target organisms by showing that the reduced residue levels in soils previously exposed to carbofuran resulted in a decrease in the percentage of cabbage maggot egg mortality. The work of Read (1983) and Felsot et al. (1981) demonstrated that the inability of carbofuran to control target pests in soils was associated with the rapid breakdown of the compound. Furthermore, microorganisms were shown to be responsible for this rapid breakdown of carbofuran. However, the question remained were we seeing an enhancement of carbofuran degradation or were we witnessing only soils that had the capacity to degrade carbofuran at rapid rates? Felsot et al., (1981) noted that soils

PAGE 30

23 collected along the fence rows next to carbofuran treated soils also exhibited accelerated degradation rates of carbofuran. To demonstrate that enhanced degradation of carbofuran resulted from the repeated applications of carbofuran to the same field, Suett (1986) determined carbofuran degradation rates in eight U.K. soils with histories of carbofuran applications, and in soils collected from adjoining fields that had no prior history of exposure to carbofuran. These soils covered a wide range of physical and chemical properties. Suett showed that the degradation of carbofuran was more rapid in soils previously treated with the pesticide, and that the "initial" lag phase for each soil was shorter in previously exposed soils as compared to soils with no history of carbofuran application. One of the soils fumigated with dazomet did not degrade carbofuran despite repeated carbofuran applications. The reduced microbial activity may reflect a general reduction in the microbial biomass due to the fumigant. Subsequently, Camper et al. (1987) and Turco and Konopka (1990) also found in South Carolina and Indiana soils with a previous history of carbofuran application, a more rapid degradation than adjoining soils not previously treated with cabofuran. Factors that Influence Enhanced Degradation Key soil physical, chemical and biological factors. Extensive efforts have been made to understand the basic mechanisms involved in the development of enhanced degradation of pesticides in soils. One question that has often been asked is: what are the characteristics of a soil that develops enhanced

PAGE 31

24 degradation from repeated applications of carbofuran? Researchers have attempted to determine the key soil factors that affect the mineralization and disappearance rates of carbofuran in soil. The key soil factors that had been studied included soil pH, organic matter content, clay content, CEC, C:N ratio, organic N content and CaC0 3 content. None of these soil factors were found to correlate with the mineralization or disappearance of carbofuran in enhanced and nonenhanced soils (Ou et ah, 1982; Charnay and Fournier, 1994). Only water content and temperature were correlated to the mineralization and disappearance of carbofuran in soil (Ou et al., 1982; Chapman et al., 1986; Chapman and Harris, 1990; Parkin and Shelton, 1994). The soil water content was found to be the most dominate factor in determining the spatial and temporal variations of carbofuran degradation rates in enhanced soils (Parkin and Shelton, 1 994). Total soil aerobic microorganisms and aerobic bacterial populations, along with amidase and urease activity, were poorly correlated with carbofuran degradation rates in enhanced and nonenhanced soils (Ou et al., 1982; Dzantor and Felsot, 1990). In addition, initial carbofuran-hydrolyzing population size was also poorly correlated with the development of enhanced degradation of carbofuran in soil (Dzantor and Felsot, 1990; Charnay and Fournier, 1994). Thus, Charnay and Fournier (1994) concluded that only a soil's exposure history to carbofuran can be correlated with the development of enhanced degradation. Enhanced degradation and chemical treatment It appears that carbofuran must be applied to soils at a certain threshold level prior for enhanced degradation to occur (Chapman et al.,1986; Hendry and Richardson, 1988;

PAGE 32

25 Chapman et al., 1990). Mineral soils were found to develop enhanced degradation at application rates of 1.0 ug carbofuran g" 1 soil (Chapman et al.,1986; Chapman and Harris, 1990), but enhanced degradation did not occur at application rates below 0.1 ug carbofuran g' 1 soil (Chapman et al.,1986; Hendry and Richardson, 1988). In organic soils, the application rate of carbofuran required to induce enhanced degradation was 10.0 ug carbofuran g" 1 soil, and at application rates below 1 .0 ug carbofuran g" 1 soil, enhanced degradation did not develop (Chapman and Harris, 1990). The higher loading rates for carbofuran to develop enhanced degradation in organic soils is attributed to the higher sorption capacity of the organic soils and, thus, the decreased bioavailability of carbofuran to the microorganisms. Further evidence supporting a threshold concentration required for triggering enhanced degradation is found in the increased spatial variability of enhanced degradation activity in no-till plots compared to conventional tillage plots (Parkin and Shelton, 1992). In no-till plots, enhanced degradation of carbofuran corresponded to the placement of carbofuran, while in conventional tillage plots the homogenizing of the soil resulted in a lack of any observed spatial distribution of enhanced degradation activities. Enhanced degradation of carbofuran is not only induced by a threshold level of carbofuran treatment, but also by structurally similar carbamate pesticides (Harris et al., 1984; Racke and Coats, 1988; Dzantor and Felsot, 1989; Morel-Chevillet et al., 1996). This phenomenon is known as "cross-conditioning." However, not all carbamate compounds induced enhanced degradation of carbofuran. Recently, a soil in France was shown to exhibit enhanced degradation toward carbofuran after pretreatment with 1 5

PAGE 33

26 different aryl methylcarbamate pesticides, but soils that were incubated with primicarb (2-dimethylamino-5,6-dimethylpyrimidin-4-yl dimethylcarbamate) and formetanate ( 3dimethylaminomethyleneiminophenyl methylcarbamate) did not develop enhanced degradation toward carbofuran (Morel-Chevillet et al., 1996). Knowing that structurally similar compounds have the potential to induce enhanced degradation of carbofuran, one could ask what is the response of soils with enhanced degradation toward other unrelated agricultural chemicals? The N fertilizer urea, when applied to enhanced soils, exhibited an inhibitory response to the mineralization of [ 14 C-CAL] carbofuran (Merica and Alexander, 1990), while other N fertilizers (sodium nitrate and ammonium nitrate) had no effect on the mineralization of [ 14 C-CAL] carbofuran in enhanced soils (Hendry and Richardson, 1988; Racke and Coats, 1990; Merica and Alexander, 1990). The addition of organophosphate pesticides to soils has been shown to have inhibitory effects on enhanced degradation of carbofuran (Racke and Coats, 1990; Talebi and Walker, 1994). In particular, the organophosphates ethoprop and paraoxon have been reported to markedly reduce carbofuran degradation in enhanced soils (Racke and Coats, 1990; Talebi and Walker, 1994). The reduction of carbofuran degradation may result from inhibition of the enzymes involved in the hydrolysis of the carbamate linkage to carbofuran (Talebi and Walker, 1994). Inhibition of the enzymes is not unexpected since it has been shown that N-methylcarbamates do inhibit phenylcarbamate hydrolyzing enzymes (Kaufman et al., 1970), and possibly some organophosphates as well.

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27 Duration of enhance degradation of carbofuran. The number of field applications of carbofuran to a soil before enhanced degradation occurs and the duration of enhanced degradation has been investigated. Several researchers reported that after one field application of carbofuran resulted in an increase in the degradation of the chemical (Getzin and Shanks, 1990; Harris et al., 1988). Racke and Coats (1990) reported that enhanced degradation may persist for different periods of time depending upon soil type and possibly soil depth. Recently, Suett et al. (1993) reported that several soils in the U.K. sustained enhanced degradation of carbofuran without being treated with the pesticide for over 5 years. Eagle (1986) found that some soils that had received a single carbofuran treatment were still able to degrade carbofuran at an enhanced rate even after 4 years without a treatment of carbofuran. Role of biomass and enzyme activity on enhanced degradation The capacity of a soil to exhibit enhanced degradation of a pesticide has been directly attributed to the ability of the soil microbial community to degrade the pesticide at increasing rates until the pesticide is no longer of any efficacious value. The following questions are often debated by scientists as to the main mechanism responsible for a soil to develop enhanced degradation: 1) does repeated exposure of the pesticide result in increased microbial biomass capable of degrading the pesticide; or 2) does repeated exposure of the pesticide result in an increase in the enzyme activity specifically toward the degradation of the pesticide? Evidence supporting the first hypothesis is based on the degradation of the herbicide 2,4-D (Ou, 1984; Holben et al., 1992; Ka et al., 1994) and the organophosphate

PAGE 35

28 isofenphos (Racke and Coats, 1987). Ou (1984) reported that soils treated with 2,4-D exhibited an increased in the degrading population with time after treatment with the pesticide. Racke and Coats (1987) found the numbers of isofenphos degraders in soils progressively increased as the number of field applications increased resulting in a progressive increase in the degradation rate of the chemical. Evidence for the second hypothesis is based on the numbers of EPTC -degrading microorganisms in soils with and without a history of EPTC field application. Moorman (1988) found that the numbers of EPTC-degraders in soils with a history of EPTC applications were not significantly different from the numbers of EPTC-degraders in soils with no history of EPTC application In addition, the number of EPTC-degraders in soils did not significantly increase 25 days after the application of EPTC despite the increased degradation of EPTC in those soils. What do these studies on carbofuran-degraders reveal about the nature of enhanced degradation of carbofuran? The answer is enhanced degradation may include both. Several studies point to an increase in the number of carbofuran-degraders in soils with enhanced degradation compared to the indigenous carbofuran-degrading microorganisms (Hendry and Richardson, 1988; Dzantor and Felsot, 1989; Dzantor and Felsot, 1990). While other studies fail to link an increase in the number of carbofuran degraders with the number of carbofuran applications (Racke and Coats, 1988; Merica and Alexander, 1990; Scow et al. 1990; Robertson and Alexander, 1994). Hendry and Richardson (1988) reported a significant increase in the number of carbofuran-hydrolyzers from 1.6 x 10 3 to 3.1 x 10 5 cells g" 1 after one treatment with

PAGE 36

29 carbofuran. Subsequent treatments of carbofuran did not result in a significant increase in the number of carbofuran-hyrolyzers in the laboratory treated soil. Racke and Coats (1988) reported that the higher number of carbofuranhydrolyzers in carbofuran treated soil was not significant to the number in the control (no carbofuran history) soil, and they concluded that the enhanced degradation of carbofuran in the treated soil could not be explained by differences in the number of carbofuranhydrolyzers in the different soils. In similar findings, Scow et al. (1990) and Robertson and Alexander (1994) concluded that the microorganisms capable of mineralizing l4 Ccarbofuran did not grow at the expense of either the methylamine or the phenyl moiety of carbofuran, rather, carbofuran was degraded by a cometabolic process. Microorganisms involved in carbofuran degradation Since enhanced degradation of carbofuran is biological, the next question to ask is what are the major groups of microorganisms involved in the degradation of carbofuran. The use of selective antibiotics allows understanding of the roles the major groups of microorganisms capable of degrading a pesticide. The use of selective antibiotics in soils with enhanced degradation toward EPTC revealed that bacteria were the predominate factor of enhanced degradation in soils ( Dick et al., 1990). The bactericides chloramphenicol and streptomycin and a fungicide cycloheximide have been used to determine whether or not bacteria or fungi are the main group responsible for the degradation of carbofuran in soils. Studies that employed the bactericides chloramphenicol demonstrated that the mineralization of [ l4 C-CAL] carbofuran was predominantly bacteria in nature (Lenvanon, 1994; Racke and Coats,

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30 1987). Wootton et al. (1993) investigated the role of fungi in the degradation of carbofuran and concluded that fungi did not play a major role in the degradation of carbofuran. These studies implicated bacteria as the major biological source of carbofuran degradation in enhanced soils. Carbofuran Degradation by Soil Microorganisms Involved in Enhanced Degradation The ability to isolate cultures of pesticide degrading microorganisms is evidence that pesticides are being metabolized in the field. Mixed cultures allow for greater understanding of the biological mechanisms involved in the degradation of pesticides in the environment. However, it is through the isolation of axenic cultures that detailed genetic and physiological mechanisms of pesticide degradation are obtained. The isolation and characterization of 2,4-D degrading bacteria (Don and Pemberton, 1981) that has allowed researchers to obtain information on the evolution (Fulthorpe et al., 1995) and the general ecology of the pesticide degrading microbial communities (Xia et al., 1995; Kaet al., 1995). The first carbofuran-degrading bacteria (actinomycete) isolated from soil degraded carbofuran in the presence of a second C source (Williams et al., 1976). The isolation of these bacteria resulted in the warning of high numbers of actinomycetes in soil may result in rapid degradation of carbofuran. The early carbofuran-degrading isolates degraded carbofuran slowly taking over 20 days for the complete degradation of carbofuran (Venkateswarlu et al.,1977; Felsot et al., 1981; Venkateswarlu and

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31 Sethunathan, 1985) Achromobacter sp. strain WM1 1 1 (Karns et al., 1986) was the first soil bacterial isolate that exhibited the degradation kinetics that were comparable to the rapid degradation of carbofuran observed in enhanced field soils. Phenotypic classification based on the metabolism of carbofuran Chaudhry and Ali (1988) classified carbofuran degrading bacteria into three different groups (I, II, III) based on their phenotypic metabolism of carbofuran (Figure 24) : Group I utilized carbofuran as the sole source of nitrogen and converted carbofuran into carbofuran phenol; Group II utilized carbofuran as a sole source of carbon and nitrogen and converted carbofuran to carbofuran phenol; and Group III utilized carbofuran as a sole source carbon and nitrogen and converted carbofuran to carbofuran phenol and further mineralized the carbofuran phenol to C0 2 and H 2 O. The majority of soil bacteria isolated from soil thus far belong to Groups I and II. Little is known about the physiology of soil isolates, the enzymes involved in the degradation and the genetics of these organisms. Group I Carbofuran-degrading bacteria belonging to Group I are capable of hydrolyzing carbofuran to carbofuran phenol and methylamine and utilizing the methylamine as a N source (Figure 2.4). Achromobacter sp. strain WM1 1 1 belongs to this group. This isolate degraded not only carbofuran but other N-methylcarbamate pesticides, including carbaryl, baygon, and aldicarb (Karns et al., 1986). WM1 1 1 degraded > 99% of the applied carbofuran within 42 hours with a doubling time of 3 hours in a N free mineral medium supplemented with glucose (7,200 mg ml" 1 ) and carbofuran (200 mg ml" 1 ). The

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0 CH ii 0 CH, Carbofuran CH 3 Group III C0 2 + H 2 0 C0 2 + H 2 0 OH S CH 3 Carbofuran phenol Group II + NH 2 CH 3 Methylamine + \ Group I Second Carbon Source C0 2 + H 2 0 Figure 2-4. Metabolic classification of carbofuran-degrading bacteria.

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enzymes for carbofuran degradation are induced in the presence of carbofuran and methylamine, but the expression of the hydrolase enzyme was inhibited when a rich source of N was introduced into the growth medium. The gene encoding for carbofuran hydrolase, mcd, was located on a plasmid and has been cloned (Tomasek and Karns, 1989). The enzyme properties have been characterized (Derbyshire et al., 1987; Karns and Tomasek, 1991). Chaudhry and Ali (1988) also reported the isolation of Group I type soil bacteria, but did not report any additional information on these bacteria. Group II The Group II carbofuran degrading bacteria are capable of hydrolyzing carbofuran to carbofuran phenol and methylamine, and utilizing the methylamine as a C and N source (Figure 2.4). The majority of the soil isolates capable of degrading carbofuran belong to this classification. One of these isolates exhibited synergistic carbofuran degradation behavior with another soil bacterium not capable of hydrolyzing carbofuran (Singh et al., 1993). Some of the isolates expressed carbofuran hydrolase activity constitutively with increased activity in the presence of carbofuran (Topp et al., 1993). Group II isolates have been found to exhibit greater hydrolase activity than Group I isolates (Chaudhry and Ali, 1988). Some of the soil bacteria in this group were found to hybridize with the mcd gene probe (Topp et al., 1993; Parekh et al., 1995). The majority of the soil bacteria in Group II failed to hybridize to probes from the mcd clone (Chapalamadugu and Chaudhry, 1992; Parekh et al., 1995). All the soil bacteria that hybridized with the mcd

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34 probe harbored plasmids greater than the size of 100 kbp (Topp et al., 1993; Parekh et al., 1994). Group III The isolates that belong to Group III are capable of hydrolyzing carbofuran to carbofuran phenol and methylamine, and further degrading carbofuran phenol to carbon dioxide, cellular components, and soluble products (Figure 2.4). More than 1 00 bacteria that have the capacity to degrade carbofuran, but to date only five of these soil bacteria are capable of degrading carbofuran phenol (Chaudry and Ali, 1988; Ramanand et al., 1988; Head et al., 1992; Feng et al., 1997a). Group III isolates generally contain multiple plasmids (Chaudry and Wheeler, 1988; Head et al., 1992; and Feng et al., 1997a). None of the soil bacteria from Group III have hybridized to the mcd probe (Chapalamadugu and Chaudhry, 1992; Feng et al., 1997a). Plasmids of the Group III isolates may harbored the genes responsible for degradation of carbofuran. However, the f degradation of carbofuran by Sphingomonas sp. CF06 was the only known case involving plasmids (Feng et al., 1997a). Carbofuran Hydrolase Enzyme. The only carbofuran hydrolase enzyme characterized to date was isolated from the Achromobacter sp. WM1 11, and the enzyme existed as a homodimer. (Derbyshire et al., 1987; Karns and Tomasek, 1991). Three other N-methylcarbamate hydrolase enzymes have been isolated from three different soil bacteria (Mulbry and Eaton, 1991; Chapalamadugu and Chaudhry, 1993; Hayatsu and Nagata, 1993). Two of these hydrolases (Mulbry and Eaton, 1991 ; Hayatsu and Nagata, 1993) were very similar to

PAGE 42

that of the carbofuran hydrolase reported by Karns and Tomasek (1991). The two hydrolase enzymes were homodimers with a broad range of N-methylcarbamate activity (Mulbry and Eaton, 1991; Hayatsu and Nagata, 1993). The hydrolase enzyme isolated by Mulbry and Eaton (1991) has also been found to have enzymatic activity toward carbofuran, but the hydrolase enzyme isolated by Hayatsu and Nagata was not tested for its ability enzyme to hydrolyze carbofuran. One of the hydrolase enzyme was found to be an esterase (Hayatsu and Nagata, 1993). The third carbaryl hydrolase enzyme exhibited no enzyme activity toward carbofuran and did not have broad specificities toward other N-methylcarbamates (Chapalamadugu and Chaudhry, 1993). Genetics of Carbofuran Degradation The genetics of carbofuran degradation by soil microorganisms is largely unknown. The mcd gene from the Achromobacter sp. strain WM1 1 1 is harbored on a large plasmid (greater than 100 kbp), and is the only gene coding for carbofuran degradation that has been cloned (Tomasek and Karns, 1989). The soil bacteria that hybridized to the mcd probe were all found to harbor the gene on large plasmids as was the case for the original strain WM1 1 1 (Topp et al., 1993; Parekh et al., 1995). A comparison of carbofuran degrading microorganisms that had sequence homology with the mcd gene probe were found to breakdown into five plasmid restriction length polymorphism patterns (RFLP) (Parekh et al., 1996). Two of the soil isolates that are from different RPLP groups and geographical areas, exhibited different chromosomal and plasmid backgrounds, indicating that the carbofuran degrading genes did not independently evolve, but rather was acquired from a plasmid already containing the set

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36 of genes from the degradation of carbofuran (Karns, 1 990). Thus, it appears that enhanced degradation of carbofuran may result from the transfer of catabolic plasmids harboring the genes for carbofuran degradation. The involvement of plasmids in the mineralization of the aromatic ring of carbofuran has largely been speculative with only plasmids present in the strains capable of completely mineralizing carbofuran as evidence (Chaudry and Wheeler, 1988; Head et al., 1992). Sphingomonas sp. strain CF06 harbors five plasmids some of which were required for mineralizing the aromatic ring of carbofuran (Feng et al., 1997a). The plasmids of CF06 contained at least six putative insertion sequence (IS) elements that were cloned (Feng et al., 1997a). They found three of the five plasmids hybridized with the IS elements. Since the acquisition of catabolic genes has been attributed to IS elements (Tomasek et al., 1989), IS elements may play a role in the evolution of carbofuran metabolism (Feng et al., 1997a). Understanding how soil bacteria acquire and express the genes responsible for enhance degradation may provide researchers potential tools to minimize the impact of enhanced degradation in agriculture fields.

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CHAPTER 3 EXPERIMENTAL PROCEDURE Field Work Site Selection and Soil Characterization Soil samples Soil samples were collected from experimental plots at the University of Florida Agricultural Experiment Station near Hastings, FL. The experimental site was located 6 miles SE of the city of Hastings off County Road 13. Soil at this site is classified as Ellzey fine sand (sandy, siliceous, hyperthermic, Arenic Ochraqualfs). The majority of this site was under potato cultivation for more than 20 years. Some experimental plots under potato cultivation had been treated with carbofuran annually from 1993 to 1996, and others had never been treated with carbofuran or any other structurally similar pesticides. Sample designations were according to cultivation and histories of carbofuran exposure. Soil samples from plots under potato cultivation and treated with carbofuran were designated Cultivated Treated (CT). Samples from cultivated plots not receiving carbofuran treatment were designated Cultivated Nontreated (CN). Soil samples from plots not in cultivation but previously exposed to carbofuran were designated Noncultivated Treated ( NT), and samples from plots with no exposure to carbofuran and 37

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38 not under cultivation were designated Noncultivated Nontreated (NN). Table 3-1 shows the histories of carbofuran applications and cultivation, and application rates of carbofuran to the plots where soil samples were collected. A schematic of the field plots layout is shown in Figure 3-1. The CT plots have been under potato cultivation for the past 10 years prior to the beginning of this project, and CN plots were in potato production for only five years. The NT soil samples came from a strip of grass along the drainage ditch adjacent to the CT plots that was assumed to have been exposed to carbofuran due to its close proximity to the CT plots. NN soil was an undeveloped grass field next to a woodland area where groundwater flowed away from the grass field toward the experimental plots of CN; the risk of exposure to carbofuran was considered negligible. In July 1994, one month after annual field application of carbofuran, surface and subsurface CT and CN soil samples were collected to a depth of 60 cm (top of the water table) at 15 cm increments. A composite of three soil cores were taken from various locations of the plot using a 10 cm diameter bucket auger. Soils with no carbofuran exposure were collected first, while those soils previously exposed to carbofuran were collected afterward. In March 1995, one month after annual field application of carbofuran surface and subsurface soils were collected as previously described for CT and NT soil samples, except this time soil cores were not composited as done previously. Also in March 1996, soil samples CT and NN, were collected exactly the same way as in March 1995. All soil samples were stored in the dark at 4C and were used within four months.

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39 Table 3-1. Description of the field plots where soil samples were collected and their histories of carbofuran exposure. Year Application rate Cultivation 1994 1995 1996 1994 1995 1996 kg ha"' Cultivated Treated Plots" (CT) 4.5 Potato 4.5 Potato 4.5 Potato Cultivated Nontreated Plots" (CN) 0 Potato/Onions Noncultivated Treated Plots (NT) 0 None Noncultivated Nontreated Plots (NN) 0 None a These plots were treated with carbofuran the first time in 1992 at a rate of 4.5 kg ha" 1 "Although these plots were not treated with carbofuran, they received fertilizers at rates similar to the CT plots.

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41 Soil characterization The soil properties that were determined included: water content, pH, particle size, and organic carbon content. Methods for particle size analysis, water content, pH, and organic carbon content were as those described in Methods of Soil Analysis, Parts I and II (American Society of Agronomy, 1986). Particle size analysis was performed following the pipette method (Gee and Bauder, 1986). Soil water content was obtained by gravimetric determination (Gardner, 1986). Soil pH was measured in a 1:1 soil extract using distilled water (McLean, 1982). Soil organic carbon content was determined following the Walkey-Black procedure (Nelson and Sommers, 1982). Carbofuran Biodegradation Potential Chemicals Technical grade carbofuran (99% purity) and uniformly ring-labeled [ 14 C-URL] and carbonyl-labeled [ 14 C-CAL] carbofuran were provided as gifts from FMC Corp. (Princeton, NJ). Radio-purity of the chemicals was verified by thin-layer chromatography (TLC). Chemicals were not used unless radio-purity was greater than 98 %. If needed, the labeled chemicals were purified by preparative TLC to greater than 98%. All other chemicals were either HPLC grade, or the highest grade commercially available. Carbofuran mineralization Mineralization of [ l4 C-CAL] and [ 14 C-URL] carbofuran was used to determine the potential of the Ellzey soils to biodegradation carbofuran. One hundred grams of soil

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42 (oven dry weight basis) were added to 250 ml Erlenmeyer glass flasks and mixed with radio-labeled and technical grade carbofuran at a rate of 1 .7 KBq and 1 mg, respectively. After hand-mixing with a sterile spatula for 5 minutes, each flask was tightly closed with a rubber stopper under which a stainless steel vial containing 0.5 ml of KOH (5.3 mol L" 1 ) was hung (Ou, 1991). For the soil samples that were treated with [ 14 C-CAL] carbofuran, KOH traps were replaced with new traps on days 1 3, 7, 14 and 28. The KOH traps used in trapping the evolved l4 C0 2 from the [ 14 C-URL] carbofuran treated samples were replaced on days 3, 7, 14, 21, and 28. At the same time, the flasks were weighed, and deionized water was added to compensate for any water loss. KOH in the trap vials was diluted (1:10) with distilled water to 5 ml. The 14 C in the diluted KOH solution (0.5 ml) was quantified by liquid scintillation counting (LSC) using a Beckman liquid scintillation counter model LS 5801 (Palo Alto, CA). Extraction of soil After 28 days of incubation, 10 g of soil was removed from the flasks and placed in 50 ml glass culture tubes with Teflon liner screw caps along with 20 ml of methanol. These tubes were shaken for one hour on a reciprocal shaker. After one hour of shaking, methanol from the soil extracts was separated from the soil by vacuum filtration using a Whatman no. 41 filter paper. Volumes of soil extracts were measured, and a small aliquot (0.5 ml) was removed for the determination of 14 C-activity. Combustion of extracted soil The extracted soil was placed in a mortar and homogenized by mixing and grinding with a pestle for 3-5 minutes. Two-tenths of a gram of the soil was used for the

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43 determination of the 14 C-activity in the extracted soil. The ,4 C-activity in the extracted soil was combusted to l4 C0 2 in a Packard Tri-Carb sample oxidizer. The I4 C0 2 was trapped in a scintillation solution containing an organic amine, and quantified by LSC. Statistical analysis The initial amounts of 14 C0 2 evolved from soils treated with l4 C-carbofuran will be compared for different soils, soil depth and number of carbofuran treatments using a Student t-test. Carbofuran loses its pesticide efficacy upon the hydrolysis of the carbamate moiety that results in the evolution of l4 C0 2 from [ 14 C-CAL] carbofuran. Therefore, the monitoring the evolution of 14 C0 2 from [ 14 C-CAL] carbofuran treated soils is probably the best indicator for evaluating enhanced degradation of carbofuran in soil. Carbofuran Disappearance and its Metabolite Formation Extraction of soil samples collected in 1994 One hundred fifty grams of soil (oven-dry weight basis) collected in 1 994 were placed in a 500 ml glass Erlenmeyer flask along with 1 .5 mg of technical grade carbofuran, 17 KBq of [ 14 C-URL] carbofuran, and 1.5 ml of deionized water and mixed for five minutes. After mixing by hand for five minutes with spatulas, each flask was tightly closed with a rubber stopper under which a stainless steel KOH trap containing 0.5 ml KOH solution (5.3 mol L 1 ) was hung (Ou, 1991). After removing the KOH traps, 10 g of soil were removed from the 500 ml flask and placed in a 50 ml glass culture tubes with a Teflon liner screw caps. Fresh KOH vials were hung in the flasks. Methanol (20 ml) was used to extract the [ 14 C-URL]

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44 carbofuran and its 14 C-metabolites from the soil in the tubes. Tubes were shaken for one hour on a reciprocal shaker. The extracts were filtered under vacuum through Whatman no. 41 filter papers. The volumes of the soil extracts (25-30 ml) were measured and 0.5 ml of the methanol extract was removed to determine the l4 C-activity in the methanol extracts by LSC. Water in the extracts was removed by anhydrous sodium sulfate, and the extracts were concentrated under a gentle stream of N 2 to approximately 0.3 ml. Extracted soils were combustion following the procedure described previously. Thin-layer chromatography (TLC) analysis Carbofuran and its metabolites in the concentrated extracts were separated on silica gel G TLC glass plates (E. Merk, Dernstdt, Germany). The plates were developed to a distance of 15 cm using a solvent system of diethyl ether and hexane (3:1). The TLC plates were exposed for 3 to 4 weeks to Kodak x-ray films (SB-5) for autoradiographical analysis (Ou et. al., 1982). Radioactive area of the TLC gels corresponding to carbofuran, metabolite standards, and unknown were scrapped, transferred to scintillation vials and quantified by LSC. Extraction of soil samples collected in 1996 A similar procedure used to extract soil samples collected in 1 994, with minor modifications, was also used to extract soil samples collected in 1996. In this study, 10 g of soil were removed from each flask and placed in a culture tube and extracted with 20 ml of methanol as previously mentioned. Soil suspensions were first filtered under vacuum through Whatman no. 41 filter papers, and then washed three times in succession with 20 ml of methanol. After the volumes of the methanol extracts were determined, the

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45 ,4 C-activity in the extracts was quantified by LSC. Water in the extracts was removed by anhydrous sodium sulfate, and the extracts were concentrated to approximately 5 ml on a roto-evaporator (Brinkman Instruments, Westbury, NY). The extracts were transferred to small glass vials and concentrated under a gentle stream of N 2 to approximately 0.3 ml. The procedures for the determination of 14 C-activity in the extracted soil samples and the determination of metabolites by a TLC-autoradiographical procedure were as described previously. Growth of Carbofuran-Degrading Populations Most-Probable-Number fMPlSO assay A 14 C-MPN technique was used to estimate the microbial population size in soil that was capable of degrading the carbamate and aromatic ring moieties of carbofuran (Ou, 1984). [ I4 C-CAL] and [ l4 C-URL] carbofuran were used to determine the population size in soil capable of hydrolyzing carbofuran and capable of mineralizing the aromatic ring structure of carbofuran, respectively. A MPN assay that determined the population size capable of utilizing a substrate for growth was used for determination of the population size capable of utilizing methylamine (Alexander, 1982). A description of soil samples, depth, incubation time, and specific degrading population is shown in Table 2-3. One hundred grams of soil (oven-dry basis) were added to a 250 ml Erlenmeyer glass flask and mixed with 1 .0 mg technical grade carbofuran. The flasks were incubated

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46 Table 3-2. Substrates, soil depths and sampling time for the MPN experiments. Year Soil Substrate Soil Depth Sampling time cm days 1994 CT, CN [ 14 C-CAL] a 0-15,45-60 0 CT,CN [ ,4 C-URL] b 0-15,45-60 0 1995 CT, NT [ ,4 C-CAL] 0-15, 45-60 0, 1,3,7,14,28 CT, NT [ 14 C-URL] 0-15, 45-60 0, 1,3,7, 14,28 CT, NT 14 Carbofuran phenol 0-15 0,1,3,7, 14,28 CT, NT methylamine 0-15 0,1,3,7, 14,28 1996 CT, NN [ l4 C-URL] 0-15,45-60 0,1,3,5, 7, 14 a [ ,4 C-CAL] = [ 14 C-CAL] carbofuran b [ 14 C-URL] = [ 14 C-URL] carbofuran

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47 in the dark at ambient temperature (23 2 C), and once a week they were checked for water loss and deionized water was added to compensate for any water loss. At predetermined time intervals (Table 2.2), 1 g of soil was removed and placed in a sterile capped MPN tube that contained 9 ml of a sterile minimal mineral medium (MMA) consisting of the following ingredients per liter: K 2 HP0 4 4.8 g; KH 2 P0 4 1.2 g; NH 4 N0 3 1.0 g; MgS0 4 0.25 g; CaCl 2 2H 2 0, 40 mg; Fe 2 (S04) 3 1 mg. In addition, 10 mg of tryptone was added to the MMA prior to sterilization. Technical grade carbofuran (10 mg) and l4 C-carbofuran or 14 C-carbofuran phenol were added after autoclaving of the MMA. The MPN medium used to enumerate carbofuran degraders contained approximately 400 dpm ml" 1 of [ 14 C-CAL] carbofuran or 800 dpm ml' 1 of [ 14 C-URL] carbofuran, and the MPN medium used to enumerate carbofuran phenol degraders it contained approximately 500 ml" 1 dpm of [ 14 C-URL] carbofuran phenol. When enumerating methylamine degraders in soil, only unlabeled methylamine hydrochloride was added to the tubes as the sole source of carbon at a rate of 100 mg L' 1 of methylamine. After adding 1 g of soil to the MPN medium, five replicates of successive 5 to 10 fold dilution were made. All tubes were incubated in the dark at 28 C for four weeks. Control MPN tubes were treated identically as above except that soil was not added to the tubes. At the end of the incubation period, 100 uL of concentrated HC1 was added to each tube mixed and let stand for 4 hours. After standing for 4 hours, 0.5 ml of the MPN media was assayed for 14 C-activity remaining in solution as determined by LSC. Tubes were scored as positive when less than 60 % of the initial ,4 C-activity remained in

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48 solution. 14 C in all the control 14 C-MPN tubes were unchanged after 28 days of incubation. For methylamine degraders, tubes were scored positive if turbidity was observed in the MPN solutions in the tubes. Controls for the methylamine MPN tubes were all devoid of any turbidity. Statistical analysis MPN numbers were calculated using the Eureka (Borland International, Scotts Valley, CA). The equations calculate the MPN number and the 95% confidence limits of the MPN numbers. Data processed into the program include dilution factors, replicates at each dilution, number of positives at each dilution, and the weight of soil (g) initially added to MPN tubes. The mean value for the various degrading microbial populations were compared for differences between soils and differences between soil depth. Growth in the various degrading microbial populations were also compared with initial degrading populations. The results were used to determine if enhanced degradation of carbofuran is a function of increased growth or increased activity of the microorganism capable of degrading carbofuran. Characterization of Carbofuran-Degrading Soil Isolates Isolation and Metabolism of Carbofuran by Soil Isolates. Isolation procedures A batch enrichment technique was used to isolate carbofuran-degrading bacteria from soil. Technical grade carbofuran was applied to 100 g CT soil at a concentration of

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49 10 mg kg" 1 The flasks were incubated in the dark at ambient temperature (232 C), and the flasks were checked for water loss once a week and deionized water was added to compensate for any water loss. Carbofuran was reapplied every 4 weeks, and after 4 months of incubating 10 g of the soil was removed and inoculated into a 250 ml Erlenmeyer flask that contained 100 ml sterile MMA and carbofuran at a rate of 400 jug ml" 1 In addition to the batch enrichment technique, soil was also inoculated directly into sterile MMA that contained 400 ug of carbofuran ml" 1 The flasks were incubated at room temperature on a rotary shaker at 1 00 rpm. Once every two weeks, 10 ml of sample was transferred to a fresh MMA containing carbofuran (400 ug ml" 1 ). After 4 successive l-to-10 transfers, liquid cultures were free of soil particles, and a red pigmented color developed along with slight turbidity in some flasks. Small amounts of the culture fluids that developed the red color were streaked onto carbofuran-MMA (400 ug carbofuran ml" 1 MMA) agar plates. Colonies that developed on carbofuran-MMA agar plates were restreaked onto fresh carbofuran-MMA agar plates. Colonies that developed on new carbofuran-MMA plates were then streaked on Luria-Bertani (LB) agar plates for checking the purity of the isolated culture. Once colonies developed on the LB agar plates and determined to be a pure culture, they were restreaked again on carbofuran-MMA agar plates. For confirming that the isolate was capable of degrading carbofuran, a small amount of biomass from a colony was inoculated into liquid carbofuran-MMA containing a small amount of [ 14 CCAL] or [ 14 C-URL] carbofuran (30 Bq ml" 1 ) (Ou and Sharma, 1989). Those bacterial

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50 isolates were considered to have the capacity to degrade carbofuran if they mineralized [ 14 C-CAL] and/or [ 14 C-URL] carbofuran. Identification of soil bacteria isolated Unknown bacterial isolates capable of utilizing carbofuran as a sole source C for growth were sent to the Plant Pathology Department at the University of Florida for analysis of their fatty acid methyl-ester signature profiles using the MIDI system. In addition, each bacterial isolate was also identified by gram stain (Difco Laboratory, Detroit, MI), motility, and biochemical properties (Oxi/Ferm Tubes II, Becton Dickinson Cockeysville, MD). Mineralization of carbofuran as sole source of carbon The purpose of subsection was to determine the mineralization rate of carbofuran by the soil bacterial isolates as related to their growth. A small amount of biomass from a colony was inoculated to a 250 ml flask that contained 50 ml of carbofuran-MMA (50 ug carbofuran ml" 1 MMA). After 24-48 hours of incubation, 5 ml of the culture fluid were transferred into a fresh carbofuran-MMA. After the second transfer, one-day old cultures were inoculated into 250 ml Biometer flasks (Bellco, Vineland, NJ) containing 50 ml of MMA, and technical grade carbofuran, and [ l4 C-CAL] or [ l4 C-URL] carbofuran at rates of 50 jag ml" 1 and 4 KBq ml" 1 respectively. The side arms of the flasks contained 5 ml of 0.5 M KOH solution for trapping l4 C0 2 At predetermined time intervals, KOH was removed from the side arms and replaced with fresh KOH. Trapped 14 C0 2 in the KOH was quantified by LSC. After 72 hours, 10 ml of culture solution was removed and vacuum filtered through 0.2 (am Nylon filters (Micron Separations, Inc., Westboro, MA).

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51 The filters were washed three times with 5 ml of MMA. l4 C-activity in filtered extracts and washed filters were quantified by LSC. All samples were done in triplicate. The Reactivity detected in the KOH solution represented the portion of l4 C-carbofuran being mineralized, while 14 C retained on the filters represented the 14 C being incorporated into the biomass and 14 C in the filtered extracts represented the 14 C-activity in the cell free medium. Growth of soil bacteria In conjunction with the sampling of the KOH traps, growth of the soil bacteria in the carbofuran-MMA was measured using a Petroff-Hasuer bacteria counter (Becke et al., 1990). During the replacement of KOH traps, 100 pi of culture solution was also removed and diluted with an equal volume of a phosphate buffer (0.1 mol L" 1 K 2 HP0 4 and 0.1 mol L" 1 KH 2 P0 4 pH 7.2). After mixing, two drops of the diluted culture solution were deposited on the surface of the counting chamber along with a glass slip cover. After allowing 1 5 minutes for bacterial cells to settle down, cells were counted according to the procedure of Becke et al. (1990). All samples were done in duplicate. Mineralization of carbofuran without supplement of an extra N source. Metabolism of carbofuran as a sole source of N was studied in an identical experimental set-up with the study on the metabolism of carbofuran as a sole source of carbon. In this experiment the mineral media (MMB) consisted of the following ingredients L' 1 H 2 0: K 2 HP0 4 0.48 g; KH 2 P0 4 0.12 g; MgS0 4 0.25 g; CaCl 2 2H 2 0, 40 mg; Fe 2 (S04) 3 1 .0 mg. In addition, carbofuran (200 pg ml' 1 ) and glucose (325 pg ml' 1 ) were added to the MMB prior to sterilization. The radio-labeled carbofuran was applied

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52 at a rate of 6 Bq ml" 1 for [ l4 C-CAL] carbofuran and 80 Bq ml" 1 for [ ,4 C-URL] carbofuran. All glassware was acid washed in a 1% HC1 solution (3 hours) and triple rinsed with distilled water prior to use. The mineralization and mass balance of the l4 C-activity followed similar procedure described in the section of "Mineralization of carbofuran as a sole source of C." Mineralization of carbofuran in soil extract The metabolism of carbofuran in soil extract was also studied in a similar procedure used for determination of mineralization of l4 C-carbofuran in MMA as a sole source of C. The soil extract was prepared by autoclaving a mixture of 500 grams of CT surface soil and 500 ml of deionized water at 121 C for 1 hour (Ou, 1991). The soil suspension was carefully transferred to centrifuged tubes, and they were centrifuged for 20 minutes at 10000 rpm. The supernatant fluid was vacuum filtered through 0.22 mm cellulose acetate filters (Corning, Inc., Corning, NY). Technical grade and radio-labeled carbofuran were added to the soil extract at 50 ug ml' 1 and 80 Bq ml" 1 respectfully. Carbofuran Degradation Pathways Degradation and metabolite formation In this experiment, the degradation of carbofuran and the formation of its metabolites were monitored by means of TLC-autoradiographical analysis. Ten ml of one-day-old cultures grown in carbofuran-MMA was inoculated into a 1 000 ml Erlenmeyer flask containing MMA (500 ml), carbofuran (50 p.g ml" 1 ), and [ 14 C-URL] carbofuran (80 Bq ml" 1 ). After inoculation, each flask was tightly closed with a rubber

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53 stopper under which a stainless steel vial containing 0.5 ml of 5.3 mol L" 1 KOH was hung (Ou, 1991). At predetermined time intervals, the stainless steel traps were replaced with new traps containing fresh KOH. The removed KOH solutions were diluted with deionized water to 5 ml, and the trapped l4 C0 2 in the KOH was quantified by LSC. While changing the traps, 1 0 ml of culture solutions was removed from the flasks, and they were vacuum filtered through 0.2 mm Nylon filters. The filters were washed three times with 5 ml of MMA. After the volumes of the filtered fluids was determined, the 14 C-activity in the fluids were determined by LSC. 14 C-activity retained on the Nylon filters were quantified by LCS. Prior to extraction, the filtered extracts were acidified with 60 p.1 of concentrated HC1. They were then extracted twice with 25 ml of ethyl acetate. Moisture in ethyl acetate extracts were removed by anhydrous sodium sulfate and evaporated to dryness using a roto-evaporator. The residue in each flask was redissolved in 5 ml of anhydrous methanol and the methanol solutions were transferred to glass vials. The volume of the methanol solutions volume to 0.3 ml by gently passing N 2 gas over the extracts. Carbofuran and its metabolites in the concentrated extracts were separated and quantified by TLC-autoradiographical analysis and LSC as described previously in the soil metabolite section. Red-colored metabolite(s). The purpose of these experiments was to isolate, purify, and identify the redcolored metabolite associated with the metabolism of carbofuran. Three bacterial isolates

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54 capable of degrading carbofuran produced a water soluble red-colored pigment(s) when grown in carbofuran-MMA or carbofuran-MMB. One of these isolates was designated to be HPL strain and was used for this study. The HPL strain was inoculated into a carbofuran-MMA solution with a carbofuran concentration of 400 [ig ml" 1 and incubated on a shaker (100 rpm) for 1 week at 28 C. After one week, the culture solution developed a dark red color, and the culture fluid was centrifuged at 10,000 rpm for 20 minutes. The supernatant was collected and acidified to pH 3. The acidified supernatant was vacuum filtrated through a C lg reverse phase column (SepPack, Waters, Milford, MA). The retained pigment in the column was eluted with anhydrous methanol into glass vials. Methanol extract was spotted on a silica gel G preparative TLC glass plate (E. Merk, Darnstadt, Germany). The plate was developed to a distance of 15 cm using the same developing solvent used for the separation of carbofuran and its metabolites as described previously. A red-colored band traveled 2.0 cm (R f = 0.12) from where the extract was originally spotted. The gel associated with the red-colored band on the plate was scrapped and placed in a glass beaker. The gel was extracted 3 times with deionized water. The water extract was acidified to a pH of 3 and then vacuum filtered through a solid phase C 18 column. Methanol was used to remove the red-colored pigment from the column. The methanol extract was spotted on a preparative TLC plate for a second purification of the metabolite. The purified methanol extract was further purified by loading the extract into a silica gel (particle size 63-200 mm) column 2.5 x 20 cm (Spectrum Chromatography,

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55 Houston, TX). The methanol extract was eluted from the column under gravity. The mobile phase was a mixture of ethyl acetate: acetic acid (100:1), and fractions collected every 10 minutes. Fractions were combined, and the ethyl acetate:acetic acid was removed by a roto-evaporator as previously described. Residues were redissolved in anhydrous methanol (dried over sodium sulfate). Samples were sent to the Stine-Haskell Research Center (DuPont Agriculture Products, Newark, DE) for liquid chromatography/ mass spectrometry analysis (HPLC/MS). Cell-free culture extracts The purpose of these experiments was to determined the carbofuran degradation pathway by the carbofuran-degrading bacterial strain TA05. The TA05 strain was chosen for the isolation of unknown metabolite(s) because it degraded carbofuran more rapidly than the strain HPL. The procedures for the isolation of the unknown metabolites in oneday-old cell-free culture extracts were similar to the previously section. In this isolation procedure, the one-day-old cell-free culture extract was not further purified on preparative TLC plates or silica gel columns. Rather, the methanol extract from the solid phase C, g column was dried by flushing with N 2 gas and redissolved into methylene chloride. The methylene chloride samples were sent to Charles Schmindt of the Environmental Engineering Science Department at the University of Florida for gas chromatography/ mass spectrometer analysis (GC/MS).

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56 Genetics of Carbofuran Degradation The purpose of this study was to determine whether the genes responsible for the degradation of carbofuran reside on the chromosome or on plasmids. One of the isolates (TA strain) capable of degrading carbofuran harbored a single plasmid. The plasmid in the TA strain has been shown to have regions of homology with the plasmids of another carbofuran degrading isolate {Sphingomonas sp. strain CF06). The CF06 strain harbored five plasmids and these plasmids were shown to be responsible for the mineralization of [ ,4 C-URL] carbofuran (Feng et al., 1997a). Plasmid curing A small amount of biomass from a colony of the TA strain was inoculated into liquid LB medium and grown at 42 C with shaking for two days. Curing and isolation of the cured TA strain was done by Drs. X. Feng and A. Ogram at Washington State University in the Crop and Soil Science Department. The cured TA05 strain was provided by Drs. Feng and Ogram. This cured strain was obtained by growing the parent strain in LB broth at high temperature (42 C). This strain lost the capacity to mineralize both the [ 14 C-CAL] and [ 14 C-URL] carbofuran. Isolation of plasmids Plasmid DNA was isolated from the TA strain using a modified procedure developed by Feng et al. (1996a). Table 3-3 shows the procedures used to isolate plasmid from the strain in a large scale (500 ml) and a small scale (5-10 ml). Purified plasmid (chromosomal free) DNA was obtained by the CsCl density gradient ultracentrifugation

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57 Table 3-3. Procedures used to isolate plasmid DNA (Feng et al., 1996a). Procedure Large scale Small scale 1 Collect cells from culture by centrifugation (1 0,000 rpm for 1 5 min.). 500 ml 5-10 ml 2. Resuspend cells in 6.7% sucrose-50 mM Tris-lmM EDTA pH 8.0. Warn to 37C for 5-10 min. 5 ml 200 ul 3. Add lysozyme (100 mg ml in 25 mM Tris pH 8.0). Mix well and incubate at 37C for 30 min. 100 pi 10 (al 4. Add alkaline SDS solution (3% SDS, 0.2 M NaOH). Mix immediately with gentle shaking and incubate on ice for 1 0 min. 8 ml 400 ul 5. Add ice cold sodium acetate (pH 4.8). Mix gently and incubate on ice for 30-60 min. 6 ml 300 ul 6. Centrifuge (1 0,000 rpm for 1 5 min.) At 4C and recover the supernatant. 7. Add 0.6 volumes 2-propanol and mix well. Spin at 12,000 rpm for 15 min. and pour off supernatant 12 ml 600 ml 8. Resuspend DNA pellet in 0.5 ml TE buffer (10 mM Tris-HCl, ImM EDTA; pH 8.0) 500 ml 500 ul 9. Add phenol:chloroform:isoamy alcohol (25:24:1). Mix and spin for 2 min. at max. speed (15,000 rpm) 750 pi 500 ul 10. Add chloroform :isoamy alcohol (24:1). Mix and spin at max. speed for 2 min. 500 ul 500 pi 1 1 Transfer the aqueous phase to another tube and add 3 M sodium acetate. 50 ul 50 pi 12. Add two volumes of ethanol (100%). Mix well and spin at max. speed for 2 min. 1000 ^1 1000 pi 13. Wash pellets by adding 75% ethanol and invert tube and let tube air dry for 30 min. 1000 ul 500 pi 14. Resuspend pellet in TE buffer with RNAase (0.1 mg ml 1 )Store at 4C. 200 ul 30 pi

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58 procedure (Sambrook et al., 1989). The plasmid DNA was stored in sterile HPLC grade water at 4C until used. Introduction of TA05 plasmid into Pseudomonas fluorescens M480R strain Competent cells Competent cells of P. fluorescens M480R strain were the host to receive the plasmid DNA isolated from the TA strain by electroporation. A single colony of M480R strain was inoculated into 10 ml of LB broth and incubated overnight at 28 C on a shaker. One ml of the overnight culture was inoculated into 100 ml of LB broth and incubated as before. Cells were grown to 0.4 to 0.5 absorbance (600 nm) (exponential growth) and immediately chilled in an ice bath for 1 -2 minutes. Cells were then centrifuged for 10 minutes at 6,000 rpm. After the supernatant was discarded, the cells were resuspended into a cold 1 0 % glycerol solution and centrifuged again. This washing with glycerol was repeated a total of 5 times. After the final washing, cells were resuspended in 400 ul of the glycerol solution and subdivided into several microcentrifuge tubes (40 ul per tube) and stored at -70 C. Electroporation The plasmid DNA isolated from the TA05 strain was introduced into the competent cells of P. fluorescens M480R strain by electroporation. The competent cells (40 ul) were mixed with 1 ug of the plasmid DNA from the TA strain and electroporated with a Gene Pulser apparatus II (Bio-Rad, Richmond, CA). Electroporated cells were initially incubated in cold LB broth for 8 to 1 0 minutes followed by incubation at room temperature for 3 hours. Transformants were selected for their ability to grow on carbofuran-MMA agar plates with carbofuran as a sole source of C or N. The positive control in the electroporation was the introduction of the plasmid

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59 pRK415 into the M480R strain. pRK415 is the plasmid that carries the antibiotic resistance to tetracycline. The negative control was the substitution of water for the TA plasmid.

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CHAPTER 4 CARBOFURAN DEGRADATION IN SURFACE AND SUBSURFACE SOILS Introduction Carbofuran is a broad-spectrum N-methyl carbamate insecticide used to control certain soil borne insects and nematodes. In 1993, carbofuran was listed as the fifth most heavily used field insecticide in the US (Gianessi and Anderson, 1995). Although carbofuran is moderately persistent in soil, exhibiting an average field half-life of 50 days (Wauchope et al., 1992), its relatively high mobility (K^ = 30) (Sukop and Cogger, 1992) and toxicity toward mammals (Fahmy et al., 1970) and aquatic organisms (Trotter et al., 1991) raises concerns over its use in agriculture. These concerns are heightened by the fact that carbofuran residues have been detected in groundwater and in surface water (Erickson and Norton, 1990; Shahane, 1994). Measurements of the biodegradation potential of most pesticides are typically done on the top 1 5-20 cm of soil. This may over estimate the biodegradation potential in the subsurface horizons. The biodegradation potential of carbofuran was reportedly lower in the subsurface soil (> 50 cm depth) compared to surface soil (Buyanovsky et al., 1993) In addition, Mallawatantri et al. (1996) reported that the mineralization of carbofuran in the A horizon was significantly greater than in lower horizons for soils with 60

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61 no prior exposure to carbofuran. This reduction in the biodegradation potential of carbofuran in subsurface soils has been attributed to a reduction in microbial activity of the subsurface environments (Buyanovsky et al., 1993). The repeated use of carbofuran in agricultural fields has resulted in its accelerated degradation in soil (Suett, 1986). A consequence of the shortened half-life of carbofuran is a reduction in its insecticidal efficacy against target pests (Read, 1983). Enhanced degradation of carbofuran has been attributed to carbofuran-degrading microorganisms that develop after repeated exposure to the pesticide (Hendry and Richardson, 1988). The purpose of this study was to characterize the degradation of carbofuran in surface and subsurface soils under various carbofuran treatment histories and cultivation practices. This study characterizes the degradation of carbofuran in surface and subsurface soils according to its biodegradation potential and solvent extractable of carbofuran using [ 14 C-CAL] and [ 14 C-URL] carbofuran. The biodegradation potential is accessed by measuring the evolution of 14 C0 2 from soil applied with [ 14 C-CAL] and [ 14 CURL] carbofuran, and the disappearance of solvent extractable [ 14 C-URL] carbofuran from soil. In addition, 14 C-metabolites and 14 C recovery in soil are also determined. Results Soils The selected properties of the soils used for this study are given in Table 4-1 The annual application of carbofuran in the cultivated treated (CT) soils began in the March,

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62 Table 4-1. Selected characteristics of the Ellzey soil samples used in this study. Depth pH Moisture content Organic Carbon Sand Silt Clay cm ml kg" 1 gkg % CT soil 0-15 5.9 99 4.2 97 1 2 45-60 5.1 159 6.2 92 5 3 CN soil 0-15 6.9 151 15.5 92 5 3 45-60 5.2 181 5.7 95 2 3 NT soil 0-15 5.7 80 5.3 97 1 2 45-60 6.2 175 6.1 92 5 3 NNsoil 0-15 6.0 208 19.5 90 8 2 45-60 5.6 172 4.5 95 3 2

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1992, and continued through March, 1996. The low organic carbon content in the surface (0-15 cm) CT soils (4.2 g kg" 1 ) reflects the continuous cultivation in this plot for over ten years, while the higher organic carbon content in the cultivated nontreated (CN) surface soil (15.5 g kg 1 ) reflects the shorter period of time this field has been in cultivation (five years). The noncultivated nontreated (NN) surface soil has never been under cultivation which is evident by the high organic carbon content (19.5 g kg" 1 ). The typical organic carbon content in the surface horizon of the Ellzey soil series is between 10-30 g kg' 1 It is interesting to note that the noncultivated treated (NT) soil has not been under cultivation, but its organic carbon content in the surface soil is similar to that of the treated soil. This may result from traffic (next to a dirt road) that could result in soil compaction and reduced biological activity. The lower organic carbon content (less than 10 g kg" 1 ) in subsurface soils (45-60 cm) for all soils samples is a characteristic of Ellzey soil series. Mineralization of [ 14 C-CAL] in Surface and Subsurface Soils Surface soil TO15 cm depth). The mineralization of [ 14 C-CAL] carbofuran was rapid in the surface CT soils collected in 1994 and 1996 (Figure 4-1 A) with none exhibiting any lag periods. The CT94 mineralized 12 % of the applied l4 C-activity in the first 24 hours and over 90% after three days. The CT-96 exhibited an even greater rate of mineralization than the CT-94 with over 70 % of the [ 14 C-CAL] carbofuran applied being mineralized within 24 hours (Figure 4-1 A). This initial mineralization rate was significantly higher (a < 0.05) for the

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64 0 5 10 15 20 25 30 Time (days) Figure 4-1. Mineralization of [ 14 C-CAL] carbofuran in surface (0-15 cm) Ellzey soil: A) treated soil; B) control soils. Error bars represent the standard deviations of analysis.

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65 CT-96 compared to that collected in 1994. The fumigated CT-94 mineralized [ 14 C-CAL] carbofuran slowly with less than 1 1% of the 14 C-applied mineralized after 28 days of incubation. This is in sharp contrast to the nonfumigated CT soil samples collected in 1994 and 1996 (Figure 4-1 A). The mineralization pattern for [ l4 C-CAL] carbofuran in the control soils was somewhat different from the treated soils. In the CN soil, there was a three day lag period prior to rapid mineralization of [ 14 C-CAL] carbofuran with greater than 80 % of the applied l4 C being mineralized after 14 days (Figure 4IB). The NN soil initially did not mineralize [ 14 C-CAL] carbofuran rapidly with less than 20 % of the applied 14 C being mineralization after 28 days of incubation (Figure 4-1 B). When comparing cumulative l4 C0 2 production, there was a significantly greater amount of 14 C0 2 evolved in the CT soils than in the CN soil after 7 days of incubation (a < 0.05). There was a significant higher mineralization rate in the cultivated soils than in the nonculitvated soil (a < 0.05). Subsurface soil (45-60 cm depth). There was a day lag period of 3 days prior to the onset of rapid mineralization in the CT subsoil collected in 1994 (Figure 4-2A), while in the CT subsoil collected in 1996 the lag period was not observed. The CT-96 also mineralized a significantly higher (a < 0.05) amount of [ I4 C-CAL] carbofuran after 3 days of incubation compared to the CT-94. The CT-96 mineralized more than 10% and 80 % of the applied l4 C in 1 and 7 days, respectively. The CT sample collected in 1994 mineralized greater than 80 % of the

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100 0 5 10 15 20 25 30 Time (days) Figure 4-2. Mineralization of [ 14 C-CAL] carbofuran in subsurface (45-60 cm) Ellzey soil: A) treated soil; B) control soils. Error bars represent the standard deviations of analysis.

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67 applied [ 14 C-CAL] carbofuran within 7 days. After 3 days of incubation, the surface CT soils mineralized significantly (a < 0.05) more [ 14 C-CAL] carbofuran than the corresponding subsurface CT soil (Figures 4-1 A and 4-2 A). In the fumigated CT soil, the lag period lasted seven days prior to the onset of rapid mineralization of [ 14 C-CAL] carbofuran (Figure 4-2A). Mineralization of [ 14 C-CAL] carbofuran was very low in the subsurface control soils (CN and NN). The mineralization in these soils was slightly more than 10% of the applied ,4 C during the 28 days of incubation. The subsurface control soils (CN and NN) mineralized a significantly (a < 0.05) smaller amount of [ 14 C-CAL] carbofuran than the CT soils. Mineralization of [ l4 C-URL] Carbofuran in Surface and Subsurface soils Surface soils (0-15 cm depth). The mineralization of [ 14 C-URL] carbofuran in all CT soils was rapid with more than 30% of the 14 C applied being mineralized in the first three days of incubation (Figure 4-3 A). Mineralization for the surface soils peaked after 14 days of incubation. There were no differences in the initial degradation rates or total 14 C0 2 evolution from any of the surface CT-94, CT-95 and CT-96. There were differences between the mineralization rates of [ 14 C-CAL] carbofuran and [ l4 C-URL] carbofuran, with the [ 14 C-CAL] carbofuran exhibiting a faster mineralization than [ 14 C-URL] carbofuran.

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68 "a a. o 6 s 100 80 60 40 20 0 73 £ 80 O 60 O' U 3 40 20 0 CT soil '94' CT soil '95' CT soil '96* B CN soil '94' NT soil '95' NN soil *96' 20 25 30 Time (day) Figure 4-3. Mineralization of [ 14 C-URL] carbofuran in surface (0-15 cm) Ellzey soil: A) treated soil; B) control soils. Error bars represent the standard deviations of analysis.

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The control soil samples (CN, NT and NN) exhibited various patterns of [ ,4 C-URL] carbofuran mineralization (Figure 4-3B). In the CN soil, there was a lag period of 3 days prior to the onset of rapid mineralization of [ l4 C-URL] carbofuran. This mineralization reached a plateau after 14 days with 56 % of the applied 14 C being mineralized at the end of 28 days of incubation. The NT soil exhibited no lag period (note: based on the mineralization curve, a short lag period of less than 3 days might exit), and 1 1 % of the applied [ 14 C-URL] carbofuran was mineralized after three days, and it also peaked on day 14, with 67% of the applied l4 C being mineralized after 28 days of incubation (Figure 43B). The NN soil mineralized [ 14 C-URL] carbofuran slowly but steadily with less than 10% of the applied l4 C being mineralized after 28 days of incubation (Figure 4-3B). There was a significantly higher mineralization of [ l4 C-URL] carbofuran in the CT soils than in the CN soil during the first 3 days of incubation. After 3 days, there was no difference between CT, CN and NT soils for cumulative mineralization of [ l4 C-URL] carbofuran. There was a significantly faster (a < 0.05) mineralization of [ 14 C-URL] carbofuran in the CT soil than in the NN soil, however. Subsurface soils (45-60 cm depth). The mineralization of the CT soil collected in 1 994 exhibited a lag period of 3 days prior to the onset of rapid mineralization of [ l4 C-URL] carbofuran (Figure 4-4A). The CT soil samples collected in 1995 and 1996 rapidly mineralized [ l4 C-URL] carbofuran without a lag period (Figure 4-4A). Total cumulative ,4 C0 2 evolution in CT94, CT-95, and CT-96 was not significantly different despite CT-94 and CT-96

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70 100 0 5 10 15 20 25 30 Time (day) Figure 4-4. Mineralization of [ 14 C-URL] carbofuran in subsurface (45-60 cm) Ellzey soil: A) treated soil; B) control soils. Error bars represent the standard deviations of analysis.

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71 mineralizing on average 22% more of the [ 14 C-URL] carbofuran. For the CT-96, the total amount of [ 14 C-URL] carbofuran mineralized throughout the 28 days of incubation was significantly larger (a < 0.05) in the subsurface soil than in the surface soil, but not significant in 1994 or 1995. The CN soil samples exhibited a seven day lag period prior to the onset of mineralization of [ 14 C-URL] carbofuran. After the lag period, the mineralization was linear from days 7 to 21, with total cumulative 14 C0 2 production being 58% of the applied l4 C at the end of 28 days of incubation. In the NT soil, a lag period of 3 days was observed prior to the onset of rapid mineralization of [ l4 C-URL] carbofuran with 64% of the total applied 14 C being mineralized in 28 days. There was little mineralization of [ 14 CURL] carbofuran in the NN soil, with about 1.5 % of the applied 14C being mineralized after 28 days of incubation (Figure 4-4B). Once rapid mineralization occurred in the CN and NT soils, there was not a significant differences in the total cumulative 14 C0 2 production in the CT soils treated with [ 14 C-URL] carbofuran than in the CN and NT soils. During the 28 days of incubation, there was a significantly (a < 0.05) faster mineralization of [ I4 C-URL] carbofuran in the CT soils than in the NN soil. The CN and NT soils also exhibited a greater mineralization of [ 14 C-URL] carbofuran than the NN soil after the onset of rapid mineralization. I4 C Recovery in soil The recovery of the 14 C-applied in the Ellzey soils treated with [ l4 C-URL] carbofuran ranged from 67.2 to 98.3 % at the end of 28 days of incubation (Table 4-2).

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72 Table 4-2. Distribution of l4 C activity in Ellzey soil treated with [ 14 C-URL] carbofuran (10 mg kg 1 ) at the end of 28 days of incubation. Depth Evolved Extractable Nonextractable Recovery i4 C q 2 u c i4 C cm % of applied ,4 C CT soil '96' 0-15 46.7( 1.0) a 10.9(0.6) 36.4(2.7) 92.3(4.2) 45-60 77.2 ( 1.7) 4.2(0.3) 16.9(0.5) 95.4(3.0) CT soil '95' 0-15 56.3( 4.2) 5.2(0.4) 27.3(2.6) 88.8(3.2) 45-60 57.6(10.5) 6.9(0.8) 32.2(7.8) 98.5(2.0) CT soil '96' 0-15 46.1(4.1) 4.0(0.6) 31.1(4.9) 85.7(2.2) 45-60 74.0(0.9) 3.8(0.8) 20.3(0.2) 98.1( 1.3) CN soil '94' 0-15 56.2(1.2) 2.8(2.1) 32.5(8.3) 89.3(13.3) 45-60 59.2 (3.2) 9.8(4.6) 28.4(3.7) 92.6( 7.1) NT soil '95' 0-15 67.6( 1.6) 5.2(1.4) 19.9(4.5) 92.8(5.8) 45-60 64.0(5.7) 5.7(1.0) 28.6(1.1) 98.3(2.1) NN soil '96' 0-15 8.6( 1.6) 56.9(4.3) 1.6(0.7) 67.2(2.9) 45-60 1.7(0.2) 86.2(3.4) 10.1(1.0) 98.0(2.2) a = values within parentheses are the standard deviations of the measurements

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73 Excluding the NN surface soil, l4 C recoveries ranged from 85.7 to 98.3%. The CT soils collected in 1994, 1995 and 1996 were the only Ellzey soils to exhibit a different pattern of l4 C distribution between its surface and subsurface soils. The CT subsurface soils mineralized between 1 to 31% (on average 20 %) more of the applied 14 C activity than the surface soils, while the CT surface soils averaged about 10% more nonextractable l4 C bound residues. The CT subsurface soils exhibited greater percentage of carbofuran mineralized than all other Ellzey soils. The CT, CN, and NT soils were capable of mineralizing the aromatic ring of carbofuran and exhibited similar trends of 14 C-activity distribution. The trends being that these soils had low recovery of extractable I4 C-activity (2.8-10.9%) with nonextractable 14 C-activity ranging from 16.9-36.4%. The NN soils were the only soils that did not mineralize the aromatic ring structure extensively. These soils had larger quantities of extractable l4 C-activity (56.9-86.2), but smaller quantities of nonextractable 14 C (1.610.1%). Carbofuran metabolism in the Ellzey soils In this study, [ l4 C-URL] carbofuran was employed to monitor the disappearance of carbofuran and its toxic metabolites 3 -hydroxy lcarbofuran and 3-ketocarbofuran in the Ellzey soils, as well as less toxic phenolic products. The total toxic residues (TTR) include carbofuran, 3 -hydroxy lcarbofuran and 3-ketocarbofuran. Determination of metabolites led to the estimation of the half-lives for TTR in the Ellzey soils and possible elucidation of the degradation pathway of carbofuran. The TLC R f values for carbofuran and its toxic metabolites 3-hydroxylcarbofuran and 3-ketocarbofuran were 0.42, 0.19 and

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74 0.37, respectively. The R f values for the phenolic metabolites carbofuran phenol, 3hydroxyl-carbofuran phenol, and 3-ketocarbofuran phenol were 0.73, 0.53, and 0.59, respectively. Degradation pathway of carbofuran in Ellzey soils The distribution of extractable I4 C-carbofuran and its 14 C metabolites varied for Ellzey soil samples collected in 1994 and 1996. In the CT soil samples collected in 1994 and 1996, carbofuran phenol was the only metabolite detected in both surface and subsurface soils (Tables 4-3 and 4-4). There was also an unknown polar metabolite(s) (R f = 0.0) detected in all soil samples except in the CN subsurface soil. Carbofuran phenol, 3-ketocarbofuran, and 3-ketocarbofuran phenol were occasionally detected in CN surface and subsurface soil samples. 3 -Hydroxy lcarbofuran and 3-ketocarbofuran were detected after 3 days of incubation in the NN surface soil samples, while carbofuran phenol and 3ketocarbofuran phenol were detected only once, and that was after a week of incubation. Carbofuran phenol and 3-ketocarbofuran were occasionally detected in the NN subsurface sample (Table 4-4). The proposed degradation pathway for carbofuran in the CT, CN, and NN of the Ellzey soils are depicted on Figure 4-5. Disappearance of carbofuran in Ellzey soils. The disappearance of the carbofuran in the CT soils followed similar pattern for the mineralization of [ 14 C-CAL] carbofuran in the CT soils. Initial rapid disappearance of carbofuran was observed in the CT surface soils collected in 1994 and 1996 with less than 1% of the applied l4 C remaining after 7 and 3 days, respectively (Tables 4-3 and 44). The subsurface CT soils sampled in 1994 exhibited a lag period of 3 days prior to

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75 Table 4-3. Carbofuran and carbofuran metabolites detected from solvent extracts in cultivated treated soils and cultivated nontreated soils in 1994. Soils were treated with [ ,4 C-URL] carbofuran. Days Carbofuran 3-Keto TTR a Carbofuran 3-Keto Unknown carbofuran phenol carbofuran R f = 0.0 phenol % of applied 14 C CT soil (0-15 cm) 1 65. 8( 0.1) b 0.0 65.8( 0.1) 0.9(1.2) 0.0 2.5(1.7) 3 19.7( 3.6) 0.0 19.7(3.6) 2.3(2.3) 0.0 9.3(8.5) 7 0.8( 0.2) 0.0 0.8( 0.2) 0.0 0.0 6.8(2.4) CT soil (45-60) 1 83.8( 3.8) 0.0 83.8(3.8) 0.0 0.0 0.0 3 78.4( 2.2) 0.0 78.4( 2.2) 2.0(0.5) 0.0 0.8(0.54) 7 0.6( 0.1) 0.0 0.6( 0.1) 0.0 0.0 7.0(1.3) CNsoil (0-15 cm) 3 37.5(14.0) 0.0 37.5(14.0) 0.0 0.0 0.0 7 3.8( 5.1) 3.1(4.7) 6.9( 0.4 ) 0.0 0.0 0.7(1.0) CN soil (45-60 cm) 3 72.7( 5.0) 0.0 72.7( 5.0) 0.0 0.0 0.0 7 71.7(22.6) 15.6(9.1) 87.2(31.7) 0.0 0.0 0.0 14 33.5(17.0) 0.0 33.5(17.0) 2.5(0.8) 3.4(0.3) 0.0 21 10.1( 5.2) 0.0 10.1(5.2) 0.0 7.2(0.6) 0.0 a TTR = total toxic residues b = values within parentheses are the standard deviations of the measurements

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o CO T3 U 76 a o .o £ o 9 "S S o ^ 1 o o O & §1 2 1 ^ i a o CO •a q >n o d S O | 1 u o o q o CN d CN q, CN o d o d o d o in t— 1 u o d d CO d o o d o d o d CN O O d o d o d o d q d o CO d o d I in -a u 1 -a > u C o o d o d oo CO d oo d O d ^3q, d o o d o d CN O d o d o d in d in — s 00 CN q oo 00 rn CN 1 o in U 1 -a o d o d o d o d ri CN O CN, d o d, in' d q CO O d Co" o d o c o d Co d d d d /— s ON cn o o> 00 s 1 I <-(— I o 1 I & T3 CO u CO 1 1 i >

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77

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78 rapid disappearance of carbofuran with less than 1% of the applied 14 C remaining after 7 days. The CT subsurface soil collected in 1996 degraded carbofuran significantly more rapidly after 3 days than the CT soil collected in 1994. In both of the years the CT soils were sampled, the surface soils degraded carbofuran significantly faster than the subsurface soils. The disappearance of carbofuran in the control CN soil (Tables 4-3) did not follow the same pattern as those of the mineralization of [ 14 C-CAL] carbofuran (Figure 41). The lag period prior to rapid disappearance of carbofuran in the surface soil appeared to be shorter than for the mineralization of [ 14 C-CAL] carbofuran, less than 3 days. The lag period prior to rapid disappearance of carbofuran in the CN subsurface soil was shorter than for the mineralization of [ 14 C-CAL] carbofuran as well. In the NN soil, the pattern was similar for the disappearance of carbofuran and mineralization of [ 14 C-CAL] carbofuran for both the surface and subsurface soils with little degradation of carbofuran occurring. The CT surface and subsurface soils collected in 1996 exhibited a significantly more rapid loss of carbofuran than any of the control soils. The CN surface soil degraded carbofuran more rapidly than the NN surface soil. Half-life values of carbofuran based on disappearance of carbofuran in Ellzey soils. The half-life values for carbofuran in Ellzey soils were estimated based on the disappearance of carbofuran using first-order kinetics. The disappearance rate constants (k,) and half-lives (t 1/2 ) are listed on Table 4-5. Most of the rates approximate first-order kinetics as indicated by correlation coefficients (r 2 ). The half-life value for carbofuran in

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79 Table 4-5. The estimated half-life values for carbofuran disappearance, total toxic residues (TTR) disappearance and the hydrolysis rates carbofuran in Ellzey soils. TTR and carbofuran Hydrolysis of disappearance carbofuran ueptn 1. k l l l/2 r 2 r cm days" 1 days days" 1 days xlO 2 xlO 2 CT soil '94' 0-15 71.1 1.0 0.990 82.1 1 0.919 45-60 74.7 1.0 0.863 25.0 3 0.885 CT soil '96' 0-15 80.0 1.0 0.829 57.9 1 0.884 45-60 83.1 1.0 0.999 25.6 3 0.965 CT soil 'fum' 0-15 ND a ND ND 0.4 170 0.989 45-60 ND ND ND 4.1 17 0.931 CN soil '94' 0-15 45.7(47.2) b 2(2) 0.971(0.979) 13.1 5 0.946 45-60 7.8(10.5) 9(7) 0.842(0.930) 1.0 68 0.891 NN soil '96' 0-15 1.0( 1.0) 70(62) 0.637(0.445) 1.0 71 0.995 45-60 0.6( 0.6) 117(117) 0.987(0.988) 0.4 176 0.983 a ND = not determined b value within the parentheses represent the half-life values for carbofuran disappearance if they are different from the TTR disappearance

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80 the NN surface soil collected in 1 996 was not followed well by first order rate kinetics, as evidenced by the poor r 2 (0.445). Due to the rapid degradation of carbofuran in the CT surface soil collected in 1 996, it was not possible to obtain good data for the half-life estimation. The half-lives of carbofuran in the CT surface soil samples ranged one to less than one day and from 1 to 3 days for the subsurface soils (Table 4-5). The CN soils that had not been previously exposed to carbofuran exhibited halflife values that are not characteristic of soils with no history of exposure to carbofuran, but rather similar to a soil with a prior exposure history. The calculated half-life values for carbofuran were 2 and 7 days in the surface and subsurface CN soil, respectively. The NN surface and subsurface soils exhibited little mineralization of [ 14 C-CAL] carbofuran (Figures 4IB and 4-2B), and this was evident in the half-life values for carbofuran being 62 and 1 1 7 days for surface and subsurface soils, respectively. Half-life values of TTR disappearance and carbofuran hydrolysis in Ellzey soils. The half-lives of TTR disappearance were estimated based on the disappearance of TTR in the Ellzey soils, and the half-lives of the hydrolysis of [ 14 C-CAL] carbofuran in the soils were estimated based on the evolution of ,4 C0 2 The hydrolysis of carbofuran and the mineralization of [ 14 C-CAL] carbofuran should coincide since both are measures the hydrolysis of the carbamate group from carbofuran. The half-life values were estimated based on the first-order kinetics for biodegradation (Table 4-5). Most of the rates approximate first-order kinetics as indicated by their r 2 values. In the CT soils, the disappearance of carbofuran and TTR were similar since none of the two toxic metabolites 3 -hydroxy lcarbofuran and 3-ketocarbofuran were detected. The half-lives of

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TTR disappearance in the CT soils was one day for surface soils, and 1 to 3 days for subsurface soils. However, for the fumigated CT soils collected in 1994, the half-life values were much larger than for the CT soils collected in 1994 and 1996. The calculated half-lives for the fumigated surface and subsurface soils were 1 70 and 1 7 days, respectively. The half-life values for the hydrolysis of carbofuran in the CN soil ranged from 2 to 5 days in surface soil and from 9 to 68 days in subsurface samples. The large variations of the half-life values in the CN samples reflects the differences in the observed lag periods between the TTR disappearance and mineralization of [ l4 C-CAL] carbofuran in the CN soil samples. The lag periods for TTR disappearance were shorter than the lag periods for the mineralization of [ 14 C-CAL] carbofuran. The NN soil samples exhibited little degradation of carbofuran and this was evident in their calculated half-lives. The half-lives of carbofuran in surface NN soils samples ranged from 70 to 71 days and from 1 17 to 175 days in the subsurface soils. Discussion Metabolism of [ 14 C-CAL and 14 C-URL] Carbofuran in Ellzev SoilMineralization of [ 14 C-CAL1 vs. [ 14 C-URL] carbofuran. As mentioned above, the mineralization of [ 14 C-CAL] carbofuran was more rapid than that of [ l4 C-URL] carbofuran in all samples of the Ellzey soil. This reflects that the linkage of the carbamate group is readily subject to biological hydrolysis resulting in the

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release of l4 C0 2 In addition, the aromatic ring structure is more resistant to microbial attack than is the carbamate group. The carbonyl carbon from the carbamate group of carbofuran is already at its highest oxidation state and thus can not be assimilated by aerobic heterotrophic organisms. At present, the mechanisms have not been identified for the enzymatic cleavage of the ring structure of carbofuran and the resulting product(s), as well as subsequent degradation pathways that lead to the formation of the final oxidation products, C0 2 and H 2 0. Lag period for carbofuran degradation. The CT soils that had been continuously treated with carbofuran exhibited more rapid mineralization of [ 14 C-CAL] carbofuran and loss of TTR than the soils CN and NN that did not receive carbofuran. The differences in the mineralization rates of the freshly applied [ 14 C-CAL] carbofuran were evident in the duration of the initial lag periods in the CT soils and those of the nontreated soils (Figure 4.1). Suett (1986) reported that the most notable losses of carbofuran from degradation were in the initial stages of the incubation for soils exhibiting enhanced degradation. Other researchers observed that the lag periods were greatly reduced in soils exhibiting enhanced degradation of carbofuran (Harris et al., 1984; Camper et al., 1987; Turco and Konopka, 1990). This lag period was correlated with the duration between the last application of carbofuran, with the most recent application exhibiting shorter lag periods (Suett et al., 1993). Reduction in the lag periods can be achieved by pre-treating soils with carbofuran (Read, 1983; Chapman et al., 1986; Dzantor and Felsot, 1989), and other aryl N-methylcarbamates (Dzantor and Felsot, 1989; Dzantor and Felsot, 1990; Morel-Chevillet, 1996).

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83 In this study, the initial lag periods prior to the onset of rapid carbofuran degradation were similar in the CT surface soils taken in 1 994 and 1 996, yet the degradation rates were significantly different. A lag period of 3 days was observed for the CN soils prior to the onset of the rapid degradation of carbofuran. The CT soil collected in 1 996 exhibited a significantly greater capacity for degrading carbofuran than soil collected in 1994. This greater capacity suggests that a larger population of carbofuran-hydrolyzing microorganisms were present in the CT soil collected in 1 996 than in 1994. This degrader population probably developed from the two additional annual field applications of carbofuran received by the CT surface soil. Hendry and Richardson (1988) observed similar trends in soils repeatedly treated with carbofuran in a laboratory study. They reported that on a soil with no previous history of carbofuran treatment, only 7% of the applied [ 14 C-CAL] carbofuran was mineralized in 3 days, but in subsequent treatments, the mineralization rate progressively increased. After the third treatment, more than 90 % of the applied [ 14 C-CAL] carbofuran was mineralized in 19 hours. The increase in the mineralization of [ 14 C-CAL] carbofuran was linked to an increase in the number of carbofuran-hydrolyzing microorganisms after each additional treatments (Hendry and Richardson, 1988). The NN soil samples never exhibited any lag period, and carbofuran was mineralized at a slow but constant rate (Figure 4. IB). The constant rate of carbofuran degradation in the NN soils may suggest that: 1) the hydrolysis of the carbamate group from carbofuran was not linked to any growth in the microbial population (Alexander and

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84 Scow, 1989); or 2) the initial microbial population capable of hydrolyzing carbofuran was too small to be capable of significantly degrading the pesticide (Alexander, 1994). Half-life Values for Carbofuran Disappearance and Hydrolysis. Surface soil (0-15 cm). Half-life values for carbofuran disappearance or hydrolysis were smaller than the published values (Felsot et al., 1981; Suett, 1986; Turco and Konopka, 1990; Parkin and Shelton, 1992). Published half-life values ranged form 3 to 10 days and only accounted for the disappearance of carbofuran, and did not include the two toxic oxidation products of carbofuran. The rapid degradation in the CT soils may reflect the number of annual field applications to the CT soils (3 to 5 times); other enhanced soils in the literature generally ranged from 1 to 3 applications of carbofuran. It is interesting to note that the half-life values (2 to 5 days) of carbofuran and TTR disappearance in the CN soil were smaller than those in the NN soils. The CN soils were not treated with carbofuran, yet rapid degradation of carbofuran occurred. This suggests that the CN soils might have been previously exposed to carbofuran or structurally similar compounds. This assumption is based on the observation that the half-lives for carbofuran in soils not previously exposed to the pesticide were much larger, ranging from 18 to 90 days (Wauchope et al., 1992). Furthermore, half-life values for carbofuran degradation in the NN surface soil were much larger (62 days). Similar rates of carbofuran degradation have been reported to occur in the fence rows near treated field plots (Felsot et al., 1981; Racke and Coats, 1990) and adjoining fields

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85 (Suett, 1986). The contamination by carbofuran residues in the CN soils may have resulted from inaccurate record keeping of pesticide application, pesticide drift during application, or contamination via farm machinery that was used in both fields. For the pesticides MBC (benzimidazol-2-yl carbamate) and EPTC (s-ethyl N,N-dipropyl carbamothioate), it has also been demonstrated in the laboratory that a small amount of soil (2% of total) from an enhanced field when inoculated into a soil not previously exposed to the pesticide can trigger a rapid increase in the degradation of the pesticide (Yarden et al., 1987; Dick et al., 1990). Thus, another possibility is that small amounts of inoculum from the CT soil could have been inadvertently transferred into the CN soils via farm equipment. Subsurface soils (45-60 cm) The CT surface soils degraded carbofuran more rapidly than the corresponding subsurface soils, yet the half-life values for the degradation of carbofuran in the CT subsurface soils (1 to 3 days) were comparable to those of other surface soils with problems of enhanced degradation (Felsot et al., 1981; Parkin and Shelton, 1992). This is the first report of enhanced degradation extending down into a shallow subsurface horizon. Previous reports on pesticide degradation in subsurface soils found no link between pesticide degradation and growth of the microbial biomass (Muller et al., 1992). Thus, degradation of pesticides in subsurface soils was considered to be a cometabolic processes (Moorman, 1990). Recent studies on the degradation of carbofuran in subsurface soils have dealt exclusively with soils having no previous history of exposure

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to the pesticide (Buyanovsky et al., 1993; Mallawatantri et al., 1996). These studies found that the degradation of carbofuran in subsurface soils decreased with depth. Source of Carbofuran Degraders in Subsurface Soils. The question arises as to the source of the microorganisms in the subsurface soil that are capable of degrading a xenobiotic chemical such as carbofuran. Do the microorganisms capable of degrading carbofuran develop in subsoils as a result of transport from the surface soil? Or, do these microbes develop independently in the subsurface soil through natural adaptation? In an aquifer, Aelion et al. (1987) linked the adaptation of p-nitrophenol (PNP) degrading microorganisms with an increase in the number of PNP degraders in the sediment. This adaptation was linked to the concentration of PNP, with the concentrations < 14.0 u£ kg" 1 soil being unable to sustain any mineralization of PNP, although adaptation developed at higher concentrations > 3 1 .0 fig kg" 1 soil. Similar findings for carbofuran degradation in soils were observed, and at concentrations below 100.0 ug kg" 1 soil enhanced degradation was not induced (Hendry and Richardson, 1988; Chapman et al., 1988). If a threshold concentration of carbofuran is needed for the development of enhanced degradation in soils, what is the concentration of carbofuran leaching into the shallow subsurface soil (45-60 cm) that triggers enhanced degradation in the soil? The computer model "Chemical Movement in Layered Soils" (CMLS) version 4.0 (Nofziger and Hornsby, 1 987) was used to simulated the mass fraction of carbofuran leaching into the subsurface soil (45-60 cm). The organic carbon contents from the CT soils coupled

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87 with the estimated half-lives from the CT soils samples collected in 1 994 along with water content at field capacity for the Ellzey fine sand were entered into the program. In addition, a carbofuran application rate of 4500 fig kg" 1 of soil was assumed based on the field application rates of carbofuran. Rainfall and evapotranspiration data were obtain from local Gainesville data collected in 1985. The computer model simulated a carbofuran concentration of 22 ug kg" 1 soil would reach the 45-60 cm depth in 8 days. From the CMLS model simulation, there is a very low probability of carbofuran concentration in the subsurface soil exceeding the threshold values needed for the development of enhanced degradation to occur. It appears that subsurface microorganisms capable of degrading carbofuran may have originated from the surface soils. Since local Gainesville rainfall and evapotranspiration data was used in the simulation, confidence in the estimated carbofuran concentration values should be taken with some skepticism. If carbofuran-degrading microorganisms in surface soil were transported through the soil profile, by what mechanisms would this movement be accomplished? The transport of microorganisms in soil has been shown to be greatly limited by the structure of the soil (Smith et al., 1985), with most of the transported occurring through macropores (Smith et al., 1985; Fontesetal., 1991). Transport through an agricultural field has been shown to be minimal since cultivation causes the soil to be structureless and devoid of large pores, thus retarding the movement of bacteria (Smith et al., 1985). In sandy soils, Wollum and Cassel (1978) and Fontes et al. (1991) measured only a small amount (< 14%) of bacteria recovered from in the effluents for a fine sand (Ellzey soil is

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88 a fine sand). While in course sand columns recoveries from the effluents was greater than 90% (Fontes et aL, 1991 ; Gannon et al., 1991). In other packed columns, PNP degrading bacteria were found to be mostly retained in the top 2.5 cm, while below the top 7.0 cm, little of the PNP degrading bacteria were detected (Kelsey and Alexander, 1995). Degradation of phenanthrene was also found to be limited to the top portion of packed columns with little degradation of phenanthrene occurring in lower portions of the columns (Devare and Alexander, 1995). Another possible mechanism for the transport of carbofuran degrading organisms from surface to subsurface soils would be through the cultivation practices used in growing potatoes. It has been shown that soils under conventional tillage exhibit little spatial variability in enhanced degradation of carbofuran (Parkin and Shelton, 1992). The lack of spatial variability is a result of homogenization of soil during tillage. In the potato fields at the Hastings site, mounds were cultivated down to a 50 cm depth (D.P. Winegarten, personal communication) and the subsurface soils were collected from 45 to 60 cm depth. Thus, it would appear that the transport of surface carbofuran-degrading microorganisms to subsurface soil would probably be achieved through conventional tillage practices and movement with percolation waters. Distribution of [ l4 C-URL] Carbofuran It was noted earlier that the CT soil was the only sample of the Ellzey soils that exhibited a different pattern of 14 C distribution between its surface and subsurface soil. These soils exhibited a greater percentage of [ l4 C-URL] carbofuran mineralized in the

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89 subsurface soil than in surface soil, but the CT surface soil had a greater percentage of soil bound residues than the CT subsurface soil. Soil bound residues of carbofuran are a result of the carbofuran phenolic metabolites undergoing oxidative coupling to organic matter and thus becoming covalently bound to the organic matter (Getzin, 1973; Willems et al., 1996). Willems et al. (1996) found that carbofuran phenol was susceptible to oxidative coupling in the presence of horseradish peroxidase and hydrogen peroxide. Other phenolic compounds have also been shown to bind readily to organic matter in soil via abiotic and biotic means (Bollag et al., 1983; Pal et al., 1994). In laboratory incubation studies, [ 14 C-URL] carbofuran degradation in surface soils has been shown to result in the formation of large amounts (59.3-94.5 %) of soil bound residue in both enhanced (Talebi and Walker, 1993) and nonenhanced (Getzin, 1972; Ou et al., 1982) soils, while in flooded soils, the degradation of [ 14 C-URL] carbofuran results in a small amount of soil bound residue, and a large accumulation of carbofuran phenol (Venkateswarlu and Sethunathan, 1979). For the Ellzey soil series, soil water contents at field capacity was 156 g kg" 1 for 0-15 cm and 54 g kg" 1 for 45-60 cm soil depth. Soil water contents of CT soil samples were determined to be 99 g kg" 1 at 0-15 cm and 155 g kg" 1 at 45-60 cm (Table 4.1). Thus, CT soil samples collected from the 45-60 cm depth would have many of its pores filled with water and soil samples at this depth would be more anaerobic. Reduced conditions in the subsurface CT soils may leave carbofuran phenol less susceptible to oxidative coupling by nonspecific abiotic and biotic reactions (Pal et al., 1994), but still susceptible to microbial attack and mineralization.

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90 The reasons are not clear as to why the CN and NT soil samples did not follow similar l4 C distribution patterns as the CT soil samples. One reason for the difference in 14 C activity distributions may be the greater organic content in the CN soil sampled. Greater organic contents would be expected to reduce the bioavailability of carbofuran and its metabolites to microbial attack (Ogram et al., 1985), but still leave it susceptible to abiotic or biotic (extracelluar enzymes) attack. The result would be greater oxidative coupling to organic matter, forming bound residues. Another possible reason may be the reduced activity of the soil microorganisms toward the mineralization of [ 14 C-URL] carbofuran in the CN and NT soil samples. Lower levels of microbial activity would result in slower mineralization of carbofuran phenol, leaving more chance for oxidative coupling with the organic matter to occur. In the NT soil samples, the lack of added fertilizers to the soil may explain reduced rates of bound residues. The NT soil samples may be nutrient limited since these soils were not in cultivation, and the growth of fungi and other organisms that produce peroxidases could be reduced. The reduced activity of these organisms would reduce the oxidative coupling of the phenolic groups of carbofuran with the organic matter of soil. This, in turn, would reduce the amount of soil bound residues of carbofuran. Metabolites of ["C-URL] Carbofuran The degradation of carbofuran exhibits different patterns of degradation for each of the Ellzey soils with various carbofuran exposure histories (Tables 4.4 and 4.5). Degradation of carbofuran, for both [ l4 C-CAL] and [ l4 C-URL] carbofuran, in the surface

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and subsurface soils were clearly enhanced. Because carbofuran phenol was only the metabolite in the soils in conjunction with rapid 14 C0 2 production from the soils treated with [ 14 C-CAL] carbofuran, hydrolysis was the only step of the initial degradation of carbofuran in the enhanced CT soils. Oxidation of carbofuran to 3-hydroxylcarbofuran did not appear to occur in the CT soils. As a result, carbofuran phenol was either mineralized after hydrolysis, incorporated into the microbial biomass, or converted to bound residues. Turco and Konopka (1990) and Talebi and Walker (1993) reported that carbofuran in enhanced soils was mainly degraded through biological hydrolysis to carbofuran phenol. Getzin (1973) found that the application of [ l4 C-URL] carbofuran phenol resulted in a rapid incorporation of ,4 C to soil organic matter (bound residue formation). Degradation of carbofuran in enhanced soils can be inhibited up to 70-80% in the presence of the organophosphates paraoxon (Talebi and Walker, 1994) and enthoprop (Racke and Coats, 1990), respectively. The monoxygenases that have cytochrome P 450 as their active center are inhibited by piperonyl butoxide. Application of piperonyl butoxide to an enhanced soil in the laboratory reduced the degradation of carbofuran by 10% (Talebi and Walker, 1994). These studies suggested that carbofuran in enhanced soils was metabolized mainly through hydrolysis of the carbamate linkage resulting in the formation of carbofuran phenol, methylamine and C0 2 Oxidation of carbofuran to 3hydroxyl-carbofuran may not occur in the CT soil, or only as a minor pathway.

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Practical Implication Prior application of the soil fumigant telone II to the CT soil inhibited the degradation of carbofuran in the soil and blocked enhanced degradation (Figure 4-1 A). Suett (1986) reported a similar effect of the fumigant diazomet despite repeated applications of carbofuran (Suett, 1986). Another pesticide susceptible to enhanced degradation is the organophosphate fenamphos (Ou et al., 1994; Ou and Thomas, 1994), but when used in conjunction with telone II enhanced degradation of fenamphos did not develop (L.-T. Ou, personal communication). The benefits of using soil fumigants, such as diazomet and Telone II, with carbofuran in enhanced soils are: 1) increased efficacy of carbofuran in the root zone; and 2) reduced risk of carbofuran contaminating the groundwater due to rapid degradation in subsurface soil.

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CHAPTER 5 INFLUENCE OF MICROBIAL POPULATIONS ON ENHANCED DEGRADATION OF CARBOFURAN IN SOIL Introduction The degradation of carbofuran in soils with no previous history of exposure to the pesticide has been shown to be both abiotic and biotic in origin (Getzin, 1973). Repeated exposure of carbofuran to field soil may result in enhanced degradation of the pesticide (Felsot et al., 1981; Read, 1983). Although this enhancement is of biological origin, attempts to correlate the degree of enhanced degradation in soil with key soil physical, chemical and biological properties have been unsuccessful (Dzantor and Felsot, 1990; Charnay and Fournier, 1994). Turco and Konopka (1990) reported a decrease in total biomass after applying carbofuran to a laboratory soil that exhibited enhanced degradation of the pesticide. The inability to link the increase in the total microbial biomass to carbofuran degradation is possibly due to a small or specialized portion of the biomass that is responsible for degradation of the pesticide in enhanced soils. This raises the following question regarding enhanced biodegradation: 1) does enhanced degradation of carbofuran result from an increase in specific microbial biomass capable of degrading the pesticide; or 2) does enhanced degradation result from an 93

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94 increase in enzyme activity specific for carbofuran degradation? Several studies suggest that increases in the number of carbofuran-degrading microorganism explains enhanced degradation (Hendry and Richardson, 1988; Dzantor and Felsot, 1989; Dzantor and Felsot, 1990). Other studies (Racke and Coates, 1988; Merica and Alexander, 1990), however, fail to link an increase in the number of carbofuran-degraders with enhanced degradation and favor the idea of increased activity of carbofuran-degrading microorganisms. At present, little information is available on the growth response of carbofuran-degrading microorganisms following the application of carbofuran to enhanced soils. The purpose of this study was to investigate possible relationships between degradation of carbofuran in soils and carbofuran-degrading microorganisms. The growth patterns of carbofuran-degrading microorganisms in soils with various exposure histories to the pesticide were investigated with a 14 C-MPN (Most-Probable-Number) technique. In addition, growth patterns of degraders microbial populations of methylamine and carbofuran phenol, the hydrolysis products of carbofuran, were determined using MPN techniques. Results Carbofuran Degrading Populations in Field Soils After four annual field applications of carbofuran to the CT soil in 1995, the number of carbofuran hydrolyzers in the CT surface soil increased significantly from 9.2

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95 x 10 4 cells g" 1 in 1994 to 1.2 x 10 7 cells g"' soil in 1995 (Table 5-1). The number of carbofuran ring degraders in the CT surface soils remained unchanged after 3, 4, and 5 consecutive annual field applications of carbofuran. The number of carbofuran ring degraders in the CT soils ranged from 1 1 to 49 cells g" 1 of soil (Table 5-2). The number of the carbofuran ring degraders in the CT subsurface soils also remained unchanged after the annual applications of carbofuran. There were no differences in the numbers of carbofuran ring degraders in the CT surface soils compared to the CT subsurface soils (Table 5-2). Numbers of carbofuran-hydrolyzers were significantly greater ( a < 0.05) than the number of carbofuran ring degraders in both the surface and subsurface CT soil. The numbers of carbofuran ring degraders in the control surface soils (CN, NT and NN) were low (< 33 cells g" 1 soil), and the degraders in the NN soils were below the detection limit (Table 5-2). Carbofuran-ring degraders were all below the limits of detection in the control subsurface soils (Table 5-2). There was no difference in the numbers of carbofuran ring degraders between the CT soils and the control soils. Higher numbers of carbofuran-hydrolyzers than carbofuran ring degraders was observed in all control soils. Methylamineand Carbofuran Phenol-Degraders in Soils. Despite the significantly higher number of carbofuran-hydrolyzers in the CT surface soil collected in 1995 than in the NT surface soil samples collected in 1995, the numbers of methylamine-utilizers in the two soils were not different (Table 5-3). In

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96 Table 5.1. The Most Probable Number (MPN) of carbofuran-hydrolyzers in various Ellzey soils. Depth MPN carbofuranLower 95% C.I. hydrolyzers Upper 95% C.I. cm 0-15 45-60 0-15 0-15 45-60 0-15 cells g" 1 CT soil '94' 9.2 X 10 4 2.8 X 10 4 2.8 X 10 4 8.4 X 10 3 CT soil '95' 1.2 X 10 7 2.3 X 10 6 CN soil '94' 1.6 X 10 5 4.8 X 10 4 2.8 X 10 4 8.4 X 10 3 NT soil '95' 7.6 X10 4 1.41 X 10 4 3.0 X 10 5 9.3 X 10 4 6.3 X 10 7 5.3 X 10 s 9.3 X 10 4 4.1 X 10 5 a C.I.= confidence interval

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97 Table 5-2. The Most Probable Number (MPN) of carbofuran aromatic ring degraders in various Ellzey soils. Depth, cm MPN carbofuran Lower 95% C.I. a Upper 95% C.I. ring degraders cm cells g CT soil '94' 0-15 49 14 162 45-60 2 1 7 CT soil '95' 0-15 11 4 26 45-60 3 1 5 CT soil '96' 0-15 18 6 55 45-60 2 1 5 CN soil '94' 0-15 33 10 109 45-60 BDL b NT soil '95' 0-15 9 3 22 45-60 BDL NN soil '96' 0-15 BDL a C.I. = confidence interval b BDL = below detection limit

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98 Table 5-3. The Most Probable Number (MPN) of methylamine and carbofuran phenol degraders initially present in CT and NT surface soil collected in 1995. Degraders MPN degraders, g' 1 Lower 95% C.I. a Upper 95% C.I. cells g" CT soil'95' Methylamine 7.9 X 10 6 2.6 X 10 6 2.6 X 10 7 Carbofuran phenol 2 1 5 NT soil '95' Methylamine 4.9 X 10 6 1.5 X 10 6 1.6 X 10 7 Carbofuran phenol 2 1 5 C.I. = confidence interval

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99 addition, there was no difference between the numbers of carbofuran phenol degraders in the surface CT and NT soils. A significantly greater number of methylamine degraders than carbofuran phenol degraders was observed in these soils. Growth of the Carbofuran-Degrading Populations Carbofuran-hydrolyzers The number of carbofuran-hydrolyzers in the CT surface soil collected in 1995 after the addition of carbofuran at a rate of 10 ug g" 1 in the laboratory remained relatively unchanged during the first 14 days of incubation (Figure 5-1). The number of carbofuran-hydrolyzers in the NT soil increased significantly 7 days after the carbofuran treatment.. The number of carbofuran-hydrolyzers in the NT soil 7 days after the carbofuran treatment was similar to the number in the CT soil. After 14 days, the number of carbofuran-hydrolyzers in the NT and CT soil remained similar. Carbofuran ring-degraders Surface soils (0-15 cm) In 1995, the initial number of carbofuran ring degraders in the CT surface soil were low (1 1 cells g" 1 soil), and 3 days after laboratory treatment of carbofuran, the number of carbofuran ring degraders increased significantly (Figure 52A). However, after 7 days of incubation, the number of carbofuran ring degraders declined to the initial levels and remained at those levels throughout the rest of the 28 days of incubation (Figure 5-2A). In 1996, the initial number of carbofuran ringdegraders in the CT surface soil again was low (18 cells g" 1 soil) and similar to the CT soil collected in

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100 Figure 5-1. Number of carbofuran-hydrolyzing microorganisms in surface (0-15 cm) Ellzey soil amended with 10 ug carbofuran g" 1 soil. Error bars represent the 95 % confidence intervals.

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101 Figure 5-2. Number of carbofuran ring degrading microorganisms in Ellzey soils amended with 10 ug carbofuran g" 1 soil: A) surface (0-15 cm); B) subsurface (45-60 cm). Error bars represent the 95 % confidence intervals.

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102 1995. After a laboratory application of carbofuran, the number of carbofuran ringdegraders increased significantly on day 3 to 295 cells g" 1 soil, and this increase continued for 2 days to 851 cells g" 1 soil on day 5. However, after 14 days of incubation, the numbers of the carbofuran ring degraders declined to initial levels. The decline in the number of carbofuran ring degraders in 1996 was not as great as in 1995 (Figure 5-2 A), and the number of carbofuran ring degrading microorganisms in surface soil was more stable in 1996 than in 1995. In the NT surface soil, the number of carbofuran ring-degraders was initially low. After treating with carbofuran, the number of the carbofuran ring degraders remained low and statistically unchanged throughout the entire 28 days of incubation (Figure 5-2A). The number of carbofuran-ring degraders in the NN soil remained below the limit of detection throughout the entire 14 days of incubation (Figure 5-2A). Subsurface soils f45-60 cm). The changes in the number of carbofuran ringdegraders in the CT subsurface soils after laboratory application of carbofuran followed trends similarly to the CT surface soils. The maximum increase in numbers of carbofuran ring degraders in the subsurface soil collected in 1995 was observed on day 3. The increase in the number of carbofuran ring degraders was small, but it was significant (Figure 5-2B). After 7 days of incubation, the number of ring degraders in the subsurface soil declined to initial levels. In the CT subsurface soil collected in 1996, a significant increase in the number of carbofuran ring-degraders was observed one day after the incubation began. The number continued to increase and reached the maximum number after 5 days (631 cells g" 1 ). After day 14, the number of degraders remained significantly

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103 greater than initial population levels. The decline in the number of carbofuran ring degrading microorganisms in the soil was not as great as that collected in 1995 (Figure 52A). The number of carbofuran ring degraders in the NT subsurface soil was below the detection limits throughout the entire incubation period of 28 days (Figure 5-2A). Methylamine degraders The CT and NT surface soils collected in 1 995 were treated with carbofuran and incubated for 14 days. During the entire incubation period, there was not a significant increase in the number of methylamine degraders in either the CT or NT (Figure 5-3). However, after 7 days of incubation, the number of methylamine degraders was significantly greater in the CT soil than in the NT soil. After 14 days, the number of methylamine degraders in the CT soil was no longer significant to the number in the NT soil. Carbofuran phenol-degraders Carbofuran was applied to CT and NT surface soils in the laboratory to determine the effect of carbofuran on the numbers of carbofuran phenol degrading microorganisms. In the CT surface soil, the initial number of carbofuran phenol degrading microorganisms was small. After three days of incubation, I observed a significant increase in the number of carbofuran phenol degrading microorganisms (209 degraders g" 1 soil) (Figure 5-4). The number of carbofuran phenol degraders declined to initial levels after 14 days of incubation and remained at that level for the remainder of the incubation (28 days). The number of carbofuran phenol degraders in the NT surface soil were initially similar

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104 Figure 5-3. Number of methylamine degrading microorganisms in surface (0-15 cm) Ellzey soil amended with 10 ug carbofuran g" 1 soil over 14 days of incubation. Error bars represent the 95 % confidence intervals.

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105 0 4 8 12 16 Time (days) Figure 5-4. Number of carbofuran phenol degrading microorganisms in surface (0-15 cm) Ellzey soil amended with 10 ug carbofuran g" 1 soil over 28 days of incubation. Error bars represent the 95 % confidence intervals.

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106 to those observed in the CT soil. There was no significant increase in the number of carbofuran phenol degraders in the NT soil throughout the entire 28 days of incubation. After 7 days of incubation, the number of carbofuran phenol degraders in the CT soil was significantly larger than in the NT soil. After 14 days, the number of carbofuran phenol degraders were similar between CT and NT soils and this similarity remained throughout the rest of the 28 days of incubation (Figure 5-4). Discussion Carbofuran-Hydrolyzers Even though the mineralization rate of [ 14 C-CAL] carbofuran in the CT surface soil collected in 1995 was not determined, it is likely that the larger number of carbofuran-hydrolyzers in this soil (Table 5-1) would result in a more rapid mineralization of [ 14 C-CAL] carbofuran than in the same soil collected in 1994. Although the number of carbofuran-hydrolyzers in the CT surface soil collected in 1996 was not determined, the number of the carbofuran-hydrolyzers in the CT soil collected in 1996 should be larger than in the same soil collected in 1994 judging from the 6 fold increase in the initial mineralization of [ ,4 C-CAL] carbofuran after 24 hours of incubation. Hence, it is likely that an increase in the number of the annual field applications of carbofuran to the CT soil would result in a progressive increase in the size of the carbofuran-hydrolyzers, resulting in progressively more rapid mineralization of

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[ ,4 C-CAL] carbofuran. After four consecutive annual field applications to the CT soil, the population of the carbofuran-hydrolyzers may have reached the plateau and additional annual applications of carbofuran will not result in an increase in the number of carbofuran-hydrolyzers. This conclusion is based on the evidence that the number of carbofuran-hydrolyzing microorganisms in the CT surface soil collected in 1 995 was initially large in size, and remained stable even after a laboratory treatment of carbofuran to the soil. In addition to the large existing number of carbofuran-hydrolyzers in the CT soil in 1995, the lack of growth of the carbofuran-hydrolyzing microorganisms after a laboratory treatment of carbofuran may reflect the fact that hydrolysis of carbofuran to carbofuran phenol and methylamine is a cometabolic process that will not generate energy for growth (assuming N is not limiting). If we assume that methylamine provides N and C for growth of carbofuranhydrolyzers in the CT soil and that carbofuran is applied to soil at a rate of 10 pg g" 1 soil, then 0.63 pg N g" 1 soil would be supplied to the soil as a N source for growth. If we assume that a bacterial cell mass typically contains a dry mass N content of 14%, the added carbofuran could yield a bacterial mass of 4.52 pg g" 1 soil, provided that other nutrients are not limiting factors. Furthermore, a typical bacterial cell has a dry mass of 2.8 10' 7 pg (Neidhardt et al., 1990), then 4.52 pg biomass g" 1 soil would be equivalent to 1 .6 x 10 7 bacterial cell g" 1 soil. Alternatively, if we assume that methylamine is the only C source for the growth of the carbofuran-hydrolyzers, and 50% of the C from methylamine is incorporated into biomass, the added carbofuran would support a bacterial mass of 0.53 pg g" 1 soil or 1.8 x 10 6 bacterial cells g" 1 soil. Based on the above

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108 two estimations, it may be that carbofiiran applied at a rate of 10 pg g' 1 soil would not support a significant growth of carbofuran-hydrolyzers in the CT soil collected in 1995. The field application rate of carbofuran was 4 lb A" 1 (D.P. Weingartner, personal communication), which is equivalent to 2 pg g" 1 soil. The growth of biomass in soil resulting from the field application rates would be 5 fold less. Additional evidence strengthens the idea that carbofuran serves as an N source for growth of carbofuran-hydrolyzers in the enhanced Ellzey soils. In the presence of carbofuran applied at a rate of 10 pg g" 1 soil, the size of the carbofuran-hydrolyzing population increased to near the theoretical maximum size (1.8 x 10 7 cells g" 1 soil) based on N utilization from carbofuran. After the carbofuran-hydrolyzers in the NT soil reached the theoretical maximum growth, the number of carbofuran-hydrolyzers maintained a constant size ( Figure 5-1). This suggests a link between the utilization of N from methylamine released from carbofuran and enhanced degradation of carbofuran. Some nitrogen fertilizers such as urea and ammonium nitrate inhibited mineralization of [ 14 C-CAL] carbofuran in enhanced and nonenhanced soils (Merica and Alexander, 1990; Rajagopal and Sethunathan, 1984). Achromobacter sp. WM1 1 1, a soil isolate that utilizes carbofuran as a sole source of N, was reported to exhibit reduced levels of carbofuran hydrolase activity when grown in N rich media (Karns et al., 1986). Carbofuran was not the only substrate to induce carbofuran degradative enzyme activity; WM1 1 1 incubated in media containing methylamine and glucose was found to express carbofuran hydrolase activity. This suggests that soils treated with methylamine may be induced to develop enhanced degradation of carbofuran.

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109 The amount of methylamine released from the hydrolysis of carbofuran is not sufficient enough to meet the C demands of the estimated carbofuran-hydrolyzing population in the CT soil. This indicates that a second C source is required for the hydrolysis of carbofuran in the CT soil. The question is "does the soil supply a sufficient amount of C to sustain a population of this size?" Reddy et al. (1982) estimated that the available dissolved organic carbon (DOC) is 1% of the total organic carbon of a soil. The amount of organic carbon supplied by CT soil (4.2 g kg" 1 soil organic carbon content) to the carbofuran-hydrolyzing population is estimated to be 42 p.g DOC g" 1 soil. A carbon supply of this amount would be adequate in maintaining the C demands of the carbofuran-hydrolyzing population. It is difficult to determine the level of methylamine-C incorporated into biomass in the enhanced Ellzey soils treated with carbofuran. Since the number of carbofuranhydrolyzers in the CT and NT soils were larger than the number of methylamine degraders, and not all methylamine degraders have the capacity to hydrolyze carbofuran, it appears that the methylamine degraders constitute only a small fraction of the total number of carbofuran-hydrolyzing microorganisms. Hendry and Richardson (1988) and Racke and Coats (1988) reported numbers of carbofuran-hydrolyzing microorganisms in enhanced soils that were closer to the predicted size that utilized methylamine-C and than that utilizing methylamine-N. Most of the reported carbofuran-degrading bacteria isolated from soil thus far have been methylamine degrading bacteria with the ability to hydrolyze the carbamate linkage of carbofuran. Topp et al. (1993) isolated a methylotrophic bacterium that was

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110 capable of hydrolyzing carbofuran and utilizing the methylamine for energy and growth. They reported that carbofuran hydrolase activity increased 7 fold in the presence of carbofuran, but when incubated in the presence of methylamine or glucose, little or no increase in hydrolase activity was observed. Chaudhry and Ali (1988) found that bacteria that utilized the methylamine of carbofuran as a sole C source exhibited higher carbofuran hydrolase activity than isolates that utilized carbofuran as a sole source of N. This suggests that a carbofuran-degrading population dominated by carbofurandegrading isolates capable of utilizing carbofuran as a source of C would be smaller than one dominated by a population that utilizes carbofuran as an N source. The findings of Merica and Alexander (1990) and Robertson and Alexander (1994) that reported no evidence of linking carbofuran degradation with the growth of the carbofuran-hydrolyzing population seem to contradict the results of this study. Merica and Alexander (1990) measured the carbofuran-hydrolyzing population only twice at two different concentration levels (10 and 50 ug g" 1 soil). At 10 ug g' 1 they did not sample until 40 days after the initial incubation; this late sampling date may have precluded any detection of an increase in the number of carbofuran-hydrolyzing microorganisms. At the higher concentration, they sampled on days 4 and 84; due to the long interval time between sampling, the patterns of growth of the carbofuran-hydrolyzing microorganisms could not be determined. Ou (1984) reported that after reaching stationary growth (7 days), the number of 2,4-D degraders in Cecil soil began to decline. This would suggest that the sampling strategy employed by Merica and Alexander (1990) may have missed any fluctuations in the number of carbofuran-hydrolyzers.

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Ill Robertson and Alexander (1994) repeatedly measured the number of carbofuranhydrolyzing microorganisms after successive treatments of carbofuran to soil over a tenday period. After an initial increase in the number of carbofuran-hydrolyzing microorganisms in the first 3 days, the population size decreased after 7 days of incubation. They also reported that growth of carbofuran-hydrolyzing microorganisms was coupled with a low mineralization rate of [ 14 C-CAL] carbofuran. The low mineralization rate coupled with little growth in the number of carbofuran-hydrolyzing microorganisms suggests that this soil was degrading carbofuran cometabolically and was not an enhanced soil such as the CT and NT soils. Carbofuran Ring-Degraders Even though the mineralization of [ l4 C-URL] carbofuran in the CT surface soil was rapid, an appreciable growth of carbofuran ring degraders was not observed. A small increase in the number of carbofuran ring degraders, in conjunction with the rapid mineralization of [ 14 C-URL] carbofuran in the CT soil, suggests that the mineralization of the [ 14 C-URL] carbofuran in the CT soils was not linked to the growth of the carbofuran ring degrading population. Hence, mineralization of carbofuran was likely to be primarily a cometabolic process. Similar findings were also observed in the surface NT soil. The NT subsurface soil exhibited extensive mineralization of [ 14 C-URL] carbofuran, but the number of carbofuran ring degraders in this soil was below the detection limit throughout 28 days of incubation.

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112 If the mineralization of [ 14 C-URL] carbofuran in the enhanced soils is linked to the growth of the carbofuran-ring degraders, one would expect that the carbofuran ring will provide C source for growth of the carbofuran ring degraders, resulting in an increase in the number of the carbofuran ring degraders depending on the concentration of carbofuran. Additional assumptions are: 1) only C from the aromatic ring is incorporated into the specific biomass; and 2) 50% and 30% of the C from the aromatic ring are mineralized to CO, and form bound residues, respectively. At the application rate of 10 p.g g" 1 soil, the aromatic ring C will support a biomass of 1 .3 p.g g" 1 soil that is equivalent to 4.6 x 10 6 bacterial cell g" 1 soil. The MPN results of this study showed otherwise. Thus, the mineralization of the aromatic ring of carbofuran maybe mainly a cometabolic process. In contrast with low MPN numbers in the CT and NT soils, a number of Sphingomonas sp. capable of mineralizing [ 14 C-URL] carbofuran and utilizing carbofuran as sole source of C and N for growth were isolated from the soils. This finding suggests that the carbofuran ring structure in the enhanced soils could support the growth of carbofuran-ring degraders, resulting in the production of C0 2 via mineralization. The question may be asked why these degraders do not increase in number in the soils as indicated from the MPN results. One possible reason is that hydrolysis is the first step of carbofuran degradation and [ ,4 C-URL] carbofuran phenol is the hydrolysis product of [ l4 C-URL] carbofuan. Carbofuran phenol is highly active in forming bound residues to soil organic matter (Turco and Konopka, 1990; Talebi and Walker, 1993). The binding of [ l4 C-URL]

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113 carbofiiran phenol to enhanced soils can be high, from 42 to 85% of the applied 14 C (Racke and Coats, 1988; Turco and Konopka, 1990; Talebi and Walker, 1993). The number of carbofuran-ring degraders in the CT and NT surface soils were low, and carbofuran phenol in the soils available for microbial attack may have been limited due to bound residue formation and sorption to soil (Ogram et al., 1985), growth of the carbofuran-ring degraders in the soils may have been limited. Merica and Alexander (1990) also concluded that cometabolism was the predominant route for mineralization of [ l4 C-URL] carbofuran in enhanced soils. Some aromatic pesticides were found to be degraded cometabolically. The N-methylcarbamate insecticide carbaryl was completely mineralized by two soil bacterial isolates (Chapalamadugu and Chaudhry, 1991). One of the isolates hydrolyzed the carbamate linkage, while the other mineralized the resulting product, 1-naphthol. Ou and Thomas (1994) demonstrated that a mixed bacterial culture enriched from an enhanced soil rapidly mineralized the insecticide-nematicide fenamiphos in the presence of soil extract. The individual isolates when separated did not have any capacity to degrade the chemical. A mixed soil bacteria culture that degraded the insecticide methyl parathion cometabolically degraded methyl parathion more rapidly than an axenic bacterial culture that utilized the chemical as a sole source of C for growth (Ou and Sharma, 1989). It must also be kept in mind that the MPN technique has a high error associated with the procedure, and coupled with the inability to culture less than 1% of the soil microorganisms leaves the exact number of carbofuran ring degraders in doubt. The fact that in this study, a number of bacteria capable of utilizing the aromatic ring structure of

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114 carbofuran as a sole source of C for growth were isolated from the enhanced Ellzey soils suggests that [ l4 C-URL] carbofuran in this soil was being mineralized by some bacteria for growth. It appears that mineralization of the aromatic ring of carbofuran maybe used by some bacteria as a sole C source for growth while others mineralize the aromatic ring of carbofuran through a comatabolic process.

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CHAPTER 6 CHARACTERIZATION OF CARBOFURAN-DEGRADING BACTERIA Introduction A number of bacteria capable of hydrolyzing carbofuran to carbofuran phenol and methylamine have been isolated from soils. The most intensively studied bacterium to date is a strain of Achromobacter sp. that hydrolyzes carbofuran and uses the hydrolysis product methylamine as a sole source of N for growth (Karns et al., 1986). Karns and coworkers later showed that the gene responsible for carbofuran degradation was located on a plasmid (Tomasek and Karns, 1989). The majority of bacterial isolates that degrade carbofuran utilize methylamine as a sole source of C and/or N. A few bacteria isolated from soil have the capacity to metabolize the aromatic ring of carbofuran for growth (Chaudhry and Ali, 1988; Ramanand et al., 1988; Head et al., 1992; Feng et al., 1997a). Sphingomonas sp. strain CF06 that was capable of mineralizing [ 14 C-CAL and 14 C-URL] carbofuran was recently isolated from a Washington soil (Feng et al., 1997a). CF06 harbors 5 plasmids at least some of which are responsible for the degradation of carbofuran. The plasmids contain insertion sequence (IS) elements that were speculated to be involved in the recruitment of the genes responsible for carbofuran degradation. 115

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116 As mentioned in Chapter 4, repeated field applications of carbofuran to the Ellzey soil at the Hastings site resulted in enhanced degradation of the chemical. Three strains of bacteria were isolated from this soil, and all had the capacity to mineralize the aromatic ring of carbofuran. These bacteria were able to utilize carbofuran as a sole source of C and N for growth. The purpose of this study is to report the isolation, metabolism, and genetics of these carbofuran-degrading soil isolates. Results and Discussion Isolation of carbofuran-degrading bacteria. Three strains of bacteria were isolated from various Ellzey soil samples collected from the Hasting site under different carbofuran application histories. All isolates were capable of utilizing carbofuran as a sole source of C and N for growth. Attempts to isolate carbofuran degrading bacteria from the NU soils failed, and no attempts were made to isolate bacteria from the CU soils capable of degrading carbofuran. All bacterial isolates were capable of mineralizing [ l4 C-CAL] and [ l4 C-URL] carbofuran. The carbofuran-degrading bacteria isolated from the CT soils in 1993 and 1995 were designated as strains HPL and TA05, respectively, and the strain CD was isolated from the NT soil in 1995. Identification and characterization of carbofuran-degrading bacteria The strain HPL was isolated from the CT soil after two annual applications of carbofuran. This strain was a nonpigmented, motile, short gram-negative rod with a long

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117 single polar flagellum (determined by scanning electron microscopy). The bacterium grew slowly on LB broth and agar plates. Colonies did not develop on LB agar plates until 3 days of incubation. Colonies measuring approximately 1-mm diameter were observed after one week. Identification of the HPL strain using the Oxi-Fem Tubes II Roche (Becton Dickinson, Cockeysville, MD) was inconclusive (Table 6-1). The MIDI, Inc. (Newark, DE) system based on the fatty acid methyl ester signature patterns identified the HPL strain as a Sphingomonas sp. with similarity indices of 0.42 (Table 61). In addition to carbofuran, the HPL strain was capable of utilizing carbofuran phenol, methylamine, carbaryl, and baygon as sole sources of C for growth. The HPL strain was not capable of utilizing anthracene, benzofuran, dibenzofuran, sodium benzoate, catechol, 1-naphthol, or naphthalene. When growing in a liquid mineral medium containing carbofuran as the only C source, an unknown water-soluble pink metabolite(s) was formed, after one day of incubation in a rotary shaker, resulting in formation of a bright red color in the medium after 2 or 3 days, and turning yellow after 2 weeks. The bacterium TA05 was isolated from the CT soil after four annual applications of carbofuran, while the strain CD was isolated from the NT soil at the same time as TA05. These two strains were pigmented yellow, motile, short gram negative rods with a single polar flagellum (determined by scanning electron microscopy). Similar to the HPL, the TA05 and CD strains grew slowly on LB broth and plates. Colonies on the LB plates were not observed until 2 days after incubation with colonies measuring

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Table 6-1 Characteristics of Sphingomonas sp. HPL 118 A. Morphology and biochemical characteristics Gram-stain Oxidase Mobility Maltose + + + Anaerobic glucose Xylose Lactose Urea + + + Aerobic + glucose Citrate + Lysine + PA Arginine dihydrolase Sucrose Mannitol B. Major cellular fatty acids FAME 14:0 20H 15:0 20H 16:0 16: w7c 16:1 w5c % of Total 8.67 0.54 11.06 8.90 0.85 FAME 17:1 w8c 17:1 w6c 18:w5c 19:0 cyclo w8c Summed 18:1 w7c/w9t/ wl2t % of Total 0.57 3.27 2.81 1.05 62.28

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119 approximately 2 mm diameter. The colonies of the TA05 strain were a dry shiny yellowish orange while the colonies of the CD strains were a mucous dull yellowish orange. The identification of the TA05 and CD strains were based on the MIDI, Inc. (Plant Pathology Department, University of Florida) system for analysis of the fatty acid methyl ester signature patterns. Both were identified as Sphingomonas sp. with similarity indices of 0.69 and 0.59, respectively (Table 6-2). The fatty acid patterns of the TA05 and CD strains indicated that these strains were similar at the species level, that and differences were mainly attributed to differences in metabolic substrate utilization (C. Hodges, personal communication, Plant Pathology, University of Florida). In addition to carbofuran, TA05 and CD were also capable of utilizing carbofuran phenol, methylamine, carbaryl, and baygon as sole sources of C for growth. The two strains were not capable of utilizing anthracene, benzofuran, dibenzofuran, sodium benzoate, catechol, 1-naphthol, and naphthalene for growth at 400 ug ml" 1 MMA. When growing in MMA containing carbofuran as the C source, after over-night incubation in a rotory shaker, an unknown water-soluble blue metabolite(s) was initially formed, resulting in formation of a red color in the medium after 1 day, and eventually turning yellow after one week. Mineralization and degradation of carbofuran by soil bacteria. Mineralization of [ l4 C-CAL] and [14C-URL] carbofuran by the Sphingomonas sp. (HPL, TA05, and CD) are shown in Figures 6-1,-2, and -3. The mineralization of [ ,4 C-CAL] carbofuran during the first 72 hours were similar for all three isolates and

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Table 6-2. Fatty acid methyl ester (FAME) analysis of Sphingomonas sp. TA05 and CD. 120 FAME % FAME % TA05 14:20H 11.64 17:1 w7c 1.68 15:0 20H 0.68 17:1 w6c 2.84 16:0 7.45 18:1 w5c 2.08 16:1 w5c 0.78 18:1 w7c/w9c/wl2t 64.55 16:1 w7c; 15:0iso2OH 8.30 CD 14:20H 9.73 17:1 w6c 2.96 15:0 20H 0.81 18:1 w5c 1.43 16:0 12.40 18:1 w7c/w9c/wl2t 61.54 16:1 w5c 1.00 16:1 w7c; 15:0iso2OH 10.13

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Figure 6-1 Growth and mineralization of [ 14 C-CAL] and [ l4 C-URL] carbofuran by Sphingomaonas sp. strain HPL in 50 ug carbofuran ml-1 MMA. Error bars represent the standard deviation of the analysis.

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122 Figure 6-2. Growth and mineralization of [ l4 C-CAL] and [ l4 C-URL] carbofuran by Sphingomaonas sp. strain TA05 in 50 u.g carbofuran ml-1 MMA. Error bars represent the standard deviation of the analysis.

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123 Time (hours) Figure 6-3. Growth and mineralization of [ 14 C-CAL] and [ l4 C-URL] carbofuran by Sphingomaonas sp. strain CD in 50 ug carbofuran ml-1 MMA. Error bars represent the standard deviation of the analysis.

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124 ranged from 83.4 to 87.8% of the applied 14 C (Table 6-3). The bacterium CF06 isolated from a Washington soil had a similar capacity to mineralize [ l4 C-CAL] carbofuran (77 % after 5 days) (Feng et al., 1997a). The three isolates, however, had a different capacity of mineralizing [ 14 C-URL] carbofuran. TA05, CD, and HPL mineralized 47.4, 46.5, and 40.8 % of the applied14 C in 72 hours, respectively. The strains TA05 and CD mineralized significantly more [ 14 CURL] carbofuran than the HPL strain (Table 6-3) in 72 hours. Pseudomonas sp. strain 50432 isolated from a Florida soil mineralized 40 % of [ 14 C-URL] carbofuran (Chaudhry and Ali, 1988), and CF06 mineralized at a similar rate, 39.8 % of the [ 14 C-URL] carbofuran (Feng et al., 1997a). An Achromobacter sp. isolated from a flooded soil was capable of mineralizing more than 92% of the total [ 14 C-URL] carbofuran recovered in 72 hours (Ramanand et al., 1988). The extensive mineralization of this Achromobacter sp. suggests that the bacterium was utilizing methylamine and/or the furan ring for growth, while at the same time the aromatic ring was being cometabolized. The degradation of [ l4 C-URL] carbofuran by the TA05 strain was rapid, with less than 1% of the applied l4 C-carbofuran remaining in solution after 24 hours (Figure 6-4B). The degradation of [ ,4 C-URL] carbofuran by the HPL strain was slower than the TA05, and 99 % of the applied 14 C-carbofuran was degraded in 96 hours after incubation (Figure 6-4A). These rates of carbofuran degradation were similar to other soil bacteria that degraded the aromatic ring of carbofuran (Ramanand et al., 1988; Head et al., 1992).

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125 Table 6-3. The distribution of l4 C-activity in culture MMA media containing 50 ug ml carbofuran and [ l4 C-CAL] or [14C-URL] carbofuran after 72 hours of incubation. Culture l4 C0 2 l4 C-Medium 14 C-Biomass 14 C-Recovery % of applied 14 C [ l4 C-CAL] Carbofuran HPL 83.4(1.2) a 10.2(1.9) 2.1(0.2) 95.7(1.9) TA05 85.4(1.1) 5.2(0.9) 2.6(0.0) 93.3(1.9) CD 87.8(2.3) 6.5(0.5) 1.1(0.2) 97.0(3.1) [ l4 C-URL] Carbofuran HPL 40.8(2.3) 33.5(2.8) 21.1(0.5) 95.4(2.4) TA05 47.4(1.5) 24.8(0.9) 27.6(1.0) 99.8(1.9) CD 46.5(0.1) 16.3(0.8) 32.6(1.0) 98.4(0.7) values within parentheses are the standard deviations of the measurements

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126 0 20 40 60 80 100 Time (hours) Figure 6-4. Degradation of [ 14 C-URL] carbofuran and distribution of 14 C activity for A) HPL and B) TA05.

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127 Growth of carbofuran degrading bacteria. The growth of the three carbofuran degrading bacteria on carbofuran as a sole source of C is shown in Figures 6-1,-2, and -3. Maximum growth was achieved in 24 hours for the strains TA and CD, while the strain HPL did not achieve maximum growth until 48 hours after the incubation. The doubling times for the strains TA05 and CD strains grown in mineral media containing 50 \xg ml" 1 carbofuran were similar; 6.8 and 7.0 hours, respectively, while the slower growing HPL strain was 12.2 hours. The doubling times of TA05 and CD were half that of the HPL strain. The doubling time for Achromobacter sp. WM1 1 1 on carbofuran (200 fig ml" 1 ) as a sole N source in mineral medium supplemented with glucose (40 mM) was 4 hours (Karns et al., 1986). A methylotrophic bacterial strain ER2 had a doubling time of 3 hours in a mineral medium with carbofuran (100 jig ml" 1 ) as sole source of C (Topp et al., 1993). Continued application of carbofuran to the Ellzey soil may have resulted in the selection of carbofuran-degrading microorganisms that are more efficient at utilizing carbofuran. The extent of carbofuran-C incorporated into the biomass of the carbofuran degrading strains was studied using [ 14 C-URL] carbofuran. I found that the CD strain had the highest amount of 14 C-activity (32.6 %) associated with its biomass after 72 hours of incubation, while TA05 was next (27.6 %), and HPL had the lowest 14 C-activity (21.1 %) associated with its biomass (Table 6-3). The amount of 14 C-associated with each strain was significantly different from the other. The carbofuran degrading bacterial strain 50432 (Chaudhry and Ali, 1988) incorporated approximately 32 % of the l4 C-activity of [ 14 C-URL] carbofuran into its biomass.

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128 Table 6-4. The distribution of 14 C-activity in three media containing 14 C-carbofuran inoculated with the TA05. The culture media were incubated on a rotary shaker for 72 hours or 1 5 days. Culture 14 CO, 14 C-Medium 14 C-Biomass 14 C-Recovery %ofappliedl4 C MMA a MMB Soil Ext. 3 MMA MMB Soil Ext. 85.4(1.1)" 80.5(9.1) 80.7(0.8) 49.5(5.8) 54.1(1.2) 50.7(1.0) [ 14 C-CAL] Carbofuran 5.2(0.9) 2.6(0.0) 93.3( 1.9) 13.9(4.8) 0.5(0.2) 94.9(11.4) 16.9(3.9) 2.4(0.5) 100.0( 2.6) [ 14 C-URL] Carbofuran 0 31.0(11.7) 19.5(6.4) 100 42.5( 1.5) 3.4(0.8) 100 21.5(2.8) 28.1(2.9) 100 a TA05 grown in MMA and soil extracts were incubated for 72 hours. The initial concentration of carbofuran in the two media were 50 jug ml" 1 carbofuran. b values within parentheses are the standard deviations of the measurements c Note: due to the significant difference in the 14 C-recoveries between the soil extract (86.3%) and MMB (106.9 %), the values for the 14 C-distribution were normalized so that the 14 C recoveries in all the culture media were 100 % for the [ 14 C-URL] carbofuran studies.

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129 TA05 was isolated from the same soil (CT) two years after the HPL strain was isolated and the soil was subject to two additional annual field treatments of carbofuran. Repeated exposure of carbofuran to the CT soil may resulted in the selection of bacteria that are more efficient in degrading carbofuran. The higher percentage of carbofuran associated with the biomass of the TA05 coupled with the faster degradation of carbofuran indicates that the repeated applications of a pesticide to a soil can result in evolution of microorganisms capable of increased degradation of the pesticide. Because of its rapid degradation of carbofuran and its harboring of only one plasmid, the strain TA05 was chosen for further study of the effects various culture media have on carbofuran degradation. The mineralization of [ l4 C-CAL] and [ ,4 C-URL] carbofuran by TA05 was investigated in three different culture media: 1 ) in mineral medium (MMA); 2) in soil extract; and 3) in a N-free mineral medium supplemented with glucose (MMB). Most of the media contained 200 ug ml" 1 of carbofuran. The extent of mineralization of [ 14 C-CAL] and [ 14 C-URL] carbofuran was not different among the three culture media (Table 6-4). When the TA05 was grown in MMA and soil extracts, the amounts of l4 C-activity associated with the biomass or cell free culture fluids were not significant (Table 6-4). Incubation of TA05 in the MMB culture medium resulted in very little of the 14 C-activity being incorporated into its biomass (3.8%). The amount of 14 C-activity incorporated into the biomass of the TA05 incubated in MMA (a < 0.1) or soil extract (a < 0.05) was significantly greater than that incubated in MMB.

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130 The metabolic controls for assimilation of carbofuran into the biomass may be repressed when the TA05 was incubated in a culture medium with a better C source (glucose) than carbofuran. The assimilation of carbofuran into the biomass may be repressed, yet carbofuran is still readily degraded by the TA05 strain in the MMB containing glucose. Chaudhry and Ali (1 988) reported that enzymes required for carbofuran degradation were not constitutively expressed but were induced by the presence of carbofuran for several soil isolates, including the strain 50432. In addition, when TA05 cells were incubated in MMA containing second C substrates (sodium acetate, sodium succinate, or glucose), the development of the blue and red metabolites in the culture medium was observed. Thus, it appears that the genes regulating the mineralization of [ 14 C-CAL] and [ l4 C-URL] carbofuran are under different regulatory controls than genes responsible for the assimilation of carbofuran-C into biomass. Degradation of Carbofuran by Soil Bacteria. Metabolite of [ 14 C-URL] carbofuran The metabolites and degradation pathway of carbofuran were investigated using the strains TA05 and HPL. The two cultures exhibited different rates of degradation and different efficiencies of carbofuran-C assimilation. In addition, a blue transient metabolite(s) was observed during the degradation of carbofuran by the TA05. Judging from the rapid 14 C0 2 evolution from [ 14 C-CAL] carbofuran, it appeared that both TA05 and HPL initially hydrolyzed carbofuran to carbofuran phenol. Subsequent degradation of carbofuran phenol resulted in the production of C0 2 and assimilation of carbofuran

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131 phenol-C into the biomass leaving little or no accumulation metabolites in the culture fluids (Table 6-5) for both strains, with the exception of an unknown water-soluble red metabolite. This red metabolite (R f = 0.12) was detected in the culture media after 6 hours of incubation with the TA05 or HPL culture. This red metabolite was also observed in cultures of HPL grown on carbofuran phenol as a sole source of C. A similar red metabolite was observed in other bacterial cultures that degraded carbofuran (Head et al., 1992; Feng et al., 1997a). In the HPL culture solution, 3-keto carbofuran phenol was also detected. Unknown polar metabolites (R f < 0.1) were detected in both the TA05 and HPL culture fluids. The number of compounds associated with the unknown polar metabolites could not be determined, but it appeared to have at least three poorly resolved radioactive spots on the TLC plates using [ 14 C-URL] carbofuran. Identification of the unknown metabolite. The unknown red metabolite(s) has been speculated to be associated the phenyl moiety of carbofuran by some carbofuran-degrading bacteria isolated from soils (Head et al., 1992). This study demonstrated for the first time a direct link to the carbofuran metabolism through TLC-autoradiographic analysis of filtered culture fluids using [ 14 CURL] carbofuran. Analysis of the purified red metabolite by LC/MS determined that the molecular weight of the red metabolite is 342. This suggested that the red metabolite was some type of dimer, possibly derived from carbofuran phenol. A direct dimer of carbofuran phenol would result in a compound with a molecular weight of around 326. The larger molecular weight of the unknown than a dimer of carbofuran phenol suggests

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132

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133 that the red metabolite was further oxidized, although, nothing can be definitively stated about its structure. Elucidation of the structure of the red metabolite will require additional purification and spectroscopic analysis. The identification of the red metabolite may shed some light on the degradation pathway of carbofuran by soil bacteria. This study has found that the red metabolite is derived from the aromatic ring of carbofuran. Analysis of the extract of one-day old cell-free culture fluid of TA05 by GC/MS. The GC/MS analysis of methylene chloride extracts of 1-day old cell-free TA05 culture fluid revealed that an unknown metabolite was detected in addition to carbofuran phenol. This unknown had a retention time of 10 minutes and a molecular weight of 1 15. The mass fragmentation pattern suggested that this unknown had a number of hydroxyl functional groups and a possible carboxyl group. In addition, based on the GC/MS analysis, the furan ring of carbofuran appeared to be split (C.E. Schmidt, Environmental Engineering Science, University of Florida, personnel communication). This is supported by the finding of Head et al. (1992) who reported that 14 C-furan labeled carbofuran was mineralized to l4 C0 2 by a carbofuran-degrading bacterium. Thus, it appeared that TA05 had the capacity to hydrolyze carbofuran, and to completely degrade the resulting products methylamine and the phenyl and furan rings of carbofuran phenol. Location of the Genes Responsible for Carbofuran Degradation by TA05 The plasmid of the TA05, pCTOOl, was transferred to a neutral host strain, Pseudomonas fluoresceins M480R, by electroporation. The putative transformant of P. fluoresceins M480R-T grew poorly, if at all, on carbofuran. This transformant degraded

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134 [ 14 C-CAL] and [ 14 C-URL] carbofuran at the same rates as the negative control. P. fluorescens M480R, with only 25% and < 0.3% of the applied 14 C being mineralized after 15 days of incubation. An electrophoresis analysis indicated that P. fluorescens M480RT appeared to harbor the plasmid pCTOOl (Figure 6-5). The gel appears to confirm the transfer of pCTOOl into P. fluorescens M480R-T, but because hybridization of pCTOOl with the putative transformant was not preformed, the results can not be definitive. The above results are somewhat surprising since the strain TA05 contained a single plasmid that was found to share extensive homology with the Sphingomonas sp. strain CF06 plasmids (Feng et al., 1997b). CF06 is rich in IS elements (Feng et al., 1997a), one of which was also present in the TA05 plasmid (Feng et al., 1997b). CF06 was found to harbor the genes encoding for the biodegradation of carbofuran, and these genes were possible distributed among all five plasmids (Feng et al., 1997a). A cured TA05 strain was incapable of utilizing carbofuran as a sole C or N source (Feng et al., 1997b). Thus, it was proposed that the pCTOOl harbored the genes required for carbofuran degradation. The lack of carbofuran degradation by P. fluorescens M480R-T may be a result of a poor of expression of the pCTOOl in the new host. Tomasek and Karns (1989) were able to subclone mcd, carbofuran hydrolase gene, into P. putida P1252015R, but expression of the gene activity was lower than the original host. They later found that other gram negative bacteria also exhibited low levels of enzyme activity with E. coli exhibiting no enzyme activity. Head et al. (1992) isolated a soil bacterium, strain M2dc, that was capable of completely degrading carbofuran. However, the cured strains were

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Figure 6-5. Agrose gel showing plasmid profiles of M480R-T (lane 1) and pCTOOl from TA05 (lane 2).

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unable to metabolize carbofuran phenol, but the cured strains retained the capacity to hydrolyze carbofuran. Attempts to transfer the plasmids of M2dc into E. coli Jm83 and P. putida PaW130 were unsuccessful. Feng et al. (1997a) also attempted to determine which of the five plasmids harbored the genes encoding for carbofuran hydrolase, but were unsuccessful. They were able to demonstrate that when all five the plasmids were transferred to P. fluorescens M480R, the new host had the capacity to mineralize [ l4 CCAL] and [ 14 C-URL] carbofuran. Thus, it appeared that the transfer of plasmids that harbored the carbofuran degrading genes, and the expression of the genes for carbofuran degradation in neutral bacteria can be complicated and not always successful. Another possible reason for the lack of carbofuran degradation in the putative transformant P. fluorescens M480R-T is that the genes encoding for carbofuran degradation are located on both the chromosomal and plasmid DNA, or all are on the chromosomal DNA. For a time, the degradation of 2,4-D was considered to be exclusively plasmid encoded, but recently it has been found that the chromosomal DNA in some bacteria harbored the genes responsible for 2,4-D degradation (Ka and Tiedje, 1994; Suwa et al., 1996). Suwa et al. (1996) reported the isolation of a soil isolate of Burkholderia sp. harboring genes for 2,4-D degradation on its chromosome. In addition, there is evidence that the 2,4-D genes located on the chromosome of the Burkholderia sp. strains RASC and TD6 were part of mobile genetic elements (Mateheson et al., 1996). The evidence for this: 1) the high similarity of tfdA gene sequence between strains; 2) more divergence between the Burkholderia sp (16S rDNA) than between tfdA sequences;

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137 and 3) the G+C content of the chromosomal tfdA genes was 6% lower than the normal value for Burkholderia sp. Since the plasmid pCTOOl of TA05 harbors at least one IS element there is also the possibility that during the curing of TA05, there was a mobilization of IS elements that inserted into a chromosomal gene responsible for carbofuran degradation. This would result in the loss of the carbofuran degrading phenotype during the curing of the TA05 strain. It appears then that the association of mobile genetic elements with a catabolic function tends to complicate our ability to determine the evolution of xenobiotics metabolism in the environment. Further analysis of pCTOOl is required before any definitive statements can be made on the location of the carbofuran degrading genes in TA05.

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CHAPTER 7 CONCLUSIONS It has been recognized since 1 98 1 that repeated use of carbofuran in soils results in the loss of pesticidal efficacy of carbofuran, which is attributed to the enhanced degradation of the pesticide in soils. My results on the mineralization of 14 C-labeled carbofuran and the disappearance of 14 C-carbofuran in soils with different histories of carbofuran exposure have led to the conclusion that the repeated exposure of carbofuran to a soil does result in an enhancement in the degradation rate of carbofuran. Evidence supporting this conclusion is based on: 1 Disappearance rates of carbofuran increased with an increase in number of annual field applications of carbofuran to a soil. 2 Soils with a previous history of carbofuran application exhibited greater degradation rates than soils with no history of exposure to carbofuran. 3. Soils with a previous history of carbofuran exposure exhibited shorter lag periods prior to the onset of rapid mineralization of [ 14 C-CAL] carbofuran than soils with no previous history of carbofuran exposure. The loss of pesticidal efficacy in enhanced soils has been attributed to carbofuran-degrading microorganisms. The data from the mineralization assays of 14 C carbofuran and the MPN assays supports the conclusion that enhanced biodegradation of 138

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139 carbofiiran is due to an increase in the number of carbofuran-hydrolyzing microorganisms resulting in an increase in the hydrolysis rate of carbofuran. Evidence supporting this conclusion is based on: 1 Soils with a previous history of carbofuran exposure resulted in increased mineralization rates of [ l4 C-CAL] carbofuran. 2. Mineralization rates of [ l4 C-CAL] in fumigated enhanced soil was significantly lower than in non-fumigated enhanced soil. 3. The number of carbofuran-hydrolyzing microorganisms in soils increased with successive annual applications of carbofuran. 4. An application of carbofuran in the laboratory to the noncultivated nontreated soil significantly increased the number of carbofuranhydrolyzing microorganisms. Enhanced degradation of carbofuran in soils previously exposed to the compound is the result of a shift in the degradation pathway of carbofuran, with the hydrolysis of the carbamate moiety being the major route. Evidence supporting this conclusion is based on: 1 The hydrolysis rate after four annual field applications increased significantly over the hydrolysis rate after two annual field applications of carbofuran. 2. There was no detection of the carbofuran oxidation products in soils with a previous history of carbofuran treatment.

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140 3. The oxidation products of carbofuran were detected only in soils not previously treated with carbofuran. Cometabolism appears to be a major route in the mineralization of the aromatic ring of carbofuran in soil. Evidence supporting this conclusion is based on: 1 After repeated field applications of carbofuran, the mineralization rate of [ 14 C-URL] carbofuran remained unchanged. 2. The initial number of carbofuran ring degrading microorganisms did not increase despite repeated field applications of carbofuran. 3. The number of carbofuran ring degrading microorganisms in soils after laboratory treatment of carbofuran increased, but the number remained small. 4. The MPN number of carbofuran ring degrading microorganisms in some soils was below the detection limit, yet the mineralization of [ 14 C-URL] carbofuran in the same soils was rapid. Soil bacteria capable of utilizing carbofuran as a sole C and N source were isolated from enhanced soils, but attempts to isolate carbofuran degrading microorganisms in soils with no history of carbofuran exposure failed. All the soil isolates were capable of mineralizing [ 14 C-CAL] and [ ,4 C-URL] carbofuran, and all harbored at least one plasmid. In addition, soil bacteria isolated after four annual field applications of carbofuran metabolized carbofuran more efficiently than the soil bacterium isolated after only two annual applications of carbofuran.

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141 Attempts to identify the degradation pathways of carbofuran by the soil isolates were unsuccessful, while attempts to determine the location of the genes encoding for carbofuran degradation were inconclusive. The association of mobile genetic elements with the TA05 strain complicated my ability to determine the location of the genes encoding for carbofuran degradation. It is hoped that future work on the ecology, physiology and genetics of the carbofuran-degrading microorganisms will yield insights into the cellular and molecular mechanisms of enhanced degradation of carbofuran by soil microorganisms. Enhanced degradation of certain pesticides does occur. Proper management practices must then be developed for the effective use of soil applied pesticides that are susceptible to enhanced degradation. The areas of pesticidal management should include: 1) determining the rate of application of a pesticide that induce enhanced degradation development; 2) the duration of enhanced degradation for a pesticide; and 3) alternative management practices used to control the development of enhanced degradation of a pesticide. Further research into the application rate that induces enhanced degradation of carbofuran is needed. Previous studies report induction of enhanced degradation as a function of a soil's bioavailability (Chapman et al., 1986). Soils with lower organic carbon contents would be expected to be more susceptible to enhanced degradation of carbofuran than soils with higher nature organic carbon contents. Thus, proper management practices of carbofuran should consider a soil's organic carbon content when

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142 formulating the application rates of carbofuran to prevent or reduce the development of enhanced degradation. Further research into the persistence of enhanced degradation is needed. No study has investigated the duration of enhanced degradation for carbofuran. If enhanced degradation is inevitable then what time period between applications should one wait before applying carbofuran to a soil again. In the U.K., it is suggested that carbofuran be used once every 3 to 4 years in order to control enhanced degradation (Eagle, 1986). What type of recommendations should be given for soils in different climatic regions? Should soils in the tropic regions that have lower organic carbon contents and warmer temperatures than U.K. soils be expected to develop and maintain enhanced degradation similarly? If enhanced degradation is a function of the bioavailability of carbofuran, would not the duration of enhanced degradation be less for soils with lower organic carbon contents? Thus, the understanding of how the bioavailability of carbofuran along with the climatic conditions of a soil should be consider when determining the frequency of carbofuran application to a given soil. Further research into alternative management practices that can control the development of enhanced degradation of carbofuran is needed. The application of urea (Merica and Alexander, 1990) and the use of soil fumigants in my study show that enhanced degradation of carbofuran can be controlled. Thus, management practices should be considered when determining the use of carbofuran in soil. More work is still needed in how soil properties and management practices influence enhanced degradation of carbofuran.

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GLOSSARY cometabolism Transformation of a substrate by a microorganism without deriving energy, carbon, or nutrients from the substrate. The organism can transform the substrate into intermediate degradation products but fails to multiply at its expense. degradation Process whereby a compound is usually transformed into simpler compounds. mineralization Conversion of an element from an organic form to an inorganic form to an inorganic state as a result of microbial decomposition. 143

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Dzantor, E.K. and A.S. Felsot. 1990. Soil differences in the biodegradation of carbofuran and trimethacarb following pretreatment with these insecticides. Bull. Environ. Contam. Toxicol. 45: 531-537. Eagle, D.J. 1986. ADAS experience of enhanced degradation of carbofuran. Aspects of Appl. Biol. 13: 101-105. Elliot, J.E., K.M. Langelier, P. Mineau, and L.K. Wilson. 1996. Poisoning of bald eagles and red-tailed hawks by carbofuran and fensulfothion in the Fraser delta of British Columbia, Canada. J. Wildlife Diseases 32: 486-491. Erickson, D. and D. Norton. 1990. Washington state agriculture chemicals pilot study: Final report. Washington State Dept. Ecology. Olymipa, WA. Fahmy, M.A.H., T.R. Fukuto, R.O. Myers, and R.B. March. 1970. The selective toxicity of new N-phosphorothioylcarbamate esters. J. Agric. Food Chem. 18:793-796. Felsot, A., J.V. Maddox, and W. Bruce. 1981. Enhanced microbial degradation of carbofuran in soils with histories of furadan use. Bull. Environ. Contam. Toxicol. 26: 781-788. Felsot, A. and A. Lew. 1989. Factors affecting bioactivity of soil insecticides: relationships among uptake, desorption, and toxicity of carbofuran and terbufos. J. Econ. Entomol. 82: 389-395. Felsot, A.S., K. Steffey, E. Levine, and J.G. Wilson. 1985. Carbofuran persistence in soil and adult corn rootworm (Coleoptera: Chrysomelidae) susceptibility: relationship to the control of damage by larvae. J. Econ. Entomol. 78:45-52. Felsot, A., and J. Wilson. 1980. Adsorption of carbofuran and movement on soil thin layers. Bull. Environ. Contam. Toxicol. 24: 778-782. Feng, X., L.-T. Ou, and A. Ogram. 1997a. Plasmid-mediated mineralization of carbofuran by Sphingomonas sp strain CF06. Appl. Environ. Microbiol. 63: 1332-1337. Feng, X., S. Trabue, H. Castor, L. Ou, and A. Ogram. 1997b. Metabolism of carbofuran encoded by pCTOOl. Q-372, p 292. In Abs. 97th Ann. Meeting Amer. Soc. Microbiol., Miami, FL. Fontes, D.E., A. Mills, G. Hornberger, and J. Herman. 1991. Physical and chemical factors influencing transport of microorganisms through porous media. Appl. Environ. Microbiol. 57:2473-2481. 147

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Fulthorpe, R.R., C. McGowan, O.V. Maltseva, W.E. Holben, and J.M. Tiedje. 1995. 2,4Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes. Appl. Environ. Microbiol. 61:3275-3281. Gannon, J., Y. Tan, P. Baveye, and M. Alexander. 1991. Effect of sodium chloride on transport of bacteria in a saturated aquifer material. Appl. Environ. Microbiol. 57:2497-2501. Gardner, W.H., 1986. Water content, pp. 493-544. In A. Klute (ed.) Methods of Soil Analysis. Part I: Physical and Mineralogical Methods (2nds). Agron. Monogr. 9. ASA and SSSA, Madison, WI. Gee, G.W., and J.W. Bauder. 1986. Particle-size analysis, pp. 383-41 1. / A. Klute (ed.) Methods of Soil Analysis. Part I: Physical and Mineralogical Methods (2nds). Agron. Monogr. 9. ASA and SSSA, Madison, WI. Getzin, L.W. 1973. Persistence and degradation of carbofuran in soil. Environ. Entomol. 2: 461-467. Getzin, L.W. and C.H. Shanks. 1990. Enhanced degradation of carbofuran in pacific northwest soils. J. Environ. Sci. Health B25: 433-446. Gianessi, L.P, and J.E. Anderson. 1995. Pesticide use in U.S. crop production: national summary report. National Center for Food and Agricultural Policy, February. Greenhalgh, R. and A. Belanger. 1981. 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. 29:231-235. Gupta, R.C. 1994. Carbofuran toxicity. J. Toxicol. Environ. Health 43: 383-418. Harris, C.R., R.A. Chapman, C. Harris, and CM. Tu. 1984. Biodegradation of pesticides in soil: rapid induction of carbamate degrading factors after carbofuran treatment. J. Environ. Sci. Health B 19: 1-1 1. Harris, C.R., R.A. Chapman, R.F. Morris, and A.B. Stevenson. 1988. Enhanced soil microbial degradation of carbofuran and fensulfothion-a factor contributing to the decline in effectiveness of some soil insect control programs in Canada. J. Environ. Sci. Health B23: 301-316. Hart, M.R. and P.C. Brookes. 1996. Soil microbial biomass and mineralization of soil organic matter after 19 years of cumulative field applications of pesticides. Soil Biol. Biochem. 28: 1641-1649. 148

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BIOGRAPHICAL SKETCH The candidate was born in North Carolina, but he was raised in Wheaton, MD, a suburb of Washington, D.C. His summers during his youth were spent outdoors on the family farm in Ohio. He completed his undergraduate studies at the University of Maryland in 1985 with a B.S. degree in biochemistry. Upon graduation, he worked for an environmental service company, Ecoflo Inc., as a field chemist. In the fall of 1988, he entered the University of Florida in pursuit of a MS degree in soil science (soil chemistry) under the supervision of Dr. Clifford Johnston. He completed his studies in the spring of 1991 and moved back to Maryland. Upon returning, he obtained employment in the Environmental Chemistry Laboratory at the USDA Agriculture Research Service, Beltsville, MD. He worked under the guidance of Drs. Jack Meisinger and Jim Starr in the area of soil nitrogen. In the fall of 1993, he returned to the University of Florida in pursuit of a Ph.D. degree in soil and water science (soil microbiology) specializing in biodegradation of pesticides in soils under the supervision of Dr. Li Tse Ou. Upon graduation the candidate will continue to work in the area of pesticide degradation in soils for DuPont in Wilmington, DE. 158

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Li-Tse Ou, Chair Scientist of Soil and Water Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. rorge A-O'Connor, ^ofessor of Soil and Water Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. CLJ^a V ffifr — Andrew V. Ogram / Assistant Professor of Soil and Water Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Palakurthi S. C. Rao Graduate Research Professor of Soil and Water Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Professor of Soil and Water Science

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy ie O. Ingram ^ Lonnit Professor of Microbiology and Cell Science This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1997 ^-^Dean, College of Agriculture Dean, Graduate School