EPA-650/1-74-002



September  1973
Environmental  Health  Effects  Research  Series


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                                     EPA-650/1-74-002
METABOLISM OF  CARBAMATE
            INSECTICIDES
                    by

              H.  Wyman Dorough

            Department of Entomology
             University of Kentucky
           Lexington, Kentucky 40506
              Grant No. R-802005
           Program Element No. 1E1078
        EPA Project Officer: Dr. R. L. Baron

       Primate and Pesticides Effect Laboratory
       National Environmental Research Center
     Research Triangle Park, North Carolina  27711
                 Prepared for

      OFFICE OF RESEARCH AND DEVELOPMENT
     U.S. ENVIRONMENTAL PROTECTION AGENCY
           WASHINGTON, D. C. 20460

                September 1973..

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This report has been reviewed by the Environmental Protection Agency
and approved for publication.  Approval does not signify that the
contents necessarily reflect the views and policies of the Agency,
nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
                                  11

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                                 ABSTRACT

The metabolism of selected carbamate insecticides and certain of their
key metabolites are reported.   Aldicarb was studied in lactating cows,
laying hens, and in boll weevils and houseflies.   Carbaryl  was investi-
gated in lactating cows and in soil.  Carbofuran  was studied in houseflies.

Effects of exposure to mixtures of pesticides on  the metabolism of car-
baryl were determined.  The fate of the carbaryl  was compared to that
in animals exposed to carbaryl alone.   Similar studies were conducted
where monoamine oxidase inhibitors were given to  the rats.   Aldicarb, an
oxime carbamate, was evaluated for its effect on  the toxicity of methyl
parathion to rats.

Radioactive 3-hydroxycarbofuran was biosynthesized and its  metabolism
investigated in rats and bean plants.   Similar studies were performed
with its glucoside and glucuronide and with 1-naphthol and  its glucoside
conjugate.

Mechanisms of glycosylation were studied in a variety of animal species.
Successful chemical syntheses of the glucosides of 1-naphthol, 4-
hydroxycarbaryl and 5-hydroxycarbaryl  were accomplished.  Acute toxicity
of the carbamate derivatives to rats was determined.

This report was submitted in fulfillment of Grant Number R-802005 by
the University of Kentucky under the sponsorship  of the Environmental
Portection Agency.  Work was completed as  of August 1973.
                                  iii

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                             CONTENTS

                                                                Page

List of Tables                                                   vi
Acknowledgments                                                  x
Sections
I    Conclusions                                                  1
II   Recommendations                                              2
III  Experimental                                                 3
       Introduction                                               3
       Metabolism                                                 3
          Aldicarb in Lactating Cows                              4
          Aldicarb in Laying Hens                                31
          Aldicarb in Boll Weevils and Houseflies                51
          Carbaryl in Lactating Cows                             56
          Carbaryl in Soils                                      83
          Carbofuran in Houseflies                              113
       Fate of Carbamate Metabolites                            120
          1-Naphthyl Glucoside in Rats                          120
          3-Hydroxy Carbofuran and its Glycosides in Rats       130
       Interactions                                             139
          Effect of Aldicarb on Methyl Parathion Toxicity       139
          Modification of Carbaryl Metabolism with MAOI's       144
          Influence of Insecticides on Carbaryl Metabolism      156
       Conjugation                                              174
          Mechanism in Rats                                     174
          Mechanism in Insects                                  189
          Factors Influencing Conjugation                       199
          Synthesis of Glycosides                               214
IV   References                                                 226
V    Publications                                               234
VI   Summary                                                    236

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                                 TABLES

 No.                                                                Page

 1.   Aldicarb and  its metabolites                                   24
 2.   Aldicarb rates and feed consumption                            25
 3.   Elimination of radioactivity by cows fed alidcarb-  C          26
 4.   Aldicarb-  C  equivalents in milk                               27
               14
 5.   Aldicarb-  C  equivalents in urine                              28
 6.   Separation of water-soluble aldicarb-  C metabolites           29
 7.     C-Residues  in tissues of cows fed aldicarb-  C               30
 8.   Treatment rates of aldicarb-  C to laying hens                 42
 9.   Nature of aldicarb-  C equivalents in feces of hens            43
10.   Elimination of aldicarb-  C residues in the feces of hens      44
11.   Aldicarb-  C  residues in feces of hens                         45
12.   Ppm aldicarb  equivalents in tissues and eggs of hens           46
13.   Residues in tissues and eggs of hens treated with radio-
      active aldicarb                       v                        47
14,   Residues in eggs of hens aldicarb in the diet                  48
15.   Ppm aldicarb-  C equivalents in tissues of hens fed
      aldicarb in the diet                                           49
16.   Residues in tissues of hens fed aldicarb in the diet           50
17.   Distribution and excretion of aldicarb in boll weevils
      and houseflies                                                 55
18.   Study plan for carbaryl-naphthyl-  C cow feeding experiment    74
19.   Chemical nature of carbaryl metabolites in cow's milk          75
                     14
20.   Total carbaryl-  C equivalents in cow tissues                  76
21.     C-Residues  in cow tissues after feeding with carbaryl-  C    77
22.   Centrifugal  fractionation of   C-residues in the liver and
      kidney of a cow fed carbaryl                                    78
23.   Extraction characteristics of   C-residues in acid-treated
      supernatant of liver and kidney                                79
24.   Extraction characteristics of   C-residues in acid-treated
      pellet of liver and kidney                                     80

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                              TABLES -  cont.
No.                                                                 Page

      14
25.     C-Residues in the precipitate of a 15,000g  supernatant
      of liver                                                       81
26.   Precipitation, and dialysis of a  15,000g supernatant
      of kidney                                                      82
27.   Radioactivity of soils fortified  with carbaryl-1-   C and
      incubated at 27°C, 85% R.H.                                   102
28.   Radioactivity in soils fortified  with carbaryl-1-   C
      and incubated at 27°C, 85% R.H.                                103
29.   Radioactivity of carbaryl-treated soils  fortified  with
                                                  14
      polar water-soluble metabolites of carbaryl-   C               104
30.   Radioactivity of soils fortified  with polar water-soluble
      metabolites of carbaryl-14C incubated at 27°C, 85% R.H.        105
31.   Fate of carbaryl-naphthyl-1-  C and 1-naphthol- C in
      carbaryl-treated soil                                          106
32.   Radioactivity of acetone and barium hydroxide trap
      solutions                                                     107
33.   14C02 from carbaryl-  C and 1-naphthol-   C during
      incubation with soil                                          108
               14
34.   Carbaryl-  C inoculated with suspension  of mixed pesticide-
      treated soil                                                  109
35.   Carbaryl-1-  C inoculated with carbaryl-treated soil
      suspensions or fungal  isolates                                110
               14
36.   Carbaryl-  C inoculated with bacterial  isolates               111
37.   Nutrient broth with carbaryl-  C  inoculated with
      bacterial isolates                                            112
38.   Nature and magnitude of carbofuran in the body and excreta
      of houseflies                                                 119
39.   Nature of residues in the 0-24 hour urine of  rats  treated
      orally with 1-naphthol and 1-naphthyl glucoside               129
40.   Influence of carbofuran on its metabolism by  rat liver         135

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                              TABLES - cont.
No.                                                                Page

41.   Effect of substrate concentrations on glucuronidation         136
42.   Effect of incubation periods on the glucuronide
      and glucoside conjugation                                     137
43.   Partitioning characteristics of metabolites in the urine
      of rats treated orally with 3-OH-carbofuran or its
      glucuronide or glucoside derivatives                          138
44.   Effect of aldicarb, aldicarb oxime, 2-PAM and atropine
      on the acute toxicity of methyl parathion to mice             143
45.   Effect of monoamine oxidase inhibitors on the excretion
      of carbaryl-  C by rats                                       152
                       14
46.   Fate of carbaryl-  C in rats which received phenelzine
      in the drinking water                                         153
47.   Nature of carbaryl-  C metabolites in urine of normal
      and phenelzine-treated rats                                   154
48.   Metabolism and excretion of naphthol-  C when administered
      with phenelzine                                               155
49.   Effects of carbofuran and Ruelene on the excretion of
      carbaryl-14C by rats                                          166
50.   Excretory pattern of carbaryl  by rats fed a diet
      containing carbofuran, Ruelene or coumaphos                   167
51.   Nature of radioactivity in the urine of rats treated
      with carbaryl-  C alone and with either carbofuran
      or Ruelene                                                    168
52.   Weight and microsomal  protein  content of livers from
      rats exposed to carbaryl, carbofuran and DDT                  169
53.   In vivo metabolic activity in  the microsomal fractions
      of liver and kidney of rats fed carbaryl, and DDT for 40 days 170
54.   TPNH oxidase activity in the microsomal  fractions of
      livers from rats treated IP with carbaryl and DDT daily
      for up to 5 days                                              171

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                              TABLES - cont.
No.                                                                 Page

55.    The effect of pre-exposure to DDT on the nature and
      magnitude of carbaryl-  C metabolites in the urine
      of rats                                                       172
56.    Excretion and metabolism of naphthol-1-  C by rats
      given daily injections of DDT for 5 days                      173
57.    Importance of conjugation in the metabolism of carbamate
      insecticides in rats                                          185
58.    Effect of insecticide synergists on conjugation of
      1-naphthol by rat liver microsomes and  intestine enzymes      186
59.    Inhibition of rat liver microsomal epoxidation of aldrin
      by certain insecticide synergists                             187
60.    Effect of reduced glucuronidation on the total metabolism
      of carbaryl-14C                                               188
61.    Reaction mixture used for the in vitro  conjugation of
      1-naphthol by tobacco hornworm midgut enzyme                  196
62.    1-Naphthol glucosylation of whole body  extracts of
      adult houseflies and tobacco hornworm larvae                  197
63.    Sensitivity of glucosylation and epoxidation enzymes
      to insecticide synergists                                     198
64.    Glucosylation by the 5,000g supernatant of different
      insects and insect tissues                                    208
65.    Glucosylation by subcellular fractions  of a housefly
      homogenate                                                    209
66.    The effect of age and sex on conjugation by susceptible
      and resistant houseflies                                      210
67.    Fate of topically applied 1-naphthol-l-  C in susceptible
      and resistant houseflies                                      211
68.    Conjugation by the microsomal fractions of livers
      from different animals                                        212
69.    61ucuronyltransferase activity in various rat tissues         213
70.    The toxicity of 4- and 5-hydroxycarbaryl and their
      respective glucosides to mice                                 225

                                   ix

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                            ACKNOWLEDGMENTS

The research reported herein is the result qf the combined efforts of
                                          i
graduate students, post doctoral scholars, research associates, and
technicians.  Sincere thanks are extended to the 'individuals  listed
below for their hard work and dedication to this research program.
Graduate Research Assistants
N. A. El-Shourbagy
B. W. Hicks
T. H. Lin
L. D. Rodriguez
Research Associates
R. B. Davis
A. E. Smith
H. E. Bryant
Post Doctoral Scholars
R. A. Cardona
S. S. Kumar
H. M. Mehendale
J. P. McManus
G. A. Reddy
M. L. Saini
R. F. Skrentny
Technicians
M. E. Blackburn
S. C. Lolmaugh
C. N. Thomas

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                             I  CONCLUSIONS

In animals, the carbamate insecticides, aldicarb, carbaryl,  and carbo-
furan are rapidly metabolized and excreted from the body.   Continuous
exposure of animals to the pesticides added to the diet do  not result
in the accumulation of high levels of residues.   However, metabolites
containing the carbamate moiety, and therefore considered as potentially
toxic, do exist in the meat, milk and eggs of treated animals.

Glucoside derivatives of carbamate insecticide metabolites  formed in
plants do not behave in the animal system in the same manner as the
aglycone.  Cleavage of the glucoside linkage yields the aglycone which
is further metabolized by conjugation to form glucuronides,  sulfates,
etc.   Little, if any, oxidation of the sugar moiety takes  place to form
the corresponding glucuronide.  Studies with naphthyl glucoside showed
that the glucosidic bond was stable enough so that about 20% of an
oral  dose was eliminated, intact, in the urine.

Mechanisms of glycosylation of carbamates and other chemicals can be
effectively investigated using in vitro techniques developed in this
program.  Also, it is now possible to critically evaluate the chemical
and toxicological properties of glycosides of certain carbamate
metabolites.  The glucosides of 4- and 5-hydroxycarbaryl were chemically
synthesized in quantities suitable for such studies.

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                             II  RECOMMENDATIONS

Emphasis should be placed on developing practical analytical methods for
monitoring residues of toxic carbamate metabolites.  Metabolites contain-
ing the carbamic acid ester  exist both in the free and conjugated forms.
However, these metabolites often go undetected because of improper ex-
traction techniques and/or of inadequate methods of detection.

Because the majority of the  terminal residues of carbamate insecticides
in animals and plants are water-soluble and/or unextracted from the
substrate, the significance  of these materials in animals must be deter-
mined.  Attempts should be made to study the toxicology of each product
individually.  However, it will be years before this is technically
feasible.  Therefore, the unknown residues should be generally characterized.
organo-soluble, water-soluble, unextracted, carbamate vs. hydrolysis
products, etc.  Then, the compounds should be separated accordingly and
their fate  in animals evaluated.  Metabolites stored or accumulated in
the body should be generated in sufficient quantities to allow 90-day
feeding studies to be performed.  If the "no-effect" level is too low,
the continued use of the parent insecticide must be reconsidered.

The development of the carbamate insecticides as commercial pest control
agents should be encouraged.  As a group, the compounds have exhibited
(1) effective insect control, (2) relatively short residual life in the
environment, (3) rapid and almost quantitative excretion from the
animal body, and (4) terminal residues that are polar in nature and are
formed by chemical  processes normally considered as metabolic detoxication.
Unless new .and better insecticides are made available within the next
3 to 5 years, it is likely that this country will suffer insect-related
catastrophes of greater consequent than ever experienced by mankind.  A
well-devised plan to facilitate the development of the carbamate insect-
icides could prevent such an occurrence.

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                           Ill EXPERIMENTAL

INTRODUCTION

The principal investigator began work on this project in 1965.   At that
time, the research was funded by the National Institute of Health and
was supported initially for a 3-year period.  The first progress report
was submitted April 3, 1967 covering the period from January 1, 1965
through December 31, 1967.  The report, 266 pages, was identified as
NIH Research Project ES-00085.  Continued funding was requested and
approved, and the metabolism of carbamate insecticides was further in-
vestigated.  The next progress report, 246 pages, was submitted
December 10, 1969 covering the period of January 1, 1968 through Dec-
ember 31, 1969.  Because of changes within NIH, the project was ident-
ified as Food and Drug Administration Grant Number FD-00273.  A renewal
of the grant was requested and approved for the period of September 1,
1970 through August 31, 1973.  On May 21, 1971, the grant number was
changed to EP00828.

Basically, the main objective of. the research proposed in 1964 has not
changed.  This objective, namely to determine the metabolic fate of
carbamate insecticides in various biological organisms, was believed
essential if the carbamates were to become major agents of pest con-
trol in this country.  It is now well established that commercial
development of the carbamates did continue and are vital to agricultural
and health programs throughout the world.  Research funded by the
current project has been instrumental in the development of the carbam-
ates.  Metabolic pathways have been established for many of these
insecticides in a variety of animal and plant species.  These data have
been relied upon heavily by regulatory officials who must determine if
the compounds can be safely used.

Initially, most of the research into the metabolism of carbamate insect-

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icides dealt with metabolites of a "free" nature.  These were materials
formed by oxidative and hydrolytic mechanisms and were generally charac-
terized as "organo-soluble" metabolites.  Subsequent work demonstrated
that the free metabolites were often converted to other materials having
vastly different extraction and partitioning characteristics.  These
metabolites are referred to as "water-solubles" and/or "bound" residues
depending upon their behavior during analyses of the substrate.  Little
is known about the chemical and toxicological properties of the water-
solubles and bound metabolites.  Consequently, much of our efforts have
been in this area during the past 2 years.

In this report, the research which has been conducted since September 1,
1970 is included in 4 different categories.  First, studies designed to
determine the metabolic fate of carbamate insecticides in a specified
organism will be presented.  Second, work conducted on the fate of
selected carbamate metabolites will be considered.  Third, results of
studies to determine the interactions of pesticides and/or some selected
drugs on carbamate metabolism will be discussed.  Fourth, a report will
be presented covering studies of the mechanisms of glycosylation of
carbamate materials, and its significance in the over-all metabolism of
these toxicants.

METABOLISM

Aldicarb in Lactating Cows^

An earlier report showed that when Aldicarb [2-methyl-2-methylthio)
propionaldehyde 0-(methylcarbamoyl) oxime], was administered to a
lactating cow as a single oral  dose, approximately 85% of the dose was
eliminated from the body within 24 hours (Dorough and Ivie, 1968).
Aldicarb equivalents in the milk were low, maximum of 62 p.p.b., and no
Aldicarb per se was detected.  The nature of the metabolites was
similar to that reported for other animals and for plants.  The data

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indicated that the rapid excretion of Aldicarb, primarily in the urine,
by dairy animals would prevent the accumulation of residues in milk and
tissues to appreciable levels.  However, this could not be stated with
certainty because continuous feeding of Aldicarb could alter its
metabolic fate, possibly resulting in higher residues and/or ones
different chemically from those found after a single treatment.

The need for long-term, continuous feeding studies stems largely from the
behavior of the chlorinated hydrocarbon pesticides.  Certain of these
toxicants appear in milk even when very low levels are in the cow's feed.
Furthermore, several weeks, and often several months, of feeding are
necessary before the concentration of residues in the milk reaches a
plateau.  For example, heptachlor epoxide residues appeared to be still
increasing at the end of a 35-day feeding period (Williams et al., 1964).
With dieldrin, endrin, lindane, and DDT, the concentration of each res-
idue in milk had reached a maximum at various times during the feeding
period.  The nature of these chemicals continues to influence the in-
vestigational format of other groups of pesticides, such as the car-
bamates, despite the fact that these newer products are different chemically,
biologically, and metabolically.  Obviously, a format based on the
chlorinated hydrocarbons would not necessarily be appropriate for all
other insecticides.  In addition to establishing the fate of Aldicarb
in cows when administered over a long period of time and correlating
dosage rates to residue levels in meat and milk, the present investi-
gation should be useful in determining experimental parameters most
desirable for future studies using similar compounds.

The concentration of Aldicarb and/or its metabolites in mature forage
crops treated with Aldicarb has not been reported.  However, sufficient
work has been done to indicate the type of metabolites that must be
considered.  Aldicarb is readily oxidized in cotton to its sulfoxide
which is then slowly metabolized to the sulfone (Coppedge et al., 1967;
Metcalf et al., 1966).  When applied to the soil, maximum uptake of

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           35
Aldicarb-S   by cotton  plants occurred within 2 weeks, with no indication
of  significant uptake thereafter.  After 6 weeks, Aldicarb sulfoxide and
Aldicarb  sulfone were present in mature leaves in about equal quantities.
No  Aldicarb was detected.  These data demonstrate that the residues likely
to  be  present in feeds  would be Aldicarb sulfoxide and Aldicarb sulfone
and that  these products should be considered when evaluating the fate
of  Aldicarb residues in dairy animals.

Because Aldicarb sulfoxide is unstable in the pure form, Aldicarb was
used in the present study.  Based on the rapidity with which Aldicarb
was converted to its sulfoxide in rats (Andrawes et al., 1967), a fate
study  of  Aldicarb would yield results comparable to those obtained should
Aldicarb  sulfoxide be the administered compound.  The rapid conversion
of  Aldicarb to Aldicarb sulfoxide in a lactating cow also was noted
(Dorough  and  Ivie, 1968).  Even in the urine, which contained over
90% of the dose given,  Aldicarb was not detected.  However, Aldicarb
sulfoxide accounted for over 50% of the total Aldicarb equivalents in
urine collected only 3  hours after treatment.  Thus, the 1 to 1 molar
ratio of Aldicarb and Aldicarb sulfone administered to the cows in the
current study was intended to represent, as closely as possible, actual
residues that could be  consumed by cows if fed crops treated with
Aldicarb for  insect control.

Radioactive Aldicarb was used in an attempt to obtain a complete
picutre of its metabolic fate in dairy cows.   Admittedly, total detect-
able residues, and consequently the number of unknown metabolites, are
probably increased over those observed had more selective analytical
methods been employed.   However,  with selective methods, the scope of
any metabolic fate or residue study is limited to a predetermined set
of conditions.  There is little likelihood that products of an unpredict-
able nature would be detected.   This could result in toxicologically
significant metabolites being overlooked in the environment and/or
in products destined for human consumption.   Radiotracer techniques do

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not assure the identity of all residues but they do assure that the
presence of most metabolites is recognized.  This way, it is possible
for them to be evaluated as to general chemical nature, concentration,
and detectability.  Depending upon these factors, judgment may be made
as to the need for additional characterization.

Methods

Animals - Holstein cows purchased from a commercial dairy were used in
this study.  One animal was used in a pilot study to determine the effect
of the high Aldicarb dose on the general health of the cow.  The animal
weighed 645 kg. and was in the latter stage of lactation.  Three
additional cows were used in the radioactive Aldicarb feeding study.
Each had calved 6 to 8 weeks previously and was in peak milk production.
The cow fed the low level of Aldicarb weighed 470 kg., that fed the
medium level 500 kg., and the one fed the high level weighed 518 kg.
at the beginning of the experiment.  It was incidental that the weights
of the cows increased with the dosage rates.  In fact, the animals were
placed in designated stalls prior to being weighed and by persons un-
familiar with the fate of the animals.

The animals were held in metabolism stalls and a 12-hour milking
schedule was maintained.  Milking was done by machine with separate
apparatus used for each cow to minimize chances of cross contamination.
The close proximity of the cows proved to be far superior to the single
isolated animal situation used in earlier investigations.  There was
little sign of nervousness by the cows, and they responded to handling
in a normal manner.  More important, there was no decline in milk
production owing to confinement as had often been the case with an
isolated cow.

For the first 10 days of the study, during which the feces and urine
were not collected, the cows were exercised daily.  However, the cows

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were held  in constant confinement during the next 14 days while being
fed radioactive Aldicarb and while the urine and feces were collected
separately.

The urine  was collected by attaching a flexible vinyl hose, 1 inch in
diameter,  to the vulva of the cow.  First, the hose was fixed to a
triangular pad constructed of vinyl tape in such a manner that the
opening of the hose could be positioned directly over the vulva.  Oils
on the skin surrounding the vulva were removed by washing with ether,
and then a layer of contact cement was applied.  Contact cement was
applied to the triangular pad also.  When the cement on both surfaces
was dry, the pad was attached to the vulva.  To prevent the hose from
falling beneath the animal when she was in a prone position, it was
suspended  from the ceiling with rubber tubing so that it was held away
from the animal and about 12 inches from the floor at the lowest point.
Collecting the urine by this method created no apparent problem for the
cows and proved sufficiently stable so that only one hose had to be
repaired during the 14-day test period.

To take samples of blood at frequent  intervals without exciting the
cows, an intravenous catheter was inserted into the jugular vein of
each animal before treatment began.  The catheter was equipped with a
receptacle for a standard syringe with which the blood was withdrawn.
As there was no pain associated with sampling the blood using this
technique, it was unnecessary to restrain the animals.

At the morning and evening mil kings each cow was given 12 pounds of
grain.   Water and alfalfa hay were provided ab libitum.  The amount of
feed consumed by each animal was recorded daily.

Treatment and Sampling - Aldicarb-S-methyl-C   and Aldicarb sulfone-S-
methyl-C   , both having a specific activity of 5 mCi. per mmole, were
obtained from the Union Carbide Chemicals Co. as were nonradioactive

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forms of these chemicals and metabolite standards (Table 1).   Equal  molar
quantities of Aldicarb and Aldicarb sulfone were dissolved in acetone
and appropriate aliquots of the solution transferred to a gelatin cap-
sule containing crushed grain.  The capsules were administered to the
cows with a balling gun twice daily at 12-hour intervals, one at the
morning milking and one at the evening milking.  For each treatment,
feeding was begun and then, after several minutes, was interrupted
briefly when the cow was given the capsule.  The cows were milked
immediately thereafter.

Dosage rates of Aldicarb were calculated on the basis of an anticipated
total feed intake of 50 pounds per cow per day.  For example, the cow
receiving 1.2 p.p.m. Aldicarb equivalents in the diet was given 0.036
mmoles, 6.8 mg., of Aldicarb and 0.036 mmoles, 7.9 mg., of Aldicarb
sulfone at each feeding.

The cow used in the pilot study was given nonradioactive Aldicarb and
Aldicarb sulfone at the rate equivalent to 1.2 p.p.m. of Aldicarb in the
diet for a total of 10 days.  Animals in the dosage series were fed
nonradioactive insecticide for 10 days and then the radioactive products
for an additional 14 days.  In all cases, treatment was continuous
throughout the indicated time periods.

Blood was taken daily from each cow while on the Aldicarb treatment.
Samples were withdrawn 6 hours after the morning milking and were
immediately assayed for plasma and red blood cell (RBC) cholinesterase
activity.  Blood from animals fed radioactive Aldicarb was also radio-
assayed to determine the total Aldicarb equivalents present.   Milk,
urine, and feces were collected at 12-hour intervals, weighed, and then
frozen until analyzed.  Eighteen hours after the last radioactive
Aldicarb treatment, the cows were slaughtered and tissue samples re-
moved for analysis.

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Assay  Procedure  -  For  all  radioactive measurements reported herein, a
Packard Tri-Carb Model  3365  instrument was used.  The scintillation
mixture consisted  of a 2 to  1 mixture of toluene and methyl cellosolve
containing  5 grams  of  PRO  per liter.  All fluid samples and extracts
were assayed directly  by counting aliquots of 0.2 to 0.5 ml.  Total
radioactivity  in blood, tissues, feces, and substrate solids after ex-
traction was determined by oxygen combustion in a Parr double-valved
bomb.  Approximately 1  gram  of whole blood was placed in a small bag
made from cellulose dialysis tubing (Kelly et al., 1961) and dried
overnight at 40°C.  The dried sample was combusted in oxygen at 25 atm.
pressure, and  the  resulting  carbon-14 dioxide was trapped in 20 ml. of
a mixture of 2 to  1 methyl cellosolve and monoethanolamine (Jeffay and
Alvarez, 1961).  A  2-ml aliquot of the trap solution was radioassayed
by liquid scintillation counting.

For determination of blood cholinesterase levels, freshly drawn
hepan'nized blood was  centrifuged to separate the RBC's from the plasma.
After the plasma was decanted, the RBC's were diluted to the original
blood volume with distilled water.  They were not washed prior to
dilution.  Cholinesterase  activity in the plasma and RBC's was measured
using a radioisotopic method (Reed et al., 1966).

Extraction - Radioactive residues were extracted from the milk using
the basic procedure of Timmerman et al. (1961).   Briefly, the method
called for the thorough mixing of whole milk, 50 ml., with acetonitrile
to precipitate the milk solids, and then for the addition of chloro-
form so that an aqueous and an organic solvent layer was obtained.   With
the original method, the residues in the two liquid phases and in the
milk solids would be quantitated at this point.   In the present study,
the aqueous phase was further concentrated to approximately 10 ml.  and
reextracted in a manner identical to the whole milk.   This allowed
more complete removal  of the solids suspended in the aqueous portion of
the milk and resulted in greater extraction of radioactive residues into
                                10

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the organic solvent fraction.  For future reference, residues in the
organic solvent fraction will be referred to as organo-extractables,
those in the water layer as water-solubles, and radioactive residues
remaining in the milk solids as unextractables.

Urine was extracted with chloroform, and the organo-extractable
metabolites were characterized using the same techniques described for
evaluating the nature of the organo-extractables from milk.  Partial
cleanup of the water-soluble metabolites from urine was accomplished on
a Sephadex column.  Sephadex LH-20, after being placed in distilled water
for 3 hours and then in acetone for 10 minutes, was added to a chromato-
graphic column 2.5 cm. in diameter, until a column bed of 10 cm. was
attained.  The column was then washed with 50 ml. of acetone.  Five
milliliters of the chloroform-extracted urine was concentrated to
approximately 0.2 ml. and transferred to the column.  One hundred
milliliters of acetone was passed through the column to elute any
organo-extractable metabolites remaining in the urine after extraction
with chloroform.  This was followed by 100 ml. of a 6 to 1 mixture of
acetone and methanol and finally by 100 ml. of a 1 to 1 mixture of acetone
and methanol.  The 6 to 1 solvent system served to remove certain
interfering materials.  Those radioactive materials eluted with the 1
to 1 solvent mixture were considered as the true-water-soluble
metabolites.

The liver was the only tissue in which attempts were made to extract
the radioactive products.  Acetone, benzene, n-butanol, ethanol,
methanol, water, and mixtures thereof were used in efforts to extract
the residues.

Thin Layer Chromatography - Techniques used to separate and identify
the radioactive organo-extractable metabolites were the same as re-
ported  earlier (Andrawes et al., 1967; Dorough and Ivie, 1968).
Metabolite designations are those used by Dorough and Ivie (1968).
                               11

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Their separation by thin layer chromatography is shown in Table 1.

After elution from the column, the water-soluble metabolites from
urine were resolved by thin-layer chromatography (TLC) using plates
prepared from Silica Gel G slurried in a 0.1M boric acid solution.
The chromatograms were developed in a 5 to 4 to 1 mixture of acetone,
n-butanol, and 0.1M boric acid.  After the radioactive areas on the
plates were located by radioautography, they were extracted from the
gel with methanol.

Analysis of Hater-Soluble Metabolites - Attempts were made to cleave
the aglycones from the urine water-soluble metabolites by acid and
enzymatic hydrolysis.  For acid hydrolysis, each metabolite(s) extracted
from the silica gel was incubated in 2N HC1 for 30 minutes at 95°C.
Similarly, each metabolite(s) was incubated in various enzyme prepar-
ations at 37°C for as long as 3 days.  Enzymes (Sigma Chemical Co.)
used in these studies and the pH of the incubation media were as
follows:  beta-glucuronidase Type H-l, pH 5.0; beta-glucuronidase
Type 1, pH 6.9; beta-glucosidase, pH 5.3; sulfatase, pH 5.0; protease,
pH 7.5; maltase, pH, 6.4; and alpha-amylase, pH 6.9.

Following incubation, the mixtures were extracted with chloroform and
the percentage conversion of water-solubles into organo-extractables
was determined.  The radioactive components of the chloroform extract
were separated by TLC and their chromatographic behavior was compared
with Aldicarb metabolite standards and with the original  organo-
extractable metabolites from the urine.

Results and Discussion

Effect on Animals - The pilot study indicated that feeding a dairy cow
a diet containing 1.2 p.p.m.  Aldicarb equivalents would not cause any
visible effects to the animal.  Moreover, the blood cholinesterase levels
                               12

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were not reduced and the quantity of milk produced by the cow remained
constant.  Hay and grain consumption, 27 pounds per day, and milk pro-
duction, 30 pounds per day, were well below that for the other animals
used in this study (Table 2).  However, these values were considered
normal for this particular cow since they were the same as the pre-
treatment figures.  The fact that this cow was in the latter stage of
lactation and was isolated from other animals during the feeding ex-
periment could account for the low feed consumption and milk production.
Urine and feces from the pilot cow were not quantitated.

Even though the pilot cow did not consume 50 pounds of feed per day, the
animal was administered that amount of Aldicarb and Aldicarb sulfone
which would be present in 50 pounds of feed containing 1.2 p.p.m. of
Aldicarb equivalents.  Since this dose of insecticide was not harmful
to the animal, the Aldicarb feeding study involving three cows was
initiated.

As observed in the pilot study, there were no apparent harmful effects
to the cows resulting from Aldicarb in the diet at 0.12 p.p.m., 0.6
p.p.m., or 1.2 p.p.m.  Blood cholinesterase levels were the same
during the time Aldicarb was being fed as they were before treatment
commenced.  Milk production, feed consumption, and quantity of excretory
products remained stable throughout the experiment.  Each animal con-
sumed approximately 47 pounds of feed per day but varied slightly in
the amount of milk produced (Table 2).  However, there was little day-
to-day variation in the amount of milk produced by an individual animal.

Elimination of total  Aldicarb Equivalents - The amount of the administered
radioactive Aldicarb eliminated from the cows in the milk, urine and
feces is shown in Table 3.  Although all samples collected were analyzed,
only those data sufficient to demonstrate the pattern of elimination are
presented.  The quantity of radioactivity eliminated by these three
routes stabilized very rapidly.  After 3 days, the concentration of
                                  13

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           14
Aldicarb-C  equivalents  in the milk, urine, and feces was very close
to the maximum detected at any time during the study.  The fact that there
was a consistent relationship between the amount of Aldicarb consumed
and the rate of elimination of residues from the body was apparent from
the level  of radioactivity detected in the blood.  After 2 days on the
                                     14
Aldicarb diet, residues of Aldicarb-C   equivalents in the blood of
the cow fed 0.12 p.p.m. were maintained at approximately 3 p.p.b.  The
blood of animals fed Aldicarb at 0.6 and 1.2 p.p.m. contained
approximately 8 and 18 p.p.b., respectively, after the second day of
feeding.

                               14
Most of the consumed Aldicarb-C   was eliminated from the cows in the
urine (Table 3).  Regardless of the level of treatment, about 90% of
the daily  dose could be accounted for in urine collected the following
day.  There was a slight, but continuous, increase in the percentage of
the dose eliminated in the urine during the feeding of radioactive
Aldicarb.  This may have resulted from an increase in efficiency of
elimination of the daily doses by the animals or from the release of
products stored from the earlier feedings.  The same type of increase
was noted  in the feces.  Whereas from 1 to 2% of the dose was voided in
                                                           14
the feces  after the first day, 3 to 3.5% of the cumulated C  -treatments
was eliminated in the feces by the 14th day.   By adding the percentage
of the dose eliminated in the milk, approximately 1.0%, to the corres-
ponding values for urine and feces, it was concluded that dairy animals
on a continuous diet containing Aldicarb would consistently eliminate
90% or more of the insecticide consumed daily.

                                      14
Milk - Parts per billion of Aldicarb-C   equivalents in the milk were
directly related to the concentration of Aldicarb in the diet, Table 4.
The level  of C  -residues in milk from the cow fed 0.12 p.p.m. of
Aldicarb ranged from 0.9 to 1.9 p.p.b.  and averaged 1.4 p.p.b.  For the
0.6 p.p.m.  feeding level, the residues in the milk ranged from 5.0 to
6.5 p.p.b.  and at the 1.2 p.p.m.  level, the residues ranged from 12.1
                                 14

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19 16.3 p.p.b.  The average concentrations of Aldicarb-C   equivalents
frt irtlk resulting from the latter two feeding levels were 5.7 and 13.3
p.0*b., respectively.  The ranges cited above do not include values
Obtained from samples collected within 12 hours of the first treatment.
lidlolabeled residues had not reached the point of stabilization at that
tltoe and did not represent a true continuous feeding situation.   The
average values include data from 28 separate milk samples for each
fitding level, two samples each day for 14 days.

The nature of the radioactive residues in the milk is shown in Table 4.
Again, analyses were performed on each milk sample collected throughout
the feeding study, but only selected data are presented because the
results were almost identical for all samples within a given treatment.
As demonstrated in Table 4, the relative concentration of metabolites
in the milk remained fairly constant during the 14-day feeding period.
These results indicated that the metabolism of Aldicarb was not altered
by continuous feeding of the insecticide or by increasing its concen-
tration in the diet from 0.12 to 1.2 p.p.m.

Oxlmes and nitriles made up approximately 50% of the radiolabeled
metabolites.  Nitrile sulfone was the major metabolite, accounting for
approximately 60% of the known hydrolytic products and 30% of all radio-
active, residues in the milk.

Aldicarb sulfone was the principal carbamate in the milk, constituting
from 15 to 19% of the radioactive products present.  Aldicarb sulfoxide
was present at about one-fourth this level.  Their combined concentrations
were 0.3, 1.0, and 2.7 p.p.b. in milk from cows fed Aldicarb at the
three dosage levels.  The parent compound, Aldicarb, was not detected in
any of the milk samples.

Collectively, the organo-extractable metabolites listed as unknowns in
Table 4 composed 15% of the total residues.  However, the maximum con-
                                 15

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centration of these unknown metabolites, even in milk of the cow fed the
high  level of Aldicarb, was only 2.2 p.p.b.

After concentrating the aqueous fraction of the milk extract and
thoroughly re-extracting it with acetonitrile and chloroform, there were
no detectable water-soluble metabolites present in the milk (Table 4).
Without this additional extraction, however, from 10 to 15% of the radio-
activity in the milk remained in the water phase.  Independent analysis
of these water-soluble products revealed that approximately 65% were
bound with the milk solids, 15% as Unknown 3, 10% as Aldicarb sulfoxide,
5% as Aldicarb sulfone, and 5% as Unknown 3a.

Radioactivity remaining with the milk solids after extraction accounted
for 15 to 20% of the total labeled-residues in the milk.  Although
sizable when considered in relation to their percentage of the total
residues, their absolute concentration was very low.  These values were
0.3, 0.9 and 2.1 p.p.b. for the three treatment rates, respectively
(Table 4).  No attempt was made to characterize the radioactive metabo-
lites of Aldicarb located in the solid fraction of the milk.

Urine - Radioactive metabolites of Aldicarb in the urine increased
proportionally with the increased levels of Aldicarb in the diet of the
cows (Table 5).   As was the case in the milk, however, the relative con-
centrations of the metabolites were strikingly similar regardless of the
levels of Aldicarb fed.

Only about 25% of the radiolabeled products in the urine was extractable
with chloroform.  Of these, approximately 40% was identified as Aldicarb
sulfoxide and Aldicarb sulfone, 50% as oximes and nitriles, and 10% was
unknown materials.  At the low feeding level, the average concentrations
of these products were 11, 14 and 3 p.p.m., respectively.   Urine from
cows fed 0.6 and 1.2 p.p.m. contained residues corresponding to the
increased dosage rates.  With the exception of Unknown 5,  found only in
                                  16

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the milk, the organo-extractable metabolites in the milk and urine were
the same.

Seventy-five per cent of the radioactivity in the urine remained in the
aqueous phase after extraction with chloroform (Table 5) and was resolved
by TLC into four distinct radioactive bands (Table 6).  When unextracted
urine was concentrated and applied to the chromatograms, two additional
bands were observed, one with a Rf value of 0.60, band 5, and the other a
Rf of 0.76, band 6.  Examination of these bands individually on TLC,
using the solvent systems for organo-extractables, showed that the Rf
0.6 material cochromatographed with Aldicarb sulfoxide and that the Rf
0.76 band was a mixture of other organo-extractable metabolites.  In this
same solvent system, the radioactive materials of bands 1 through 4
(Table 6) stayed at the origin.

TLC analysis of chloroform-extracted urine disclosed that small amounts
of bands 5 and 6 were still present, demonstrating that the partitioning
characteristics of the organo-soluble metabolites make it virtually im-
possible to attain complete extraction.  Although their combined concen-
tration was only 1 to 3% of the .radioactivity in the extracted urine, it
was sufficient to interfere with subsequent analysis of the water-
soluble metabolites.  This problem was solved by removing the organo-
extractable radioactivity from the urine by Sephadex column chromatography.
That material eluted from the column with a 1 to 1 mixture of acetone and
methanol contained only the four radioactive bands shown in Table 6.
The organo-extractable materials were eluted beforehand with acetone.
In addition to allowing complete separation of the two classes of
metabolites, the Sephadex column removed much of the interfering material
from the radioactivity and transferred the water-soluble metabolites to
an organic solvent.  The latter could be more readily reduced in volume
for application to TLC and gave improved separation of metabolites.

Water-Soluble Metabolites - Cleavage of water-soluble metabolites of

                                 17

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Aldicarb from urine  by enzymatic means was almost totally unsuccessful.
The maximum conversion of water-soluble metabolites to organo-extract-
able materials came when the incubation mixture consisted of beta-
glucuronidase Type H-l in pH 5 buffer.  After 3 days at 37°C, 7% con-
version had taken place.  Increasing the amount of enzyme and/or the
period of incubation failed to increase the quantity of organo-
extractable materials.  Two-dimensional TLC chromatography (Dorough and
Ivie, 1968) of the aglycones showed the presence of three products, one
chromatographing with Unknown 3 of the original organo extractables, one
chromatographing with Unknown 3a, and the other chromatographing with
oxime sulfone.  Exact quantitation and identification were not
possible because of the small quantity of radioactivity.

Acid hydrolysis of the water-soluble metabolites yielded much higher
amounts of organo-solubles than did the enzymatic method.  For these
studies, radioactive bands 1 through 4 (Table 6) were incubated separately
in 2N HC1 at 95°C for 30 minutes.  Each of the bands yielded several
organo-extractable products after being resolved by TLC.  However, there
was a single major product produced in every case.  With band 1, 72%
of the water-solubles was cleaved by the acid, and 95% of these were in
the form of an unknown which was designated as Unknown A.  Band 2 was
hydrolyzed 80% by the acid and 96% of the aglycones was as a material
designated Unknown B.  Acid hydrolysis of band 3 gave only 45% cleavage
of the water-soluble materials while 73% of band 4 was cleaved.  Both band
3 and 4 aglycones were in the form of Unknown B in excess of 95%.

Unknowns A and B were products which did not cochromatograph with any
of the metabolite standards of Aldicarb.  Thus, the watersolubles in the
urine were not conjugates of the free Aldicarb metabolites or the free
metabolites, if formed by acid hydrolysis, were unstable in the incu-
bation medium.  Incubating the Aldicarb metabolite standards in acid and
then examining the products extracted with chloroform showed that
Aldicarb and the standards were highly unstable under these conditions.
                                18

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This made it impossible to identify the aglycones as they existed as part
of the water-soluble metabolites.

Since the enzymatic cleavage of the water-soluble metabolites failed
and the acid cleavage resulted in the destruction of the aglycones,  only
indirect evidence is available concerning the identity of the Aldicarb-
contributing portion of the conjugate metabolites.  When Unknown A and
Unknown B, formed by acid hydrolysis of the conjugates, were compared
with the unknowns produced from the Aldicarb standards upon acid
hydrolysis, certain products were identical chromatographically.  The
major acid degradation product of oxime sulfone cochromatographed with
Unknown B.  That oxime sulfone was the only standard yielding Unknown B
suggested that it could be the major component of the water-soluble
metabolites of Aldicarb in the urine.  In fact, over 95% of the radio-
activity in bands 2 through 4 (Table 6) was degraded to the same
product by acid hydrolysis as was oxime sulfone.  Only band 1, which
remained at the origin of the TLC, was degraded to a large extent to
Unknown A.  This unknown was not the principal degradation product of any
of the Aldicarb standards when placed in the acid hydrolysis conditions.
However, it did cochromatograph with Unknown 3 of the organo-extractable
metabolites.  It is possible, therefore, that approximately 5% of the
urine water-solubles was conjugates of an unknown aglycone and that
which remained was conjugates of oxime sulfone.  While positive ident-
ification of the conjugate metabolites of Aldicarb must await further
evaluation, these data offer strong evidence that Aldicarb, Aldicarb
sulfoxide, and Aldicarb sulfone, which are of obvious toxicological
importance, do not directly contribute to their formation.

Tissues - Of 27 different tissue samples analyzed for total Aldicarb-
C   equivalents, detectable residues were observed in 22 tissues from
the cow fed 1.2 p.p.m. Aldicarb in the diet, in 20 tissues from the
cow fed 0.6 p.p.m. Aldicarb, and 1 sample taken from the cow fed 0.12
p.p.m. Aldicarb (Table 7).  Even at the high feeding level, residues
                                19

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were absent  in muscle tissue, fat, and bone.  With the exception of those
in the  liver, all of the residues in the tissues from the cow fed 0.6
p.p.m.  Aldicarb were considered as trace quantities since they were
present at levels only slightly above the limit of sensitivity of the
analytical method.  Generally, the same was true for tissues from the cow
fed the high level of Aldicarb.  Only the liver and lungs contained re-
sidues  in excess of fourfold the 4 p.p.b. limit of sensitivity.  In this
animal, the  lungs contained 35 p.p.b. Aldicarb-C   equivalents and the
liver 164 p.p.b. Aldicarb-C   equivalents.

The liver from the animal fed the highest concentration of Aldicarb was
the only tissue in which the radioactive content was sufficient to warrant
extraction and characterization of the residues.  However, attempts to
extract the radioactive residues failed, and nothing of their nature was
determined.  The fact that they resisted extraction so successfully might
suggest that the residues in the liver were not Aldicarb-like at all
but were naturally occurring products containing a mere fragment of the
Aldicarb molecule.  It is unlikely that products other than these would
remain with the solid liver residue rather than being in the organic
solvent or water phase after extraction with water, acetone, methanol,
n-butanol, or hot ethanol as was found to be the case.

Analytical Considerations - The present study showed that the parts per
million level of total  residues in the milk of cows fed Aldicarb were
approximately 1/100 that level  in the diet (Table 4).  This relation-
ship held true for feeding levels varying from 0.12 to 1.2 p.p.m., and
in animals where the average milk production varied from 41 to 58 pounds
per day.  Because of this, one should be able to predict with a high
degree of accuracy the concentration of residues in milk when the
level  of Aldicarb in the diet is known.   However, the actual quantitation
of Aldicarb equivalents in milk may be difficult.

Since the residues in milk of cows fed Aldicarb-contaminated feed were so

                                20

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low, it is unlikely that they would be detected by conventional  analytical
methods.  Even on a total Aldicarb-equivalent basis,  a method would  have
to be sensitive below the 0.01-p.p.m.  level  to detect residues in milk
of animals fed 1 p.p.m. Aldicarb in the diet.  To detect only the known
carbamate materials, Aldicarb sulfoxide and  Aldicarb  sulfone, the
sensitivity of the method would have to be greater than 0.002 p.p.m.
Lower feeding levels of Aldicarb would obviously demand even greater
sensitivity if residues were to be quantitated.

For monitoring purposes and for certain investigational uses, it would
be possible to use the urine as an indicator of residue levels in the
milk.  The p.p.m. Aldicarb equivalents in urine (Table 5) were approx-
imately 100 times greater than those in the  milk, and the same as the
p.p.m. Aldicarb fed in the diet.  It would be possible to detect combined
Aldicarb sulfoxide and Aldicarb sulfone in urine if cows were fed
Aldicarb in the diet at concentrations as low as 0.12 p.p.m.  This
would require a sensitivity of only 0.01 p.p.m.  If the two compounds
were detected, it would indicate that their  combined  concentration in
the milk (Table 4) was about one-fiftieth that observed in the urine.
If total residues were detected, the indicated residues in the milk
would be 1/100 that in the urine.

Comparison of Single-Dose and Continuous-Feeding Studies - Generally,
there was good agreement between results reported by  Dorough and Ivie
(1968) and those obtained in the current tests.  In the earlier study,
          35
Aldicarb-S   was given as a single oral dose at a rate equivalent to
approximately 3.5 p.p.m. in the diet.   During the first 24 hours after
treatment, 83% of the dose was eliminated in the urine and 1% in the
milk.  These values are very close to that amount eliminated 24 hours
after the first Aldicarb-Aldicarb sulfone doses (Table 3).  The feces
                 35
of the Aldicarb-S   treated cow contained a  lower percentage of the
dose after 24 hours than did the Aldicarb-Aldicarb sulfone treated
animals, 0.6% as compared with about 2%.  However, the total
                                  21

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eliminated by this route after the single treatment, 2.9% of the dose,
was almost identical to the average daily values observed in the con-
tinuous feeding study.

The similarities between the results of the single- and continuous-
Aldicarb feeding studies were maintained to a large degree when the residues
in milk were considered in detail.  The average concentration of
Aldicarb equivalents in milk collected during the first 24 hours after
              35
the Aldicarb-S   treatment was 39 p.p.b., three times the 14-day
average value in milk from the cow treated with Aldicarb plus Aldicarb
sulfone at 1.2 p.p.m.  The threefold increase was coincident with the
higher treatment rate, 3.5 p.p.m. Aldicarb in the diet, used in the
single treatment study.

There were two differences noted when the chemical  nature of residues in
the milk from the single- and continuous-Aldicarb feeding studies were
compared.  First, the 24-hour milk from the Aldicarb-S   study had 67%
of the radioactivity in the organo-extractables, 23% in the aqueous
milk phase, and 10% in the milk solids.  Corresponding values for the
Aldicarb-Aldicarb sulfone feedings were approximately 80, 0, and 20%.
These differences may be explained by incomplete extraction and
separation of the milk phases in the initial  study.  As pointed out
earlier, improved extraction techniques were utilized in this experiment.

The second difference in the chemical  nature of residues in milk from
the two studies was the presence of metabolite Unknown 3a in the latter
study (Table 4).   This material  was not detected in the Aldicarb-S
test but was evident in milk from all  three animals treated with.
Aldicarb-Aldicarb sulfone-C   for 14 days.   It also was detected in the
urine of these cows.   The fact that Unknown 3a was  detected in samples
of milk and urine collected within 1  day after treatment began shows
that the metabolite did not result from continuous  treatment.   There-
fore,  the metabolite must be an  initial metabolic product of Aldicarb
                                 22

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and/or Aldicarb sulfone.  Recent studies in our laboratory on the metab-
olism of Aldicarb in chickens showed that metabolite Unknown 3a was
present in milk and urine of the cow treated with a single dose of
          35                        14
Aldicarb-S   and Aldicarb-S-methyl-C  .   These data indicate that Un-
known 3a was present in milk and urine of the cow treated with a single
                  35
dose of Aldicarb-S  .   Possibly, its presence was not detected because
of the frequent intervals (3, 6, 12, and 24 hours after treatment) in
which the samples were collected.  Sampling in this manner could pre-
vent the accumulation of a relatively slowly-formed metabolite to a
detectable level.
With only minor exception, then, it is apparent that the continuous ex-
posure of dairy animals to Aldicarb in the diet does not significantly
alter its fate as compared to a single exposure.  Therefore,  long-term
feeding studies to determine the relationship of levels in the diet to
residues in animal products when dealing with rapidly metabolized and
rapidly excreted compounds such as Aldicarb are not required.   Also,
it is evident that a single-dose study, usually designed to determine
the general metabolic fate of a compound, can be very useful  in esti-
mating the concentration and nature of residues which might occur in
consumable products of animals receiving insecticides of this  type in
the diet.
                                 23

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TABLE 1.  DESIGNATIONS USED FOR ALDICARB AND ITS METABOLITES

                   AND THEIR SEPARATION BY TLC

Designation3
Aldicarb-C14
Aldicarb su If oxide
14
Aldicarb sulfone-C
Aldicarb oxime
Oxime sulfoxide
Oxime sulfone
Nitrile sulfoxide
Nitrile sulfone
Unknown 1
Unknown 2
Unknown 3
Unknown 3a
Unknown 5

1
0.67
0.05
0.21
0.78
0.18
0.56
0.31
0.62
0.05
0.10
0.11
0.36
0.47
Rf Values5
2
0.94
0.18
0.64
0.95
0.22
0.78
0.70
0.93
0.11
0.24
0.34
0.41
0.86
a Metabolite designations from Dorough and Ivie (1968)

  Two dimensional TLC.  First solvent system, 2:1  ''ether-
  hexane + 20% acetone and second system, 2:1 methylene
  chloride-acetonitrile.
                        24

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TABLE 2.  TREATMENT RATES AND FEED CONSUMPTION FOR DAIRY COWS FED

                          ALDICARB FOR 24 DAYS3

Average per day values/cow no.

Aldicarb equivalents in
feed, p. p.m.
Aldicarb equivalents,
nig/ kg (body wt)
Feed consumption, Ibs.
Milk production, Ibs.
Pilot
1.2
0.042
27
30
1
0.12
0.006
46
41
2
0.6
0.027
48
50
3
1.2
0.052
47
58
  The pilot cow was given nonradioactive aldicarb for 10 days while
  the other animals received the insecticide for a total of 24
  days, 10 days on nonradioactive material  and 14 days on radio-
  labeled products.
                                 25

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ro
       TABLE 3.  ELIMINATION OF RADIOACTIVITY BY COWS FED ALDICARB-C14 FOR 14 DAYS AT RATES OF 0.12, 0.6
                                               AND 1.2 P.P.M. IN THE DIET

Percent of total dose for each treatment level in
Days fed
insecticide
1/2
1
2
3
7
10
12
14
Milk, p. p.m.
0.12
0.9
0.8
0.7
0.7
0.8
0.8
0.9
0.9
0.6
0.5
0.7
0.8
0.8
0.9
0.9
0.9
0.9
1.2
0.7
1.1
1.2
1.2
1.3
1.3
1.3
1.3
Urine, p. p.m.
0.12
68.7
82.0
86.0
90.1
90.7
90.9
93.1
93.8
0.6
75.6
81.9
85.1
89.7
88.5
90.5
91.2
91.6
1.2
74.4
83.6
85.8
89.7
90.8
90.4
91.0
92.1
Feces, p.p.
0.12
1.3
2.0
3.1
3.3
3.4
3.5
3.5
3.5
0.6
0.5
1.1
2.0
2.4
2.5
2.8
2.9
3.0
m.
1.2
0.8
1.6
1.9
2.3
2.6
2.8
2.8
2.9
       a Calculations based on total dose consumed and total radioactivity eliminated by each indicated time.

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TABLE 4.  RADIOACTIVE COMPONENTS IN MILK OF COWS FED ALDICARB-C14 AT

                RATES OF 0.12, 0.6, AND 1.2 P.P.M.  FOR 14 DAYS

Metabolites
Organo-extractables
Aldicarb sulfoxide
Aldicarb sulfone
Oxime sulfoxide
Oxime sulfone
Nitrile sulfoxide
Nitrile sulfone
Unknown 1
Unknown 2
Unknown 3
Unknown 3a
Unknown 5
Water-solubles
Milk solids
Total
P.
Cow #1 (0.12)
14-day avg.

0.06
0.21
0.05
0.07
0.05
0.44
0.01
0.04
0.08
0.08
0.02
0
0.28
1.39
P. B. /14-day average
Cow #2 (0.6)
14-day avg.

0.22
0.85
0.27
0.34
0.17
2.22
0.04
0.12
0.29
0.22
0.06
0
0.91
5.71

Cow #3 (1.2)
14-day avg.

0.40
2.24
1.13
0.94
0.61
3.71
0.08
0.27
0.74
0.96
0.11
0
2.06
13.25
a Metabolites extracted from whole milk with acetonitrile and
  chloroform.
                                27

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TABLE 5.  RADIOACTIVE COMPONENTS IN URINE OF COWS FED ALDICARB-C14 AT
                RATES OF 0.12, 0.6, AND 1.2 P.P.M. FOR 14 DAYS

Metabolite
Organo-extractabl esa
Aldicarb sulfoxide
Aldicarb sulfone
Oxime sulfoxide
Oxime sulfone
Nitrile sulfoxide
Nitrile sulfone
Unknown 1
Unknown 2
Unknown 3
Unknown 3a
Water-solubles
Total
P
Cow #1 (0.12)
14-day avg.

4.4
7.3
7.7
4.9
1.0
1.5
0.1
0.6
1.1
1.2
96.0
125.8
. P. B./ 14-day average
Cow n (0.6)
14-day avg.

25.6
35.9
34.7
32.3
3.6
6.7
0.6
2.4
7.9
3.6
465.2
609.5

Cow #3 (1.2)
14-day avg.

43.6
66.5
96.4
43.6
8.0
10.3
1.1
6.8
8.0
16.0
847.2
1147.5
  Metabolites extracted from urine with chloroform.
                               28

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TABLE 6.  THIN LAYER SEPARATION3 OF RADIOACTIVE WATER-SOLUBLE METABOLITES

                                  IN URINE

Percent of water solubles/days
Radioactive band
1
2
3
4
Rf
0
0.1
0.17
0.39
3
5.1
42.2
42.8
9.9
7
5.8
40.4
46.4
7.4
10
3.1
46.7
42.0
8.2
14
5.0
38.9
40.9
15.2
a TLC plates prepared with Silica Gel G in 0.1M boric acid.  Solvent
  system consisted of a 5:4:1 mixture of acetone, n-butanol, and 0.1M
  boric acid.
                                29

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TABLE 7.  RADIOLABELED RESIDUES IN TISSUES OF COWS FED ALDICARB-C14 FOR

                                  14 DAYS3

Tissues
Liver
Lungs
Kidney
Bile
Adrenal glands
Abomasum
Omasum
Large intestine
Ovaries
Rumen
Udder
Pancreas
Spinal cord
Gall bladder
Heart
Reticulum
Spleen
Skin
Small intestine
Brain
Neck muscle
Tongue
P.P.B. aldicarb equivalents
feeding level0
Cow #1 Cow #2
(0.12 p. p.m.) (0.6 p. p.m.)
29 123
7
6
9
6
4
4
5
6
5
6
5
_
4
6
6
5
4
5
4
-
4
at indicated
Cow #3
(1 .2 p. p.m.)
164
35
16
16
12
11
11
10
10
10
10
9
9
8
8
8
8
7
7
6
6
6
  Animals slaughtered 18 hours after last treatment.

  Residues were not detected (below 4 p.p.b.) in the  following tissues:
  foreleg muscle, hindleg muscle, omental fat, subcutaneous fat,  and
  rib bone.

  - indicates residues below 4 p.p.b.

                                 30

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Aldicarb in Laying Hens

Recent reviews of aldicarb metabolism (Dorough, 1970; Kuhr, 1970)
pointed out that sulfur oxidation and hydrolysis of the carbamate  ester
were of major importance in the metabolism of this carbamate.   Subse-
quent work supported this view and has extended the study of aldicarb
metabolism to include the synthesis and identification of highly polar
products not considered previously (Bartley et al., 1970; Durden et al.,
1970).  The metabolic pathway for aldicarb presented by Bartley et al.
(1970) is the most complete of any reported thus far.  It is likely that
this pathway is representative of the type of metabolism which aldicarb
undergoes in the other biological systems tested.

It was the intent of the current study to determine if laying hens
metabolized aldicarb in the same manner as reported for other organisms.
In addition, these investigations were designed to provide evidence for
the levels and chemical nature in residues in eggs and tissues of  hens
which consume aldicarb residues in the diet.  The sulfone analog was
included since it is a common metabolite in plants which may serve as
poultry feed (Bartley et al., 1970).

Methods

                                 14                                14
Insecticides - Aldicarb-S-methyl-  C and aldicarb sulfone-S-methyl-  C
                                                           35
each with a specific activity of 5 mCi/mmol, and aldicarb-S  , 45.7
mCi/mmol, were supplied by the Union Carbide Corp., as were a series
of metabolite standards.  The radioactive materials contained less than
1% radioactive impurities as determined by thin-layer chromatography
(tic) and radioautography.

Treatment and Sampling - In the first experiment, ten White Leghorn
                                                              35
laying hens were treated with a single oral dose of aldicarb-S   at a
rate of 0.7 mg/kg.  The birds, each weighing approximately 1.5 kg  were

                                 31

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administered the radioactive aldicarb via a 5 grain gelatin capsule which
contained a small amount of laying mash.  Two hens were sacrificed at
6 hr, and 1, 3, 5, and 10 days after treatment.  Eggs and feces were
collected at 6-hr intervals during the first 12 hr and then at 24-hr
intervals thereafter.  In this and all other tests described herein,
the birds were maintained in air-conditioned housing under continuous
lighting.  Water and laying mash were provided ad libitum.

In another test, six laying hens received a 1:1 molar ratio, single
                      14                       14
oral dose of aldicarb-  C and aldicarb sulfone-  C.  Doses of the
insecticides were prepared in gelatin capsules so that each hen received
0,7 mg/kg of aldicarb equivalents.  The birds were sacrificed 6 hr, 1
day, and 3 days after the carbamates were administered.  Eggs and feces
were collected as described above.

To study the fate of aldicarb residues when consumed by hens for an ex-
                        14                       14
tended period, aldicarb-  C and aldicarb sulfone-  C, 1:1 molar ratio
doses, were administered to hens every 12 hr for 21 days.  Based on an
average feed consumption of 80 g per bird per day, the treatment levels
corresponded to aldicarb levels in the diet of 0.1, 1.0 and 20.0 ppm.
Six hens were used for each treatment level, and another six birds serv-
ed as control animals.  Nonradioactive insecticides were given to the
birds for 7 days prior to initiating the radioactive feeding.  Each
capsule contained half the total amount of aldicarb equivalents requir-
ed in 1 day's ration to obtain the desired ppm level in the diet.  Eggs
and feces were collected twice daily just prior to administering the
radioactive aldicarb and aldicarb sulfone.  Three hens from each feed-
ing level were sacrificed 12 hr after the last treatment and the remaining
three hens were killed on the seventh day following the last treatment.
Tissue samples were collected and frozen until  analyzed.

Radioassay - Radioactive measurements were accomplished on a Packard
Tri-Carb Model  3380/544 liquid scintillation counter.   The
                                 32

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scintillation mixture and the details of counting liquid and solid samples
were the same as described in a similar study with carbofuran (Hicks
et al., 1970).  The blood was radioassayed by oxygen combustion tech-
niques, as were all solids, after 1 g of blood was evaporated to dry-
ness in a bag made from dialysis tubing (Andrawes et al., 1967).

Extraction of Residues - Feces, 20 g, were extracted by blending with
40 ml of water, followed by the addition of 150 ml of acetonitrile with
continued blending for about 1 min.  The homogenate was filtered and
the feces solids were extracted with 60 ml of a 2:1 mixture of
acetonitrile and water.  The combined homogenates were washed five times
with 80 ml portions of hexane which was discarded because the hexane
contained only negligible amounts of radioactivity.  The acetonitrile-
water phase was extracted thoroughly with chloroform and the aqueous
and organic solvent layers were radioassayed.  The latter phase was
decolorized with a small quantity of activated carbon, filtered, and
concentrated for spotting on tic.

Egg whites, 40 g, were extracted three times; each time the homogenates
were filtered and the solids returned to the blender for further homo-
genization.  The first solvent was 60 ml of acetonitrile, the second was
90 ml of a 2:1 mixture of acetonitrile and water, and the third was
60 ml of hexane.  All extracts were combined, shaken, and the layers
allowed to separate completely.  The acetonitrile-water extract was re-
moved and washed twice with 40-ml portions of hexane.  Chloroform was
added to separate the acetonitrile and water, and the procedure contin-
ued as described above for the feces.                      '

Egg yolks (20 g) were homogenized in 40 ml of water, 60 ml of acetonitrile
were added, and homogenization continued for 3 min.  The remainder of
the extraction and cleanup was identical to that used for the egg whites.
Equal extraction efficiency, but an acetonitrile extract with less
oils, was obtained when the egg yolks were extracted, using the more
                                 33

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involved method described below for tissues.

The various tissues were chopped into small pieces and a 40-g subsample,
or all available, was analyzed for residues.  The subsample was homo-
genized in 60 ml of a 2% potassium oxalate solution for 5 min.  Ethanol
(50 ml) was added to the homogenate and the mixture was homogenized
for 3 min.  The entire homogenate was transferred to a separatory
funnel containing 100 ml of ether and 50 ml of pentane, mixed, and the
water layer removed.  The water was again extracted with the ether and
pentane mixture.  After separating the layers, 100 ml of acetone were
added to the aqueous phase and shaken thoroughly.  The solids, formed
on shaking with acetone, were removed by filtration and 10 ml of acetone
and 100 ml of ether were used to wash the solids.  The filtrate was trans-
ferred to a separatory funnel, shaken, and the layers were separated.
All organic solvent extracts were combined, dried with anhydrous sodium
sulfate, and concentrated to an oily residue.

The residue was transferred to a separatory funnel with 30 ml of hexane
and 30 ml of acetonitrile.  The funnel was shaken, phases were separated,
and the hexane was extracted twice more with acetonitrile.  The com-
bined acetonitrile extracts were washed with 20 ml of hexane.  Follow-
ing this final wash to remove the oils, the acetonitrile extract was
prepared for tic analysis.

Thin-Layer Chromatography - Because tic was used to separate the
metabolites of aldicarb, and cochromatography of the unknown products
with authentic standards was the major means of metabolite identification,
great care was taken in developing several efficient tic systems.   The
supports consisted of silica gel  plates coated 0.3 mm thick and Chromar
500 thin-layer sheets (Mallinckrodt,  St.  Louis, Mo.).   A series of two-
dimensional solvent systems was utilized in achieving complete separ-
ation of metabolites and for establishing cochromatography in multiple
solvent systems.   The basic system was a 2:1  ether-hexane + 20%
                                 34

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acetone for the first direction and a 2:1 methylene chloride-
acetonitrile mixture for the second dimension (Dorough and Ivie,  1968).
Organic extracts of the various substrates were spotted on the tic
along with a mixture of metabolite standards.  Following development of
the tic, the plates, or sheets, were exposed to iodine vapors or  spray-
ed with a 1% potassium permanganate solution to visualize the
standards.  Radioautography was used to locate the radioactive areas
on the tic's.

When there was an indication that one of the standards cochromatographed
with an unknown metabolite, the metabolite was isolated and additional
tests for cochromatography were conducted.  For these experiments, var-
ious combinations of solvent systems were used to confirm or rule out the
two-dimensional cochromatography of the two materials.  The solvent
systems were as follows:  5:1 ethyl acetate and methanol-, 1:1 dioxane
and hexane; 9:1 dioxane and methanol; and a 5:1:1 mixture of chloroform,
ethyl acetate, and hexane.

Results and Discussion

Effect of Treatments on Hens - There were no symptoms of carbamate
poisoning in any of the hens treated either with the aldicarb, per se,
or with the combination of aldicarb and its sulfone analog.  As shown
in Table 8, the 21-day treatment of hens with aldicarb and aldicarb
sulfone had no deleterious effects on the birds.   When compared to the
control animals, the treated birds showed no appreciable differences
in body weight, food consumption, egg production, or quantity of fecal
matter voided from the body.

Quantity and Nature of Aldicarb Equivalents Excreted from the Body - The
pattern of excretion of the aldicarb doses was similar when hens  were
treated with aldicarb-S   alone, or with a mixture of aldicarb-  C and
aldicarb sulfone-  C (Table 9).  In both tests, the hens eliminated
                                 35

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approximately 80% of  the dose  in 2 days.  Extending the collection of
feces from the aldicarb-S   treated animals to 10 days showed that a
total of 90% of the single oral dose had been excreted from the body.
The pattern of elimination of aldicarb from the hens was not altered a
great deal when the insecticide was administered over a 21-day period
(Table 10).  After 8 to 10 days of treatment, there was an equilibrium
formed between the amount of material consumed and the amount excreted.
At this point, an equivalent of 80 to 85% of each daily dose was de-
tected in feces excreted during the following 24 hr.  These values were
lower at the 20-ppm feeding level, 60 to 65%, although there was no
apparent reason for this discrepancy in the amount of the daily doses
eliminated.  Removing the source of aldicarb resulted in almost 90% of
the total doses consumed being excreted from the body within 1 week.
Such rapid and thorough elimination of the carbamate would likely pre-
vent the accumulation of large quantitites of residues in the tissues

The chemical fate of aldicarb in laying hens when administered as a
single dose or for an extended period of time is shown in Tables 9 and
10.  Approximately half of the radioactive residues in the feces
consisted of unknown water-soluble metabolites.  These materials were
formed very rapidly by the hens, as evidenced by the results of the
analysis of feces collected 6 hr after a single dose was administered
(Table 9).  No attempts were made to identify these metabolites.  How-
ever, it is unlikely that the carbamate moiety was intact on these
materials, since conjugation and/or degradation of aldicarb to
alcohols and acids, processes which yield water-soluble metabolites,
would be preceded by hydrolysis of the carbamate ester (Bartley et al.,
1970).   The unextractable radioactive metabolites, 8 to 10% of the
residues, may be similar in chemical  nature to the water-soluble
metabolites.

Of the metabolites which were identified, aldicarb sulfoxide was the

                                 36

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only product detected following a single dose that also was not found
in the feces of birds treated repeatedly.  It was apparent from the
single-dose study that the sulfoxide was formed and excreted rapidly.
Only trace amounts were detected in feces collected after 6 hr.  Assum-
ing that the birds on the continuous treatments did excrete aldicarb
sulfoxide in the feces shortly after dosing, it must have been trans-
formed into another material before the feces were collected and fro-
zen.  The same may have been true for the aldicarb sulfone, since it
was present in higher amounts in the 6 hr feces (Table 9) than in the
feces of hens used in the continuous feeding study (Table 11).

The only other metabolite of the intact carbamate identified was the
N-hydroxymethyl analog of aldicarb sulfone.  This product accounted for
8 to 9% of the radioactive residues in the feces of hens fed aldicarb
for 21 days.  Based on its relatively low content in the hens treated
with a single dose of aldicarb or aldicarb sulfone, aldicarb-hydroxy-
methyl-sulfone was probably formed rather slowly in the hens.  It
reached a maximum concentration after the hens were exposed to the
insecticides for approximately 8 days.

Hydrolytic products accounted for most of the other residues in the
feces of the treated hens.  The sulfone forms of these metabolites were
predominate, with the nitrile sulfone present in highest amounts in feces
of hens treated for a total of 21 days.  Nitrile sulfone was also the
major identified metabolite in feces of hens given a single oral dose
of aldicarb-C  .  However, the oxime sulfone concentration was greater
                                                     14
in the feces of hens given a single dose of aldicarb-  C and aldicarb
sulfone-  C.  This was the only marked difference noted in the quantity
of individual metabolites in feces of hens receiving the two single
treatments.

The two unknown metabolites, designated Unknown 4 and 5 based on
earlier studies (Dorough and Ivie, 1968) were present in the feces of
                                 37

-------
all  tested  hens.   Unknown  5 was  always  present  in greater quantities
than Unknown  4,  and  in  some cases  accounted for 6% of the radioactive
residues  in the  feces.   Neither  Unknown 4 or  5  cochromatographaed with
any  of  the  available  standards or  was present in sufficient quantity
for  more  detailed  evaluation of  their chemical  nature.

Eggs  -  Adding aldicarb  sulfone to  the aldicarb  dose resulted in higher
levels  of residues in the  eggs than when only aldicarb was given as a
single  oral dose (Table 12).  In neither case did the total aldicarb
equivalents exceed 0.2  ppm in the  egg yolks or  whites.  However, max-
imum  aldicarb equivalents  of 0.18  ppm were observed when the two
insecticides  were  administered together as compared to a maximum of 0.07
ppm when  aldicarb was the  only component.  Whereas the radioactive
residues  in the whites  had declined markedly  by the third day, the re-
sidues  in the yolks were similar or showed a  slight increase by the
third day.  By 10  days,  residues in eggs of hens treated with the
          35
aldicarb-S    were 0.014 ppm in the yolk and 0.007 ppm in the whites.

Although  these preliminary experiments  were not designed to yield
sufficient eggs for extensive analysis, the nature of the majority of
the residues  was  tentatively projected (Table  13).  Eggs containing
the aldicarb-S   residues  showed the presence of a number of products
not detected  in the eggs of hens treated with aldicarb-  C and aldicarb
sulfone-  C.  This was  because the sulfur-35 material was of a very
high specific activity  and the sensitivity for  detecting the individual
metabolites was much greater than with  the carbon-14 insecticides.

The data  (Table 13) show that none of the carbamate metabolites of
aldicarb were at detectable levels in the egg yolks or whites.   The
water-soluble metabolites  and nitrile sulfone were the predominant
products in the eggs.  Other hydrolytic metabolites were detected in
                                oc
the egg whites of the aldicarb-S   treated birds.   Although the data
were not as complete as desired, these  tests  indicated the types of

                                 38

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metabolites which could be expected in the eggs of hens consuming
residues of aldicarb in the diet.  The tests also suggested that the
aldicarb sulfone contributed more to the residue content of the eggs
than did aldicarb.  In the yolks this increase was expressed as water-
soluble metabolites, while in the whites there was a noted increase in
the amount of nitrile sulfone.

Continuous exposure of aldicarb residues to hens resulted in some
very interesting patterns of residue levels in the egg.  At feeding
levels of 1.0 and 20.0 ppm aldicarb equivalents in the diet, it was
observed that the total   C residues in the yolk did not reach a plateau
until after 12 to 15 days of feeding.  This occurrence was not noted
at the 0.1 ppm feeding level because the amount of residues in the
eggs was only slightly above the level of sensitivity, which was 0.005
ppm total aldicarb-  C equivalents when analyzing 1 g of sample.

Residues in the egg whites stabilized on the sixth or seventh day of
                                             14
treatment.  In this case, the total aldicarb-  C equivalents reached a
plateau of approximately 0.006, 0.06 and 0.7 ppm for the three feeding
levels.  Although there were day.-to-day variations in the quantities
of residues in the egg whites, there were no significant increases or
declines in the residue levels until the insecticide source was re-
                                                  14
moved.  Once the treatments stopped, the aldicarb-  C equivalents in
the egg whites dropped rapidly and by 7 days were almost nondetectable.
Unlike the residues in the egg yolks of hens treated with single doses of
aldicarb, there was a portion of the radioactive materials in the egg
yolks of these hens which partitioned into the hexane fraction (Table
14).  This was an unexpected occurrence because the use of the
hexane in the cleanup procedure was a means of removing the fats and
oils from the acetonitrile extracts as had been done with the feces.

The identity of the hexane-soluble radioactivity from the yolks was

                                 39

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not determined.  Attempts to separate these products from the oils by
tic and extractions of various kinds were unsuccessful, and it may be
that they were naturally occurring materials synthesized from S-methyl-
14
  C fragments from the carbamate materials.  They were certainly not
the typical types of aldicarb metabolites usually encountered.

Subtracting  the hexane-soluble radioactivity from the total aldicarb-
  C equivalents in the egg yolks revealed that the pattern of accumu-
lation of the remaining residues in the yolk was very similar to that in
the egg whites.  These residues reached a plateau on the sixth or seventh
day of treatment and did not vary greatly until the carbamate treat-
ments were terminated.  Once the treatments were stopped, the non-
hexane-soluble residues in the yolks declined as did those in egg whites.

With the exception of the increasing hexane-soluble radioactive
materials, the relative concentrations of aldicarb metabolites remained
fairly constant.  For this reason, the nature of the residues in the
eggs is present in Table 14 as averages of the data gathered on eggs
laid the fifth through the 21st days of treatment.  The N-hydroxymethyl
analog of aldicarb sulfone was the only carbamate material  identified in
either the yolks or the whites and then only in eggs of hens at the
20-ppm feeding level.   As was the case with the feces and eggs from the
single-treatments studies, hydrolytic and unknown water-soluble
metabolites accounted for most of the residues.  The very low quantities
of aldicarb-  C equivalents in the eggs and their chemical  nature
suggested that small  levels of aldicarb residues in the diet of laying
hens would not result in toxicologically significant levels of residues
in the eggs.

Tissues - The situation in regards to the levels and nature of residues
in the tissues of the hens was very much like that described for the
eggs.   Feeding a combination of aldicarb and aldicarb sulfone appeared
to cause slightly higher residues in the various tissues than did the
                                 40

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treatment with just aldicarb (Table 12).   Residues in the tissues de-
clined sharply as the carbamates were eliminated from the body,  and by
3 days the levels had fallen from a high  of about 0.6 ppm aldicarb
equivalents to levels generally below 0.1 ppm.   By 10 days the radio-
active residues were less than 0.03 in all the tissue analyzed.

Small quantities of the residues in the kidney and breast of hens
killed 6 hr after treatment with a single dose of the insecticide were
identified as aldicarb sulfoxide.  Similarly low levels of aldicarb sul-
fone were detected in the liver and breast.  All other detectable
metabolites were hydrolytic products of known identity or unknown
metabolites in the water fraction or were unextractable from the tissues.

Generally, the magnitudes and nature of residues in the tissues  of hens
treated 21 days with aldicarb and aldicarb sulfone were not too
different than what had been observed with the single treatments (Tables
15 and 16).  The liver and kidney contained the highest levels of
aldicarb-  C equivalents and the majority of these were hydrolytic
products, mainly nitrile sulfone, and water-soluble unknowns.  The only
carbamate identified was a trace amount of aldicarb sulfoxide in the
liver of birds sacrificed 12 hr after the last treatment.

There were considerable quantities of hexane-soluble products in the
gizzard, liver, and kidney.  As with the  eggs, these materials were not
present in hens other than those on the continuous treatments.  Tissues
of hens killed on the seventh day after receiving their last aldicarb
treatment contained only nitrile sulfone  in the acetonitrile fraction
of the tissue extracts.  The remainder of the residues was distributed
among the hexane, water, and in the tissue solids as unextractable
metabolites.  As pointed out for the eggs, the data gathered by this
investigation do not indicate that low levels of aldicarb residues in
the diet of poultry would result in harmful levels of residues in
the meat.
                                 41

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TABLE 8.   TREATMENT RATES AND  CRITERIA FOR DETERMINING THE  EFFECT OF  FEEDING EQUIMOLAR DOSES OF
                   ALDICARB-14C AND ALDICARB  SULFONE-14C TO LAYING HENS  FOR 21  DAYS.

Group numbers9

Treatment rates
Aldicarb equivalents in feed, ppm
Aldicarb equivalents, mg/kg of body wt
Body weight, kg
0 Day, range, and average
21 Day, range, and average
Feed consumption
g/hen/day, range, and average
Egg production
Average/day/hen
Feces eliminated
g/hen/day
I
0.1
0.005
1.3-1.5(1.4)
1.3-1.6(1.4)
64-112(74)
0.70
108
II
1.0
0.05
1.3-1.7(1.5)
1.3-1.6(1.4)
77-103(84)
0.50
109
III
20.0
1.0
1.4-1.8(1.5)
1.3-1.7(1.5)
66-106(82)
0.58
118
Control
0
0
1.4-1.7(1.5)
1.3-1.7(1.5)
67-101(79)
0.63
98
  Six hens in each group.
  Based on an average daily food intake of 80 g for each bird.
  Based on an average body weight of 1.5 kg for each bird.

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TABLE 9.  NATURE OF RADIOACTIVITY IN FECES OF HENS TREATED WITH A SINGLE
                 ORAL DOSE OF ALDICARB-S35 (A) OR AN EQUIMOLAR
                   DOSE OF ALDICARB-14C AND SULFONE-14C (B)a

Percent of total radioactivity in
Metabolites
Aldicarb suit oxide
Aldicarb sulfone
Aldicarb-NCH2OH
sulfone
Oxime sulfoxide
Oxime sulfone
Nitrile sulfoxide
Nitrile sulfone
Alcohol sulfoxide
Alcohol sulfone
Unknown 4
Unknown 5
Water-solubles
Unextractables
Cumulative % of
dose excreted

A
5.1
3.9
1.0
5.2
2.8
2.3
7.2
1.8
1.7
1.3
5.9
50.0
11.8
50.2
6 hr
B
13.3
1.1
1.6
2.6
14.6
4.2
4.2
1.4
1.6
0.3
1.9
44.0
9.2
48.0
1
A
0.0
0
0.9
1.8
4.1
2.3
9.2
2.0
1.7
1.2
3.3
65.1
8.4
74.3
day
B
0.6
0
5.8
2.5
10.6
1.3
5.4
1.2
3.0
0
2.0
57.5
10.1
76.1
sample after
1
A
0.0
0
2.7
4.5
5.6
2.4
16.3
5.9
7.7
3.8
0
44.6
6.5
84.5
day
B
0.0
0
5.5
4.5
9.9
2.9
10.7
2.8
4.9
1.6
0.9
45.1
11.2
79.3
a Dosage rate = 0.7 mg/kg
  Values on day experiments terminated:  A = 90% after 10 days;
  B = 82% after 3 days.
                                  43

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TABLE 10.  ELIMINATION OF RADIOACTIVITY  IN THE  FECES OF HENS  FED EQUIMOLAR
                            :ARB-14C AND  ALDICARB SUl
                             IN THE DIET  FOR 21  DAYS
DOSES OF ALDICARB-14C AND ALDICARB SULFONE-14C

Days fed
insecticides
1/2
1
3
11
19
21
21 -day avg.
Days after last
treatment
1
2
4
7
Percent of consumed
0.1 ppm
76.0
75.3
77.5
87.9
83.3
84.9
82.2


85.3
85.9
86.2
86.2
doses excreted
1.0 ppm
76.2
69.8
71.9
88.6
82.7
82.6
80.3


86.1
86.4
86.8
87.3
in the feces3
20.0 ppm
64.7
62.7
64.9
69.8
70.1
71.0
67.5


72.4
72.6
72.8
73.3
a Based on total dose consumed and total radioactivity eliminated by
  indicated time after first treatment.
  Average of all daily values over the 21-day feeding period.
                                 44

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TABLE 11.  RADIOACTIVE RESIDUES IN FECES OF HENS  FED  EQUIMOLAR  CONCEN-
                     CENTRATIONS OF ALDICARB-14C  AND  ALDICARB
                       SULFONE-14C IN THE DIET FOR 21  DAYS

Percent of total radioactivity in feces at
indicated feeding levels
Metabolites
Aldicarb sulfone
Aldicarb-NCH20H
sulfone
Oxime sulfoxide
Oxime sulfone
Nitrile sulfoxide
Nitrile sulfone
Alcohol sulfoxide
Alcohol sulfone
Unknown 4
Unknown 5
Water-solubles
Unextractables
0.1 ppm
0.0
9.1
0.0
4.3
0.0
10.7
5.1
8.2
0.0
5.8
48.1
8.7
1 . 0 ppm
0.0
8.1
1.7
8.4
1.5
12.0
2.4
9.6
0.6
3.7
43.8
7.6
20.0 ppm
0.9
8.7
0.8
4.9
1.0
9.6
3.8
11.5
1.4
4.9
43.3
9.2
  Values are averages of analysis of feces collected 1, 2,  4,  12,  17,
  and 21 days after initiating feeding of the radioactive insecticides.
                                 45

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TABLE 12.   PPM ALDICARB EQUIVALENTS  IN  TISSUES  AND EGGS  OF HENS  TREATED WITH A SINGLE ORAL DOSE OF
           ALDICARB-S35 (A)  OR WITH  AN  EQUIMOLAR DOSE OF ALDICARB-14C AND ALDICARB SULFONE-14C (B)a

Ppm (wet wt) aldicarb- C equivalents after
Tissue
Kidney
Li ver
Heart
Gizzard
Skin
Breast
Thigh
Leg
Blood
Brain
Fat
Egg yolk
Egg white
6
A
0.59
0.53
0.28
0.26
0.22
0.20
0.20
0.20
0.18
0.18
0.08
c
c
hr

0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.

B
73
68
35
31
25
32
29
27
28
27
06
14
16


0
0
0
0
0
0
0
0
0
0
0
0
0
1
A
.19
.26
.12
.11
.08
.08
.08
.08
.11
.08
.04
.01
.03
day
B
0.21
0.31
0.09
0.08
0.08
0.08
0.08
0.07
0.07
0.09
0.07
0.13
0.18
3 days
A
0.09
0.10
0.07
0.04
0.04
0.03
0.04
0.04
0.06
0.04
0.03
0.07
0.06

0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
B
11
14
05
06
05
05
04
04
04
04
05
18
07
10 daysb

0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
A
020
034
010
020
004
006
008
005
014
008
004
014
007
  Dosage rate =0.7 mg/kg.
  Experiment involving treatment with B was terminated after 3 days.
  No eggs laid.

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TABLE 13.  RESIDUES IN TISSUES AND EGGS  OF HENS TREATED WITH A SINGLE  ORAL  DOSE  OF  ALDICARB-S35  (A)  OR
                     WITH AN EQUIMOLAR DOSE OF ALDICARB-14C  AND ALDICARB  SULFONE-14C  (B)a

Ppb in indicated tissue
Liver
Metabolites
Aldicarb sulfoxide
Aldicarb sulfone
Oxime sulfoxide
Oxime sulfone
Nitrile sulfoxide
Nitrile sulfone
Alcohol sulfoxide
Alcohol sulfone
Water-solubles
Unextractables
a Dosage rate =0.7
b _. ..
A
0.0
30.2
8.0
27.0
7.4
28.1
4.8
5.8
353.0
65.7
mg/kg
, .
B
0.0
0
14.3
102.0
20.4
17.7
0
0
439.9
85.7


Kidney
A
7.7
0
9.4
63.7
20.1
50.8
0
0
373.5
64.9


B
48.9
0
0
39.4
53.3
70.0
7.3
0
379.6
131.5


Gizzard
A
0.0
0
1.3
9.4
74.6
23.4
1.3
2.1
105.6
42.4


B
0.0
0
9.9
54.9
6.8
14.9
7.8
0
182.0
33.8


Breast
A
0.0
3.8
7.0
25.2
4.6
27.4
0
5.4
83.4
42.8


B
7.4
0
10.8
45.1
4.2
20.8
0
7.4
160.7
60.0


Egg
A
0.0
0
3.2
2.1
0
30.9
0
0
28.1
5.7


yolkc
B
0.0
0
8.2
0
0
44.3
0
9.4
57.6
5.7


Egg
A
0.0
0
10.6
3.6
3.6
7.0
2.5
3.1
24.2
5.5


white0
B
0.0
0
0
0
0
126.9
0
0
29.3
23.8


0 A, analysis of eggs  laid  third day after treatment; B, eggs laid first day after treatment.

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TABLE 14.   RESIDUES IN EGGS  OF HENS  FED EQUIMOLAR CONCENTRATIONS  OF ALDICARB-14C AND ALDICARB
                    SULFONE-14C AT RATES OF 1.0  AND 20.0 PPM IN THE DIET  FOR 21  DAYS

Metabolites
Acetonitrile solubles
Aldicarb-NC^OH sulfone
Oxime sulfoxide
Oxime sulfone
Nitrile sulfoxide
Nitrile sulfone
Alcohol sulfoxide
Alcohol sulfone
Unknown 5
Hexane-solubles
Water-solubles
Unextractables
Total


Yolk

0.0
1.3
1.8
0.3
21.1
0.0
0.2
0.3
52.8
6.6
4.0
88.4
Ppb
1 . 0 ppm
White

0.0
3.2
1.6
0.4
28.5
0.2
0.5
0.5
0.0
14.3
4.1
53.3
at indicated feeding level3

Total

0.0
2.5
1.7
0.4
25.7
0.1
0.4
0.4
17.8
10.9
2.8
62.7

Yolk

0.0
14.1
4.3
42.7
304.5
8.3
11.6
6.0
495.4
192.4
82.5
1161.8
20 ppm
White

4.8
40.0
21.3
27.3
417.4
12.0
16.5
12.0
0.0
223.8
89.6
864.7

Total

3.0
29.7
13.5
33.0
374.5
10.1
14.3
10.0
189.6
211.9
87.0
976.6
  Average of eggs laid 5-21  days  of feeding  radioactive insecticides.

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TABLE 15.  PPM ALDICARB-14C EQUIVALENTS IN TISSUES OF HENS KILLED 12 HR
                (A) AND 7 DAYS (B) AFTER THE BIRDS WERE FED EQUIMOLAR
                    CONCENTRATIONS OF ALDICARB-14C AND ALDICARB
                           SULFONE-14C IN THE DIET FOR 21  DAYS

Tissues
Liver
Kidney
Heart
Brain
Gizzard
Blood
Leg
Skin
Thigh
Breast
Fat
1.0
A
0.14
0.12
0.07
0.07
0.07
0.07
0.06
0.06
0.06
0.06
0.05
ppm
B
0.02
0.03
0.02
0.02
0.02
0.03
0.03
0.02
0.02
0.02
0.02
20.0
A
1.40
1.38
0.92
0.90
0.81
0.76
0.71
0.70
0.70
0.68
0.52
ppm
B
0.36
0.39
0.35
0.40
0.33
0.34
0.30
0.36
0.31
0.28
0.22
a Residues in tissues of hens fed 0.1 ppm were below the level of
  sensitivity, 0.005 ppm, except in the following tissues taken 12 hr
  after the last treatment:  blood, 0.015 ppm; kidney, 0.012 ppm; and
  liver, 0.011 ppm.
                                  49

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      TABLE 16.   RESIDUES  IN  TISSUES  OF  HENS AFTER A  21-DAY  PERIOD OF FEEDING EQUIMOLAR CONCENTRATIONS OF

                       ALDICARB-14C AND  ALDICARB  SULFONE-14C AT A RATE OF 20.0 PPM IN THE DIET
en
O

Metabolites
Aldicarb sulfoxide
Oxime sulfoxide
Oxime sulfone
Nitrile sulfoxide
Nitrile sulfone
Alcohol sulfoxide
Alcohol sulfone
Unknown 5
Hexane-solubles
Water-solubles
Unextractables

Breast
0.0
9.3
19.7
8.1
412.6
2.5
3.0
6.4
5.3
80.0
138.7
12
Gizzard
0.0
6.4
23.1
5.7
410.4
2.4
3.9
6.1
13.3
98.5
242.8
hr
Liver
3.0
2.6
14.4
10.1
418.0
0
10.6
7.2
95.4
160.3
681.9

Kidney
0.0
4.1
22.0
8.2
401.6
0.7
10.6
7.1
96.5
321.6
503.3
7
Breast
0.0
0
0
0
144.1
0
0
0
4.0
22.7
112.4
days
Gizzard
0.0
0
0
0
74.2
0
0
0
12.0
,15.1
231.5

Liver
0.0
0
0
0
184.5
0
0
0
19.8
29.0
129.3

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Aldicarb In Boll Weevils and House-flies

The current study was conducted so that the metabolism of aldicarb
in insects could be compared with that reported for mammals.  Boll
weevils, Anthonomus grandis Boheman, and houseflies, Musca domestica
L., were used as test insects in order that species differences
among insects also could be considered.

Methods

Insects used in this investigation were from insecticide-susceptible
strains which had been maintained in the laboratory for several years.
Only female houseflies were selected for testing, but both male and
female weevils were included.  All insects were utilized within 3 to
7 days after they had reached the adult stage.

For metabolism studies, aldicarb-carbonyl-C   was applied topically
to the insects in 1 microliter of acetone.  Technical grade, non-
radioactive aldicarb was used in toxicity tests and in cholinesterase,
ChE, assay experiments.  The ChE assay procedure of Simpson et al.
(1964) was employed.

Following treatment of the flies or weevils with aldicarb-carbonyl-
C   at a dosage rate of 0.1 microgram per insect, groups of 20
insects were placed in 250 ml Erlenmeyer flasks until analyzed.  Each
flask was fitted with a side arm to allow a continuous flow of air
into the chamber.  Air was drawn through the flask and a carbon
dioxide trap.  This allowed the collection and quantitation of carbon-
14 dioxide which was indicative of the rate of hydrolysis of the
insecticide.  At predetermined times after treatment, the radio-
activity on the surface of the insects, that located in the body and
that excreted was determined.  Methods of extraction, chromatographic
purification and identification of metabolites were the same as those
                                 51

-------
 described  by  Andrawes  et  al.  (1967).

 Results  and Discussion

 There was  a rapid  disappearance  of  aldicarb-carbonyl-C   from the
 surface  of boll weevils and houseflies  following  topical application
 of  the insecticide (Table 17).   By  6  hours after  treatment, about 6%
 of  the applied dose was detected in the external  wash of the houseflies;
 thereafter, no radioactivity  could  be detected.   With the  boll
 weevils, approximately 30% of the dose  remained on the surface 6 hours
 after treatment.   Subsequent  analysis showed that trace amounts of
 radioactivity could still  be  detected in the surface wash of weevils
 after 24 hours.

 Some of the loss of insecticide  from  the surface  probably resulted
 from mechanical removal as the insects  came in contact with the
 holding containers.  Thus, a  portion  of the aldicarb, per se, found
 in  the containers  may  have come  from  this source  and not actually have
 been excreted by the weevils  or  flies.

 Data in Table 17 show  that the amount of aldicarb-C   equivalents
within the insects  remained relatively  constant during the first 6
 hours after treatment.  This  indicated  that the rate of excretion was
 sufficient in both  insect  species to  prevent large build up of the
 insecticide when applied at a sub-lethal dose.

Considering all the distribution data,  it was evident that houseflies
absorbed and excreted  the carbamate at a faster rate than the boll
weevils.   The reason for the faster excretion rate in houseflies
was evident by the  nature of the radioactivity in the excreta.   For
                              14
example,  26% of the aldicarb-C   equivalents in the excreta of flies
were in the form of water soluble metabolites after 6 hours;  in boll
weevils,  they constituted less than 4%.   Although the water soluble
                                 52

-------
metabolites are listed as unknown metabolites in Table 17, it is
suspected that they are conjugates of some type (Andrawes et al.
1967).  This type of metabolite represents a near end-point in the
metabolism of carbamate materials and is the form most easily elimin-
ated from the body.  The greater ability of the flies to convert
aldicarb to water soluble metabolites also was evident by their con-
centrations in the internal extracts of the insects.

Aldicarb sulfoxide was the major non-conjugated metabolite formed in
boll weevils and houseflies.  Further oxidation yielded lesser amounts
of the sulfone derivative.  Hydrolytic products were  not detected in any
of the extracts in either of the insect species.  This was not un-
expected since the amount of carbon-14 dioxide produced was very
minute.  The percentages of the applied doses liberated as carbon-
14 dioxide were 0.5 and 1.0 after 6 hours in the weevils and flies,
respectively.

The inability of the boll weevils and houseflies to rapidly hydrolyze
aldicarb and its carbamate metabolites was the major  difference ob-
served in the metabolism of aldicarb by insects and rats.  Hydrolysis
was the predominant pathway of metabolism in rats treated orally with
the carbamate.  In fact, over 50% of the dose was hydrolyzed within
8 hours after treatment (Andrawes et al. 1967).  Consequently, most
of the metabolites formed in rats were non-toxic derivatives of
aldicarb such as oxime sulfoxide and nitrile sulfone.

Since aldicarb sulfoxide was formed very rapidly and  its concentration
remained relatively constant in the bodies of the insects over a
6-hour period, attempts were made to estimate its contribution to the
insecticidal activity of aldicarb.  First, the LD5Q of aldicarb and
aldicarb sulfoxide to boll weevils and houseflies when applied
topically was determined.  With the boll weevil, the  LDgQ value was
10 micrograms per gram for aldicarb and 150 micrograms per gram for
                                53

-------
aldicarb sulfoxide.  LD5Q values for the 2 compounds with houseflies
were 5 and 12.5 micrograms per gram, respectively.  These data demon-
strated that aldicarb sulfoxide could contribute to the insect
toxicity of the parent compound.

Studies of the anticholinesterase activity of aldicarb and aldicarb
sulfoxide against housefly-head ChE and boll weevil whole-body ChE
indicated that the toxicity of aldicarb sulfoxide might be more
important than indicated by the LDrQ tests.  Whereas the I™ values
of aldicarb against fly ChE was 4.7 x 10  M, the value was
3.0 x 10" M for aldicarb sulfoxide.  The values obtained for boll
weevil ChE were about the same.  It is evident, then, that the
aldicarb sulfoxide is a much more potent ChE inhibitor than the parent
compound and that it should be more active as an insecticide.  That
this was not demonstrated by topical applications to the insects may
be that the sulfoxide degrades on the surface of the insects faster
than does aldicarb, or that the penetration rate is much slower than
that of aldicarb.   In either case, the compound could not exhibit  its
full toxic potential when applied topically.  However, it may be more
insecticidal  than aldicarb when formed in the body as a result of
enzymic metabolism.
                                54

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      TABLE 17.  DISTRIBUTION AND EXCRETION OF ALDICARB AND ITS METABOLITES IN BOLL WEEVILS (W) AND HOUSE-
                                  FLIES (F)  TREATED TOPICALLY WITH ALDICARB-CARBONYL-C
                                                                                      14
en
en

Metabolites
Aldicarb
Aldicarb sulfoxide
Aldicarb
Aldicarb sulfoxide
Aldicarb sulfone
Unknown
Aldicarb
Aldicarb sulfoxide
Aldicarb sulfone
Unknown


W
57.6
5.4
11.2
2.8
1.0
1.2
12.3
6.5
0.6
0.7

1
F
13.6
5.4
18.7
16.1
6.1
15.8
12.2
5.1
0.7
6.3
Percent of

W
39.9
5.5
11.1
6.3
2.8
2.8
12.3
13.5
3.0
2.8
recovered radioactivity3/ hours
2
F
13.7
5.9
15.2
16.5
6.4
16.2
10.4
5.5
1.8
9.4

W
Surface
25.5
7.8
Internal
12.5
5.6
4.1
5.6
Excreted
13.7
15.0
6.8
3.4
4
F
11.8
6.5
14.0
13.1
7.8
17.7
10.4
5.6
1.8
11.3

W
19.6
9.8
13.5
6.6
5.4
5.9
13.4
14.3
7.6
3.9
6
F
4.2
2.4
8.9
7.2
4.5
14.6
10.0
13.3
9.3
25.6
        The recovery of the applied dose ranged from 83 to 98%,  with an average of 92%.

        Radioactive metabolites in the water phase after partitioning between water and  chloroform.

-------
 Carbaryl  in  Lactating  Cows

 The  evaluation  of the  residual  nature  of  carbaryl  in  cattle has been a
 major  source of progress  in  defining the  metabolic  fate of carbamate
 insecticides.   Even  before the  realization  that oxidative and hydro-
 xylative  mechanisms  played a significant  role  in the  metabolism of
 carbaryl,  some  of the  more complete fate  studies with this carbamate
 were performed  with  cows  (Gyrisco et al., 1960; Claborn et al., 1967;
 Whitehurst et al., 1963).  Although the analytical  methods used by
 these  scientists  were  later  reported to be  inadequate for the de-
 tection of total  carbaryl residues in  meat  and milk (Dorough, 1967)
 they did  establish that carbaryl was rapidly metabolized and excreted
 by dairy  animals.

 In 1964,  a report on the metabolism of carbaryl indicated that the
 biochemistry of this carbamate, and probably carbamates in general,
 was  a  very complex subject (Dorough and Casida, 1964).  These authors
 demonstrated the  presence of certain carbaryl metabolites in the
 milk of a  treated goat that  were neither carbaryl or  1-naphthol  nor
 any  product  which could be converted into 1-naphthol  by alkaline
 hydrolysis.  Subsequent studies on the metabolism of  carbaryl in
 dairy  cows were  instrumental  in confirming  the identity of some
 carbaryl metabolites and in  suggesting the  chemical nature of others
 isolated  for the  first time  (Baron et  al.,1968, 1969; Dorough, 1967).
 Progress  towards the complete elucidation of the metabolic fate of
 carbaryl  in  cows and other animals has recently been  reviewed
 (Dorough, 1970).

 General Study Plan

 A summary of the overall study is presented in Table  18.   One Hoi stein
 cow was used at each of the  feeding levels  indicated.   All  animals
were in a medium stage of lactation,  and production was  considered
                                56

-------
satisfactory for a commercial dairy.  They were housed in metabolism
stalls, which permitted separate and quantitative collection of urine
and feces.  Non-radioactive carbaryl was administered to the animals
for a two-week period before feeding of the   C-labelled carbamate
began.  Collection of samples for analysis began 12 hr after the
               14
first carbaryl-  C treatment.

The 5 p.p.b. level of sensitivity for carbaryl-  C equivalents was
determined on the basis of the specific radioactivity of the parent
compound, which was 1.2 mc/mmole, and the size of sample radioassayed.
For milk, 0.5 g was routinely assayed, and 1 g dry weight of tissue
was assayed.  The sensitivity was increased when necessary by assaying
larger samples.  The basic procedures used for quantitation of
residues and for their isolation and identification were similar to
those described in a study involving aldicarb (Dorough et al., 1970).

Elimination of Carbaryl
As expected from the earlier studies on the metabolism of carbaryl in
cows, there was very rapid elimination of the doses from the animals.
It was evident that an equilibrium between 'intake' and 'output' of
carbaryl was established in rather fast order.  Generally, the per-
centage of the previously applied dose present in the excreta remain-
ed fairly consistent after three or four days of feeding the radio-
active compound.

Milk from cows fed 10, 30 or 100 ppm carbaryl contained about 0.2 per
cent of the consumed carbamate.  There were no indications that the
pattern of excretion was altered significantly by increasing the
dose tenfold.  It was noted that the residues in the milk of the cow
fed 100 ppm did not reach their maximum levels as quickly as they
did in animals fed the two lower doses.  However, this may have been
caused by some biochemical difference in this particular animal
                                57

-------
               14
since carbaryl-  C equivalents in the urine and feces exhibited a
similar excretion pattern.
Most of the administered carbaryl doses were eliminated from the body
in the urine.  From 70 to 85 per cent of the consumed carbamate was
detected in the urine.  There was a correlation between dose and
percentage of dose excreted in the urine, with more complete elimin-
ation occurring as the dose decreased.  The opposite pattern was
indicated in the feces, where 5 to 11 percent of the dose was ex-
creted.  This type of excretion suggested that the metabolism of
carbaryl may have been hindered somewhat by the larger levels of
toxicant consumed.  However, it is important to note that the total
elimination of carbaryl equivalents exceeded 80 percent of the ad-
ministered doses regardless of the level fed to the animals.

         14
Carbaryl-  C equivalents in the milk as ppm - Having established that
the percentage of the carbaryl doses eliminated in the milk remained
at a fairly constant level during the 14-day study, it was expected
that the same would hold true when the residues were converted into
ppm in the milk.  Generally this was found to be the case.  However,
there was some day-to-day variation in milk production, which re-
sulted in slight variations in the ppm level observed in the milk.
These minor variations were expected but were not considered too
important since there was no indication that carbaryl-  C equivalents
were increased as the time of feeding was extended.
Therefore our investigations showed that the level of total carbaryl-
  C equivalents in the milk of cows on a continuous diet containing
carbaryl was approximately 1/400 of that level in the diet.  The non-
accumulative nature of this carbamate in milk was likewise observed
when aldicarb was fed to cows for 14 days (Dorough et al., 1970).

Extraction of carbaryl metabolites from milk - From a practical and

                                 58

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toxicological viewpoint, the numbers defining the total  carbaryl
equivalents in milk have little significance.  What is important,
however, is the amount of those equivalents that were toxic or which
should be considered toxic until proven otherwise.  Generally, the
                  \
latter would include any metabolite having the carbamate moiety
still attached to the naphthyl ring and any other metabolites of
unknown identity.

In the previous studies on the metabolism of radioactive carbaryl in
dairy cows, the milk was extracted with organic solvent and the total
residues in the organic extract, water phase and milk solids were
quantitated.  Only those metabolites appearing in the organic solvent
phase have been subjected to rigorous testing to establish their
identity.  Such techniques were effective in identifying, or tentatively
identifying, less than one-half of the total residues (Dorough, 1967).

A major portion of our efforts in the current study was devoted to
the development of an extraction procedure that would remove all
residues from the water and solid phases of milk.  Finally, a proced-
ure that accomplished this goal was perfected.

Acetone was added to 50 ml of milk contained in a glass-stoppered,
250 ml Erlenmeyer flask and the contents were thoroughly shaken
before addition of the acetonitrile.  Reversing the order, or adding
the two solvents together, coagulated the milk proteins so rapidly
that residues were trapped in the solids, and additional extractions
with either acetone or acetonitrile were ineffective in their removal.
The acetonitrile, after the acetone, coagulated the milk proteins
without trapping the carbaryl residues and allowed the proteins to
be removed by filtration.  However, the flask and solids were
thoroughly washed with acetonitrile.  Analysis of 1 g of the dried
milk solids by combustion techniques revealed that they were free of
radioactive residues.
                                59

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The filtrate was added  to a  separatory  funnel and extracted with
hexane  to  remove the  fats and oils.  After back-extracting the com-
bined hexane with 20  ml of acetonitrile,  the hexane was discarded.
The acetonitrile was  returned to  the separatory funnel containing
the milk extract.

                  14
Once all carbaryl-  C equivalents were  removed from the milk solids,
the most important step in this procedure was the removal of the water
while retaining the residues in the organic solvent phase.  This was
done by adding sodium chloride to the extraction mixture, slowly
with shaking, until two layers were formed.  Chloroform (50 ml) was
then added and the funnel shaken  vigorously, and then the layers
again were allowed to separate completely.  Chloroform was necessary
in this procedure because considerable  water was present in the
acetonitrile-acetone  layer when chloroform was not used.  Without the
addition of the salt, the more polar metabolites were only partially
extracted.

The water layer was extracted twice more with acetonitrile and chloro-
form and the water phase then discarded.  No radioactive residue was
detected in a 1 ml aliquot of the water, and only traces were evident
when the entire water layer was concentrated and radioassayed
directly.  Thus all the carbaryl equivalents had been extracted from
the milk and were present in an organic solvent.

The extract of the milk was then dried with anhydrous sodium sulphate
and concentrated just to dryness on a rotary evaporator.  Small
washes of acetonitrile were used to transfer the residue to a 15 ml
centrifuge tube.   If  the extract appeared to contain too much oily
material for application to a thin-layer chromatogram, the acetonitrile
was extracted with 2-4 ml  of hexane.  This step was usually
necessitated by impure solvents rather than by oils from the milk.
                                60

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Recent work has shown that the butterfat of milk is free of carbaryl
metabolites.  Therefore skim milk can be used without any changes in
results and with less oil  to be removed by the hexane washes.

Separation and isolation of residues in milk - A series of thin-layer
chromatographic (tic) analyses, on Chromar 500 sheets, was used to
separate and help identify the radioactive residues from the milk.
The entire extract was applied to a chromatog ram and developed two-
dimensionally.  Radioautograms of the tic revealed that six radio-
active components were resolved by the solvent systems used.  Each
of the areas above the origin was isolated separately and analysed
in several two-dimensional solvent systems.  These analyses demon-
strated that each radioactive area, II-VI, was composed of a single
material. Metabolites II-VI are those referred to in previous  studies
as the 'organo-solubles'.   The radioactive materials remaining at
the origin of tic no. 1 were a combination of the 'water-solubles'
and 'solids or unextractables' that were obtained with extraction
procedures reported earlier (Dorough, 1967).

The tic no. 1 origin material was extracted with methanol and then
applied to a new tic and developed in ethyl acetate.  When this was
done, the radioactive material was resolved into two distinct areas
(tic no.  2).  Since such polar metabolites of carbaryl and other
carbamates have been shown to be conjugates of some type (Dorough,
1970), metabolites VII and VIII were extracted from the tic no. 2
and subjected to Glusulase enzyme.  This enzyme contained both 6-
glucuronidase and sulphatase and therefore would cleave the metabolites
if they were glucuronide or sulphate conjugates.

To determine if cleavage would occur, an aliquot of the methanol
extract of either metabolite VII or VIII was added to a 25 ml
Erlenmeyer flask and the solvent removed by evaporation.  Citrate-
phosphate buffer pH 5.0 (4 ml), and 0.5 ml of the Glusulase solution,

                                61

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as received  (Endo Lab.  Inc., Garden City, New York 11530), was added
to the flask.  A drop of toluene was added to retard microbial
growth and the flasks were  incubated, with shaking, at 37°C for 24 hr.

After incubation the contents of the flask were transferred to a
separatory funnel with  water washes of the flask until the final
volume was approximately 20 ml.  Extraction of the radioactive pro-
ducts from the water was accomplished by using the procedure described
for the extraction of milk.

The concentrated extract was applied to a tic and developed  in
methylene chloride-ethyl acetate (1:1).  Radioautographic analysis
of the tic's showed that metabolite VII yielded two aglycones, VII A
and VII B, and that metabolite VIII yielded only one product, VIII A.
Cleavage of metabolites VII and VIII by Glusulase enzyme was about
60 to 70 percent during the 24 hr period of incubation.  Repeated
isolation of the non-cleaved material and additional incubation with
Glusulase enzyme showed that metabolites VII and VIII could be con-
verted almost quantitatively into the aglycones.

Over 90 percent of the applied radioactivity was routinely recovered
from tic no. 1 and no. 2.  Therefore the metabolites on these tics
were not unusually volatile, and no special precautions were required
for good recovery and re-chromatography.  However, metabolite III
was often converted almost completely into metabolite IV.  This con-
version was kept to a minimum by using very pure solvents and main-
taining metabolite III under slightly acid conditions whenever possible.

Poor recoveries of metabolites VII A and VIII A from tic no. 3 were
often encountered when attempts were made to isolate them for futher
study.  This was primarily a function of volatility during the
evaporation of solvents since direct counting of the radioactive areas
of the tic yielded recoveries exceeding 80 to 90 percent of the
                                62

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applied materials.

Chemical nature of residues in milk - Once techniques for separating and
isolating carbaryl metabolites from milk had been accomplished, each
material was subjected to a variety of tests to determine its identity.
The small concentrations of certain of the metabolites precluded
detailed analysis of their chemical nature.  When the same metabolite
also was present in cow urine, but at much higher levels, that
material was isolated and used for identification purposes.  This was
the case for metabolite II ( and its hydrolytic product not detected
in milk) and metabolites III and  IV.

Results of the characterization studies of the metabolites in milk
are shown in Table 19.  A summary of the data supporting these
findings is presented below.

Metabolite III- This product is considered first because it was the
major metabolite of carbaryl in the milk and because its chemical
characteristics were important in the identification of metabolite
II.  Moreover, it has been tentatively identified in a number of
organisms in the past, and its identity after isolation from milk
has been recently confirmed (Baron et al., 1968, 1969).

The basis on which our particular metabolite III from milk was ident-
ified as 5,6-dihydro-5,6-dihydroxy-l-naphthyl methylcarbamate were:
(a) metabolite II cochromatographed with authentic sample synthesized
by Union Carbide chemists; (b) both the material from milk and the
synthetic product were identical and supported the proposed structure.

Metabolite II - This metabolite and its hydrolytic product were found
in rather large quantities in the urine.  Therefore, these materials
were isolated from the urine  in sufficient quantity for mass spectral
analysis.  Since 3,4-dihydro-3,4-dihydroxy-l-naphthyl methylcarbamate
                                63

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has not been synthesized,  identification of metabolite II as this
product was based  largely  on  its similarity to the 5,6-dihydrodi-
hydroxy derivative of carbaryl.  In addition to the fact that the
mass spectral data were consistent with the proposed structure,
metabolite II could be dehyrated to yield 4-hydroxy-l-naphthyl
methylcarbamate.  This reaction was comparable to that already
mentioned in reference to  metabolite III.

Metabolites V and VI - Each of these two materials was present at
low levels in the milk, and the evidence for their structures was
based primarily on cochromatography with authentic samples.  There
was sufficient quantity of metabolite VI to establish that it yielded
1-naphthol upon hydrolysis, which further supported its identity as
carbaryl.

Metabolite VIII - This metabolite was identified as 1-naphthyl sulphate.
Before its incubation with Glusulase it cochromatographed with a
standard sample of 1-naphthyl sulphate (potassium salt).   That this
product exists interchangeably as a number of salts was point out
by Paulson et al., 1970.   These salts chromatograph differently on
tic and can lead to confusion about the identity of the material.
This can be avoided by converting the compound into the sulphate
by the addition of acid,  or by converting it into a single salt by
adding any one salt in excessive amounts.   Glusulase enzyme cleavage
of metabolite VIII yielded the aglycone, 1-naphthol  (metabolite VIII A
on tic no. 3), in the same manner as the standard sample.

Metabolite VII - This material represents an entirely different type
of carbaryl  metabolite and one which has not been previously isolated.
It is a carbamate and is  formed by two types of conjugation, namely,
sulphate conjugation and  methylation.   Data to support its identity
as l-methoxy-5-(methylcarbamoyloxy)-2-naphthyl  sulphate are as
follows:  (a)  It is a conjugate metabolite.   This is  based on the
                               64

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polar nature of the metabolite and the fact that Glusulase enzyme
treatment yields two aglycones, metabolites VII A and VII B.   About
99 percent of metabolite VII is a conjugate of VII A.  (b) It is a
sulphate conjugate.  Sulphatase hydrolysed twice as much as metabolite
VII as did B-glucuronidase.  By inhibiting the sulphatase activity
in the glucuronidase preparation, the B-glucuronidase did not
hydrolyse any of the conjugate metabolite,  (c) Aglycone VII  A is a
ring-modified carbamate metabolite of carbaryl.  If aglycone  VII A
was not a ring-modified derivative of carbaryl, alkaline hydrolysis
would yield 1-naphthol.  However, hydrolysis of VII A yielded a
product that cochromatographed with VII B.  Mass spectral analysis of
aglycone VII A showed a loss of methyl isocyanate, which also demon-
strated that the ring was modified and, more important, showed that
the metabolite was definitely a carbamate material.  Other peaks of
the mass spectrum suggested the presence of an hydroxyl group and a
methoxy group.  Mass spectral and i.r. data on 5-methoxy-6-hydroxy-l-
naphthol methylcarbamate synthesized by Union Carbide chemists were
identical with that of aglycone VII A.

Aglycone VII B is probably the hydrolytic product of VII A.  However,
the yield of VII B from the enzyme hydrolysis of metabolite VII is so
small that detailed studies have not been possible.  It is known
that alkaline hydrolysis of VII A does yield a product that co-
chromatographs with aglycone VII B.  These data suggest the possibil-
ity that VII B is not present in milk as a conjugate but may form
from aglycone VII A during the enzymatic cleavage of metabolite VII.

Residues in Meat - The total carbaryl-  C equivalents in various
tissues of cows fed with carbaryl for 14 days are shown in Table 20.
The animals were slaughtered and the tissues taken approximately 18 hr
after the last treatment with the insecticide.  Total residues in
each sample were determined by combusting 1 g of dry tissue and
                                14
collecting the radioactivity as   C-labelled carbon dioxide.
                                65

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All of the tissues from cows fed with carbaryl at 30 and TOO ppm con-
tained detectable levels of residues.  At the 10 ppm feeding level,
all of the tissues contained residues except the fat.  A good correla-
tion existed between the level of pesticide fed and that which appeared
                                14
in the tissues.  Total carbaryl-  C ei
1/1000 of that level fed in the diet.
                                14
in the tissues.   Total  carbaryl-  C equivalents in the muscle were
Although the total radioactive content of the tissues was quite low,
attempts were made to determine the nature of the residues in all
tissues except the fat.  Tissue (25 g) was homogenized with 50 ml of
water and then 100 ml of acetonitrile was introduced and blending
continued.  Again, the blender was stopped, 25 ml of acetone added
and homogenization continued for about 1 min.  The acetone aided
protein coagulation and facilitated filtering.  After being filtered
off on Whatman No. 1 paper, the solids were extracted once more.  The
extracts were combined and analysis was continued by using a procedure
similar to that described for milk.  Unlike those in milk, however,
some radioactive residues remained in the tissue solids after extraction
and in the water phase after the addition of sodium chloride and
partitioning with acetonitrile and chloroform.  The concentration of
these unknown materials and those residues that were identified are
shown in Table 21.

Carbaryl was detected in all tissues except the blood.  It accounted
                               14
for 17 percent of the carbaryl-  C equivalents in the muscle but
constituted a lesser proportion of the total residues present in other
tissues.

The only other identified carbamate metabolite from the tissues was
5,6-dihydro-5,6-dihydroxy-l-naphthyl methylcarbamate.  It was a major
constituent of the residues in the muscle, heart and blood.   Small
quantities of the hydrolytic product of this metabolite were found in
other tissues except the muscle and lungs.
                                  66

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Naphthyl sulphate was identified as 52 percent of the carbaryl-  C
equivalents in the blood.   The same material  accounted for about 30
percent of the residues in the kidney and lung but was low in the liver
and heart; none of this product was evident in the muscle tissue.

Extraction of the liver and kidney with various solvents and for
different periods of time  failed to remove more of the radiocarbon than
did the acetonitrile-water-chloroform system.   Homogenization in water
alone appeared to extract  more of the   C-residues from the tissues,
however, the addition of any organic solvent caused a precipitate to
form which contained large quantities of radioactivity.

Ultrasonic disintegration  of the tissues (Brinkmann Polytron) in
ethanol, followed by overnight soxhlet extraction with the same solvent
removed 20% from the kidney.  Soxhlet extraction for another 24 hrs
using acidified ethanol was ineffective.

Separation of liver and kidney homogenates into various centrifugal
fractions was conducted and the distribution of radioactivity among
the cellular components evaluated.  Homogenization of the tissue, 25  g
in 200 ml of water, was accomplished using a Virtis homogenizer.  The
homogenate was centrifuged at 15,000g for 30 min., the supernatant de-
canted and the pellet resuspended in 100 ml of water.  After centrifu-
gation at 15,000g for 30 min and the supernatant decanted, the pellet
was again suspended in water and centrifuged.  The supernatant fractions
from each centrifugation was radioassayed separately by liquid scintill-
ation counting. The first supernatant was subjected to additional centrifu-
gation at 105,000g for 1 hr and the supernatant decanted and radioassayed.
Both the 15,000g pellet and the 150,000g pellet were radioassayed by
combusting the solids in a Beckman Biological  Materials Oxidizer.

Distribution of the   C-residues among the various cellular fractions
of liver and kidney are shown in Table 22.  In each case, from 80 to
                                67

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85% of the  radioactivity was  in the 15,000g soluble fraction.  Centri-
fugation at 105,000g did not  remove any of the radiocarbon from the
15,000g solubles of the liver, but 1.4% of the radioactivity in the
kidney was  detected in the microsomal  (105,000g pellet) fraction.

Numerous modifications of the procedures used in the centrifugal frac-
tionation of the liver and kidney were attempted.  However, the results
were always similar to those  presented in Table 22.  For example, centri-
fungation of the tissue homogenates at 5,000g or 15,000g showed that
fhe resulting pellet contained essentially the same levels of radio-
activity.   Re-homogenation of the pellets, as opposed to washing by
                                                               14
suspending  the pellets in water, did not increase the level of   C-
residues in the supernatant fractions.  Also, phosphate buffer, pH 7.0,
was shown to work as well as water, but no better.  The use of the
buffer would be desired when the supernatants are to be exposed to
enzymatic treatment.

Repeated ultrasonic homogenization of the tissues in water or buffer
did not alter the distribution pattern of the   C in the liver and
kidney.  However, the procedure is faster than homogenization in the
Virtis.  Three successive 40-sec. homogenizations (lOg in 80 ml) of
the tissue yielded results almost identical to those presented in
Table 22.

Organic solvent extraction of the supernatant and pellet - Combined
supernatants and pellets of the liver and kidney were extracted using
the acetonitrile-chloroform procedure previously described.

Following extraction of the liver supernatant, 40.1% of the   C in the
whole tissue was detected in the precipitate which formed upon addition
of the organic solvents.   The water phase contained 19.8% and the
acetonitrile-chlorofrom phase contained 21.4%.  In the kidney, 12.8% of
    14
the   C in the whole tissue was in the precipitate or solids, 34.8% in
                                 68

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the water and 30.8% in the organic solvent phase.   The distribution of
radioactivity among the 3 phases was changed only  slightly by refluxing
the supernatants for 3 hrs. after adjusting to 3N  HC1  (Table 23).
Recent studies have indicated that a weaker acid will  convert more of
the radiocarbon to the chloroform-acetonitrile layer.

The extraction characteristics of the   C-residues associated with the
15,000g pellets of the liver and kidney are shown  on Table 24.  These
data were obtained from tests where the pellets were suspended in  3N
HC1 and refluxed for 3 hrs.  However, distribution of the radioactivity
among the 3 phases was essentially the same when the acid treatment
was omitted.

Combined results of the extraction of the supernatant (Table 23) with
those of the extraction of the 15,000g pellets (Table 24) are as
follows:
                                     of total   C  in sample
Liver
Kidney
When compared with the data obtained upon direct extraction of the
tissues, it may be seen that the final extraction characteristics were
                            14
unchanged by separating the   C-residues according to cellular fraction.
However, these latter studies do show that a maximum of 9% of the
radiocarbon in the liver and 5% of that in the kidney cannot be re-
moved from components of the cell found in the 15,000g pellet.  The
remainder of the   C-residues was extracted from the tissues and may be
generally classified as organo-extractables and water solubles.  The
total organo-solubles after acid treatment accounted for approximately
30% of the radiocarbon in the liver and 45% of that in the kidney.
Water soluble   C-residues constituted 63% of the radiocarbon in the
liver; about 47% of these (30% of total 14C in liver) was as materials
                                 69
Solids
45
11
Water
27
46
Organo-extractabl es
27
44

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precipitated  from the  15,000g  supernatant with solvent, 43% (27% of
total   C) as materials remaining in solution after addition of solvent,
and  10% (3% of total   C) as materials removed from the 15,000g pellet
by extraction with acetonitrile and water.  Water soluble   C-residues
constituted 52% of the radiocarbon in the kidney; about 11% (6% of
total   C) was as materials precipitated from the 15,000g supernatant
with solvent, 74% (40% of total) as materials remaining in solution
after addition of solvent to the 15,000g supernatant, and 15% (7% of
total) as materials removed from the 15,000g pellet by extraction with
acetonitrile and water.  Based on these data and the report by Dorough
(1971), a preliminary hypothesis relative to the nature of the   C-
metabolites in the liver and kidney might be suggested as follows:

Nature of                                     % of total   C in tissue
  C-metabolite	Liver	Kidney
Free:  Carbaryl, dihydrodihydroxy-
       carbaryl, etc.                           16             10
Conjugated:
       0-Glucuronides, 0-Sulfates, etc.         12             33
       Glutathione conjugates   .                27             46
Protein bound and/or incorporated               36              6
Bound to cellwall and/or membranes               9              5

Protein precipitation and dialysis- In order to prepare the 15,000g
supernatant for more detailed analysis such as tic, electrophoresis,
etc., it will be necessary to remove most of the protein from the
solution.  This was attempted with the liver 15,000g supernatant using
several methods which would precipitate the proteins (Table 25).  All
of the methods which precipitated the majority of the proteins (heat,
acetonitrile, ammonium sulfate) resulted in 40% or more of the radio-
carbon in the precipitate.  Acetone, up to 30%, precipitated most of
the protein without loss of any of the radioactivity from the super-
natant.  However, precipitation with loss of radiocarbon to the solids
                                  70

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occurred during the process of removing the acetone by evaporation.
Because of the limited amount of liver available, no further studies
of this nature have been performed with this tissue.  It is  of interest,
however, that heat, acetonitrile and 40% ammonium sulfate resulted
in 40 to 48 percent of the radiocarbon in the supernatant being pre-
cipitated with the proteins.  This is equivalent to approximately 35%
of the total   C-content of the liver and agrees very well with the
level, 36%, hypothesized to be bound and/or incorporated with protein.
Further studies of protein precipitation using the kidney 15,000g
supernatant showed that 65% of the radiocarbon which was precipitated
with ammonium sulfate could be dialyzed (Table 25).   Therefore,  it is
unlikely that ammonium sulfate or the other methods  of precipitation
selectively precipitated only that radiocarbon bound to or incorporated
into various proteins.

Tic analysis of the supernatant after ammonium sulfate precipitation
showed 4 distinct bands (EM precoated silica gel plates, F-254,
0.25 mm, developed in 75:15:10 chloroform-methanol-acetic acid).   Fifty-
six percent of the radioactivity applied to the plate remained at the
origin, 28% chromatographed identical to naphthyl  glucuronide, 9%
chromatographed identical  to naphthyl sulfate and 7% moved to the
solvent front as did standards of carbaryl, 1-naphthol and other free
metabolites.  The fact that certain of the radioactivity chromatographed
                                                               14
the same as naphthyl glucuronide and sulfate suggests that the   C-
materials were of this nature.  However, it is not suggested that the
compounds were only naphthyl glucuronide or only naphthyl sulfate.
While this tic system has been shown to effect separation of sulfate
and glucuronides, it has not been effective in separating mxitures of
sulfates or mixtures of glucuronides.

The dializate from the 75-95% ammonium sulfate precipitation (Table 26,
76% dialyzed) was also analyzed by tic as described above.  In this
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case,  27% of  the  radioactivity  remained at  the origin, 16% chromato-
graphed  identical  to naphthyl glucuronide,  45% identical to naphthyl
sulfate, 5% as a  band just above the sulfate, and 7% moved to the
solvent  front.  These precipitation and dialysis data demonstrate the
care which must be taken  in future studies  if complete separation of
the metabolites is to be  achieved.

Sephadex column chromatography  - A Sephadex G-100/120 column was pre-
pared  by suspending 20 g  of the gel in 400 ml of distilled water and
allowing to swell  for 72  hours.  A 3 cm, id, column was packed to a
height of 46  cm with the  Sephadex and equilibrated with phosphate
buffer, pH 7.0.

The 15,000g supernatants  of liver or kidney were added to the column
(homogenate of 20-25 g of tissue concentrated to 10 ml) and eluted
with 500 ml of phosphate  buffer.  Ten to 15 ml fractions were
collected and 0.5  ml aliquots from each fraction radioassayed.

With both the liver and kidney, only 2 radioactive peaks were eluted
from the column, with recovery of the added radiocarbon being greater
than 95%.  The first radioactive peak (I) began to elute from the
column in the 8th  fraction and continued through the 23rd fraction,
with the maximum concentration occuring in the 17th fraction.   Peak
II was eluted from the column in fractions 26 through 36, with
fraction 31  containing the greatest concentrations of radiocarbon.

Of the total  radiocarbon  recovered from the column, peak I  contained
49% in the case of the liver and only 13% in the case of the kidney.
The remainder, 51  and 87% respectively for the liver and kidney, was
contained in  peak  II.   The nature of these materials have yet to be
critically'evaluated.   However,  it could be that the peak I materials
are the same  as the "Unextractable Unknowns" reported by Dorough.
(1971) and the peak II  was a combination of the "free metabolite"
                                72

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and "water-soluble unknowns".  This is based on the fact that the
                     14
distributions of the   C-residues in the liver and kidney were very
similar when examined by the 2 different methods.   For example, the
"Unextractable Unknowns" represented 48 and 18% of the radiocarbon in
the liver and kidney when extracted with acetonitrile-chloroform,
whereas peak I represented 49 and 13% of the radiocarbon in the 15,000g
supernatant of the 2 tissues.

When a portion of peak II from the kidney was extracted directly with
acetonitrile-chloroform, 22% of the radioactivity was recovered in
the organic solvent phase.  Tic analysis (EM F-254 plates developed
in 4:1 methylene chloride-acetonitrile) showed that approximately 15%
of the radioactivity remained at the origin and 85% chromatographed
identical to a 5,6-dihydrodihydroxycarbaryl standard.  No cochromato-
graphy of the 2 materials was attempted.  Acid treatment of another
portion of peak II (IN HC1, 37°C for 16 hrs) prior to extraction
resulted in equal portions of radioactivity in the water and organic
solvent phases.  Tic of the organo-solubles showed 3 radioactive
bands, 1 in the area of 5,6-dihydrodihydroxycarbaryl (15%), 1 in the
area of 5,6-dihydrodihydroxynaphthol (15%) and 1 chromatographing in
the area of carbaryl.  The use of standards to indicate location
of the radioactive bands is not intended as tentative identification
but only to point out the general behavior of these materials on tic.
Many analogs of carbaryl chromatograph similar to the standards
mentioned here.  This is especially true for the area where carbaryl,
per se, is located.  Detailed examination of the tic characteristics
of the materials in a number of solvent systems must be accomplished
before even tentative identification can be made.
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TABLE 18.  STUDY PLAN FOR CARBARYL-NAPHTHYL-14C COW FEEDING EXPERIMENT.
Dietary dose (ppm)              0.10,30,100
Treatment method                Gelatin capsule (one every 12 hr)
Treatment schedule              14 days on 'cold'  carbaryl followed by
                                14 days on carbaryl-naphthyl-l4C
Sampling                        Milk, urine,  feces:   every 12 hr,
                                Tissues:  18  hr after last dose
Sensitivity                     0.005 ppm carbaryl-   C equivalents
                                74

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TABLE 19.  CHEMICAL NATURE OF CARBARYL METABOLITES IN COW'S MILK AND
           THEIR AVERAGE CONCENTRATIONS AFTER FEEDING WITH CARBARYL
                        (TOO PPM IN THE DIET FOR 14 DAYS)

Metabolite
No.
VI
II

Carbaryl
3,4-Dihyd
Chemical nature

rodihydroxy-1-naphthyl
Amount in
milk (ppb)
17

% of
total
6

              methylcarbamate                       13           6
  III       5,6-Di hydrodi hydroxy-1-naphthyl
              methylcarbamate                       94          34
   V        5-Hydroxy-l-naphthyl methylcarbamate     3           1
   IV       5,6-Dihydrodihydroxynaphthalene          9           3
  VIII      1-Naphthyl sulphate                     72          26
  VII A     1-Methoxy-5-(methylcarbamoyloxy)-2-
              naphthyl sulphate                     63          23
  VII B     5-Methoxy-l,6-naphthalenediol            7           2
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TABLE 20.  TOTAL CARBARYL-14C EQUIVALENTS IN COW TISSUES AFTER FEEDING
                  WITH CARBARYL-NAPHTHYL-14C (10, 30 AND TOO
                            PPM IN THE DIET FOR 14 DAYS)

                             Carbaryl-  C equivalents (ppb)
Tissues
Kidney
Liver
Lung
Muscle
Heart
Fat
Blood
10 ppm
0.095
0.033
0.020
0.009
0.012
0.000
0.008
30 ppm
0.531
0.100
0.064
0.031
0.038
0.015
0.036
100 ppm
1.003
0.411
0.027
0.104
0.095
0.025
0.141
                                 76

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TABLE 21.
RADIOACTIVE RESIDUES IN COW TISSUES AFTER FEEDING WITH CARBARYL-14C (100 PPM IN THE DIET
                                      14 DAYS)


Metabolites
Carbaryl (No. VI)
5 , 6- Di hydrodi hydroxy
carbaryl (No. Ill)
5, 6-Di hydrodi hydroxy
naphthol
Naphthyl sulphate (No. VIII)
Water-soluble unknowns
Unextractable unknowns

Kidney
3.3
4.5
1.8
29.3
43.2
17.9
% of
Liver
9.2
3.0
4.1
4.1
32.9
46.7
total radioactivity in
Lung
2.1
8.8
0
27.3
47.5
14.3
Muscle
17.0
38.6
0
0
30.6
13.8
sample
Heart
3.7
31.3
4.9
4,0
41.8
14.3

Blood
0
22.0
2.0
51.8
7.1
17.1

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TABLE 22.  CENTRIFUGAL FRACTIONATION OF 14C-RESIDUES IN THE LIVER AND
           KIDNEY OF A LACTATING COW FED 100 PPM CARBARYL-1-NAPHTHYL-
                          14C IN THE DIET FOR 14 DAYS

Distribution, % of
Fraction
15,000g supernatant, I
15,000g supernatant, II
15,000g supernatant, III
Total 15,000g supernatant
105,000g supernatant
105,000g pellet
15,000 pellet
Recovery
Liver
75.1
6.8
2.6
85.1
85.1
0
14.7
99.8
total 14C in tissue3
Kidney
66.7
12.3
3.8
82.8
81.4
1.4
19.3
102.1
  Comparable results were obtained when tissues were homogenized  in
  distilled water or in phosphate buffer, pH 7.0.
                                78

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TABLE 23.  EXTRACTION CHARACTERISTICS OF 14C-RESIDUES IN ACID-TREATED


                15,000g SUPERNATANT OF LIVER AND KIDNEY


                     HOMOGENATES (SEE TABLE 22)a
                                                          14
                                 Distribution, % of total    C in
supernatant
Fraction
Chi orof orm-acetoni tri 1 e
Water
Solids
Recovery
Liver
30.5 (26.0)
27.2 (23.1)
42.0 (35.7)
99.7
Kidney
45.9 (37.4)
47.9 (39.0)
7.4 (6.0)
101.2
a Supernatant adjusted to 3N HC1 and refluxed for 3 hrs.

b                                       14
  Numbers in parenthesis represent % of   C in tissue.
                                 79

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TABLE 24.  EXTRACTION CHARACTERISTICS OF 14C-RESIDUES IN ACID-TREATED
                   15,000g PELLET OF LIVER AND KIDNEY
                       HOMOGENATES (SEE TABLE 22)a
                                                          14
                                 Distribution, % of total    C in
Fraction
Chi orof orm-acetoni tri 1 e
Water
Solids
Recovery

Liver
8.1 (1.2)
28.6 (4.2)
60.3 (8.9)
97.0
pelletb
Kidney
38.1 (7.4)
36.7 (7.1)
23.3 (4.5)
98.1
  Pellet placed in 3N HC1 and refluxed for 3 hrs.
b                                       14
  Numbers in parenthesis represent % of   C in tissue.
                                80

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TABLE 25.  14C-RESIDUES IN THE PRECIPITATE OF A 15,000g  SUPERNATANT OF
            LIVER HOMOGENATE TREATED IN VARIOUS WAYS  TO  EFFECT  PRE-
                            CIPITATION OF THE PROTEINS
                                        14
                                          C in precipitate,  %  of  total
                                             14
Conditions for precipitation	C in  supernatant	
Sodium chloride, saturated                           25
Heat, 90°C for 0.5 hr                                48
Acetonitrile, 20%                                    39
Acetonitrile, 40%                                    39
Acetone, 20%                                          0
Acetone, 30%                                          0
Acetone, 35%                                         12
Ammonium sulfate, 40% of saturation                  46
Ammonium sulfate, 80% of saturation                  68
Ammonium sulfate, 100% of saturation                 73
                                 81

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00

ro
      TABLE 26.  AMMONIUM SULFATE PRECIPITATION, AND DIALYSIS OF THE PRECIPITATE, OF A 15,000g SUPER-


                                            NATANT OF KIDNEY HOMOGENATE

(NH4)2S04 %
of saturation
0-40
40-60
60-75
75-95
95-100
Total
%14C
precipitated
10.5
13.2
12.7
18.6
2.9
57.9
mg
protein ppt
382.9
397.8
359.7
288.6
36.0
1465.0
Specific
activity of
protein, dpm/mg
29
35
38
68
85
42
Specific
% of
dialyzed after
68.1
61.3
58.3
68.8
76.0
64.7 (37.5)a
activity
protein
dialysis
7.8
13.1
16.1
17.9
21.9
13.6
                    14
           of  total   C  in supernatant.

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Carbaryl In Soils

Decomposition of carbaryl (1-naphthyl N-methylcarbamate) by a soil
bacterium was reported by Tewfik and Hamdi (1969).  Four unidentified
metabolites were produced, one which was possibly salicylic acid.  In
Bacteriological Proceedings of 1970, Bollag and Liu described bio-
degradation of carbaryl by soil microbes.  All microorganisms isolated
hydrolyzed carbaryl to 1-naphthol.  One-naphthol was metabolized rap-
idly by Fusarium solani, degraded gradually by a Gram (-) coccus and
accumulated in a Gram (+) rod.  Disappearances of ring-labeled 1-
naphthol and carbaryl labeled on the carbamate moiety were compared
with IF. solani and the coccus singly and mixed.  The fungus effected an
almost complete loss of radioactivity from the carbaryl  incubation,
but only 20% of the radioactivity in the 1-naphthol preparations.  The
coccus attacked carbaryl more effectively than £. solani.  The mixture
degraded both carbaryl and 1-naphthol, suggesting that complete bio-
degradation was a result of combined growth.

More recent work on the metabolism of carbaryl by soil fungi showed that
several species of the genera Aspergillus, Fusarium, Gliocladium, Mucor,
Penicillium and Rhizopus produced varying amounts of 1-naphthyl N-
hydroxymethylcarbamate and the 4- and 5-hydroxylated metabolites
(Bollag and Liu, 1971).  Gliocladium roseum was selected for further
study and the three metabolites firmly identified by ultraviolet, in-
frared and mass spectroscopy.  Aspergillus terreus produced minute
amounts of what were tentatively identified as the ring-hydroxylated
metabolites of the carbamate insecticide (Liu and Bollag, 1971b).
Two metabolites, the formation of which followed the same pattern over
a period of 8 days of incubation in yeast extract-nutrient broth,
were identified as 1-naphthyl N-hydroxymethylcarbamate and 1-naphthyl
carbamate.  A. terreus grew on media containing either of the two
metabolites or 1-naphthol.  The authors postulated that degradation
proceeds from 1-naphthyl N-hydroxymethyl carbamate to 1-naphthyl
                                83

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carbamate to 1-naphthol.  Since the medium controls contained more 1-
naphthol than did A. terreus cultures, the fungus was assumed to
actively degrade 1-naphthol (Liu and Bollag, 1971a).

In our study, radiolabeled carbaryl was added to non-sterile and auto-
claved soils and to culture media inoculated either with soil sus-
pensions or with pure cultures of microorganisms selectively isolated
from soil.  Polar water-soluble metabolites of radiolabeled carbaryl
produced in bean plants were also added to soil.  After incubation, the
soils and culture media were assayed to determine the fate of the
radiolabeled material.  The microorganisms which were capable of
attacking the carbaryl molecule to a demonstrable degree under the
experimental conditions employed were identified.

Methods

Soils - Three samples of Maury soil of the type common to the Inner
Bluegrass region but with different histories of pesticide treatment
were employed in the soil studies.  These soils have been designated as
Untreated. Carbaryl-treated, and Mixed Pesticide-treated soils.   The
Untreated and Carbaryl-treated soils were taken from neighboring plots
in the same tobacco field.  They were similar as to color, consistency
and amount of organic material.  The former had received no recorded
pesticide treatment of any kind, while the latter had received 4 lb/
        p
A Sevin   granules six months prior to the sampling date.  This  level
of treatment approximates 4 ppm if uniform distribution to a depth of
6 2/3 inches is assumed (Bollen, 1961).   The Mixed Pesticide-treated
soil  was taken from a home orchard floor which had been subjected for
fifteen years to various treatments including runoff from foliar sprays
of insecticides (including carbaryl) and fungicides applied to the
trees, soil  treatments, and herbicide drift.   This soil was observed
to contain more organic material than the other two.
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The samples were taken from the upper six inches of topsoil  and con-
tained 18-22 percent moisture when brought into the laboratory.  They
were stored in heavy plastic bags, tightly closed, at room temperature
until used in the soil studies.
Chemicals - The radiolabeled compound employed was carbaryl-naphthyl-
  14                                            1
1-  C of specific activity 0.6 mc/mM or 6.6 x 10  dpm/ug.   In addition,
radiolabeled polar water-soluble metabolites of carbaryl  prepared from
bean plants were used.  Polar water-soluble metabolites of carbaryl  (PMC)
                                                 14
were prepared by injecting carbaryl-1-naphthyl-l-  C into  young bean
plants and, after a suitable interval, extracting the water-soluble
metabolites produced in the plants.   Eleven-day old Cranberry bush
bean seedlings were injected with carbaryl-l-naphthyl-1-   C at the
rate of 50 ug/plant in 50 ul of a 3:1 mixture of water and acetone.
The solution was injected into the stem just above the soil  line using
a 50 ul syringe.  Prior to the injection, a small pinhole  was made in
the stem about 2 inches above the injection site to permit escape of
air.  Following injection, the holes were closed with silicone stop-  -
cock grease.
Dorough and Wiggins (1969) established optimal  harvest time for pro-
duction of water-soluble metabolites and their precise location in
Contender variety beans.  On the basis of their findings, a single
Cranberry bush plant was harvested 9 days after injection and the
epicot leaves extracted.  Since water-soluble metabolites were found in
amounts comparable to those that had been found in Contender variety,
the plants were harvested at 10 days after injection.   The epicot leaves
were removed and stored at -10°C until extracted.   A pair of epicot
leaves from a plant was homogenized 5 min at medium-high speed in a
Vir-Tis 45 Homogenizer in 100 ml of 90 percent acetone in water.   The
homogenate was filtered with suction through Whatman no.l filter paper,
and the marc rinsed with acetone.  The filtrates from three pairs of
leaves were combined, then fractionated into water-solubles and
                                 85

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organosolubles by shaking in a 1000-ml separatory funnel with an equal
volume of chloroform.  The organic layer was washed twice with 25 ml of
water, then the aqueous layers were combined and washed twice with an
equivalent volume of chloroform.  The foregoing procedure was the
same as that of Wiggins and Weiden (1969) except that they used five
chloroform rinses.  The aqueous layer containing the PMC was concen-
trated by lyophilization.

The nature of the organic moieties (aglycones) contained in the PMC was
determined by enzymatic hydrolysis, extraction of the hydrolysate, and
tic analysis of the extract.  In the hydrolysis procedure used a sample
containing PMC in amounts up to 100,000 dpm was adjusted to a volume
of 2.2 ml in water and placed in a 25-ml Erlenmeyer flask.  To this
was added 20 ul of glusulase, 0.08 ml of isopropanol, 4.0 ml of citrate-
phosphate buffer at pH 5.0, and 1 drop of toluene.  The mixture was
incubated  in a water bath with shaking for one hour at 37-38°C, after
which it was transferred to a 60-ml separatory funnel and extracted
three times with 25-ml amounts of methylene chloride.  The combined
extracts were dried with sodium sulfate, filtered through Whatman no.
1 filter paper and evaporated to a volume suitable for tic.

For preparation of Miles (Miles et al., 1969) carbaryl  agar, the salts
and agar were dissolved in distilled water and the pH was adjusted to
7.0 with IN potassium hydroxide.   The medium was then autoclaved and
cooled to 45-50°C.  Carbaryl dissolved in ethanol  was then added in
amounts calculated to give 1 ppm and 1 percent of carbaryl and
ethanol, respectively, in the Miles carbaryl agar.  Twenty-five ml
amounts were then poured into sterile petri plates.   Carbaryl-treated
soil  was suspended in distilled water in different proportions and
1  ml  of each spread over the surface of a plate.  The plates were
incubated at 25°C.  As isolated colonies of fungi  appeared, they
were transferred to fresh plates, either by center transplant or by
successive streaks 2 cm apart across the plate.   Bacterial  colonies were
                                 86

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transferred by conventional streakouts to plates or slants of Miles
carbaryl agar.  As in the silica gel procedure, repeated transfers were
made when necessary for purification.

Soil studies - Radiolabeled carbaryl, 1-naphthol and plant metabolites
were added to soil samples along with autoclaved soil samples for
controls.  The resultant preparations were incubated under controlled
conditions.  Immediately and after various periods of incubation por-
tions of the soil preparations were removed for analysis of the
radioactive components.   Total radioactivity was determined by direct
count or by assaying trapped carbon dioxide following combustion of
the soil or by making an acetone-water extract of the soil, fraction-
ating the extract with chloroform into water-soluble and organosoluble
portions and determining the radioactivity of each extract, as well as
that of the residue remaining after extraction.  The organosoluble por-
tions were subjected to tic analysis for separation and identification
of metabolites formed from carbaryl and from PMC.  The production and
nature of volatile metabolites produced during incubation was invest-
igated by the use of trapping systems.

In making soil preparations, the soil was sieved through hardware cloth
of 1/4 inch mesh, and moisture was determined by drying duplicate
samples at 65°C for 48 hours.  An aliquot of soil comprising about 10
percent of the total amount to be incubated was oven-dried.  The
appropriate amounts of carbaryl or 1-naphthol dissolved in acetone or
of PMC in water (filter-sterilized if to be used in autoclaved soil
preparations) were added to the oven-dried soil.  When acetone was used,
the soil and acetone were stirred constantly until the acetone odor
was no longer detectable.  The radioactive soil was then combined with
the remaining soil to be incubated, distilled water added to provide
17-20 percent moisture, and the whole soil mass mixed thoroughly.
Zero-time samples taken immediately by picking up randomly selected
small amounts of soil and radioassaying by combustion established
                                 87

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that carbaryl was distributed in a reasonably homogeneous fashion
throughout the soil.  The soil preparations were incubated in cotton-
capped Erlenmeyer flasks at 27°C and 85 percent relative humidity in
an environmental chamber.  Samples for analysis were removed at desig-
nated intervals and stored at -10° until examined.

A 10-g soil sample was placed in 50 ml of a 5:1 acetone-water solution
and shaken by hand for 5 min in a tightly-stoppered 250-ml Erlenmeyer
flask.  The suspension was filtered with suction through 2 layers of
Whatman no. 1 filter paper.  The solids were rinsed with a 5:1 acetone-
water, air-dried, weighed, and saved for radioassay.  The filtrate was
partitioned with 60 ml of chloroform and the water layer re-extracted
with 25 ml of chloroform.  The organosoluble layer was radioassayed,
dried with sodium sulfate, evaporated under reduced pressure to a
volume of 4 or 5 ml, and further concentrated under a gentle air stream
to a volume suitable for tic.

For the soil preparations with plant metabolites added the procedures
of extraction and fractionation were modified.   Ten grams of soil were
placed with 50 ml of 1:1 acetone-water in a 250-ml flask and shaken
vigorously by hand for 5 min.  By this procedure 70 percent of the PMC
were recovered from zero-time soil.  The suspensions were filtered and
the soil residues dried for radioassay.   The filtrates were partitioned
with volumes of chloroform equivalent to those of acetone in the
filtrates.  Both aqueous and organic extracts were evaporated under
reduced pressure, and further concentrated under a gentle air stream
to volumes suitable for tic.

Total  radioactivity in the soil  preparations was determined in two ways:
(1) by radioassay of an aliquot of soil  following combustion in either
Parr oxygen bombs or the Beckman Biological  Materials Oxidizer (BMO),
or directly; (2) in the cases where soil  was subjected to extraction and
fractionation procedures, by addition of the values for radioactivity in
                                88

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the organosoluble extract, the water-soluble layer, and the extracted
residue.

Liquid extracts and concentrates were radioassayed by direct count:   a
suitable aliquot was pipetted directly into a counting vial, 10 ml  of
scintillation fluid were added, the vial capped, shaken, and placed in
the spectrometer to be counted.  Aliquots consisted of 0.2 to 0.5 ml
for organosoluble extracts, 0.02 ml for organic concentrates, and
0.2 ml for water-soluble materials.

Chromatography and identification - For separation of carbaryl from its
metabolites, and the concentrated organosoluble extract was first applied
as a band to a thin layer chromatogram which was 'developed one-
dimensionally.  The chromatograms were made on either Silica Gel G 0.25
to 0.5 mm thick on a 20 x 20 cm glass plate or ChromAR 500 thin-layer
sheets (Mallinckrodt Chemical Works).  The solvent systems for one-
dimensional chromatography were  ether-hexane combinations in propor-
tions varying from 2:1 to 9:1.  A short band of carbaryl was applied to
each chromatogram as a standard.  Radioautographs were prepared by
exposing the developed tic plates to x-ray film at -10°C for a minimum
of seven days.  If unknown carbaryl metabolites appeared as bands on the
radioautographs, the bands were radioassayed in one of two ways depend-
ing on the apparent intensity of the band.  If the band represented a
metabolite of sufficient quantity for further chromatographic analysis,
the metabolite was extracted from the chromatogram with 1:1 acetone-
methanol or with acetone alone.  An aliquot of the extract was then
counted.

PMC as prepared from bean  plants were separated directly by applying
a small amount of the aqueous concentrate as a band to a thin layer
plate of Silica Gel F-254 0.5 mm thick and developing the chromatogram
one-dimensionally in 65:25:4 chloroform-methanol water (Mumma et al.,
1971).  After PMC were extracted from incubated soil and the extract
                                89

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was fractionated, direct separation of the metabolites in the aqueous
layer was not possible because of interfering substances present there-
in.  The organosoluble fraction of the extract was dried with sodium
sulfate and concentrated to tic volume.  The concentrate was applied
as a spot to Silica Gel F-254 and the chromatogram developed two-
dimensional ly, the solvent systems being 2:1 ether-hexane followed by
7:2 methylene chloride-ethyl acetate.

Rr values were computed for unknown carbaryl metabolites appearing as
bands on chromatograms and these were compared to Rf values of known
carbaryl metabolites in the same solvent system.  If present in
sufficient quantity, the unknown metabolite was extracted as described
above and the extract concentrated to tic voluem.  It was then compared
by tic with one or more known metabolite standards having similar Rf
values.  Non-radioactive carbaryl standards were visualized by examination
of the chromatograms under UV light and/or by spraying the chromatograms
with 15 percent sodium hydroxide followed by exposure to iodine
vapors in a tightly closed glass chromatography tank.

Determination of volatile metabolites - Soil preparations containing
carbaryl-1-naphthyl-l-  C, 1-naphthol-l-  C, and, in one experiment,
with carbaryl-carbonyl-  C, were incubated in systems for the
determination of the evolution of volatile metabolites such as 1-
naphthol and carbon dioxide.  The 1-naphthol soil preparations were
included in the studies on the basis of the simultaneous adaptation
theory of Stanier (1947).  According to this theory, if a micro-
organism can utilize a compound as a carbon source and metabolize it
through a particular pathway, it possesses all  the enzymes of the
pathway and thus should be able to attack any intermediate in the
pathway and utilize it likewise.   If the soil preparation metabolizes
carbaryl through hydrolysis, it also should be able to attack the
intermediate 1-naphthol.
                                 90

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For these studies, soil preparations were held at laboratory temperature
and ambient humidity (24°C and 60% R.H.).  Using this type of system,
Carbaryl-treated soil, both non-sterile and autoclaved, was incubated
for three days with carbaryl-1-  C and also with 1-naphthol-  C.  At
intervals, air was bubbled through the system to drive volatile
metabolites through the gas dispersant tubes into 20 ml of the organic
trap solution.  In a corollary test, Carbaryl-treated soil was incu-
bated with carbaryl-1-  C in a system having four 250-ml flasks set up
in series.  The first two flasks contained saturated barium hydroxide
to clear carbon dioxide from the air before it entered the third flask
containing the soil preparation.  The fourth flask contained barium
hydroxide to trap the radioactive carbon dioxide arising from the soil
preparation.  In both tests, zero-time and terminal samples were re-
moved for extraction and radioassay.  When barium hydroxide served as the
trap solution, barium carbonate formed during incubation was collected
by filtration washed with distilled water, air-dried on the filter
paper and radioassayed following combustion in the Parr bomb.  Efficiency
of combustion of barium carbonate was ascertained by combusting a
                         14
known amount of carbaryl-  C in the presence of non-radioactive barium
carbonate on filter paper.  Seventy five percent recovery was obtained.
Values obtained for radioactive samples were adjusted accordingly.
For detection of water-soluble materials such as the barium salt of
1-naphthol, the filtrate was radioassayed by direct count.

In another study, an acetone trap preceded the outlet barium hydroxide
trap.  Volatile organic metabolites if present would be detected in
the acetone trap, and carbon dioxide would pass through the acetone to
be trapped by barium hydroxide.  The Carbaryl-treated soil preparation
was incubated with carbaryl-1-  C for 13 days.  During the day, an
air stream was passed through the system at 20 ml/min.  The trap
solutions were removed, refrigerated, and replaced with fresh solut-
ions three times during the course of the study.  Aliquots of acetone
was evaporated under reduced pressure and aliquots again counted to

                                 91

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determine  low  levels  of  radioactivity.  Barium carbonate was assayed
as described above.   Total  radioactivity  in the zero-time and 13-day
soil samples was  determined following combustion in the Biological
Materials  Oxidizer.

In the final study of the series, duplicate 250-ml Erlenmeyer flasks
of Carbaryl-treated and  Untreated soils were incubated for 56 days with
each of the following labeled materials:  carbaryl-l-naphthyl-1-  C,
1-naphthol-l-  C  and  carbaryl-carbonyl-   C.  The 12 flasks were
connected  through a manifold assembly to  a common inlet air stream
pre-cleared of carbon dioxide by being bubbled through the organic trap
solution.  Outlet tubes  from the flasks were attached to gas dispersant
tubes immersed in 20  ml  amounts of the same trap solution.  Air was
passed through the system during the day  at a flow rate sufficient
to keep the slowest unit bubbling continuously.  At intervals, the trap
solutions  were removed,  aliquots radioassayed, and the traps filled
with fresh solutions.

Culture media studies -  Culture media containing carbaryl-1-naphthyl-
1-  C were inoculated with  suspensions of Carbaryl-treated and Mixed
Pesticide-treated soils and with pure cultures of bacteria and fungi.
After incubation, the media were extracted and assayed for carbaryl
and its metabolites.

Results and Discussion

Soil  studies - In a 120-day study carried out to determine the fate of
carbaryl  in non-sterile and autoclaved Untreated,  Carbaryl-treated and
Mixed Pesticide-treated soils, carbaryl-1-  C was  added at 7.0-10.0 ppm.
A striking difference was seen in the rates of dissipation of carbaryl
from the non-autoclaved soils with three histories of pesticide treat-
ment (Table 27).   Apparently more than 60 percent  of the radioactivity
had disappeared completely after four days in the  Carbaryl-treated
                                 92

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soil.  At the same time the other soils had lost less than 7 percent.
During the first 14 days of incubation the radioactivity disappeared
from Untreated and Mixed Pesticide-treated soils at about the same
rate.  Beyond that time, dissipation proceeded more slowly in the
latter.  After 120 days, the Untreated, Carbaryl-treated and Mixed
Pesticide-treated soils still  retained 23, 16 and 30 percent, respect-
ively, of the initial radiocarbon.

Autoclaved soil  did not retain total sterility throughout 120 days.
Fungal development was detected in 14 days in Untreated and Carbaryl-
treated soils, and at 120 days in Mixed Pesticide-treated soil.   How-
ever, in any given soil, radioactivity disappeared much faster from
non-autoclaved than from autoclaved soil.   This fact suggests that
increased dissipation rates in non-autoclaved soil reflected biological
action of soil microorganisms.

Organosoluble radiocarbon in the zero-time soil ranged from 90 to 97
percent and represented essentially the extraction efficiency of the
acetone procedure (Table 28).   More than 90 percent of the total organo-
soluble radiocarbon added to Carbaryl-treated soil was lost after 4
days.  This pattern was not seen in the other two soils, where a
comparable loss was noted only after 42 days.  Tic analysis of organo-
soluble extracts from all the timed samples in these soils revealed that
the radioactivity was composed entirely of carbaryl-1-  C.  Water-
soluble metabolites remained rather low (less than 2 percent) throughout
the study.  Unextracted   C-residues increased in quantity initially
but later decreased in the Untreated and Carbaryl-treated soils.
In order to determine more exactly the time of early dissipation of
carbaryl from Carbaryl-treated soil, a corollary 7-day study was con-
ducted, using non-sterile soils in conditions identical to those of
the 120-day study.  Analysis of samples removed at 0., 1, 2, 3, 4 and
7 days confirmed the difference in dissipation rates of radioactivity
                                 93

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from the three soils found  in the 120-day study.  The most rapid loss
of label (44 percent) occurred between the first and second day.

The fate of polar water-soluble metabolites of carbaryl  (PMC) in soil
was investigated in 2 studies.  In the first, PMC were added at
2650 dpm/g to non-autoclaved and autoclaved Carbaryl-treated soils, which
were then incubated at laboratory temperature and ambient humidity
(approximately 24°C, 60% R.H.) for 14 days.

After 7 days of incubation  with the non-autoclaved soil, about 50 per
cent of the initially added PMC had been dissipated (Table 29).  No
further loss occurred in the period between 7 and 14 days.  In auto-
claved soil, no such dissipation occurred.  In the second study, PMC
were added at 9074 dpm/g to non-autoclaved Untreated, Carbaryl-treated
and Mixed Pesticide-treated soils, which were then incubated for 120
days in the environmental chamber.  After the first 14 days, 55, 64
and 50 percent of the radiocarbon remained in the Untreated, Carbaryl-
treated and Mixed Pesticide-treated soils, respectively.  Little
further loss occurred from  14 to 120 days (Table 30).  This indicated
that the biological attack of soil microorganisms on PMC occurred
early in the incubation period, and that residues left after the
initial attack were generally untouched by biological action thereafter.

After prolonged incubation the PMC residues were largely unextractable
by the procedures available.  Only 15 percent of the total PMC still
present were recovered in the extract.  This finding necessitated
pooling of the samples in order to have enough material in the
extracts for characterization of the aglycones.   Samples taken between
7 and 28 days, and those taken between 42 and 120 days of incubation
were each treated as one sample for extraction purposes.

Before incubation with soil, the aglycone moieties resulting from
enzymatic hydrolysis of the PMC were identified by cochromatography
                                 94

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with known standards as 5,6-dihydro-5,6-dihydroxycarbaryl, hydroxymethyl
carbaryl, 7-hydroxycarbaryl, 4-hydroxycarbaryl (or 6-hydroxycarbaryl,
the two being inseparable in the solvent systems used), 5-hydroxycarbaryl,
carbaryl and 1-naphthol.  Small amounts of three aglycones appeared
in the organic fractions of the extracts of Mixed Pesticide-treated
soil, and the same three plus an additional one in the organic
fractions of the extracts of Untreated and Carbaryl-treated soils.
The positions to which these four aglycones migrated when the organic
concentrate was applied to a two-dimensional chromatogram indicated that
they were different than those prepared by enzymatic hydrolysis of the
PMC before incubation.  Amounts of the four aglycones recovered were
too small  to make positive identifications; however, it was noted that
one metabolite migrated to the region of the chromatogram where
naphthalenediols are generally found.

When Carbaryl-treated soil preparations incubated with carbaryl-1-  C
                 14
and 1-naphthol-l-  C were connected to trap solutions, carbon-14 dioxide
was trapped  (Table 31).  In 3 days, 18.3 percent and 3.1 percent of the
total radiocarbon from carbaryl and 1-naphthol, respectively, appeared
in the organic trap solution.  Comparable total percentages of   C
equivalents were recovered from carbaryl and 1-naphthol.  With the
latter, however, less radiocarbon was found in organosolubles and
carbon dioxide and more in water-solubles and unextractables than was
the case with carbaryl.  No carbon dioxide was ever detected in trap
solutions attached to autoclaved soil incubated for 3 days with either
carbaryl-1-  C or 1-naphthol-l-  C.  Radioassay of the soil after
3 days of incubation indicated that the labeled materials added
initially had not dissipated at all.  Another flask culture, incubated
with carbaryl-1-  C for 7 days attached to a barium hydroxide trap
gave off 22.6 percent of the initial label as carbon dioxide.  In these
studies, carbaryl was dissipated less rapidly when incubated at
laboratory temperature and ambient humidity that it had been in the
earlier persistence studies carried out in the environmental chamber.
                                 95

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When Carbaryl-treated soil was incubated for 13 days with carbaryl-1-
  C with acetone and barium hydroxide traps attached in series, less
than 1 percent of the radiocarbon was accumulated in the acetone traps
as volatile materials other than carbon dioxide.  This was a
negligible amount, compared to 30 percent trapped as carbon dioxide in
the barium hydroxide traps (Table 32).  No radioactivity was ever de-
tected in direct counts of aliquots of filtrates from the barium hydroxide
trap solutions.  This precluded the presence of water-soluble materials
such as barium salts of organic metabolites.  It was thus established
that the volatile radioactive materials arising from soils incubated
                14
with carbaryl-1-  C were essentially carbon dioxide.

The last soil study conducted was designed to compare carbon dioxide
production from Untreated and Carbaryl-treated soils incubated with
ring-labeled carbaryl, carbonyl-labeled carbaryl, 1-naphthol-l-  C
(Table 33).  It was observed that consistently throughout the incubation
period with any radiocarbon source, more carbon dioxide was trapped
from Carbaryl-treated than from Untreated soil.   In the Untreated soil,
                                                                 14
the highest percentage of carbon dioxide arose from 1-naphthol-l-  C,
especially in the early days of.the study.  In the Carbaryl-treated soil
the same effect was noted in the first three days of incubation;
comparison of the total  carbon dioxide liberated, however, shows that
comparable percen
labeled carbaryl.
comparable percentages were lost from 1-naphthol-l-  C and carbonyl-
At the time this study was conducted, nearly 1 1/2 years had elapsed
since the soils were brought into the laboratory and the first
persistence study was initiated.  During the entire 56-day incubation
period, 7 percent of the initial radiocarbon was trapped from 1-
naphthol-1-  C and carbonyl-labeled carbaryl and 4 percent from
ring-labeled carbaryl in the Carbaryl-treated soil.  Decreased de-
gradation of carbaryl is believed to have resulted from the fact that
the soil  had been stored in the laboratory for 1 1/2 years.  Bartha
                                96

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(1971) found that air-drying of soil severely reduced its capacity to
metabolize propanil.

Culture media studies - When AA medium with 3.3 ppm carbaryl-1-  C and
1 percent glucose was inoculated with a suspension of Mixed Pesticide-
treated soil and(incubated at 30°C with continuous shaking for 96 hours,
73 percent of the carbaryl was recovered unchanged from the medium, as
compared to 77 percent from the medium control (Table 34).  In the soil
cultures, organosoluble, water-soluble and unextractable radioactivity
increased with time, while in the medium control  only the first two
fractions increased.  Water-soluble radioactivity, as used here,
included both radioactivity found in the aqueous  medium after extract-
ion and radioactive material which did not migrate away from the
origin of the thin-layer chromatograms made from the organic concen-
trates.  In the soil cultures, the percentages of radioactive water-
solubles given in Table 34 represent predominantly material found in
the aqueous layer rather than on the chromatograms, while in the
medium control the reverse was true.

The nature of the organosolubles in the soil cultures differed from
that of those found in the medium control.  In the cultures, small
amounts of one organosoluble metabolite appeared after 24 hours of
incubation and a second appeared after 48 hours.   By 96 hours the
second had accumulated to a greater extent than the first.  This
suggests that the first metabolite may have served as a precursor for
the second in the degradation pathway of carbaryl.  The quantity of
the first metabolite was not sufficient for extraction and two-
dimensional chromatography.  Its Rf value one-dimensionally resembled
that of hydroxymethylcarbaryl.  The second metabolite had an Rf value
similar to those of the hydroxylated carbaryl metabolites in one-
dimensional chromatograms, but did not cochromatograph with any of
them two-dimensionally.  Other than the parent compound, the only
organosoluble metabolite found in the medium control was 1-naphthol,
                                 97

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which did not appear in any of the cultures.  Two possible explan-
ations may be suggested.  Carbaryl is subject to chemical hydrolysis
at alkaline pH and to photolysis; in cultures grown on glucose, acid
production may have prevented chemical hydrolysis and/or turbidity in
the incubation flasks may have hindered photolysis by interfering
with transmission of light.  It is also possible that free 1-naphthol
was produced in the cultures and conjugated too rapidly into water-
soluble metabolites to be detected in the organosoluble fraction.
When Liu and Bollag (1971a) found more 1-naphthol in medium controls
than in Aspergillus terreus cultures incubated with carbaryl, they
assumed that A. terreus was actively degrading the 1-naphthol.
Recovery after incubation of more than 90 percent of the initial radio-
activity indicated that in these cultures no more than negligible
amounts of radioactive carbon dioxide escaped.

In studies using Carbaryl-treated soil suspensions and bacterial and
fungal isolates as inocula, incubation proceeded for 14 days at
laboratory temperatures and no attempt was made to follow the time
course of metabolite production.  The results in Table 35, as well as
those in Tables 36 and 37, represent averages of the results of the
designated number of duplicate experiments.  When a suspension of
non-sterile Carbaryl-treated soil was used as the inoculum in Miles
medium, only 19 percent of the radioactivity could be accounted for by
unchanged carbaryl; other organosolubles accounted for less than 1
percent, water-solubles for 30 percent, and unextractables 23 percent,
of the original radiocarbon (Table 35).  In the medium inoculated
with autoclaved Carbaryl-treated soil, 71 percent of the initial label
was in unchanged carbaryl, 3 percent in other organosolubles, 9 per-
cent in water-solubles and 6 percent in unextractables, with a total
recovery of 89 percent.  In the medium control, 92 percent of the
initial radiocarbon was recovered:  76 percent as the parent compound,
6 percent as other organosolubles, 8 percent as water-solubles, and
2 percent as unextractable radiocarbon that remained on the filter
                                98

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paper during the filtration step in the extraction procedure.  The
drastically reduced percentage of unchanged carbaryl when the medium
was inoculated with the non-sterile soil indicated biological action
on the carbaryl molecule.  However, except for traces of 1-naphthol in
one flask, organosoluble metabolites were absent from media inoculated
with the non-sterile soil.  Dissipation of 28 percent of the total
label from such media compared to only 11 percent from media inoculated
with autoclaved soil could well indicate some loss from the former as
carbon dioxide.  No attempt was made to trap volatile radiocarbon in
the culture medium studies.

The fungal cultures gave some evidence of attack on the carbaryl
molecule.  The percentages of unchanged carbaryl found with fungal
isolates, except for PeniciIlium implicatum and SF-10, were similar to
those found with autoclaved soil and in the medium control (Table 35).
P.. implicatum showed an increase in total water solubles and SF-10,
which had been identified tentatively as a non-sporulating variant of
Aspergillus terreus, an increase in total organosolubles and unextract-
ables.  The percentages of total radioactivity recovered after incu-
bation with fungal isolates indicated little if any loss of carbon
dioxide.  Although some of the fungal strains produced no demonstrable
change in total radioactivity, all of them produced some metabolites.

SF-10 and P_. implicatum produced a metabolite which was identified by
two-dimensional cochromatography as hydroxymethylcarbaryl.  SF-10 and
the three Fusarium species produced an organosoluble metabolite that
had an R^ value in 2:1 ether-hexane similar to those of the hydroxy-
lated metabolites of carbaryl that migrate beyond hydroxymethylcarbaryl
in this system.  However, extensive efforts at cochromatography in
two-dimensional systems established that the metabolite was neither
one of the common hydroxylated compounds nor 1-naphthylcarbamate.  P_.
implicatum also formed a metabolite which had an Rf value in 2:1 ether-
hexane similar to those of 1-naphthylcarbamate and 4-methoxycarbaryl.
                                 99

-------
In two-dimensional chromatography it formed 2 spots which approximated
the spots of carbaryl and 1-naphthol.  Penicillium lilacinum and
and Aspergillus elegans produced very small amounts of a metabolite
having an Rf value in 2:1 ether-hexane similar to that of 5,6-dihydro-
5,6-dihydroxycarbaryl.

When three strains of bacteria isolated from Carbaryl-treated soil were
incubated with carbaryl in Miles medium no evidence of any action on
the carbaryl molecule was found (Table 36).  These strains proved
incapable of growth in nutrient broth and were dropped from further
consideration.

The bacterial species isolated from Mixed Pesticide-treated soil were
transferred several times on trypticase soy agar slants before incu-
                       14
bation with carbaryl-1-  C.  These species attacked carbaryl effectively
when cultured in nutrient broth (Table 37).  After 14 days of incu-
bation, with all except Arthrobacter tumescens, less than 10 percent
of the initial carbaryl was recovered as the parent compound.
Pseudomonas acidovorans, Nocardia flava and Arthrobacter sp. converted
carbaryl largely to water-soluble metabolites, while Xanthomonas sp.
and Bacillus sphaericus produced relatively more organosoluble
metabolites.  J3. sphaericus also grew on Miles medium but degraded
carbaryl less effectively (Table 36) than in nutrient broth.  In con-
sidering the nutrient broth culture results it should be mentioned
that in the medium control only 60 percent of the initial carbaryl-1-
14
  C was recovered as parent compound, and 24 percent of the radio-
activity was found in the water layer.   This was not a case of lesser
extraction efficiency of the acetonitrile-chloroform procedure on
                                14
nutrient broth; when carbaryl-1-  C was added to medium and re-
extracted without incubation, 93 percent and 95 percent were recovered
from Miles medium and nutrient broth, respectively.   Hence, the
fundamental  conditions in nutrient broth brought about greater de-
gradation of carbaryl  than those in Miles medium.
                                100

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Two organosoluble metabolites with R^ values in 2:1 ether-hexane great-
er than that of  the parent compound appeared in extracts of cultures
of EL sphaericus and Xanthomonas sp.  The two metabolites did not
separate completely in one dimension and did not migrate cleanly in
the two-dimensional system generally used in these studies for ident-
ification of metabolites (2:1 ether-hexane followed by 7:2 methylene
chloride-ethyl acetate).  The best separation was achieved two-
dimensional ly on Silica Gel F-254 with 5:1 hexane-ether used in both
dimensions.  One of the metabolites was identified as 1-naphthol while
the other migrated beyond 1-naphthol in non-polar solvent systems
and was not identified.  Traces of a third metabolite were produced by
Arthrobacter sp., Nocardia flava and Xanthomonas sp.  Its R-: value
in 2:1 ether-hexane was the same as one of the fungal metabolites
which was not identified.
                                101

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TABLE 27.  RADIOACTIVITY OF SOILS FORTIFIED WITH CARBARYL-l-14Ca AND

                        INCUBATED AT 27°C, 85% R.H.

Days
of
Incubation

4
7
14
28
42
120
Soils
Carbaryl-
Untreated
NA
93.5
89.0
60.8
33.7
30.2
23.1
A
-
80.1
76.6
75.4
75.9
46.7
treated
NA
38.5
33.7
24.8
22.1
22.7
16.0
A
-
84.1
58.7
67.8
63.0
62.8
Mixed Pesticide-
treated
NA
94.6
102.2
62.6
53.6
38.4
29.9
A
-
109.8
90.0
97.0
113.1
60.1
3 10 ppm in non-autoclaved and 7 ppm in autoclaved soils.

  Percent of initial radioactivity
A = autoclaved soil; NA - non-autoclaved soil.
                                 102

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o
CO
      TABLE 28.  RADIOACTIVITY IN ORGANOSOLUBLES (OS), WATER SOLUBLES (WS) AND UNEXTRACTABLES (U) IN SOILS

                               FORTIFIED WITH CARBARYL-1-14C AND INCUBATED AT 27°C, 85% R.H.

Days
of
Incubation
0
4
7
14
28
42
120
Soils
Untreated
OS
94. 2a
67.9
61.0
34.1
14.6
3.6
1.2
US
0.3
0.5
0.6
1.0
1.2
1.5
1.7
U
5.5
21.1
22.5
27.0
23.3
24.6
17.0
Carbaryl -Treated
OS
89.7
3.6
1.7
1.7
1.0
0.7
0.6
WS
0.6
1.0
1.0
1.1
1.5
1.1
0.9
U
9.7
23.6
20.8
20.2
13.5
16.6
12.6
Mixed-Pesticide treated
OS
96.8 .
81.2
67.4
50.5
20.7
7.4
1.5
WS
0.2
0.4
0.5
0.7
0.7
1.5
2 .0
U
3.0
15.4
17.1
22.0
28.6
30.5
26.9
      a Percent of initial  radioactivity.

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TABLE 29.  RADIOACTIVITY OF CARBARYL-TREATED SOIL FORTIFIED WITH POLAR
                WATER-SOLUBLE METABOLITES OF CARBARYL-1V
                    AND INCUBATED IN THE LABORATORY

Days of
incubation
7


14


Fraction
Extractables
Unextractables
Total
Extractables
Unextractables
Total
Soils
Non-autoclaved
11. 7b
39.7
51.4
6.7
50.5
57.2

Autoclaved
74.3
32.3
106,7
74.2
24.9
99.1
a Level of radioactivity in soil 2,650 dpm/g.
  Percent of initial radioactivity.
                                 104

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TABLE 30.  RADIOACTIVITY OF SOILS FORTIFIED WITH POLAR WATER-SOLUBLE
                   METABOLITES OF CARBARYL-14Ca AND
                      INCUBATED AT 27°C, 85% R.H.

Days of
Incubation
0
7
14
28
42
56
84
120

Untreated
100.05
63.7
54.9
57.9
60.5
62.6
63.2
54.2
Soils
Carbaryl-
treated
100.0
64.8
63.9
56.3
60.6
66.2
55.1
53.6

Mixed
Pesticide- treated
100.0
67.2
49.7
71.2
61.8
44.2
58.7
57.4
a Level of radioactivity in soil 9,074 dpm/g
  Percent of initial radioactivity.
                                  105

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TABLE 31.   FATE OF CARBARYL-NAPHTHYL-1-14C AND  1-NAPHTHOL-14C  IN CARBARYL-TREATED SOIL HELD  UNDER
                                        LABORATORY  CONDITIONS



Substance Days of Organo-
added Incubation solubles
Carbaryl-14C 3a
l-Naphthol-14Ca 3a
Carbaryl-14C 7b
45.6
24.2
35.3
a Air passed from soil preparation through
2-methoxyethanol and 2-aminoethanol .
Percent of
Water
solubles
0.4
16.9
0.8
trap solution
added radioactivity present as
Unex-
tractables
25.1
48.4
17.1
consisting of a
Carbon
Dioxide
18.3
3.1
22.6
2 to 1 mixture
Total
Recovery
89.4
92.6
75.8
of

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TABLE 32.  RADIOACTIVITY OF ACETONE AND BARIUM HYDROXIDE TRAP SOLUTIONS

            AFTER INCUBATION OF CARBARYL-TREATED SOIL PREPARATIONS WITH

                          CARBARYL-1-NAPHTHYL-1-14C ADDED3

Days
of
Incubation
0-3
4-6
7-10
11-13
Cumulative
as percent
Acetone Trap
0.1
0.2
0.3
0.4
radioactivity expressed
of initial radioactivity
Barium Hydroxide Trap
10.3
21.7
26.6
29.5
a Carbaryl-  C added to soil at the 1.5 ppm level  and air passed from
  the preparation through acetone and then barium hydroxide solution.
                                 107

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      TABLE  33.   CARBON-14 DIOXIDE LIBERATED  FROM CARBARYL-14C AND  1-NAPHTHOL-14C  DURING INCUBATION WITH


                                                         SOIL
o
00

Untreated soil
Material
Incubated3
Carbaryl-l-naphthyl-l-14C
Carbaryl -carbonyl - C
l-Naphthol-l-14C
Days
0-3
0.2b
0.8
3.7
of incubation
4-7
0.1
0.3
0.6
8-56
0.2
0.7
0.9
Total
0.5
1.9
5.2
Carbaryl
-treated soil
Days of incubation
0-3
2.5
2.9
4.7
4-7
0.5
1.8
0.8
8-56
0.6
2.1
1.1
Total
3.6
6.8
6.6
^     b
Added at 1.5 ppm.


Percent of initial radioactivity.

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TABLE 34.  RADIOACTIVITY OF CULTURE MEDIUM WITH CARBARYL-14Ca  INOCULATED
                WITH SUSPENSION OF MIXED PESTICIDE-TREATED SOIL

With soil inoculum:
Carbaryl
Organosolubles
Water solubles
Unextractables
Total
Uninoculated
Medium Control :
Carbaryl
Organosolubles
Water solubles
Unextractables
Total

12
100. 9b
0
0.5
0
101.4


83.2
0.8
0.6
0
84.6
Hours of
24
88.4
0
1.6
0
90.0


83.2
2.4
0.7
0
86.3
incubation
48
90.1
0.5
4.0
0.6
95.2


86.0
3.7
1.7
0
91.4

96
73.2
2.3
8.8
6.7
91.0


76.8
5.5
9.1
0
91.4
a Carbaryl-14C at 3.3 ppm with 1% glucose.
  Percent of initial radioactivity.
                                  109

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TABLE 35.
RADIOACTIVITY OF MILES MEDIUM WITH CARBARYL-l-14Ca INOCULATED WITH CARBARYL-TREATED SOIL
                SUSPENSIONS OR FUNGAL ISOLATES AND INCUBATED 14 DAYS

Radioactivity as
Inoculum
Medium control
Non- sterile soil
Autoclaved soil
Fusarium sol am'
Fusarium episphaeria
Fusarium rigidiusculum
Penici Ilium lilac inum
Penicillium implicatum
(green)
Penicillium implicatum
(white)
Aspergillus elegans
SF-10
Carbaryl
75. 5b
19.1
70.8
79.0
75.6
74.1
74.4
65.9
79.2
73.4
54. 3C
Water-
solubles
7.6
29.6
8.7
4.7
2.1
5.3
6.7
18.5
10.8
7.4
5.2
Organo-
solubles
6.5
0.4
3.1
5.4
2.0
3.4
5.2
5.7
2.0
3.4
16.1
Unex-
tractables
2.4
23.4
6.5
2.0
1.3
3.0
4.8
2.2
2.0
1.6
14.6
Total
92.0
72.5
89.1
91.1
81.0
85.8
91.1
93.3
94.0
85.8
90.2
a Carbaryl-  C added to medium at 1  ppm in 1% ethanol.
  Percent of initial  radioactivity (average of duplicate experiments).
c Percent of initial  radioactivity (average of three experiments).

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TABLE 36.  RADIOACTIVITY OF MILES MEDIUM WITH  CARBARYL-UCa  INOCULATED


               WITH BACTERIAL ISOLATES AND INCUBATED  FOR  14  DAYS

Radioactivity as

Inoculum

Medium control
Bacillus spha_ericus_
SB-1
SB-2
SB- 3

Carbaryl
h
66. r
79.1
83.2
84.9
78.9
Water-
solubles

13.6
13.9
2.1
4.5
6.6
Organo-
solubles

4.9
6.7
6.0
5.4
3.6

Total

84.6
99.7
91.3
94.8
89.1
a          14
  Carbaryl-  C added to medium at 1  ppm in 1% ethanol.


  Percent of initial radioactivity (average of 2 experiments).
                                 Ill

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TABLE 37.  RADIOACTIVITY OF NUTRIENT BROTH WITH CARBARYL-14Ca, INOCULATED
               WITH BACTERIAL ISOLATES AND INCUBATED FOR 14 DAYS

Radioactivity as
Inoculum
Medium control
Arthrobacter tumescens
Arthrobacter sp.
Nocardia flava
Pseudomonas acidovorans
Xanthomonas sp.
Bacillus sphaericus

Carbaryl
59.6b
53.5
2.4
1.0
0.9
9.1
Water-
soluble
24.4
33.1
83.2
77.0
76.9
59.3
27.1
Organo-
soluble
3.9
5.7
9.2
8.4
1.1
29.7
41.7
Total
87.9
92.3
94.8
85.4
79.0
89.9
77.9
           14
a Carbaryl-  C added to medium at 1 ppm in 1% ethanol.
  Percent of initial radioactivity (average of 2 experiments),
                                 112

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Carbofuran In Houseflies

The development of resistance to insecticides by insect pests has  re-
sulted in the use of greater quantities of toxic chemicals,  the use of
materials that are more toxic to man and, in some cases, a near complete
inability to control certain pests.  There is an ever-increasing need
to more fully evaluate the whole phenomenon of insecticide resistance
so that systematic approaches to developing control  measures for these
insects without increasing environmental  and public  health hazards may
be formulated.  Such evaluation can, and must, include a variety of
investigational formats and emphases.

The insecticidal activity of carbamate insecticides  is attributed largely
to inhibition of the enzyme cholinsterase.  When this enzyme is inhib-
ited with a carbamate insecticide, the reaction is reversible and the
enzyme can be reactivated.  This happens in insects, and if recovery
of the enzyme is too rapid, the insecticidal action  of the carbamate is
negated.  This characteristic of carbamate inhibition of insect
cholinesterase could play a part in the development  of resistance to
these chemicals by the pests.  It is evident that if one population of
insects could restore cholinesterase activity faster than another
population, then their susceptibility to cholinesterase inhibitors
would be less.

By demonstrating a difference in the metabolism and  fate of an insect-
icide in susceptible and resistant populations, mechanisms responsible
for these differences may be proposed and/or definitely defined.  This
then, would provide a basis on which to attack the problem of resis-
tance.  Should differences not be demonstrated, the  need for greater
attention to other areas such as enzyme-inhibitors stability would be
apparent.
                                 113

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Methods  and Materials

Carbofuran  (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methylcarbamate-
carbonyl-  C)  having a specific activity of 2 millicuries per milli-
mole was applied  topically  to  6 day  old houseflies at a dose of 0.04
micrograms per insect.  The susceptible insects were from a laboratory
strain of flies which previously  had not been exposed to any insecticide.
Resistant houseflies were originally taken from the susceptible strain
but had  been selected for resistance over a 2 year period by exposing
pach generation to  increasing  doses of the carbamate insecticide,
Baygon.  LD5Q  values for carbofuran against the susceptible and resis-
tant populations  were 0.1 and  1.3 micrograms per insect, respectively,
at the time these experiments  were initiated.

One-hundred each  of the susceptible and resistant flies were treated
                         14
with carbofuran-carbonyl-   C for each of the time intervals selected
for analysis.  Each experiment was replicated 3 times.  At the appro-
priate interval after treatment, the insects were anesthetized with
carbon dioxide and  the carbofuran remaining on the surface of the
flies was removed by rinsing them with acetone.  Internal radioactivity
was extracted  by  homogenizing  the flies in a 1:1 mixture of acetone and
water.  The radioactivity in the extract was partitioned into organo-
soluble and water-soluble metabolites by the addition of chloroform.
To determine the amount of  carbofuran-  C equivalents excreted by the
insects, the holding vials were rinsed with acetone and the metabolites
partitioned between chloroform and water.

The organo-soluble metabolites and the aglycones recovered from the
water-soluble conjugates after acid hydrolysis were resolved by thin
layer chromatography.   Identification of the metabolites was based on
comparative chromatographic analysis of the unknown materials and
authentic samples of compounds considered  as possible metabolites.   The
details  of these techniques have been reported (Dorough 1968).
                                 114

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The heads of laboratory strains of insecticide-susceptible and carbamate-
resistant flies served as the cholinesterase source for all  studies
unless otherwise stated.  The resistant strain had been selected with
                     D
the carbamate Baygon   for a 2-year period.   LD5Q values for Baygon
against susceptible and resistant flies were 0.7 and 14.0 micrograms per
insect, respectively.

Cholinesterase activity was determined by the colorimetric method of
Ellman (1961).  The inhibitory ability of carbaryl and Baygon was
established by incubating the insecticides with the enzyme in phosphate
buffer, for various periods at 30°C.   The I5Q values were determined
after a steady state had been reached.

For a typical run, the insecticide, in acetone, was added to a 500-ml
Erlenmeyer flask, and the acetone was removed by evaporation.  Then
100 ml of the enzyme preparation, 0.03 fly-heads per ml of phosphate
buffer, was added, and the incubation was initiated.  At designated
times, 3-ml samples were removed, and the level of cholinesterase
inhibition was determined.

To determine the rate of reactivation of carbamate-inhibited cholines-
terase, a concentrated enzyme solution, 48 fly-heads per ml, was incu-
bated with sufficient insecticide to inhibit about 70-80 percent
activity after 30 minutes.  An aliquot was then removed and diluted
800-fold with the phosphate buffer, and the incubation was continued.
This dilution prevented further enzyme inhibition by the insecticide
and allowed the enzyme already inhibited to recover.  The rate of
recovery was determined by assaying 3-ml aliquots of the diluted
solution at 10 min intervals once dilution had taken place.

Results and Discussion

It should be noted that the position of the carbon-14 atom in the

                                 115

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 carbofuran molecule  prevented  detection of  ring-containing hydrolytic
 products.  Upon  hydrolysis,  the  carbon-14 atom would be released as
 carbon-14 dioxide  in the  respiratory  gases.  This may have been
 responsible  for  the  decrease in  the percentage of the dose recovered
 as  the  time  after  treatment  increased.  By  4 hours after treatment, total
 recovery was  approximately 75% of  the dose  while it was in excess of
 90% after only 1 hour.  Other  factors such  as incomplete extraction of
 the residues  from  the  flies  also may  have contributed to the loss.

 The data demonstrated  that carbofuran was absorbed faster by the sus-
 ceptible flies.  This  was evident  by  the levels of carbofuran on the
 surface at each  time of analysis and  by the corresponding levels of
                     14
 carbofuran-carbonyl-  C equivalents within  the insects.  At each inter-
 val, the surface wash  of the resistant flies contained a greater per-
                                        14
 centage of the dose, while the internal   C-residues were always less
 than in the susceptible insects.
Of particular interest was the resistant insect's ability to excrete
the absorbed carbofuran at a more rapid rate than the susceptible ones
Even though more of the dose was absorbed by the susceptible flies, a
greater portion of the dose was eliminated by the resistant flies.  This
enhanced ability to excrete any absorbed insecticide, combined with a
slower absorption rate in the first place, would obviously result in
less toxicant in the body of the resistance flies.  Therefore a re-
duced toxic effect would be expected.

In this study, the two factors just mentioned were the only observations
made which would explain, at least in part, the mechanism of carbofuran
resistance in houseflies.  The metabolism studies did not indicate that
metabolic degradation was an important factor (Table 38).  These data
did, however, show that the reduced internal residues and increased
excreted residues in the resistant flies were primarily a function of
the parent compound, carbofuran, and not of its metabolites.   The
                                  116

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data suggest that the resistant flies can selectively excrete carbofuran
more efficiently than the susceptible flies.

The concentration of either carbaryl or Baygon necessary to inhibit house
fly-head cholinesterase was found to be the same in susceptible and
carbamate-resistant house flies.  Baygon was the more potent cholin-
esterase inhibitor as indicated by a (Irn)cc value of 4 x 10"9M; the
                                      n3U SS
(I50)ss value for carbaryl was 9 x 10  M.  These data demonstrated that
carbamate resistance was not related to a variation in the sensitivity
of the enzyme to the insecticides.

The recovery rates of the susceptible and carbamate-resistant house fly
cholinesterase after being inhibited at about the 80-percent level
with Baygon and carbaryl were identical.  Twenty-six minutes were re-
quired for 50 percent of the inhibited enzyme to be reactivated.  Thus,
our hypothesis that carbamate resistance could result, in part, from
an enhanced ability of these insects to reactivate inhibited cholines-
terase was not supported by the experimental data.

In order to prove that the techniques utilized were capable of showing
differences in reactivation rates of carbamate-inhibited cholinesterases
if such did occur, the enzyme from two other sources was subjected to
the same tests as described above.  Commercial bovine acetylcholines-
terase inhibited with carbaryl and Baygon recovered at the same rate as
the house fly-head cholinesterase.  However, a 10-fold greater
concentration of the insecticide was required to produce a (150)55 with
the bovine acetylcholinesterase.  Cholinesterase from boll weevils
show a different rate of reactivation than the enzyme from other
sources.  Once inhibited, 99 minutes was required before 50 percent of
the enzyme was reactivated.  Therefore, these experiments did show that
differences could be detected if they occurred and also that rates of
reactivation of carbamate-inhibited cholinesterase do vary in different
insect species.  The (I50)ss values for carbaryl and Baygon against

                                 117

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boll weevil cholinesterase were essentially the same as for the house
fly-head cholinesterase.

During the above investigations, a definite trend toward higher enzyme
levels in the resistant flies was noted.  Reviewing all assays, which
included over 50 individual runs, it was found that the enzyme level of
resistant flies was about 10 percent higher in 85 percent of the assays.
As a result of these observations, experiments were conducted whereby
the cholinesterase levels in the two strains of flies were compared
directly.
                                             3
The susceptible fly-heads hydrolyzed 8.6 x 10  micrograms of substrate,
acetylcholine, per minute per gram of weight.  Approximately 34 per-
cent greater cholinesterase activity was obtained with the resistant
fly-heads which hydrolyzed 11.5 x 10  micrograms of substrate per
minute per gram.  This degree of difference in enzyme activity was not
apparent in earlier tests because the enzyme concentration was expressed
on a fly-head per ml basis rather than on a weight basis.  It was later
found that the susceptible fly-heads weighed about 12 percent more
than the resistant ones although the total  body weight of flies from
the two strains was the same.
                                 118

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TABLE 38.   NATURE AND MAGNITUDE  OF  CARBOFURAN  AND  ITS  METABOLITES  IN  THE  BODY AND  EXCRETA  OF  SUSCEPT-
        IBLE (S)  AND CARBAMATE RESISTANT  (R) HOUSEFLIES  FOLLOWING  TOPICAL APPLICATION  OF THE  INSECTICIDE

Percent of applied dose at indicated hours
Body
Metabolites
Organo-extractabl es
Carbofuran
3-OH-Carbofuran
3-OH-NCH2OH-Carbofuran
3-Keto-Carbofuran
Unknown Ia
Total
Water-solubles
3-OH-Carbofuran
3-OH-NCH20H-Carbofuran
NCH20H-Carbofuran
3-keto-Carbofuran
Unknown I
Unknown II
Total

S

9.3
5.4
0.9
4.3
0.2
20.1

10.0
1.3
0.9
0.8
0.4
1.7
16.0
1
R

5.5
1.9
0.7
2.3
0.1
10.5

7.9
0.3
0.9
0.3
0.4
1.9
11.7

S

6.4
1.9
0.3
0.9
0.2
9.7

10.4
0.8
1.5
0.4
1.0
4.2
18.3
4
R

4.4
1.3
0.4
0.5
0.1
6.7

6.5
0.4
1.2
0.3
0.4
2.6
11.4

S

5.3
0.1
0
0
0.1
5.5

1.1
0
0
0
0.1
0.2
1.4
Excreted
1
R

8.3
0.2
0
0
0.1
8.7

2.0
0.1
0.4
0
0.2
0.6
3.3

S

6.2
1.6
0.5
0
0.1
8.4

0.2
0.5
1.5
0.2
1.3
4.6
8.3
4
R

12.7
1.0
0.9
0
0.1
14.7

0.2
0.6
1.9
0.2
1.0
4.2
8.1
  Radioactivity remaining  at the  origin after  development of  the  tic.
  Metabolites in the aqueous layer following acid  hydrolysis  and  extraction with chloroform.

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FATE OF CARBAMATE METABOLITES

1-Naphthyl Glucoside  In  Rats

Based upon persistence and potential harmful effects from chronic expo-
sure, the conjugate metabolites of the carbamate insecticides may prove
to be quite significant  in animals.  To date, little information exists
on the fate of these  conjugates in various species and nothing is
known of their chronic toxicity.  One of the principal reasons that so
little information is available is that the compound requires unique
methods of isolation, identification and synthesis.  Unkike the apolar
insecticide metabolites, the conjugated materials can not be extracted
into an organic solvent  and subjected to conventional tic, glc, etc.

This study was conducted to develop techniques basic to the study of
the chemistry and metabolism of conjugate metabolites.  1-Naphthol was
selected as a model compound, although it is also a metabolite of the
insecticide carbaryl.

Methods

Chemical  synthesis of 1-naphthyl glucoside - The glucoside of 1-naphthol
was prepared by reacting glucose pentaacetate with 1-naphthol to form
l-naphthyl-tetra-O-acetyl-3-D-glucopyranoside.   Details of the synthe-
sis procedures were as follows:

Glucose pentaacetate  (3.9 g, 0.01  mol) was mixed with 4.3 g (0.03 mol)
of 1-naphthol  and 0.2 g of p-toluenesulfonic acid.   The mixture was
heated, under vacuum, for 1 hr in an oil  bath at 100°C.  Following
cooling to room temperature, the melt was dissolved in 100 ml of
benzene and extracted twice with 20 ml-portions of 2% sodium hydroxide.
The benzene was dried with anhydrous sodium sulfate and then concen-
trated to dryness.  Crystallization from ethanol  and water gave 4.5 g

                                 120

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of the 1-naphthyl-tetra-O-acetyl-3-D-glucopyranoside, mp 177-178° (Lit.
178-179°).  Deacetylation was accomplished by passing dry ammonia gas
through a methanol solution of the product (2.0 g/10 ml) for 1  hr at
0° and then holding the solution at 5° for an additional 3 hrs.  The
methanol was evaporated and the residue crystallized from a mixture of
ether and ethanol.  l-Naphthyl-3-D-glucopyranoside,  1.2 g, was  recovered
as a white powder, mp 171-175°.  The mass spectrum (Finnigan Model
1015C) contained a weak molecular ion at m/e 306 while the base peak
occurred at m/e 144 due to 1-naphthol.  Peaks at m/e 116 and 115 re-
presented the base peak minus CO and CHO, respectively.  The sugar
moiety was evidenced by peaks at m/e 163 (CgH^Og +) and 162 (CgH1Q05  + ).

Biosynthesis of radioactive 1-naphthyl glucoside and glucurom'de - For
                              __i
the synthesis of 1-naphthyl-1-  C-B-D-glucopyranoside, an in vitro
glucosylation system using house fly enzymes was employed (Mehendale
and Dorough, 1972).  Each incubation mixture consisted of 0.5 uCi of
l-naphthol-l-14C (specific activity 19.6 mCi/mmol),  2.4 ml of Tris-HCl
buffer (pH 7.2), 1 ml of 0.1 M magnesium chloride, 0.5 ml of 5 mg/ml
solution of UDPG dissolved in buffer, and 1 ml (100 mg tissue equiva-
lents) of a 9,000 g supernatant.of a house fly homogenate. Incubations
were for 15 min at 37°C.

The incubation mixture was extracted thoroughly with ether to remove
the unreacted 1-naphthol and the water layer was concentrated to a
volume suitable for application to thin layer chromatograms.  After
development, the 1-naphthyl-l-  C glucoside was detected on the
chromatograms by radioautography and the compound recovered by extract-
ion of the gel with methanol.  Approximately 75% of the radioactive
1-naphthol was converted to the glycoside.

l-Naphthyl-3-D-glucopyranoside-  C also was synthesized enzymatically
using the basic procedure just described.  However,  an excess of 1-
naphthol, 1 mg/flask, was used to achieve maximum reaction with the
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 1  uCi  of  undine diphosphate glucose  [D-glucose-  C  (U), sp. act. 227
 mCi/mmole]  added to each  incubation  mixture.  Under these conditions,
                                                             14
 35% of the  radiocarbon was  recovered  as  1-naphthyl glucoside-  C.
Radioactive  1-naphthyl  glucuronide was prepared enzymatically using rat
liver  homogenates as the enzyme source and UDPGA as the co-factor
(Mehendale and Dorough, 1971).  The 1-naphthol-  C used in these exper-
iments was the same as  that used to prepare the glucoside.  1-Naphthyl
             14
glucuronide-  C was synthesized from uridine diphosphate glucuronic
acid [D-glucuronic-  C  (U)] having a specific activity of 238 mCi/mmol.

Acetylation of biosynthesized 1-naphthyl glucoside - 1-Naphthyl-  C
glucoside was synthesized using house fly enzymes as described earlier
                             14
and the unreacted 1-naphthol-  C removed by extracting the incubation
mixture with ether.  The water phase, containing the radioactive
naphthyl glucoside, was concentrated to dryness on a rotary evaporator.
The dried residue was dissolved in 1 ml of acetic anhydride and cooled
to 0°C in an ice bath.  Two drops of 70% perchloric acid were added and
the solution allowed to return to room temperature slowly over a period
of 2 hr.  At this stage, the solution was held overnight and then trans-
ferred to a separatory funnel containing 4 ml of cold water.   The
water was extracted twice with 4 mi-portions of ether which removed
85% of the radioactivity, presumably the acetylated naphthyl  glucoside.
Its identity was confirmed by cochromatography on tic with the chem-
ically synthesized material.
Deacetylation of the product with ammonia converted 93% of the radio-
activity to a water soluble material.  Tic analysis demonstrated that
this material was identical to an authentic sample of naphthyl glucoside.

Chromatography - 1-Naphthol was purified on silica gel F-254 chromato-
plates (0.25 mm, EM Lab, Elmsford, N. Y.) using a 7:3 mixture of
chloroform and acetone as the developing solvent.  In addition to
                                 122

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Providing a means of purification, this system afforded the separation
of 1-naphthol; 1 ,3-naphthalenediol ; 1 ,4-naphthalenediol ; 1 ,5-naphthalene-
diol and 1 ,6-naphthalenediol .   These compounds constituted  the more likely
free metabolites of 1-naphthol resulting from treating rats with 1-
naphthyl glucoside.  Other solvent systems used to establish co-
chroma tography of an unknown with one of these metabolite standards
were:  petroleum either-ethyl  ether 2:1; methylene chloride-ethyl  acetate
2:1; hexane-acetone 7:3.

Chroma tographic isolation of acetylated naphthyl glucoside  (1-naphthyl-
tetra-0-acetyl-beta-O-glucopyranoside) was accomplished on  silica  gel
chromatoplates developed in 10:1 petroleum ether-acetone.  The acetylated
naphthyl glucoside had an Rf of approximately 0.6 while the naphthyl
glucoside remained at the origin.  Changing the solvent to  8:2:1 chloro-
form, methanol and acetic acid moved the acetylated naphthyl glucoside
to the solvent front but resulted in an R^ of 0.6 for the naphthyl
glucoside.  Naphthol glucuronide had an R^ of 0.4 in this system while
UDPG and UDPGA remained at the origin.  This allowed the separation of
the   C-labeled biosyn
radioactive cofactors.
the   C-labeled biosynthesized glycosides from their corresponding
Non radioactive 1-naphthol and its hydroxylated analogs were detected
on the tic plates by viewing under ultraviolet light.  To locate the
glycosides, the chromatograms were sprayed with concentrated sulfuric
acid and heated at 110°C for 5 min.  Radioautography (Kodak no-screen
medicat x-ray film) was used to detect radioactive areas on the gel.
Quantitative radioassay were conducted by liquid scintillation counting.

Stability of 1-naphthyl glucoside and glucuronide -  The stabilities
of 1-naphthyl-  C glucoside and 1-naphthyl-  C glucuronide were eval-
uated under various conditions commonly used for evaluating the
chemical and/or biological fate of insecticidal compounds.  Each of
the radioactive glycosides were purified on tic prior to use in these
                                 123

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experiments.   The  situations  under which  the glycosides were held for
stability  determination were:   (1) in citrate-phosphate buffer, pH
ranging  from  2.2 to  8.0,  for  up  to 6 hr at room temperature.   (2)  In
Tris-HCl buffer, pH  7.0,  at temperatures  of 25, 0 and -20°C for up to
1 week and at 90°C for 30 min.   (3) In methanol at 0.25 and 65°C for
12  hrs and at -20°C  for 5 days.   (4) In methanol and refluxed  for
30  min.  At the designated time,  the incubation mixtures were  con-
centrated  for tic  analysis.

Enzyme studies - The ability  of  $-glucosidase and 3-glucuronidase
(Sigma Chemical Corp. St. Louis,  Mo.) to  hydrolyze naphthyl glucoside
and glucuronide was  determined.   The naphthyl-  C glycosides were
incubated  separately with each of the enzymes in citrate-phosphate
buffer,  pH 7.0.  The incubation mixtures  containing approximately
1.0 x 10   dpm of the glycoside and 4 ml of buffer, were preincubated,
with shaking,  for  10 min  at 37°C  and then 0.4 mg of enzyme in  0.2 ml of
buffer were added.   The same  amount of enzyme was added 4 more times
at  6 min intervals with the total incubation time being 30 min.  Each
reaction mixture was extracted twice with 5 ml  portions of ether and
both the aqeuous and organic solvent phases were radioassayed.   The
quantity of radiocarbon in the ether was  indicative of the efficiency
of  the enzyme  to cleave the glycoside,  thus yielding radioactive
1-naphthol.

To  determine if whole rat urine affected the enzymatic cleavage of
naphthyl  glucoside or naphthyl glucuronide, the reactions described
above were run in the presence of 0.5 ml  of urine.   In this case,
however,  the initial  buffer volume was  increased to 5 ml  and the pH ad-
justed to 7.0  if required after adding  the urine.   In addition, each of
the glycosides were treated in succession with  the  2 enzymes,  first with
glucosidase and then with glucuronidase and vice versa.   This was  done
to determine if the sequential treatment of a  single urine sample  with
the enzyme would produce  results comparable to  treatment  of the sample
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with either of the enzymes alone.

Fate of 1-naphthyl glucoside in rats - Female rats weighing approximately
250 g were treated orally with 1-naphthol-  C in corn oil  or with
water solutions of carbon-14 1-naphthyl glucoside.  For the latter
studies, the 1-naphthyl glucoside was labeled in 2 different positions.
One batch was labeled on the naphthyl ring while the other batch was
labeled on the sugar moiety.  The animals were held in metabolism
cages and the urine collected for 24 hr and the nature of the radio-
carbon determined.

Results and Discussion

Stability of 1-naphthyl glucoside and glucuronide - Both naphthyl-  C
glucoside and glucuronide were stable when held in citrate-phosphate
buffer at pH values ranging from 2.2 to 8.0 for 6 hr and at a pH of
7.0 when heated in a water bath at 90°C for 30 min.  The same was true
when the compounds were dissolved in methanol and stored at -20, 0 and
25°C for 5 days, or when the solutions were refluxed for 30 min.
Storage at -20, 0 and 25°C in Tris-HCl buffer for 1 week did not degrade
the naphthyl glucoside.  The glucuronide derivatives were similarly
stable at the two lower temperatures but 30% degradation did occur at
25°C.  At the latter temperature, the radiocarbon not associated with
glucuronide was identified by tic analysis as 1-naphthol.

The data indicate that no particular problems would be encountered in-
sofar as stability is concerned during extraction of the compounds from
biological media.  Their stability in methanol is important since this
is an excellent solvent for extracting polar insecticide metabolites
from plant and animal tissues.  Enzyme studies, such as the cleavage
of the conjugates to form the aglycones, must be conducted using the
appropriate control if specificity of the enzyme is to be considered.
Since some cleavage of the naphthyl glucuronide did occur when held in
                                 125

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Tris-HCl buffer, pH 7.0, at 25°C, non-enzymatic cleavage must be con-
sidered a possibility at incubation temperatures above room temperature,
especially  if the  incubation time is of long duration.  The citrate-
phosphate buffer may be the preferred buffer in such incubation since
both glycosides were stable when held in this buffer for 6 hr at 25°C.

Enzyme studies - Incubation of 1-naphthyl-  C glucoside with 3-glucosidase
for 30 min  at 37°C cleaved 91% of the conjugate.  3-Glucuronidase treat-
ment yielded only  3% cleavage which was comparable to a control incu-
bation containing  all constituents except the enzyme.  1-Naphthyl
glucuronide was hydrolyzed in excess of 95% by 3-glucuronidase and 7%
by 3-glucosidase.  The latter enzyme did not cleave the conjugate to
any greater degree than that observed during incubation with no enzyme.

Whole rat urine, 0.5 ml, added to the enzyme preparation did not de-
crease the amount of substrate hydrolyzed, nor did it alter the
specificity of the enzymes.  Also, 3-glucuronidase in the incubation
mixture did not affect the action of B-glucosidase on 1-naphthyl
glucoside.  The same was true when the situation was reversed.

From this study, it appears that 3-glucosidase and 3-glucuronidase
treatment of a biological  extract could be used as a convenient tool
for determining the ability of an organism to metabolize a glucoside
to a glucuronide.  For example, a rat fed 1-naphthyl glucoside might
be expected to convert the material  to a glucuronide and excrete the
compound in the urine.   Treatment of the urine with 3-glucosidase,
followed by 3-glucuronidase, would allow one to rapidly establish the
quantity of the dose eliminated or the administered compound and the
quantity metabolized to form the glucuronide.   This would be an
immediate indication of the animal's ability to attack, biochemically,
plant-derived glucosides of the chemical in question if consumed in
the diet.   Identification  of the resulting aglycones would give
virtual  proof of the intact conjugates as they exist in the plant and
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animal system.

                               14
Fate in rats - When 1-naphthyl-  C glucoside was administered as a single
oral dose to rats, 67% of the dose was eliminated in the urine after
24 hrs (Table 39).  Approximately 90% of a dose of 1-naphthol-14C was
eliminated during the same period.

A rather surprising aspect of the study with 1-naphthyl-  C glucoside
was that 19% of the dose was eliminated as the administered compound.
Thus, this glucoside metabolite did withstand the acid conditions of
the stomach and the biochemical mechanism of degradation in the body.
That the sugar moiety was the same as administered (as opposed to
cleavage of the conjugate to yield 1-naphthol, followed by re-conjugation
as a glucoside in the animal) was confirmed in 2 ways.  First, 1-naphthol-
14
  C treatment yielded only trace amounts of radioactivity in the urine
which corresponded to 1-naphthyl glucoside.  Secondly, 16% of a dose
                        14
of 1-naphthyl glucoside-  C was in the 0-24 hr urine as the administ-
ered compound.  These data clearly showed that the rat does not form
glucosides, at least to any appreciable extent, and that the 1-
naphthyl  glucoside in the urine represented the administered compound.

Cleavage of the 1-naphthyl glucoside in the rat to yield 1-naphthol
was a major metabolic pathway.  About 10% of the dose was in the
urine as 1-naphthol, 24% as 1-naphthyl glucuronide and 10% as 1-
naphthyl  sulfate.  The relative concentrations of these metabolites
                                                         14
were quite different than in the urine of the 1-naphthol-  C treated
rats.  Only 1% of the dose was as the free 1-naphthol, 73% as the
glucuronide and 15% as the sulfate.

                                            14
Treatment of rats with 1-naphthyl glucoside-  C confirmed that the
1-naphthyl glucuronide in the urine of rats administered the glucoside
did not result from the oxidation of the glucoside.  Only a small amount,
1% of the dose, of radioactivity corresponded to 1-naphthyl glucuronide,
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a level so small that confirmation of its identity was not possible.
This demonstrates that the glucoside was first cleaved to yield 1-
naphthol, which was then conjugated as a glucuronide in the animal
system.

This study with 1-naphthyl glucoside does not represent a metabolite
of high potential for significance.  However, the study has allowed us
to develop techniques that will assist us in studying more potentially
significant metabolites, such as glucosides of metabolites of carbaryl
containing the carbamate moiety.  More important, the study did estab-
lish that there is a definite need to evaluate the significance of the
conjugates, per se, rather than relying entirely on information ob-
tained with the corresponding aglycone.
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TABLE 39.   NATURE OF RESIDUES IN THE 0-24 HOUR URINE OF RATS TREATED ORALLY
                    WITH 1-NAPHTHOL AND 1-NAPHTHYL GLUCOSIDE

                                       % of dose when treated with
                                                 1-Naphthyl  glucoside
Metabolites	l-Naphthol-l-14C     Naphthy1-14C   Glucoside-14C
1-Naphthyl Glucoside           0.1                18.7           15.9
1-Naphthyl Glucuronide        73.2               23.6            1.1
1-Naphthyl Sulfate            14.9               10.3            0
1-Naphthol                     1.4                9.5            0
Unknown (tic origin)           0.7                5.2            3.1
TOTAL                         90.3               67.3           20.1
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3-H.ydroxy  Carbofuran and  its  Glucuronide and Glucoside  in Rats

Many investigators  have demonstrated that 3-hydroxy carbofuran is a
major metabolite of carbofuran  in most biological systems.  In mammals,
the 3-hydroxy carbofuran  is further metabolized by conjugation to the
corresponding glucuronide.  In  plants, a glucoside is formed.  These 3
metabolites constitute a  large  proportion of the terminal residues in
the environment resulting from  the introduction of the  pesticide carbo-
furan.  Because the 3-hydroxy analog of carbofuran is highly biologically
active, it is important to know the fate of this metabolite in animals
and plants.

Methods and Materials

Chemicals - Carbofuran, 3-hydroxy carbofuran and carbofuran-carbonyl-  C
(17 uCi/mg) were supplied by  Niagara Chemical Division, FMC Corp.,
Middleport, N. Y.   Each of the  chemicals was chemically pure as eviden-
ced by the existence of only  one spot on thin layer chromatography.
TPNH and UDPG were  purchased  from Sigma Chemical Company, St. Louis,
Missouri.

Metabolism by rat liver enzymes - A 50% rat liver homogenate (w/v) in
0.05 M Tris-HCl  buffer (pH 7.0) was prepared and separated into two
fractions.   One fraction was  prepared by centrifuging the homogenate
at 15,000g for 30 minutes; the  supernatant was used as the enzyme
source for the oxidative metabolism of carbofuran.  The other fraction
was prepared by centrifuging  the 15,000g supernatant at 105,000g for
1  hour.  The particulate fraction (microsomes) was used as the enzyme
source for conjugation of 3-hydroxy carbofuran after dispersal  in a
volume of Tris-HCl buffer that made the suspension equivalent to the
original  50% homogenate.

The 3-hydroxy carbofuran-carbonyl-  C was produced from the oxidation
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                                14
in vitro of carbofuran-carbonyl-  C.  In a typical incubation, 1 ml of
the enzyme source and 2 ml of the pH 7.4 Tris-HCl buffer containing 4
umoles of TPNH were added to a 25 ml Erlenmeyer flask containing carbo-
furan-carbonyl-  C.  After incubation at 37°C in a water bath shaken
for 30 minutes, the contents of each flask were extracted three times
with 5 ml portions of ether, and the ether extracts were combined and
dried with anhydrous sodium sulfate.  Aliquots from both the water
and organic solvent phases were radioassayed using liquid scintillation
technique.  Ether extracts were evaporated to about 0.2 ml and spotted
on tic plates (0.25 mm thick).  The plates were developed in a 3 to 1
ether-hexane mixture, exposed to x-ray film for 3 days, and the areas
of the gel corresponding to darkened areas on the radioautogram were
extracted into ether from the gel.

To determine the maximum quantity of carbofuran metabolites produced, the
15,000g soluble fraction was incubated with concentrations of carbofuran
                                                               14
ranging from 20 to 1000 ug.  The 3-hydroxy carbofuran-carbonyl-  C was
isolated from the tic and identified by cochromatography.
The glucuronide and glucoside conjugation of 3-hydroxy carbofuran were
investigated using the liver microsomes and housefly homogenates.  For
glucuronide conjugation, the incubation mixture contained 3-hydroxy
carbofuran-carbonyl-  C, MgCl2 (100 umoles, in 1 ml Tris-HCl buffer of
pH 7.0); UDPGA (2 umoles, in 1 ml Tris-HCl buffer of pH 7.0) and liver
microsomes (equivalent to 250 mg liver).  The incubation was carried out
aerobically at 37°C for 20 minutes.  Free metabolites were extracted
into ethyl ether and the percent radioactivity remained in the water
phase served as a measure of the formation of 3-OH-carbofuran-carbonyl-
  C glucuronide.  To investigate the effects of substrate level and
incubation period on conjugation, various concentrations of 3-OH
carbofuran (0.1 to 1.1 umole) as well as three different incubation
times were employed.  The same parameters and method was used for
glucoside conjugation of 3-OH carbofuran-carbonyl-  C except that a
                                  131

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housefly homogenate  (15,000g soluble, equivalent to 250 mg wet weight)
and UDPG (50 mg of 1 ml tris-HCl buffer, pH 7.0) were used in place of
the liver microsomes and UDPGA.

To determine if the rat liver enzymes could alter carbofuran  glucuronide,
the material was incubated in the presence of the enzyme source, TPNH
and UDPGA.  After incubation, the organosoluble materials formed, if any,
were extracted into ethyl ether.  The ether soluble radioactivity was
condensed to 0.2 ml and applied to a silica gel G tic and developed in
a 3 to 1 ether-hexane mixture.  The water soluble radioactivity was
applied to a silica gel G tic and developed in 3:1 chloroform-methanol
 containing 10% acetic acid.

Fate in rats - Female Sprague-Dawley rats, weighing approximately 200 gm,
were given oral doses of 3-hydroxy carbofuran-carbonyl-  C in corn oil
or 3-OH carbofuran-carbonyl-  C glucuronide or glucoside in water.  The
urine, feces and   C02 were collected for up to 144 hours after the
single doses.  The feces were dried at 50°C and ground and a 100-mg
aliquot collected at each period was combusted in a Beckman Biological
                       14
Material Oxidizer; the   C09 produced was trapped in a solution of
                                                 1/1
methyl cellusolve and 2-aminoethanol (2:1).  The   (XL produced from
respiration was collected from the animals using the same trap solution.

The metabolites in the urine collected within 32 hours of treatment were
also analyzed.   The urine was extracted 4 times with a 3 to 1 mixture
of chloroform-acetonitrile.  The organosoluble extracts and the water
layer were concentrated and analyzed by tic as described earlier.

Results and Discussion

The optimal concentration of carbofuran-carbonyl-  C required for the
formation of 3-hydroxy-carbofuran-carbonyl-  C by the rat liver homo-
genate was 50 to 100 micrograms (Table 40).

                                  132

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The formation of glucuronide and glucoside of 3-hydroxy carbofuran-
carbonyl-  C by the liver microsome preparation (105,000 x g) and house-
fly hotnogenate (15,000 x g) did not exceed about 20% regardless of the
parameters (Tables 41, 42).
When the enzymatically synthesized glucuronide conjugate of 3-hydroxy
carbofuran was subjected to enzyme hydrolysis with 3-glucuronidase at
37°C, 85% of the radioactivity was recovered as 3-hydroxy carbofuran.

Metabolism of 3-hydroxy-carbofuran-carbonyl- 4C and glycosides in rats -
It is generally thought that a compound with a polar group or groups is
metabolized and excreted faster from animals than less polar compounds.
The results of this investigation were in agreement with the principle.
When the glucuronide of 3-hydroxy-carbofuran was administered orally to
rats, 80% of the dose was excreted in the urine within 48 hours, 16% in
                    14                                         Id
the feces and 4% as   CO,.  With 3-hydroxy carbofuran-carbonyl-  C, 34%
                                                         1 A
of the dose was in the urine, 28% in the feces and 4% as   C02 by the
end of 48 hours after the treatment.  The glucuronide conjugate of
3-hydroxy-carbofuran was also excreted faster than its aglycone.  After
48 hrs, the urine contained 42% of the dose, the feces 18% and about
4% was as 14C02-

The organo-extractable and the water soluble metabolites from the
urines collected 32 hours after treatment were analyzed using thin layer
chromatography.  Water soluble metabolites were the most portion of
the radioactivity in the urine (Table 43).  The percentage as water
solubles was 61% in urine from rats receiving 3-hydroxy-carbofuran and
82% in the urine of rats receiving the glucuronide and 91% in the urine
of rats treated with the glucoside.  In the chloroform extract, only one
radiolabeled metabolite was detected and this was identified as 3-hydroxy-
carbofuran.

Metabolism of 3-hydroxy-carbofuran-carbonyl-  C in bean plants - Bean
                                  133

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leaves immersed in an acetone-water (1:20) solution containing 3-hydroxy-
                    14
carbofuran-carbonyl-  C  for 6 days were extracted and partitioned as
described for the rat urine.  Thirty-five percent of the radioactivity in
the leaves, were extracted into the chloroform-acetonitrile and consisted
of 3-hydroxy-carbofuran.  Of the 65% radiocarbon in the water, only one
metabolite was detected on the thin layer chromatograms.  This spot
occupied a higher position on the thin layer chromatogram than the
glucuronide of 3-hydroxy-carbofuran and was tentatively identified as
                              14
3-hydroxy carbofuran-carbonyl-  C glucoside.  Upon enzymatic hydrolysis
with glucosidase, 3-hydroxy-carbofuran-carbonyl-  C was recovered in
the ether extract.
                                 134

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TABLE 40.  INFLUENCE OF VARIOUS LEVELS OF CARBOFURAN ON THE METABOLISM OF
                   CARBOFURAN-CARBONYL-14C BY THE 15,000g
                        SOLUBLE FRACTION OF RAT LIVER

Micrograms
of
carbofuran
20
50
70
100
200
300
500
700
1,000
Percent
3-OH
carbofuran
41.3
42.8
42.8
43.0
35.2
38.2
37.4
28.9
27.8
of added radioactivity as metabolites
3-keto
carbofurafT
1.6
2.1
2.1
2.8
2.5
1.4
1.5
1.0
2.1
carbofuran
37.2
36.0
41.0
42.4
51.2
40.6
46.3
59.6
61.8
Water
solubles
19.8
16.1
14.1
11.2
11.1
13.6
10.8
10.4
8.1
                                  135

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TABLE 41.   EFFECT OF SUBSTRATE CONCENTRATIONS ON GLUCURONIDATION OF
                      3-HYDROXY-CARBOFURAN-CARBONYL-14C
                    BY THE 105,000g FRACTION OF RAT LIVER

   Micrograms
3-OH carbofuran                           Percent glucuronidation
Background9
23
70
117
165
210
260
4.6
20.7
18.0
16.3
14.7
14.4
14.3
  Without microsomes.
                                  136

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TABLE 42.  EFFECT OF VARIOUS INCUBATION PERIODS ON THE GLUCURONIDE AND
           6LUCOSIDE CONJUGATION OF 3-HYDROXY-CARBOFURAN-CARBONYL-14C
                BY A RAT LIVER (15,000g SOLUBLE) AND A HOUSEFLY
                            HOMOGENATE (15,000g SOLUBLE)
Incubation           	% 3-QH-carbofuran converted to
period, nrin.	Glucuronide	Glucoside
    20                       21.5                         6.8
    40                       18.8                        19.4
    60                       18.9                        14.3
                                 137

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TABLE 43.  PARTITIONING CHARACTERISTICS OF METABOLITES IN THE 0-32 HOUR

           URINE OF RATS TREATED ORALLY WITH 3-OH-CARBOFURAN OR ITS

                      GLUCURONIDE OR GLUCOSIDE DERIVATIVES3
                    	Percent of radioactivity in sample as

Treatment	Organosolubles	Watersolubles
3-OH-carbofuran
3-OH-carbofuran
glucuronide
3-OH-carbofuran
glucoside
38.6
18.2
9.5
61.4
81.8
90.5
  Approximately 60,000 dpm of each material administered to the rats.
  The glucuronide was biosynthesized using rat liver microsomes and
  the glucoside isolated from 3-OH-carbofuran-treated bean plants.
                                 138

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INTERACTIONS

Effect of Aldicarb on Methyl Parathion Toxicity to Mice
                                       •  '!'•
Aldicarb, a carbamate insecticide, showed only additive toxic effects
to mice when administered along with methyl parathion, an organo-
phosphorus insecticide.   The oxime hydrolytic product of aldicarb,  which
is nontoxic, did not alter the toxicity of methyl  parathion in any  way,
nor was the protective action of the antidotes, atropine and 2-PAM, in-
fluenced by the addition of the oxime derivative of aldicarb.

Atropine and 2-PAM (2-pyridine aldoxime methiodide) are the two antidotes
commonly employed in the treatment of poisoning by anti-cholinesterase
agents such as methyl parathion (Hayes, 1963).  2-PAM is one of the
several oximes which have the unique capability of promoting the re-
lease of enzymes bound by the toxicants.   Hayes reports that in most
cases of parathion poisoning in man a single dose of 2-PAM was suffi-
cent to produce dramatic improvement within 30 minutes.

Aldicarb [2-methyl-2-(methylthio)propionaldehyde 0-(methylcarbamoyl)
oxime] is a carbamate insecticide and a very potent cholinesterase
inhibitor.  As the chemical name shows, it also is an oxime derivative,
making it somewhat similar in chemical configuration to the antidote.
2-PAM.  While aldicarb obviously does not act as an antidote against
itself, the possibility exists that it could antagonize the toxic
effect of other cholinesterase inhibitors.  The idea is made even
more intriguing by the fact that the hydrolysis of aldicarb yields
aldicarb oxime [2-methyl-2-(methylthio) propionaldehyde-oxime].  This
is a non-toxic oxime derivative and is formed from aldicarb in mammalian
systems (Andrawes et al., 1967).  The similarity of aldicarb, and
especially aldicarb oxime, to the chemical configuration of 2-PAM
provided the stimulus for the study reported herein.
                                  139

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Methods

Female Yale  Swiss mice  approximately  4 weeks old and weighing from 20 to
25  grams were  used  in all  tests.  The animals were  housed under continuous
light at a temperature  of  78°F  prior  to and during  the toxicity deter-
minations.

For both oral  treatment and  interperitoneal, I.P.,  injections of the
compounds into the  mice, a total  volume of 0.1 ml was administered.  Re-
fined cotton seed oil served as the carrier for methyl parathion, aldicarb
and aldicarb oxime.  Atropine sulfate and 2-PAM were dissolved in water
for administration.  In all experiments, the mice were treated first with
methyl parathion and then  20 minutes  later with the other compound(s).
The percentage mortality of the mice  was recorded 24 hours after treat-
ment.  Ten animals  were treated at each dosage level, and all experi-
ments were replicated 3 times.

Results and  Discussion

In  order to  obtain  a baseline from which to work, it was necessary to
determine the  toxicity  of  all compounds used in the study to the mice
when administered alone.   It was  found that aldicarb oxime and 2-PAM
given at a dose of  25 mg/kg and atropine at a dose of 50 mg/kg did
not cause any  ill effects  to the  mice.  This was true for both oral
and  I.P. treatments.

Methyl parathion and aldicarb were toxic to the mice and the dose of
each required  to kill 50%  of the  animals, LD50, was determined to be
as  follows:
                             	LD50	
                                   Oral                I.P.
Methyl parathion                 40 mg/kg           10 mg/kg
Aldicarb                        1.5 mg/kg          0.3 mg/kg
                                  140

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These data were used as the basis for expressing the effects of the
various compounds on the toxicity of methyl parathion to the mice.
For example, the data in Table 44 shows that the effect of aldicarb on
the toxicity of methyl parathion was "additive".  This means that
treatments of the mice with one-half the LD5Q dose of methyl parathion and
one-half the ID™ dose of aldicarb gave approximately 50% mortality.
If less than 50% mortality occurred, the situation was referred to  as
"antagonistic" and if more than 50 percent mortality occurred, it was
referred to as "synergistic" action.  The latter case was not observed
with any of the combinations used in these studies.  An effect listed
as "none" in Table 44 means that the observed toxicity to the mice  was
at the level expected from one-half the LD5Q dose of methyl  parathion.

Whereas the carbamate insecticide, aldicarb, had an additive effect on
the toxicity of methyl parathion, aldicarb oxime did not in any way in-
fluence the toxicity of the organophosphorus material.  It was evident
at this point that neither aldicarb nor its oxime possessed the antidotal
properties of the oxime, 2-PAM.  There remained, however, the possibility
that aldicarb oxime might enhance the antidotal action of 2-PAM and
atropine.

When methyl parathion and 2-PAM were administered by I.P. injection,
there was an 8-fold antagonistic action observed.  In other words , 8
times the LDrn dose of methyl parathion was required to kill 50% of the
animals pre-treated with 2-PAM.  There was only a slight antagonistic
effect, less than 2 fold, when methyl parathion was administered
orally and 2-PAM administered either orally or by I.P. injection.
This same low level of protection was provided by 2-PAM containing
aldicarb oxime, suggesting that aldicarb oxime did not act to enhance
the action of 2-PAM.  Atropine alone gave only a 5-fold antagonistic
action against the toxic effects of methyl parathion.  Aldicarb oxime
did not increase its capability as an antidote.  Results of a test
not included in Table 44 showed that 2-PAM did not affect the
                                  141

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toxicity of aldicarb to the mice.

The most efficient antagonist of methyl parathion toxicity to mice was
a mixture of atropine and 2-PAM.  From 80 to TOO times the ID™ dose
of methyl parathion was required to kill 50% of the mice receiving pre-
treatments of this mixture.  It was clear that atropine and 2-PAM did
interact to provide greater protection to the mice than when either
was given alone.
                                 142

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TABLE 44.   EFFECT OF ALDICARB,  ALDICARB  OXIME,  2-PAM,  ATROPINE AND  CERTAIN MIXTURES OF THESE  COMPOUNDS
                             ON  THE  ACUTE  TOXICITY  OF METHYL PARATHION  TO MICE

Methyl  Parathion (A)           Dose  of  B,              Route of                     Effect of B
    Plus (B)	mg/kg	administration	on  toxicity of A
Aldicarb3                       1/2  LD5Q              A, B  -  Oral                Additive
Aldicarb oxime                     25               A, B  -  Oral                None
Aldicarb oxime                     25               A  - Oral, B-I.P.           None
2-PAM                              25               A, B  -  Oral                Antagonistic  (< 2-fold)
2-PAM                              25               A, B-I.P.                  Antagonistic  (8-fold)
2-PAM                              25               A  - Oral, B-I.P.           Antagonistic  (< 2-fold)
2-PAM + Aldicarb oxime           25+25            A  - Oral, B-I.P.           Antagonistic  (< 2-fold)
Atropine                           50               A  - Oral, B-I.P.           Antagonistic  (5-fold)
Atropine + 2-PAM                 50 + 50            A, B-I.P.                  Antagonistic  (100-fold)
Atropine + 2-PAM                 50+50            A  - Oral, B-I.P.           Antagonistic  (5-fold)

a Methyl parathion and aldicarb administered at one-half  their LD   doses.

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 Modification  of  Carbaryl Metabolism with Monoamine Oxidase Inhibitors

 Selective  control of  the metabolic fate of insecticides in mammalian and
 insect  systems provides one of the most promising methods of improving
 and maintaining  the efficacy and safety of these chemical toxicants.
 By inhibiting metabolic detoxication mechanisms in the pest species or
 enhancing  the mechanisms in the nontarget species through use of other
 chemicals, an ineffective compound can become an effective one, a non-
 specific toxicant can become more selective, and the level of toxicant
 required to control the pest species may be lessened considerably.
 Currently, many  compounds are referred to as insecticidal synergists
 and have been used for years with limited success to accomplish these
 desirable  features.

 Insecticidal  synergists are thought to enhance the toxic action of
 insecticides  as  a result of their ability to inhibit  the mixed-function
 oxidase system of microsomes.  This explanation for the mode of action
 is a rather recent development and followed the successful use of
 insecticidal  synergists by many years.  The action of insecticidal
 synergists and a thorough discussion of their history and potential
 were reported by Casida (1970).  In insecticidal synergists in use
 today, the active compounds were discovered first and their action
 described  later.  Obviously, if certain compounds can produce desirable
 effects by inhibiting a specific type of metabolic reaction,  namely
 oxidation, probably other compounds exist which would produce a
 similar effect by inhibiting any important metabolic process.  Other
 processes which are of importance in the metabolism of foreign com-
 pounds include hydrolysis, reduction, and conjugation (Williams 1963).

 Because conjugation is so important in the metabolism of the  carbamate
 insecticides  (Dorough, 1970; Kuhr, 1970),  this mechanism of
metabolism was selected for inclusion in studies designed to  determine
the effect of certain chemicals on metabolic processes.   Monoamine
                                144

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oxidase inhibitors were chosen because of their known interactions with
other compounds (Brodie et al. 1958; Stockley 1969 a,b) and, more
specifically, because of their reported ability to inhibit the conju-
gation of o-aminophenol by rat liver slices and rabbit liver homogen-
ates (Hargreaves, 1968).  Seven of these drugs were shown recently to
be inhibitors of carbaryl metabolism by a 15,000g soluble fraction of
rat liver (Culver et al., 1970).  The present study was undertaken to
determine in vivo effects of several monoamine oxidase inhibitors on
carbaryl metabolism and to gain-a more complete understanding of the
general process of conjugative metabolism.

Methods

Treatment of animals - Rats weighing ca. 200 g were administered oral
doses of 1 uCi of carbarylnaphthol-1-  C or naphthol-  C, each with a
specific activity of 15 uCi/mmole, in 0.1 ml of corn oil.  Some of the
animals were treated simultaneously with 50 or 100 mg/kg of a
monoamine oxidase inhibitor.  Other animals were maintained on drinking
water containing phenelzine, 80 mg/liter, for 25 days and then treated
orally with the carbaryl-  C.  Actual intal
from the total daily consumption of water.
orally with the carbaryl-  C.   Actual  intake of the drug was calculated
Monoamine oxidase inhibitors evaluated for their effect on the excretion
of carbaryl from rats were:  (A) tranylcypromine (trans-2-phenyl
cyclopropylamine); (B) isoniazid (isonicotinic acid hydrazide);
(C) isocarboxazid (5-methyl-3-isoxazoledecarboxylic acid 2-benzyl-
hydrazide); (D) phenelzine (2-phenylethylhydrazine); (E) harmaline
(2,3-dihydro-7-methoxy-l-methyl-9-pyrido (3,4b) indole); (F) iproniazid
(2-isopropyl-l-isonicotinyl hydrazine); and (6) pargyline (N-methyl-
1-N-propargylbenzyl amine).  For all other studies reported herein,
phenelzine was the only monoamine oxidase inhibitor used.

Collection and analysis of the excreta - Feces and urine were collected
                                  145

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 at  12-hr  intervals  following  treatment  of  the rats and assayed immed-
 iately  for  radioactive  content.   Aliquots  of urine,  up to 1 ml, were
 counted directly, whereas  feces were  combusted  first and aliquots of
 a C02 trap  were  assayed for radioactivity  (Andrawes  et al., 1967).
 Urine samples, used for more  complete analysis, were frozen in plastic
 bags.   Feces were discarded after the initial radioassay.

 To  extract  the urine, it was  diluted  2-fold with distilled water and
 partitioned repeatedly  with diethyl ether.  The 2 solvent phases were
 radioassayed, and the ether soluble products were analyzed further by
 tic on  Chromar 500  sheets  (Mallinkrodt, St. Louis, Mo.).  Analytical
 standards of carbaryl and  1-naphthol  were  added to the ether extract
 prior to  spotting.   The sheets were developed first  in a 1:1 mixture of
 ether and hexane, and then in the 2nd direction in a 1:1 mixture of
 methylene chloride  and  ethyl  acetate.   Radioautography with Kodak no-
 screen  medical x-ray film was used to locate radioactive areas on the
 Chromar sheets.  Carbaryl and 1-naphthol standards were visualized by
 placing the sheets  under UV light.  Quantitation of  the radioactivity
 on the  tic  included that corresponding  to  carbaryl,  that corresponding
 to 1-naphthol, and  the  total of all other  radioactivity on the sheet.

                                                               14
 In vitro conjugation of 1-naphthol -  Conjugation of  naphthol-1-  C
was evaluated by using  rat intestinal mucosal glucuronyl transferase
 (Howes  and  Hunter,  1968).  Small  intestines were removed from rats
 immediately after they  were killed, and kept cool in ice cold 0.15
M KC1.  Intestines  were  opened, were  washed thoroughly with the cold
KC1  solution, and the mucosa was  collected by scraping with a scalpel.
Enzyme  preparations were obtained by  homogenizing the mucosal
scrapings in 0.15 M  KC1   buffer for 4  min.  A typical  reaction mixture
included 0.1 uCi  of  naphthol-l-14C, 0.1  ml  of 0.1 M MgCl2, 1.0 of
phosphate buffer (pH 7.4), 0.5 ml  (1.34 umole) of uridine diphospho-
glucuronic acid,  potassium salt (Nutritional Biochemicals), and 2.5
ml  of the enzyme.   Reactions were run at 37°C in a metabolic shaker
                                146

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for 2 hr.  The reaction mixture was extracted twice with ether and
aliquots of both the organic and aqueous layers were radioassayed.
The percent increase or decrease in the radioactivity partitioning into
the aqueous phase was calculated with reference to the presence of
the monoamine oxidase inhibitor.

Radioassay - A Packard Tri-Carb Model 3380/544 liquid scintillation
counter was used for all quantitative radioassays.  For counting up to
0.4 ml of water and up to 1.0 ml of organic solvents, the scintillation
mixture consisted of a 2:1 toluenemethylcellusolve mixture containing
5 g/liter PRO, 0.1 g/liter POPOP, and 60 g/liter of naphthalene.
Scintillation grade PPO and POPOP were used, although Fisher-purified
grade solvents and naphthalene proved equal in performance to more ex-
pensive materials.  Fifteen ml of the scintillation mixture was used
in each counting vial.  For aqueous samples containing low radioactivity,
                                                            P
aliquots up to 1.0 ml were radioassayed in 10 ml of Aquasol   (New
England Nuclear, Boston).  Background counts were determined by counting
aliquots of identical size derived from untreated animals.

Results and Discussion

                                                              14
Excretion - The rate of excretion of an oral dose of carbaryl-  C from
rats was reduced by the simultaneous administration of a monoamine
oxidase inhibitor (Table 45).  Tranylcypromine reduced the rate of
excretion to a greater degree than did the other drugs when given at
                                              14
a dose of 50 mg/kg.  Only 33% of the carbaryl-  C dose was eliminated
in the urine by 48 hr after treatment, whereas animals which did not
receive a monoamine oxidase inhibitor excreted 71% of the dose in the
urine during the same period after treatment.

Low excretion of the carbaryl-  C dose in the feces of the tranyl-
cypromine-treated rats, 0.4% of the dose, was caused in part by re-
duction in the total  feces excreted by the animals following treatment
                                147

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 with  the  drug.   With  tranylcypromine,  animals treated with 50 rag/kg
 were  hyperactive for  3-6  hr  after  the  inhibitor was administered, and
 they  showed  a  reduced food intake  for  ca. 36 hr.  Animals treated with
 100 mg/kg of tranylcypromine died  within 12 hr.  This was the only drug
 tested which caused death of rats  treated at the 100 mg/kg level.
 Higher doses of  the other monoamine oxidase inhibitors simply enhanced
 their effects  on excretion (Table  45).

 The effect of  the other monoamine  oxidase inhibitors on carbaryl-  C
 excretion from rats was not  so profound as with tranylcypromine.  Iso-
 m'azid and isocarboxazid  reduced overall excretion by ca. 30%, phenel-
 zine  and  harmaline by ca. 20%, and iproniazid and pargyline by 10-15%.
 The relative effect of the drugs on carbaryl-  C excretion by rats was
 vastly different from their  effect on  the in vitro metabolism of this
 carbamate by a 15,000g soluble fraction of rat liver (Culver et al.
 1970).  In that  study, phenelzine  and  pargyline were better inhibitors
 of carbaryl  metabolism than  tranylcypromine.  Phenelzine appeared to
 have  the  greatest effect  on  in vitro and in vivo metabolism of
 carbaryl.  Because of this and because rats treated at 50 mg/kg with
 phenelzine showed no  toxic symptoms at all, this monoamine oxidase
 inhibitor was selected for additional  study.

 The data  in  Table 45  are  condensed so  that the effects of the monoamine
 oxidase inhibitors on  carbaryl can be compared readily.  Actually, ex-
 creta of  all  treated  rats were collected every 12 hr and radioassayed
 separately.  Complete  data are not presented,  because the pattern of
 elimination was  the same with each drug, and variation existed only  in
magnitude  of effect.

Chronic exposure  of phenelzine to  rats for 25 days by dissolving the
monoamine oxidase inhibitor  in drinking water reduced the rate of
excretion of a single oral dose of carbaryl-  C (Table 46).   The
greatest  reduction in excretion of the carbamate occurred during the
1st 24 hr. Total  excretion of the carbaryl-  C dose was about the same
                                148

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in the phenelzine-treated and control animals.  These tests indicate
that chronic exposure to the monoamine oxidase inhibitors could alter
normal carbaryl metabolism and excretion in mammals.   However, the level
of phenelzine exposure would have to be rather excessive, since adding
it to the drinking water of rats at 8 mg/liter for 25 days had only a
very slight effect on the fate of the carbaryl-  C.

Nature of metabolites - Comparing the chemical nature of carbaryl-  C
equivalents in the urine of phenelzine-treated and untreated rats gave
some insight into why monoamine oxidase inhibitors decreased the rate
of carbaryl excretion (Table 47).  Only 48% of the carbaryl dose were
eliminated as conjugates in the urine of rats receiving 50 mg/kg phenel-
zine.  Without phenelzine, 64% of the dose were present in the 48-hr
urine as water-soluble conjugates.   This decrease in  conjugation was
coincident with an increase in 1-naphthol  and with a  decrease in other
organo-soluble metabolites which resulted from oxidative metabolism
(Dorough and Casida, 1964).

It appears, then, that both oxidative and conjugative mechanisms of
metabolism were affected by the monoamine oxidase inhibitor.  This is
the same conclusion drawn by the in vitro studies of  Culver et al.
(1970).  Increase in 1-naphthol was a predictable occurrence, since
the efficiency of conjugation was lessened considerably.  Likewise,
total excretion of carbaryl-  C equivalents was less  because the car-
bamate and its metabolites were not converted to polar conjugates as
rapidly in phenelzine-treated animals.

The potential is clear for increasing the quantity of toxic carbamate
material in the animal's body because of continued combined exposure
of the insecticides and drugs.  However, the indication that certain
foreign compounds may interfere effectively with conjugative mechanisms
could prove beneficial rather than harmful.  For example, compounds
which would selectively inhibit conjugation in insects or in resistant
insects but not susceptible ones would reduce the hazard of the

                                 149

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 chemical  to mammals  by  enhancing  the  toxicity of the material to the
 pest  species.   Existence of  such  compounds  is conjecture at the present
 time,  however.

 Because  1-naphthol can  be conjugated  directly, without first having to
 undergo  some type of metabolism as  is the case for carbaryl, it was
 administered orally  to  rats, and  the  effects of phenelzine on its
 excretion and conjugation were determined.  This monoamine oxidase
 inhibitor only  slightly reduced the excretion rate of 1-naphthol from
 the animals (Table 48).  However, phenelzine did reduce the amount of
 1-naphthol excreted  in  urine as water-soluble conjugates.  Cumulatively,
 conjugation was decreased by ca.  20%  during the 48-hr period after
 treatment as compared with animals  which did not receive the phenelzine
 treatment.  These data  prove that in  vivo conjugation is inhibited by
 the monoamine oxidase inhibitors  as was indicated by the in vitro
 studies of Culver et al. (1970).

 Having established that conjugation could be specifically inhibited,
 attempts were made to use an in vitro system for detailed studies of
 conjugative metabolism  and to determine the effects of various chemicals
 on this metabolism mechanism.  Our  preliminary data show that a rat-
 intestine enzyme system fortified with UDPGA will conjugate 1-naphthol
 but will not in any manner metabolize the carbamate, carbaryl.  There-
 fore, this system will  be quite useful in studying conjugation mechanism
 independently of other  types of biochemical reactions.

 Of 3 monoamine oxidase  inhibitors evaluated for in vitro conjugation
 of 1-naphthol, phenelzine was the most effective.  This material in-
 hibited almost 65% of the naphthol conjugated in the absence of the
monoamine oxidase inhibitors.  This degree of inhibition was nearly
twice that reported for the inhibition of 1-naphthol  conjugation by
a NADPH2-fortified rat  liver enzyme system (Culver et al.  1970).
Tranylcypromine and isoniazid were equally effective in inhibiting the
conjugation by both the NADPA2- and UDPGA-fortified enzyme systems.
                                150

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Studies are continuing on the refinement of the in vitro conjugation
system and on evaluating the importance of conjugation in the
action of pesticides and other chemicals.
                                  151

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TABLE 45.  EFFECT OF SEVERAL MONOAMINE OXIDASE INHIBITORS (MAOI)  ON
                  THE EXCRETION OF A SINGLE ORAL DOSE OF
                      CARBARYL NAPHTHYL-14C BY RATS3

% of administered radioactivity
eliminated by 48 hr
MAOI
Control
Tranylcypromine
Isoniazid
Isocarboxazid
Phenelzine
Harmaline
Iproniazid
Pargyline
Urine
70.8
32.8
47.0
49.9
56.1
59.9
60.7
68.8
Feces
8.7
0.4
1.4
4.0
6.9
3.5
4.8
3.0
Total
79.5
33.2
48.4
53.9
63.0
63.4
65.5
71.8
  MAOI administered at a dose of 50 mg/kg simultaneously with carbaryl
                                152

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TABLE  46.  FATE OF A SINGLE ORAL DOSE OF CARBARYL-14C IN RATS WHICH

          HAD RECEIVED PHENELZINE IN THE DRINKING WATER FOR 25 DAYS3

Time after
carbaryl
treatment
12 hr
Control
Treated
24 hr
Control
Treated
48 hr
Control
Treated
% of administered
radioactivity eliminated
Urine

61.2
47.3

74.6
59.4

75.7
74.1
Feces

1.8
0.2

3.2
0.9

5.8
4.0
Total

63.0
47.5

77.8
60.3

81.5
78.1
a Phenelzine in drinking water at 80 mg/liter; avg.  daily intake of
  1.3 mg/day.
                                  153

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TABLE 47.  NATURE OF CARBARYL-14C METABOLITES IN URINE OF NORMAL AND
                          PHENELZINE-TREATED RATS3
                                % of administered carbaryl  dose in
Metabolite
Carbaryl
Naphthol
Organo-solubles
Conjugates0
Total
Normal rats
0.4
1.3
9.0
63.8
74.5
Treated rats
0.1
4.8
3.3
47,9
56.1
a Analysis of urine from rats collected 48 hr after treatment with a
  single oral dose of carbaryl-14c or with carbaryl-l^c + phenelzine
  at a dose of 50 mg/kg.
  Combined organo-soluble metabolites, excluding carbaryl and naphthol
c Total water-soluble metabolites.
                                 154

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TABLE 48.  METABOLISM AND EXCRETION OF NAPHTHOL-14C WHEN ADMINISTERED TO
          RATS SIMULTANEOUSLY WITH AN ORAL DOSE (50 MG/KG)  OF PHENELZINE

Time after
treatment
12 hr
Control
Treated
24 hr
Control
Treated
48 hr
Control
Treated
Cumulative
Urine

39.2
43.8

56.2
50.9

71.4
59.8
% of dose
Feces

0.4
0.9

1.3
2.0

4.8
4.9
in the
Total

39.6
44.7

57.5
52.9

76.2
64.7
% of dose
conjugated3

27.8
39.2

55.4
46.1

70.0
48.6
a These values reflect the percentage of the dose present in the urine
  as water-soluble metabolites.
                                  155

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 Influence of Selected  Insecticides on Carbaryl Metabolism

 Investigating the metabolism of a compound in a complex environment con-
 taining other compounds may aid in understanding the ways various com-
 pounds affect one another in the biotransformation processes in organisms.
 This was attempted in  the present  investigation to gain some knowledge
 of changes in the metabolism of a carbamate insecticide in the rafe which
 also were treated with other types of insecticides.

 Methods and Materials

 Standards - Carbaryl-1-naphthyl-  C (specific activity, 6.56 mc/mM) was
 supplied by Union Carbide Corporation, Olefins Division, South
 Charleston, West Virginia.  Pure 1-naphthol-  C (15.2 mc/mM) was
 purchased from Amersham Searle (Chicago, 111).  Several analogs of car-
 baryl considered as possible metabolites were provided by Union Carbide
 Corporation.

 Analytical standards of aldrin, dieldrin, heptachlor, and heptachlor
 epoxide were purchased from Unilab Research Corporation, Berkeley,
 California.  The purity of each chemical was over 99% as determined by
 gas chromatography.

 Technical insecticidal chemicals used were carbofuran, aldrin, heptachlor,
 DDT, Ruelene, and coumaphos.  All  were purchased from City Chemical
 Corporation, New York.

 Treatment and sampling - Rats were treated with a single dose or
multiple daily doses of insecticides via various routes and for various
 periods.

 For investigating the effect of a  carbamate or an organophosphate
 treatment on the metabolism of carbaryl  in the rat, three groups of
 rats (3 rats in each group) were administered orally a single dose of
                                 156

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carbaryl-  C (50 mg/kg) only, and in combination with carbofuran (0.5
mg/kg) or Ruelene (4.6 mg/kg).  The insecticides were administered in
corn oil.  The rats were placed in metabolism cages where the urine and
feces were collected at 12-hr intervals for 7 days and radioassayed.
The urinary metabolites of carbaryl were analyzed using thin layer
chromatography.

For evaluating the effects of multiple daily doses of carbaryl and a
                                                              14
carbamate or an organophosphate on the metabolism of carbaryl-  C, rats
were administered orally a daily dose of carbaryl-  C 260,000 dpm, (50
rog/kg) for 4 days.  Concurrently, rats were continuously provided in-
secticide-free rat chow or rat chow fortified with carbofuran (5 ppm)
or Ruelene (46 ppm) or coumaphos (15 ppm).  The animals were maintained
individually in metabolism cages and provided the diets and water for
a period of 2 weeks.  Urine and feces were collected at 24-hr intervals
for 2 weeks, radioassayed and the nature of metabolites determined.

The effects of injecting rats with DDT, carbaryl, carbofuran and Rue-
lene on various enzyme activities in the liver and kidney microsomes were
investigated.  Rats were administered intraperitoneally a daily dose of
DDT (25 mg/kg) or carbaryl (50 mg/kg) or a mixture of carbaryl plus DDT
(50 mg and 25 mg/kg) or carbofuran (0.5 mg/kg) for up to 5 days.  Three
rats were used for each treatment and for the control.  Insecticides
were dissolved in corn oil and the control rats were given corn oil at
the same volume as for the treated rats.

Rats also were given rat chow containing DDT (500 ppm), carbaryl
(1,000 ppm), DDT plus carbaryl (500 + 1,000 ppm) or carbofuran (5 ppm)
for a period of 40 days.  Animals from these treatments were given a
single oral dose of carbaryl-  C 24 hours after the last exposure to the
insecticide via injection or in the diet.  All rats, upon completion
of the insecticide administrations, were weighed and sacrificed.  The
livers, kidneys and spleen were removed for assays of microsome
enzyme activities.
                                  157

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 For  investigating  the  effect  of  pre-exposure to  DDT on the metabolism of
            14
 1-naphthol-  C  in  the  rat,  three rats were  given intraperitoneally a
 daily  dose  of DDT  (50  mg/kg)  for 5  days and a single oral dose of 1-
 naphthol-   C 24 hours  later.  Urine and feces were collected for 48 hours
 after  treatments and radioassayed.  The metabolites of carbaryl and
 naphthol in the urine  were  analyzed using thin layer chromatography.
Analysis of urine - Urines  from  rats collected for 60 hours after the
respective treatments were  analyzed.  The sample was added to an equal
amount of distilled water,  mixed well and transferred to a 125-ml separ-
atory funnel.   It was saturated with sodium chloride and then directly
extracted 5 times with a triple  volume of a 3:1 mixture of chloroform-
acetonitrile.   The wall of  the separatory funnel was rinsed with 5 ml
of distilled water and extracted twice with the chloroform-acetonitrile
mixture.  The organosoluble and aqueous phase were separated and radio-
assayed.

The chloroform-acetonitrile extract (organosoluble phase) was dried with
anhydrous sodium sulfate and filtered.  The extract was concentrated and
spotted on a 8  x 8 inch ChromAR 500 tic sheet for analysis of the
organosoluble metabolites.

The aqueous phase obtained after extraction of the urine sample was
filtered and concentrated to 1 to 2 ml in a rotary evaporator, then
transferred to  a 15-ml centrifuge tube.  After concentrating to about
0.5 ml under a  stream of air, the final concentrate was spotted as a
band on a 8 x 8 inch of ChromAR 500 sheet for separating the water
soluble metabolites.

Ascending thin  layer chromatography using 8x8 inch ChromAR 500 sheets
(Mallinckrodt Chemical Works, St. Louis, Missouri) was utilized for
resolving carbaryl  and its metabolites.  For separation of organo-
soluble carbaryl metabolites, the ChromAR sheet was developed two-
dimensional ly, first in a 4 to 1  ether-hexane mixture then in a 3 to 1
                                  158

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chloroform-acetonitrile mixture.  For separation of water soluble metabolites,
the tic was developed in a 2:1 mixture of ethyl  acetate and isopropanol.

Radioactive areas on the chromatograms were located by radioautography
after exposure to x-ray film for 3 to 5 days.   The radioactive area of
the tic was extracted with ether, while the water solubles were extracted
with distilled water.  The radioactivity in each extract was determined.

Metabolite identification - The identity of each of the carbaryl
metabolites was tentatively determined by cochromatography with standard
carbaryl and its derivatives.  The water soluble metabolites of carbaryl
were also identified according to their aglycones after acid hydrolysis.
For convenience, carbaryl and 1-naphthol were quantitated separately,
while the other metabolites were combined and referred to as extractable
carbamate metabolites (Dorough and Casida, 1964).  The unhydrolyzed
material was considered as the unknown water soluble metabolites.

Enzyme studies -  Upon completion of insecticide administrations, the
animals were sacrificed and their livers, and kidneys immediately
excised and placed in ice cold Tris-HCl buffer (0.05 M, pH 7.0) contain-
ing 8.6% sucrose.  The liver or kidney was macerated and homogenized in
Tris-HCl buffer (50% by weight).  The homogenate was centrifuged at
15,000g for 30 minutes at 3°C in an ultracentrifuge (Beckman L2-65B).
The supernatant was further centrifuged at 105,000g for 60 minutes.
The final pellet was washed once with a 1.2% KC1 solution, centrifuged
and resuspended thoroughly in Tris-HCl buffer (0.05 M, pH 7.0) so that
1 ml of buffer contained 500 mg tissue equivalents.  A portion of the
105,000g preparation was used immediately for oxidative enzyme assay
while the remainder was stored at -20°C for conjugative enzyme assay.

Microsomal oxidative and conjugative enzyme activities were determined
by measuring the rates of TPNH oxidation, epoxidation of aldrin and
heptachlor, total metabolism of 1-naphthyl-  C-carbaryl, and glucuronide
conjugation of naphthol-  C.  The method for carbaryl metabolism was
                                  159

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similar  to  that  reported  by Dorough et al.,  (1964) and Oonnithan et al.,
(1968).  The microsomal conjugating enzyme activity was determined by
the  in vitro method of Mehendale and Dorough  (1971) while the TPNH
oxidase  activity was measured according to Hart and Fouts (1965).
Epoxidase activity was determined by measuring the epoxidation of
aldrin to dieldrin and of heptachlor to its epoxide.  For these studies,
a Varian Aerograph gas chromatograph was employed.  It was equipped
with an  electron capture  detector and nitrogen as the carrier gas.  The
glass column, 6' x 1/8",  contained 4% SE on Anakrom ABS, 80/90 mesh,
and was  operated at a column temperature of 200°C, detector temperature
215°C, injector  temperature 205°C and inlet pressure of 10 psi.

Lowry's method (1951) was used for protein determination with the
standard curve constructed using bovine serum albumin crystalline
(Nutritional Biochemical  Corporation, Cleveland, Ohio).

Results and Discussion

The urinary and  fecal excretion patterns of an orally administered dose
of carbaryl-  C  equivalents to rats are given in Table 49.  Statistical
analyses revealed that there were no significant differences in the
excretion when carbaryl was administered alone or in combination with
Ruelene.   However, when carbofuran was administered along with carbaryl-
  C the excretion of carbaryl  equivalents in the urine was significantly
reduced.   The depression was most evident during the first 24 hours after
the treatment.   The average percentage reduction of excretion of carbaryl
equivalents caused by administration of carbofuran was 12.2% in the urine
and 3.4% in the  feces over a 7-day period.

The excretion of carbaryl  equivalents in urine and feces of rats follow-
                                   14
ing 4 daily oral  doses of carbaryl-  C given at 24-hr intervals was
quite different from that following a single oral  dose of the carbaryl
(Table 50).   Treatment with the 4 consecutive doses of carbaryl  reduced
the excretion of carbaryl  equivalents in the urine and feces by about
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20% during the first 72 hours after the first dose compared to that from
rats dosed with a single oral dose of carbaryl.   Similarly, a 12% de-
crease after 72 hours was exhibited by animals which received daily oral
doses of carbaryl for 4 days while on a diet containing 5 ppm of carbo-
furan.  Conversely, excretion of carbaryl equivalents by rats receiving
the daily doses of carbaryl while on a diet containing Ruelene was in-
creased as compared with that when animals were given carbaryl alone.
The effect of Ruelene on increasing the excretion rate of carbaryl
equivalents was not as remarkable as that of carbofuran on reducing the
excretion rate of carbaryl equivalents.  Ten days after the first dose
of carbaryl, the percentage excretion of carbaryl equivalents in urine
plus feces was 90% for animals receiving 4 doses of carbaryl alone,
86% for those which received carbaryl and fed on the carbofuran diet,
and almost 100% for animals receiving carbaryl and feeding diets con-
taining either Ruelene or coumaphos.

It was possible that the reduction in the excretion of carbaryl equivalents
after treatment with repeated doses of carbaryl  or carbofuran was
caused by an overloading and increased toxicity of the carbamates to the
animals.  This was supported by the finding that during the period of
consecutive dosing of carbaryl the excretion of carbaryl equivalents
was slowed.  This was also suggested by Dorough (1967) who reported that
increasing the carbaryl dose to cows decreased slightly the excretion of
carbaryl in urine and feces.

The slight increase in the excretion rate of carbaryl equivalents in
the urine of rats continuously feeding diet containing Ruelene indicated
that there was an acceleration in the metabolic rate of carbaryl in the
animal after repeated doses with Ruelene in the diet.  Stimulation of
biotransformation of carbaryl by Ruelene in the rat could be possible.

Partitioning the radioactive metabolites of carbaryl in the urine of
rats treated with a single oral dose of carbaryl alone or in combination
with carbofuran or Ruelene showed that the percentage distribution of
                                 161

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metabolites  between  chloroform  and water was dissimilar for all animals
 (Table  51).   Of  the  total  radioactivity in  the 0-12 hour urine,
approximately 15% was  organo-extractable for animals treated with carbaryl
plus  carbofuran  while  22%  was organo-extractable for animals treated
with  carbaryl  plus Ruelene.  For  rats treated with carbaryl only the
comparable value was 20%.  As expected from the excretion data (Table
49),  the  carbaryl-carbofuran treatment resulted in less total metabolites,
both  organo-extractable and water soluble,  in the urine when expressed
on a  "percent of dose" basis.

Route,  timing of administration and dosage  levels may effect different
responses of organisms to  a toxic compound; it was therefore decided
to investigate the effects of preadministrations with insecticides on
the metabolism of carbaryl in rats.  For in vitro liver microsomal
enzyme  activities were evaluated  for any induction or inhibition caused
by exposure  to the insecticides.

The liver weight of rats which received 500 ppm p,p'-DDT in the diet
for 40  days  increased 30%  compared to the livers of control animals
(Table  51).   There was no  change  in the weight of the kidney of treated
rats.   The results were in agreement with those of Wasserman (1969)
who showed that  rats receiving 200 ppm of p,p'-DDT in the drinking
water for 35  days showed an increase in the liver weight by 14% com-
pared with that of the control  animals.

Carbaryl given intraperitoneally  or in the rat feed, or carbofuran given
intraperitoneally,   did not change the weights of the liver and the
kidney.

Accompanying the increase  in the  liver weight after DDT treatment was
an increase  in the liver microsome protein content (Table 52).   The
rat liver microsome protein content increased 53% after intraperitoneal
DDT treatment for 5 days (25 mg/kg daily)  compared to the liver microsome
protein content in the DDT-free rats.   The protein content in the
                                 162

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kidney microsomes was not altered by DDT.  The increase in the liver
microsome protein content after DDT treatment could be the result of
induction of protein biosynthesis by DDT in rats as suggested by a
report that DDT at 0.5 ppm in He!a S cell culture medium increased the
incorporation of   C-leucine into cellular protein (Chung et al., 1967).

Carbaryl or carbofuran when given intraperitoneally, 50 mg/kg daily and
0.5 mg/kg daily, respectively for 5 days, caused a decrease by 30% of
the liver microsome protein content in the treated rats.  The result
indicates that carbaryl and carbofuran inhibited protein biosynthesis
in rats.

The change in the liver or the kidney microsome protein content correlated
with its metabolizing enzyme activities.  DDT treatment caused significant
increases in the liver or kidney microsomal enzyme activities for TPNH
oxidation, epoxidation of heptachlor and aldrin and total metabolism of
carbaryl (Table 53).  At 500 ppm for 40 days, DDT caused a 133% increase
in the TPNH oxidase activity; 1270% increase for epoxidation of
heptachlor; and over 800% for carbaryl metabolism.  DDT, given intra-
peritoneally at 25 mg/kg daily for 5 days, also caused an increase in
the liver microsome enzyme activity for TPNH oxidation (Table 54).
However, treatment with DDT, at 500 ppm for 40 days, did not enhance
the jn vitro liver microsomal enzyme activity for glucuronidation of
1-naphthol.

Carbaryl, given in the feed (1,000 ppm for 40 days) or given intra-
peritoneally (50 mg per kg for 5 days), did not cause significant
increases in the liver or kidney microsome enzyme activities for TPNH
oxidation, heptachlor or aldrin epoxidation, carbaryl metabolism and
1-naphthol conjugation (Tables 53 and 54).

Pre-exposure to DDT (25 mg/kg daily) for 3 days induced slight increases
in content of the organosoluble and water soluble metabolites of
carbaryl in the 48-hr urine of rats (Table 55).  DDT preadministrati on

                                  163

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 (50 mg/kg daily) for 5 days also enhanced the urinary excretion of 1-
 naphthyl glucuronide and 1- naphthyl sulfate when the rats were treated
 with naphtho1-14C  (Table 56).
The increases in microsomal enzyme activities for TPNH oxidation,
heptachlor or aldrin epoxidation, carbaryl hydroxylation and conjugation
in the rat after DDT pretreatments demonstrated a positive induction or
stimulation by DDT of the metabolizing enzyme systems in the mammal.
Increased enzyme activities were probably the result of induction on
syntheses of enzyme proteins in the animal.  These results were generally
in agreement with reports in the literature.  For instance, Hart and
Fouts (1965) observed an increase of liver microsomal TPNH oxidase
activity in rats receiving chlordane.  Gillett et al., (1968) reported
that the epoxidation of aldrin to dieldrin and heptachlor to its epoxide
were greatly enhanced by microsomal preparation from rats which received
DDT.

The failure of carbaryl or carbofuran to induce significant increases
in the liver or kidney microsome enzyme activities for oxidation of
TPNH, heptachlor and carbaryl, and for conjugation of carbaryl  metabolites
might be the result of inhibition of the microsomal protein biosynthesis
in the liver by the carbamate.  Furthermore, carbamates are generally
unstable and easily metabolized in animals, and thus might not persist
in the body long enough to produce an effect.  Therefore, the carbamate
did not appear as a microsome metabolizing enzyme inducer.

Preadministration of Ruelene (4.6 mg/kg daily) for 3 days slightly in-
creased the urinary organosoluble and water soluble carbaryl  metabolites
in the treated rats.   Simultaneous administration of Ruelene in the feed
(46 ppm)  also slightly hastened the excretion of carbaryl  equivalents
in the urine as mentioned earlier.   This suggests that Ruelene  at the
dose applied was possibly a weak microsomal  enzyme inducer.

In order to stimulate significantly the liver microsomal  enzyme

                                 164

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activities, it may require increases in the dose rates of the carbamates
and/or the organophosphorus insecticides.   As Fouts (1970) suggested,
test substances that are rapidly excreted, metabolized or sequestered
in non-hepatic tissues are not usually good enzyme inducers unless they
are given repeatedly or at a very high dose.
                                  165

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cr>
en
      TABLE 49.  THE EFFECTS OF CARBOFURAN AND RUELENE ON THE EXCRETION OF CARBARYL-14C ADMINISTERED TO RATS

                                                  AS A SINGLE ORAL DOSE3

Hours after
treatment
12
24

48

72
96
120

168
Avg. SD


Urine
28.8
67.3

75.0

76.6
77.5
77.6

79.7
5.6
Cumulative
Carbaryl
Feces
1.3
4.0

7.8

9.4
9.8
9.9

9.9
1.3
percent of dose
Carbaryl +
Urine
21.5
51.7
*
60.9
*
63.4
64.8
65.8
*
67.5
4.2
after indicated
Carbofuran
Feces
0.8
2.6

4.8

5.9
6.4
6.4

6.5
1.3
treatment
Carbaryl
Urine
28.8
61.8

77.5

77.5
78.0
79.2

80.2
6.9

+ Ruelene
Feces
0.8
3.0

4.7

5.9
6.6
6.6

6.7
1.6
       a  Naphthyl-1-carbaryl-C dose of 50 mg/kg; carbofuran, 0.5 mg/kg, or Ruelene, 4.6 mg/kg, administered
         simultaneously with carbaryl.
         Asterisk  indicates a significant difference at the 5% level from the carbaryl treatment as  deter-
         mined with t-test.

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en
       TABLE  50.  EXCRETORY PATTERN OF CARBARYL WHEN ADMINISTERED AS A DAILY SINGLE ORAL  DOSE TO  RATS  FOR

                                 4 DAYS WHILE FED A NORMAL RATION AND WHEN FED A DIET

                                      CONTAINING CARBOFURAN, RUELENE OR COUMAPHOS3

Cumulative percent of carbaryl dose when
Hours after
first carbaryl
treatment
12

24

72

96

120

168
Avg. SD
Carbaryl +
Control
Urine
22.7

30.3

58.7

66.4

80.7

87.0
4.5
Feces
0.6

1.0

7.4

9.0

9.2

10.8
1.4
Carbofuran
Urine
29.9

30.7

49.6
*
55.6
*
72.0
*
75.4
1.5
Feces
0.9

1.1

7.6

8.7

9.0

9.6
1.4
diet contained
Carbaryl +
Ruelene
Urine
36.2
*
48.0
*
74.5
*
89.0
*
90.2

90.2
7.0
Feces
0.7

0.8

9.7

9.8

9.8

9.8
1.2
Carbaryl +
Coumaphos
Urine
32.3

42.3

64.0

69.4

85.9

86.1
6.4
Feces
0.8

0.9

6.3

7.0

9.3

10.0
1.4
       a  Naphthyl-1-carbaryl-  C dose of 50 mg/kg; levels in the diet for carbofuran, Ruelene and coumaphos
         were 5, 46, and 15 ppm, respectively.  Insecticide fortified diets were provided from the time of
         the first carbaryl treatment until termination of experiment.
         Asterisk indicates a significant difference at the 5% level from the carbaryl treatment as deter-
         mined with t-test.

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00
      TABLE 51.  NATURE OF RADIOACTIVITY IN THE URINE OF RATS TREATED WITH A SINGLE ORAL DOSE OF NAPHTHYL-

                                1-CARBARYL-14C ALONE, AND WHEN ADMINISTERED SIMULTANEOUSLY

                                               WITH EITHER CARBOFURAN OR RUELENE3

Percent of dose when treated with:
Nature of
metabolites
Orga npex t ra c t abl e
carbamate metab-
olites
Hydrolysis Pro-
ducts
Unknowns
Watersolubles0
Carbamate metab-
olites
Hydrolysis Pro-
ducts
1-Naphthyl sul-
fate
Unknowns

0-12
5.8
1.9
3.2
0.7
23.0
2.5
2.9
5.2
12.4
Carbaryl
hr 12-48 hr
7.8
2.5
4.2
1.1
38.4
1.1
1.4
8.3
27.6
Carbaryl
0-12 hr
3.2
0.9
2.2
0.1
18.3
1.4
0.6
3.7
12.6
+ Carbofuran
12-48 hr
7.3
3.7
2.9
0.7
28.5
2.2
2.7
5.1
18.5
Carbaryl
0-12 hr
6.4
2.2
3.4
0.8
22.4
2.4
2.9
4.8
12.3
+ Ruelene
12-48 hr
11.1
2.8
7.4
0.9
32.6
2.0
2.6
6.8
21.2
        Dosage rates same as given in Table 49.
        Metabolites in urine which were extractable into organic solvent.  Carbamate metabolites refer to
        those materials with the carbamate ester linkage still intact.
        Nature of metabolites based on aglycones produced by acid treatment of the watersolubles.  Unknowns
        include those materials remaining in the water after acid treatment.

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TABLE 52.  WEIGHT AND MICROSOMAL PROTEIN CONTENT OF LIVERS FROM RATS EX-
                   POSED TO CARBARYL, CARBOFURAN AND DDT

Treatment method            Liver weights, percent   Protein content,  mg
and duration	of body weight3   protein per gram of liver3
IP injections for 5 days
Control (corn oil)                 4.4+0.3              20.9+2.0
Carbaryl, 50 mg/kg/day             3.5+0.2              15.9+1.8
Carbofuran, 0.5 mg/kg/day          4.0 + 0.3              15.4 + 1.7
DDT, 25 mg/kg/day                  4.6+0.5              32.4+5.1*
DDT + Carbaryl                     4.1+0.4              19.3+3.7
In diet for 40 days
Control                            3.7 + 0.3              17.7 + 1.4
Carbaryl, 1000 ppm                 3.2+0.1              20.9+1.4
DDT, 500 ppm                       4.8+0.3*             19.3+1.3
DDT + Carbaryl                     4.0+0.1              18.2+1.4

a Asterisk indicates a significant difference at the 1% level compared to
  the control animals as determined with t-test.
                                  169

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TABLE 53.   IN VIVO METABOLIC ACTIVITY IN THE MICROSOMAL FRACTIONS OF LIVER AND KIDNEY OF RATS FED

                            DIETS CONTAINING CARBARYL,  AND DDT FOR 40 DAYS

Metabolic activity
Treatment
and rate9
Control
Carbaryl, 1000 ppm
DDT, 500 ppm
DDT + Carbaryl
Control
Carbaryl , 1000 ppm
DDT, 500 ppm
DDT + Carbaryl
% TPNH
oxidation

3.5 + 0.16
4.1 + 0.15
8.0 + 0.26*
5.8 + 0.85*
1.8 + 0.90
3.5 +0.53*
4.6 + 0.90*
4.3 + 0.37*
% Heptachlor
epoxidation

3.7 + 0.04
4.8 + 0.25
50.9 + 3.87*
59.7 + 0.25
0
0
18.3 + 0.3*
17.4 + 0.8*
% Carbaryl
metabolism
Liver
3.6 + 0.05
6.3 + 0.18
33.2 + 1.57*
28.5 + 3.71*
Kidney
0
0.7 + 0.05
0.4 +_ 0.04
0.8 +_ 0.02
% Naphthol
conjugation

81.9 + 1.2
93.4 + 0.9
86.5 + 2.3
86.4 + 1.8
81.9 + 5.5
95.0 + 0.1
89.3 + 0.2
87.4 +_ 0.5
  Treatment rates for DDT + carbaryl  were the same as individual rates shown.

  Asterisk indicates a significant difference at the 5% level  compared to the controls as deter-
  mined with t- test.

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TABLE 54.  TPNH OXIDASE ACTIVITY IN THE MICROSOMAL FRACTION OF LIVERS FROM
                  RATS TREATED INTRAPERITONEALLY WITH CARBARYL
                         AND DDT DAILY FOR UP TO 5 DAYS

TK*PA "f mont anH
daily dose
Control

Carbaryl , 50 mg/kg

DDT, 25 mg/kg
DDT + Carbaryl
Percent TPNH oxidized/days on
1
2.5 + 0.23

3.0 + 0.37

3.1 +_ 0.42
3.4 + 0.10
3
3.6 + 0.16

3.6 + 0.05

4.1 +0.01
3.0 + 0.58
treatment9

4.2

3.1

11.1
4.2
5
+ 0.16
*
+ 0.53
*
+ 0.37
+ 0.53
  Asterisk indicates a significant difference at the
  controls as determined with t-test.
level  from the
                                  171

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TABLE 55.   THE EFFECT OF PRE-EXPOSURE  TO  DDT (IP,  25 MG/KG/DAY)  FOR 3  DA'.S ON THE NATURE AND MAGNITUDE
                 OF CARBARYL  METABOLITES IN THE  URINE OF RATS TREATED WITH A SINGLE ORAL
                                        DOSE OF  NAPHTHYL-1-CARBARYL-14Ca


Nature of metabolites
Prgaooextractable
Carbamate metabolites
Hydrolysis products
Unknowns
Watersolublec
Percent of dose and (
Control rats
7.7 (10.6)
3.9 (5.4)
0.4 (0.5)
3.4 (4.7)
64.8 (89.4)
% of total urine content)
DDT-treated rats
11.3 (13.4)
5.7 (6.8)
1.1 (1.3)
4.5 (5.3)
73.2 (86.6)
a Naphthyl-1-carbaryl-  C dose of 50 mg/kg given 24 hours after the last injection of insecticide.
  Metabolites in urine which wereextractable into organic solvent.   Carbamate metabolites refer to
  those materials with the carbamate ester linkage intact.
c Metabolites remaining in the water phase after extraction with organic solvent.

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00
      TABLE 56.  EXCRETION AND METABOLISM OF A SINGLE ORAL DOSE OF NAPHTHOL-1-14C BY RATS GIVEN DAILY
                                           INJECTIONS OF DDT, 50 MG/KG FOR 5 DAYS

Route of excretion/nature
of urine metabolites3
Excretion
Feces
Urine
Urine metabolites
Total organosolubles
Total watersolubles
1-naphthyl glucuronide
1-naphthyl sulfate
Unknowns
Percent
Control rats
1.14 + 0.23
58.43 + 6.52
8.18
50.25
15.24
0.39
34.62
of naphthol-1- C administered
DDT- treated rats
1.20 +. 0.23
66.41 + 7.10
11.65
54.76
29.51
13.91
11.34
      3 Based on collection of total  excreta during first 24 hours after treatment with naphthol.
        Materials remaining at the origin after tic analysis.

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CONJUGATION

Mechanism  in  Rats

Insecticidal  carbamates may be subjected to conjugative metabolism even
more extensively than other groups of insecticides  (Dorough, 1970).  In
fact, some are almost quantitatively transformed to, and excreted as,
conjugate materials.  Table 57 lists several carbamate insecticides which
were voided primarily as conjugate materials by rats.  Most of these
metabolites were glucuronides or ethereal sulfates as evidenced either by
comparison with chemically synthesized standards or by subjection of
these materials to specific hydrolytic enzymes.

Formation of  glycosides is the predominant form of conjugation in animals,
insects, and  plants.  In animals, glucuronides are formed by a reaction
catalyzed by  uridine diphosphoglucuronyl transferase (EC 2.4.1.17) and is
characterized by the utilization of uridine diphosphoglucuronic acid.
Present evidence indicates that the enzyme activity is associated in the
particulate fractions of cell-free preparations (Parke, 1968).  Different
substrates are conjugated with glucuronic acid by different enzymes.

Much of the recent literature in the area of insecticide toxicology and
metabolism deals with the initial oxidative modification of the insect-
icide chemical.  This is understandable since many insecticides are anti-
cholinesterase agents and consideration of the reactions responsible for
the initial metabolism, and its effect on anticholinesterase activity,
are of obvious toxicological  significance.   Also, biochemical  oxidation
was envisioned as a target to control  the toxicity of this type of
compound.  For example, it has been shown that the mechanism of action
of insecticide synergists involves their ability to inhibit the microsomal
oxidative enzymes (Casida, 1970).  These very important consequences of
insecticide bioalterations may not be restricted to the oxidative phase
of metabolism.
                                  174

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It is possible that the action of insecticide synergists,  the mechanism
of insect resistance, and the selective nature of some insecticides  could
be related to conjugative metabolism.   The possibility that such could
be the case had been suggested earlier (Mehendale and Dorough, 1971).
While one study (Boose and Terriere, 1967)  showed no apparent increase
in conjugative ability in chlorinated  hydrocarbon-resistant houseflies,
Culver et al., (1970), reported the inhibition of naphthol  conjugation,
as well as the total metabolism of carbaryl, by certain monoamine oxidase
inhibitors.  Later, it was demonstrated that,the same drugs inhibited  the
conjugation of 1-naphthol by a rat intestine enzyme system (Dorough, et al.
1972).  When administered orally to rats some of the drugs reduced the
rate of elimination of carbaryl from the animals and caused minor
changes in the nature and the quantity of individual metabolites in  the
excreta.

Since it was established that conjugation was a major metabolic pathway
for carbamates in animals and that the pathway was sensitive to various
foreign compounds, a detailed study directed toward establishing a well-
defined in vitro conjugative system was conducted.  The hydrolytic product
of carbaryl, 1-naphthol, was selected  as the substrate for studying  the
enzyme system responsible for glucuronidation by rat liver and intestine
enzymes since it could be conjugated directly.

Methods and Materials

Substrate and enzyme - 1-Naphthol-1-  C (15 mCi/mmole) was obtained  from
Amersham Searle Co., Des Plaines, 111, while radiolabeled carbaryl (1-
naphthyl-1-  C-N-methylcarbamate) (5.6 mCi/mmole) was supplied by
Union Carbide Corporation, Charleston, West Virginia.
Livers and small intestines of female albino rats weighing approximately
200 g were used as the enzyme souces in all studies.  All rat tissue
preparations were made so that each milliliter of buffer contained the
                                  175

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enzyme  from  250  mg  of  tissue.   Each centrifugal  fraction was tested for
glucuronyl transferase activity,  but only the  15,000g solubles and
microsomal fractions were  sufficiently active  for use in routine enzyme
assays.

A  second  source  of  enzyme  from  the rat was the small intestines.  In
preliminary  experiments, homogenates of the mucosal scrapings from
cleaned rat  intestines were used  as the enzyme source.  Later, it was
discovered that  the 12,000g soluble fraction of  homogenates from cleaned,
whole gut tissue could be  used  just as effectively.  Therefore, whole
intestines were  homogenized in  4  vol of 0.15 M KC1 solution and the 12,000g
soluble fraction used  as the enzyme source.  The enzyme activity in the
12,000g soluble  fraction was increased 3-fold  by adding 1% digitonin to the
KC1 solution.

Enzyme assay - For  routine enzyme assays, 1 umole 1-naphthol-  C (diluted
with unlabeled 1-naphthol  such  that 1 umole was equivalent to 2 x 10  dpm)
was introduced into a  25-ml Erlenmeyer flask and the solvent evaporated
just to dryness.  The  other constituents were  added to the flask in buffer
solutions.  After the  flasks were transferred  to a metabolic shaker with
the water temperature  at the desired level, the cofactor was added, and
incubation time  was recorded from that point.  Reactions were terminated
by the addition  of 2.0 ml of diethyl ether to  the flask.

Extraction - Unreacted  1-naphthol-  C was extracted by transferring the
reaction mixture to a  15-ml glass-stoppered centrifuge tube and ex-
tracting it twice with  2 vol of diethyl ether.  These extractions yielded
a recovery of over 95% of the free 1-naphthol  and, therefore, further
extraction was not necessary.   After separation of the ether and water
phases, the ether layer was adjusted to 10.0 ml and a 1.0 ml  aliquot
removed for radioassay by liquid-scintillation counting.  An aliquot of 0.2
ml  of the aqueous layer was radioassayed to determine the water-soluble
naphthyl glucuronide formed.

                                  176

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Nature of the reaction products - Ether extracts were concentrated and
applied to Chromar 500 thin-layer chromatographic sheets and developed
two dimensionally.  The first solvent system was a 5:1 mixture of hexane
and ether and the second system a 2:1 mixture of methylene chloride and
ethyl acetate.  A standard sample of 1-naphthol was used for co-
chroma tography.

The aqueous phase was concentrated and subjected to beta-glucuronidase for
24 hr (24), and the reaction mixture was extracted twice with 2 vol of
ether.  The two solvent phases were radioassayed and the ether extract
examined on tic to determine the nature of the aglycone.

Inhibition of conjugating enzyme - The chemical being tested for its
effect on the conjugating enzyme systems was introduced into a reaction
flask as an ether solution just after the substrate was added.  The
ether was evaporated using a gentle jet of air.  Other constituents of
the reaction mixture were added in the manner described before and the
reactions carried to completion.  Changes in the amount of conjugation
of 1-naphthol were determined and the degree of reduction of 1-naphthol
conjugation was expressed as percentage of inhibition as compared to
that of a control sample.

Metabolism of carbaryl - The metabolism of carbaryl by the microsomal
fraction of the rat liver was investigated.  NADPH2 was introduced into
the flasks at the rate of 2 umoles per flask, but the other components
were the same as the standard conditions for conjugation of 1-naphthol.
These flasks were incubated for 2 hr to achieve maximum metabolism of
carbaryl.

Epoxidation of aldrin by rat liver microsomes - To determine the relative
effectiveness of certain insecticide synergists on oxidative and con-
jugative metabolism, a limited number of experiments were conducted on
the microsomal epoxidation of aldrin.  A typical reaction mixture
                                  177

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contained the following ingredients:  20 ug of aldrin in 100 ul of methyl
cellosolve; 2 umoles of NADPH2 in 0.5 ml of buffer; 2.4 ml of Tris-HCl
(0.05 M) buffer, pH 7.4; 1.0 ml of rat liver microsomes (250 mg tissue
equivalents); and 0.1 ml of ethanol containing the desired amount of
synergists.  The mixtures were incubated for 1 hr at 37°C in a metabolic
shaker.  At the end of the incubation period, the reactions were termin-
ated by adding 4.0 ml of a 2:3 mixture of isopropanol and hexane and
then extracted twice with 2 vol of hexane.  The organic solvent phase was
analyzed for aldrin and dieldrin by gas chromatographic means using a
Varian Aerograph Model 1700 instrument equipped with an electron-capture
detector.  Operating parameters were as follows:  column, glass, 6 ft x
1/8 in. i.d. packed with 10% DC 200 on Anakrom  ABS, 80-90 mesh; carrier
gas, nitrogen, 45 ml/min.; temperature, column 195°C, injection port
200°C, detector 215°C.  Retention times for aldrin and dieldrin were
3.8 and 9.6 min., repsectively.

Results and Discussion

Optimum conditions for in vitro glucuronidation - For preliminary inves-
tigations into the glucuronidation mechanisms in rats, a liver preparation
commonly used for the metabolism of carbamates and other chemicals was
employed (Knaak et al., 1965).  This basic system was  modified and re-
fined until the optimum conditions for the glucuronidation of 1-naphthol-
14
  C by enzymes from rat liver and small intestine were established.  The
parameters for obtaining 1-naphthyl glucuronide in greatest quantity were
essentially the same for both enzyme sources and were as follows:

      Substrate              l-Naphthol-l-l4C, 1.0 umole
      Buffer                 Tris-HCl, 0.05 M, pH 7.0, 1.0 ml
      Metal ion              MgCl2,100 umoles in 1 ml buffer
      Enzyme                 250 mg tissue equivalents in 1  ml  buffer
      Cofactor               UDPGA, 1.4 umoles in 1  ml buffer
      Incubation             37°C, for 15 min, with shaking.

                                 178

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Generally, the conjugating enzymes were not too sensitive to temperature
variations between 35°C and 50°C.  Temperatures outside this range re-
duced the enzyme activity considerably, especially as the incubation
temperature exceeded 50°C.  Careful examination of the enzyme activity as
it related to small temperature changes showed that the greatest activity
was obtained when the enzyme preparations were incubated at 37°C.  At
this temperature, incubation of the flasks for longer than 15 min did not
significantly increase the amount of ocnjugation.

The type of buffer used for incubating the enzyme with 1-naphthol proved
to be important in maximizing the enzyme activity.  The pH of the buffer
was less critical.  With all buffers, the optimum pH was 7.0 but the
Tris-HCl buffer increased the enzyme activity about 20 and 30% over
that of the phosphate and Tris-maleate buffers, respectively.

The most important single factor in the glucuronidation of 1-naphthol was
the presence of the correct cofactor.  With the microsomal fraction of
the liver, the conjugation of the substrate occurred only when the co-
factor was UDPGA.  Even the very similar compound, UDPG, did not elicit
a trace of 1-naphthol conjugation by the microsomes.  The intestine
enzyme, being a 12,000g soluble fraction, probably contained some
endogenous UDPGA since a small amount of conjugation of the substrate was-
noted when cofactors other than UDPGA were used.  When these other co-
factors were used, however, the degree of conjugation was increased by
adding a greater quantity of cofactor.  This was unlike the response to
UDPGA where an increased concentration cuased in increase in the amount of
substrate conjugated.  To maintain the amount of 1-naphthol conjugation
between 75 and 85% during routine assays, a standard concentration of
1.4 umoles of UDPGA per incubation were used.  Mixing various cofactors
with UDPGA did not enhance the enzyme activity but, in fact, caused a
decrease when the added cofactor was either NADPH2, UTP, or ATP.

With all other conditions as stated previously, the percentage conjugation

                                   179

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was  80  to  85% when  the  1-naphthol concentration was 2.5 x 10   M.
Addition of greater amounts of  substrate gave smaller percentage conju-
gation  as  expected.  The  data suggested that substrate inhibition may
occur at high 1-naphthol  concentrations with the liver microsomal pre-
paration,  while  enzyme  saturation alone was observed with the intestine
enzymes.   The importance  of using larger amounts of substrate is readily
apparent when one considers this in vitro enzyme system as a means of
synthesizing certain glucuronides in sufficient quantity for chemical
and  toxicological evaluations.

A series of metal ions  was tested for their effect on the conjugating
activing of the  liver microsome and intestine enzymes.  Without the
addition of any  metal ion, and with all other parameters equal, only
                      14                                        -4.
20%  of  the 1-naphthol-  C was conjugated.  Magnesium at 1.3 x 10  M
concentration enhanced  the amount of 1-naphthol conjugated by about 20%.
With all other metals,  there was a marked inhibition of enzyme activity.
However, the degree  of  inhibition of the enzyme varied according to the enzyme
source  and  metal involved.  The greatest differences were noted with
cobalt  and  sodium where these metals inhibited the intestine enzyme more
than the enzyme  from the microsomes.  The other metals inhibited the micro-
some enzyme  activity to a greater degree.

Since magnesium was  the only metal  which enhanced conjugation, its con-
centration  for maximum enzyme activity was determined.  The addition of
              -4
up to 2.5  x  10  M concentration of magnesium, 100 umoles of magnesium
chloride per flask, enhanced the activity of the enzymes from the micro-
somes.  This level of magnesium inhibited the intestine enzyme by about
10% its maximum activity.

Stability of the stored enzymes - Both enzyme sources were stored at
-20°C as buffer solutions containing 250 mg tissue-equivalents per
milliliter.  The rat liver microsomal  enzyme was more stable under
these conditions than enzyme from the  intestine; half-lives  of the
enzymes under storage conditions were  25 days and 2 days,  respectively.

                                 180

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This difference in stability during storage may suggest that the conju-
gating enzymes in the microsomes were different from those in the intest-
ine.  However, the intestine enzyme preparation was only slightly more than
a crude homogenate and may have contained certain components which denatured
the enzyme at a rather rapid rate.

Inhibition of glucuronidation - The scientific basis for establishing
the mode of action of insecticide synergists considered only those
techniques which measured their effect on oxidative metabolism (Casida,
1970).  This situation prompted an evaluation of their effect on the
rat liver glucuronidation system reported herein.  All of the synergists
inhibited the conjugation of 1-naphthol by the liver microsomes and
intestine enzymes (Table 58).  However, a greater concentration was re-
quired to inhibit 50% of the microsomal enzymes.  These data demonstrate
that certain insecticide synergists do inhibit glucuronidation in an in
vitro system; although, the significance of this inhibition, insofar as
the effectiveness of insecticide synergists is concerned, can not be
fully evaluated at this time.  A series of insecticides including DDT,
parathion, carbaryl, and rotenone did not inhibit glucuronidation at
      _3
the 10  M concentration.

Based on the data presented in Table 59, it would be justified to suggest
that the inhibition  of conjugation does contribute to the action of insect-
icide synergists.  These data show that while certain of the synergists,
MGK-264 and piperonyl butoxide, are much greater inhibitors of oxidative
metabolism than conjugative metabolism, other materials are approximately
equal in their effectiveness.  For example, the I5Q of Tropital against
the glucuronyl transferase of the microsomes was 1.0 x 10" M, but was
        _o
8.0 x 10  M against  the microsomal enzymes which oxidized aldrin to
dieldrin.  These differences in the degree of effectiveness of the
insecticide synergists in inhibiting metabolic processes offer encourage-
ment to our quest for compounds which are better and more selective in-
hibitors of conjugation than those evaluated thus far.
                                  181

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Effect of controlled glucuronidation on the in vitro metabolism of
carbaryl - Whether the inhibition of conjugative mechanisms of metabolism
have any practical significance in the action of insecticides depends upon
the effect of conjugation on the normal detoxication of the parent
insecticide and/or of its toxic metabolites.  This facet of glucuronidation
was tested indirectly using carbaryl, a carbamate insecticide which is
conjugatively metabolized by most organisms.

First, it was established that carbaryl-naphthyl-  C was not metabolized
by rat liver enzymes unless the cofactor NADPH2 was added to the system,
even though UDPGA was at its standard concentration of 1.4 umoles per
incubation (Table 60, control sample).  With no UDPGA added, but with 2
umoles of NADPH2, 17% of the carbaryl was metabolized to organoextractable
products or nonconjugates, and 9% to conjugated materials.  This latter
enzyme preparation was typical of the type of in vitro system used for
the microsomal oxidation of insecticides.

Controlling the degree of glucuronidation by limiting the quantity of
UDPGA in the system gave some insight into the effect of inhibition of
conjugation on the metabolism of carbaryl.  At the 5-umole level of UDPGA,
50% of the carbamate was conjugatively metabolized.  As the cofactor
level decreased, there was a corresponding decrease in conjugation, a
corresponding increase in the amount of unmetabolized carbaryl, and no
noticeable difference in the quantity of organoextractable metabolites
produced (Table 60).   Although carbaryl was metabolized  by the micro-
somal enzymes to the extent of about 25% even when UDPGA was not added
(Table 60), washing the microsomes reduced the metabolism of the car-
bamate to less than 10%, apparently by reducing the quantity of endo-
genous UDPGA.  The failure of the organoextractable products to
accumulate was somewhat surprising because they are composed of free
hydrolytic and oxidative metabolites of carbaryl.  Since the NADPH2
concentration, and all  other conditions except the UDPGA levels, were
kept constant and were suitable for the continuation of oxidative
                                  182

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metabolism, an accumulation of the oxidative metabolites would have been
a likely response to decreased conjugation.  That this is not the case,
combined with the decreased total metabolism of carbaryl, suggest that
oxidative and hydrolytic metabolism of the carbamate cannot continue un-
                                           /
less the intermediate metabolites are converted to conjugated products.
These data support the type of action reported for certain monoamine
oxidase inhibitors on the metabolism of carbaryl in rats.

The substrate specificity of the microsomal conjugating system using a
selected number of carbaryl metabolites was determined.  Naphthol and
1-naphthyl N-hydroxymethylcarbamate were conjugated at about the 90%
level while 40% of the 5-hydroxy- and 6-hydroxy derivatives of carbaryl
were conjugated by the same enzyme preparation.  The 5,6-dihydro-5,6-
dihydroxy-1-naphthyl N-methylcarbamate did not form a glucuronide.
According to Sullivan et al.  (1972), the glucuronide of this product was
a major metabolite in the urine of rats treated with carbaryl.  This
being the case, it was unclear as to why the in vitro glucuronidation of
5,6-dihydro-5,6-dihydroxycarbaryl was unsuccessful.

Carbaryl, per se, was not conjugated by the microsomal glucuronidation
system.  As pointed out earlier, the cofactor NADPHj, was necessary for
this material to be metabolized in any manner by the microsomes.  The
same was true for the compound Banol, the N-methylcarbamate of 6-chloro-3,
4-dimethylphenol.  Although Baron and Doherty (1967) reported that as
much as 18% of an oral dose of carbonyl-  C-Banol to rats was excreted in
the urine as a metabolite thought to be Banol-N-glucuronide, this product
was not formed by the rat liver glucuronidation system.  Glucuronide con-
jugates were formed by the microsomes only when NADPH2 was added to the
incubation mixtures.

Glucuronidation is but one of many forms of conjugative mechanisms which
operate in the animal body.  However, it is a very important mechanism,
and one that is very much involved in the metabolism of insecticides.
The current study provides a basis for considering glucuronidation, and

                                   183

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maybe conjugation in general, as a means by which the toxic action of chem-
icals in the body might be controlled.  For example, increasing conju-
gative activity through the use of certain chemicals could cause a more
effective detoxication and elimination of the toxicant from the body.
In essence, such chemicals would act as antidotes.  Contrarily, compounds
which would inhibit conjugative metabolism would likely enhance the
action of a toxicant or drug, an effect which could be either disastrous
or advantageous.

An intriguing possibility which could arise from an intensive investigation
of the comparative conjugation mechanisms in insects and higher animals
is the phenomenon of selective toxicity.  Because glucuronides are formed
by mammals while glucosides are formed by insects, there is a basic bio-
chemical difference in the conjugative mechanisms of the two animal
groups.   Thus, a means of inhibiting insect glucosylation without in-
hibiting mammalian glucuronidation could serve to make a toxicant exhibit
selective toxicity toward the insect species.
                                  184

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TABLE 57.  IMPORTANCE OF CONJUGATION IN THE METABOLISM OF CARBAMATE

                          INSECTICIDES IN RATS

Compound
Banol
Carbaryl
Carbofuran
Formetanate
Meobal
Mo bam
Percentage of dose conjugated3
88.8
82.6
91.0
67.2
85.0
78.0
a Percentage of radioactive dose excreted as water-woluble metabolites
  in the urine by 48 hr after treatment.
                                   185

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TABLE 58.  EFFECT OF CERTAIN INSECTICIDE SYNER6ISTS ON CONJUGATION OF 1-

           NAPHTHOL BY RAT LIVER MICROSOMES AND INTESTINE ENZYMES3


Compound
Isosafrole
M6K-264
Piperonyl butoxide
n-Propyl isome
Safrole
Sesamex
Su If oxide
Tropital
Molar concentration
Micro somes
10.0 x 10~3
3.0 x 10"3
6.0 x 10"3
7.0 x 10"3
9.0 x 10"3
5.9 x 10"3
0.3 x 10"3
1.0 x 10"3
for 50% inhibition
Intestines
4.7 x 10"3
1.0 x 10"3
1.7 x 10"3
2.0 x 10"3
4.2 x 10"3
1.9 x 10"3
1.2 x 10"3
0.8 x 10"3
3 Conditions for conjugation described previously.
K                                                        9
  The 150 values for the following compounds exceeded 10~ M concentration:
  RO-5-1557, RO-5-1923, and RO-7-0165.
                                 186

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TABLE 59.  INHIBITION OF RAT LIVER MICROSOMAL EPOXIDATION OF ALDRIN BY

                        CERTAIN INSECTICIDE SYNERGISTS
Inhibitor	Ispa	


MGK-264                                     0.1  x 10~3
                                                    _3
Piperonyl butoxide                          0.1  x 10

Sulfoxide                                   0.5 x 10"3
                                                    _3
Sesamex                                     1.0 x 10

Tropital                                    8.0 x 10"3



a Conditions same as described previously except that 2 umoles of
  NADPH? was substituted for UDPGA and 20 ug aldrin for 1  umole of 1-

  naphthol.
                                 187

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TABLE 60.  THE EFFECT OF REDUCED GLUCURONIDATION ON THE TOTAL METABOLISM

               OF CARBARYL-NAPHTHYL-14C BY RAT LIVER MICROSOMES

WITH
NADPH2a




umoles
UDPGA
Control5
5
3
1
0.3
0
Percentage


Carbaryl
88.8
23.4
28.9
34.3
48.9
63.9
of radioactivity
Organo-
extractable
metabolites
0.0
15.4
18.3
16.3
17.5
16.8
recovered as

Glucuronide
conjugates
0.0
49.8
42.0
36.9
22.8
9.2
  Carbaryl-  C, 30 ug,  incubated for 2 hr under same conditions  described
  previously, except that 2 umoles of NADPH2 were added  to  all flasks
  but the control.

  No NADPH2 added  but containing 1.4 umoles of UDPGA.
                                 188

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 Mechanisms in Insects

Carbamate insecticides are conjugatively metabolized in insects with the
major conjugates existing as glucosides, sulphates, and phosphates (Kuhr,
1970).  Although individual glucosides have not been quantitated or
identified, it is commonly suspected that glucoside formation is an im-
portant conjugation mechanism in insects.

Much of the literature in the area of insecticide metabolism deals with
the initial oxidative modification of insecticidal chemicals.  Although
many investigators have reported the formation of a variety of glucoside
conjugates, the mechanisms of their formation, their toxicological
significance, and their relationship to important phenomena such as
synergism, insecticide resistance, and the selective nature of insect-
icides have not been investigated.

Because conjugation is a major pathway for toxicants and this pathway is
sensitive to certain chemicals, a detailed study was undertaken to
develop and standardize an in vitro system for glucosylation reactions
in insects.  Tobacco hornworms (Manduca sexta Johan) were routinely used
for insect tissue preparations.  An easily conjugated material, 1-
naphthol, was selected as a suitable substrate for studying the enzyme
responsible for glucosylation in insects.

Methods and Materials

Enzyme assay - Early fifth instar, actively feeding tobacco hornworms
were obtained from a culture maintained on an artificial diet.  The
worms were bled by cutting the horn and the blood collected.  Fat body
and fore-, mid-, and hindgut tissues were collected from the dorsally
opened worms.  The gut contents were removed and all tissues were
homogenized in 5 vol. of Tris-HCl (0.05 M, pH 8.5) buffer.  Crude
homogenates were used to assay the UDP-glucosyl transferase activity.
                                  189

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Later,  low centrifugation  supernatants and other centrifugal fractions
were tested  for activity.  When houseflies were used, whole insects were
homogenized  and utilized as whole body homogenates or fractionated by
centrifugation and then the various fractions assayed for enzyme activity.

The reactions were carried out in 25 ml Erlenmeyer flasks.  Naphthol-1-
  C was  introduced into the flasks in 0.2 ml of chloroform and the
solvent  removed by a gentle jet of air, leaving a uniform layer of sub-
strate  in the bottom of the flask.  Other constituents of the reaction
mixture  were added to the  flask as buffer solutions.  After adding the
co-factor, the flasks were transferred to a metabolic shaker with the
water temperature adjusted to the desired level.  The reactions were
shaken for the desired length of time and the reactions terminated by
the addition of 1 ml of diethyl ether.

Extraction and radioassay - Extraction of unreacted 1-naphthol-l-  C was
accomplished by transferring the reaction mixture into 15 ml glass-
stoppered centrifuge tubes.  The reaction mixtures were extracted twice
with 2 vol. of ether, which gave a recovery of free 1-naphthol of 95
percent.  After combining the ether extracts and drying them with anhy-
drous sodium sulphate, the volume was adjusted to 10 ml  and 1  ml  aliquots
were radioassayed by liquid scintillation counting.  An aliquot of 0.2
ml of the aqueous layer was radioassayed to determine the water soluble
naphthyl glucoside formed.

Ether extracts were concentrated and chromatographed on Chromar 500
sheets and developed two-dimensionally in various combinations of the
following solvent systems:  1:5 ether-hexane; 2:1 methylene chloride-
ethyl  acetate; 4:1 ether-hexane.   The aqueous phase was concentrated and
subjected to beta-glucosidase for 24 hr (Kuhr anct Casida, 1967) and the
reaction mixtures extracted twice with 2 vol. of ether.   The two solvent
phases were radioassayed and the ether layer examined on tic to deter-
mine the nature of the aglycone.
                                 190

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Effect of various chemicals on glucosylation- The chemical  being  tested
for its effect on the conjugating enzyme was introduced into  a  reaction
flask as an ether or buffer solution,  just after the substrate  was
added.  When ether was used, the solvent was evaporated by  using  a  gentle
jet of air.  Other constituents of the reaction mixture were  added  in  the
manner described earlier and then the  reactions carried to  completion.
Changes in the amount of conjugation of 1-naphthol  were determined  and
the degree of reduction of 1-naphthol  glucoside formation was expressed
as percentage inhibition as compared to that of control sample.

Epoxidation of aldrin by tobacco hornworm enzymes - To determine  the
relative effectiveness of certain insecticide synergists on oxidative
and conjugative metabolism, a limited  number of experiments was con-
ducted on the epoxidation of aldrin by hornworm midgut enzyme.   A
typical reaction mixture contained 20  ug of aldrin in 100 ul  of methyl-
cellusolve; 2 umoles of NADPH2 in 0.5  ml of buffer; 2.9 ml  of Tris-HCl
(0.05M) buffer, pH 8.5; 0.5 ml of tobacco hornworm midgut,  5000g
supernatent (200 mg tissue equivalents), and 0.1 ml of ethanol  contain-
ing the desired amount of the inhibitor.  The mixtures were incubated
for 1 hr at 37°C in a metabolic shaker.  At the end of the  incubation
period, the reactions were terminated  by adding 4 ml of 2:3 mixture of
isopropanol-hexane and extracted twice with 2 vol.  of hexane.

The organic solvent phase was analysed for aldrin and dieldrin  by gas
chromatographic means using a Varian Aerograph Model 1700 instrument
equipped with an electron capture detector.  Operating parameters were
as follows:  column: glass, 6 ft x 1/8 in. i.d., packed with  10% DC 200
on Anachrom ABS, 80-90 mesh; carrier gas: nitrogen, 45 ml/min;  temperature:
column 195°C, injection port 200°C, detector 215°C.  Retention  times for
aldrin and dieldrin were 3.8 and 9.6 min, respectively.
                                  191

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 Results  and  Discussion

 Parameters for  conjugation  -  The  conditions  necessary to obtain maximum
 conversion of 1-naphthol-   C  to 1-naphthyl glucoside by tobacco horn-
 worm midgut  enzymes  are  shown in  Table  61.   Under these conditions about
 60  percent of the  1-naphthol  was  conjugated, yielding a material which
 could  not be extracted from the aqueous  reaction mixture with ether.  Tic
 analysis of  the radioactivity extracted  into the ether demonstrated that
 over 95% was the starting material.  Occasionally, 2 minor components
 other  than 1-naphthol were  observed.  However, they were not present in
 quantities sufficient for identification.  It is possible that they were
 diol derivatives of  1-naphthol.

 Treatment of the water-soluble material  with beta-glucosidase or with
 2N  HC1 yielded  a single  ether extractable product identified as 1-naphthol
 Total  recovery  of  the radioactivity originally in the water layer
 exceeded 92  percent.  These data  showed  conclusively that 1-naphthol was
 conjugated directly with glucose  supplied by the cofactor UDP6 without
 undergoing any  other type of  chemical modification.  Because of the
 extraction characteristics  of the conjugate and aglycone, simple
 extraction of the  enzyme reaction mixture with ether, and subsequent
 radioassay of the  two phases,  were all that was required to determine
 the amount of substrate conjugated.  The optimum conditions for conju-
 gation shown in  Table 61 were  established using this technique.

The correct cofactor proved the most critical component in the in vitro
system used  to conjugate 1-naphthol by the hornworm midgut enzyme.   Of
6 different cofactors tested,  only UDPG  supported the conjugation of the
substrate.   Minute quantities  of conjugation observed with the other
substrates were  attributed to  the presence of endogenous UDPG.   This
was supported by the fact that only with UDPG did an increased amount of
cofactor, cause  a corresponding increase in conjugate formation.

Although not as  critical  as the cofactor, the substrate concentration

                                 192

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was an important factor in its conjugation.   Maximum production of naphthyl
glucoside occurred when the 1-naphthol concentration was 2.5 x 10  M.
At levels above this concentration, the rate of glucoside formation was
lessened considerably.
Enhancement of the midgut enzyme activity by the addition of metal  ions
occurred only if the metal was magnesium.  Several  metals, particularly
cadmium, mercury, and zinc, were potent inhibitors  of the conjugating
enzymes when their concentration in the final incubation mixture was as
low as 1.3 x 10" M.  Increasing the concentration of magnesium up to
        _2
2.5 x 10  M resulted in a linear increase in the production of naphthyl
glucoside.  However, higher amounts of the metal inhibited the conju-
gation reaction.  Of the four buffers used in the study, Tris-HCl
supported maximum glucoside formation.  Glycine-sodium hydroxide was
almost as effective, while phosphate and Tris-maleate were the poorest.
With all buffers, the optimum pH was 8.5.  Hence, Tris-HCl, pH 8.5
(0.05M), buffer was used for all standard incubation.

Reacting the enzyme preparations at temperatures from 20 to 79°C
demonstrated that best results were obtained at 37°C.  Glucoside for-
mation was reduced at temperatures less than 30°C or higher than 50°C.
Incubations carried out for varied lengths of time  showed that over
95 percent of the total naphthyl glucoside was formed after only 15 min
of incubation.  Therefore, all standard incubations were made at 37°C
for 15 min in a metabolic shaker.

Stability of the enzyme - Enzyme activities of the  hornworm midgut
enzyme and fat body homogenate were compared during 50 days of
storage.  The enzyme preparations were stored at -20°C as buffer sol-
utions and thawed immediately prior to use.  Although fresh fat body
preparations were more active than the midgut, this activity dropped
precipitously upon storage.  After the initial drop in activity from
the fat body, both the enzyme sources lost activity at a comparable

                                  193

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rate.  When blood, fat body and foregut and hindgut tissues from tobacco
hornworms were compared for naphthyl glucosylation, the fat body was more
active as calculated on the basis of the fresh weight of the tissue.
However, since this activity was reduced considerably by storage and
centrifugation, the midgut was used in this study as the major enzyme
source.

To determine the centrifugal fraction containing the greatest enzyme
activity, homogenates of both tobacco hornworms and adult houseflies were
assayed for naphthyl glucosylation activity (Table 62).  The whole body
homogenates and the 15,000g supernatant fraction of the hornworms were
equally active while the latter fraction from the houseflies was less
active than the crude homogenate.  Further centrifugation showed that
most of the activity was located in the 15,000g pellet of the houseflies.
The activity in the hornworm was contained in the 105,000g soluble
fraction.  That enzyme activity in the 15,000g supernatant from flies
was associated entirely with the microsomal pellet following centrifu-
gation at 105,000g.

Effect of synerqists - A number of synergists were tested for their effect
on naphthyl glucoside formation by tobacco hornworm enzyme (Table 63).
Sulphoxide was the most active of the compounds tested.  Tropital,
MGK-264, and piperonyl butoxide were other compounds found to be inhibitory
         _3
at the 10  M concentration, while Sesamex was a poor inhibitor of conju-
gation.  Other compounds tested (inosafrole, n-propyl  isome, RO-5-1923,
                                                       _o
RO-7-0165, and RO-5-1557) were required in excess of 10  M concentration
for 50 percent inhibition of naphthyl  glucosylation.
Comparison of the ability of these synergists to inhibit a conjugative
and an oxidative reaction was made using the tobacco hornworm midgut
enzyme.  Aldrin epoxidation was selected as the oxidation reaction.   As
evident by the data in Table 63, all the synergists tested were much better
inhibitors of aldrin epoxidation than naphthyl glucosylation.
                                 194

-------
Sulphoxide and piperonyl butoxide were the most effective inhibitors  of
aldrin epoxidation.

This preliminary investigation into glgcosylation in insects had estab-
lished parameters for studying this important metabolic mechanism.
Additionally, it demonstrated that the glucosyl transferase enzyme  is
sensitive to a number of compounds recognized as insecticide synergists.
Although these particular synergists were more potent inhibitors of
aldrin epoxidation, there may be other compounds which would more select-
ively inhibit the conjugation reactions.  It is now possible to more
accurately evaluate the nature of the glucosyl transferase in insects
and to attempt to utilize this system for more desirable methods of in-
sect pest control.
                                  195

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TABLE 61.  COMPOSITION OF THE REACTION MIXTURE USED FOR THE IN  VITRO  CON-
            JUGATION OF 1-NAPHTHOL BY TOBACCO HORNUORM MIDGUT ENZYME
Substrate
Buffer
Metal
Cofactor
Enzyme
Incubation
l-Naphthol-l-14C, 0.1  umole (2.5 x 10"5M)*
Tris-HCl, 0.05M, 8.5 pH, 1.0 ml
MgCl2, 100 umoles in 1.0 ml buffer (2.5 x 10~2M)*
UDPG 1.5 umole in 1.0 ml buffer  (3.8 x 10"4M)*
100 mg equivalent tissue in 1.0  ml buffer
37°C for 15 min, with shaking
  Molar concentration in final  incubation mixture of 4 ml.
                                 196

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TABLE 62.  1-NAPHTHOL GLUCOSYLATION ACTIVITY OF WHOLE BODY EXTRACTS  FROM
                 ADULT HOUSEFLIES AND TOBACCO HORNWORM LARVA

                         	Conjugation of 1-naphthol  (%)
Fraction	Tobacco hornworm	Housefly
Crude homogenate                    50.5                    48.8
15,000g Supernatant                 51.2                    21.1
15,000g Pellet                       7.3                    45.8
105,000g Supernatant                39.9                     6.0
105,000g Pellet                      5.8                    24.3
                                  197

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TABLE 63.  SENSITIVITY OF 1-NAPHTHOL GLUCOSYLATION AND ALDRIN  EPOXIDATION
                    ENZYMES FROM TOBACCO HORNWORM MIDGUT TO
                         SOME INSECTICIDE SYNERGISTS

                          Molar concentration for 50% Inhibition of	
Compound	1-Naphthol conjugation	Aldrln epoxidation
MGK-264                     5.5 x 10"3                 1.0 x TO"5
Piperonyl butoxide          6.3 x 10"3                 4.4 x 10~6
Sesamex                     1.0 x 10"2                 7.5 x 10"5
Sulphoxide                  1.2 x 10"3                 8.8 x 10"6
Tropital                    5.3 x 10"3                 3.0 x 10"5

a Same as in Table 61  except that UDPG was replaced with 2 umoles of
  NADPH2 and 1-naphthol  was substitured by 20 ug of aldrin and  incu-
  bated for 1 hr.
                                  198

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Factors Influencing Conjugation

Having established that conjugation is a major metabolic pathway for
carbamates in animals, a study was undertaken to further characterize the
conjugative enzymes in various mammals and insect species using a well
defined in vitro conjugating system.  Also, the effect of age, sex,
resistance and toxicity of other chemicals on conjugation was evaluated.

Methods and Materials

Animals - Mammalian livers were obtained from St. Louis Serum Company,
114-120 St. Claire Ave., East St. Louis, Illinois or removed from rats
maintained in the laboratory.  The multi-resistant and susceptible
strains of the housefly were obtained from the United States Department
of Agriculture, Entomology Research Division, Gainesville, Florida.

In vivo metabolism of 1-naphthol by houseflies - Adult houseflies, 5 days
old, were anesthized with carbon dioxide and then treated topically with
1-naphthol-l-  C at a rate of 0.022 ug/per fly.  This amounted to about
5,085 dpm per insect and was adequate for evaluating conjugation since
100 flies were processed as a single replicate.  A detailed examination
of 1-naphthol conjugates was conducted 4 hours after treatment of the
flies.

Radioactivity in the flask (excreta) plus the radioactivity remaining on
the surface of the flies was removed by rinsing the flask and insects
several times with water and then with ether.  Free 1-naphthol in the
ether layer was determined and the 1-naphthol conjugates in the water was
evaporated to dryness, dissolved in a small amount of methanol, and
spotted on tic.  Internal radioactivity was extracted by homogenizing
the treated flies in a 1:1 acetone-water mixture.  The filtered extract
was analyzed in the manner described for the excreta.

In vitro glucosylation of 1-naphthol - Various insect species were used

                                  199

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as an enzyme  source to compare their ability to conduct glucosylation
reactions.  The standard enzyme for glucosylation was prepared by
homogenizing  the whole insect in 0.05 M Tris-HCl buffer, pH 8, to yield
a 20% (w/v) homogenate.  The homogenate was filtered through two layers
of cheesecloth to remove gross debris and the filtrate homogenized in a
Potter Elvehyem type cell homogenizer.  The crude homogenate was cen-
trifuged at 5,000g for 10 minutes using a Beckman Model L2-65 untra-
centrifuge and, the supernatant was used as the enzyme source.

The conjugative abilities of different insect tissues also were examined.
Early fifth instar, actively feeding tobacco hornworm larvae were bled
by cutting the horn and the blood collected.  Fat body, midgut, and
malphigian tubules were collected from the dorsally opened worms.  The
gut contents  were removed and all tissues were homogenized in 5 volumes
of Tris-HCl (0.05 M, pH 8) buffer, centrifuged, and the 5,000g super-
natant assayed for enzyme activity.

The distribution of glucosyltransferase in subcellular fractions of
housefly whole-body homogenates was examined by separating the homogenates
into various  centrifugal fractions.  The first fraction assayed was the
crude homogenates, while the second and third fractions consisted of
the supernatant of the crude homogenates after centrifuging at 5,000g
for 10 minutes.  The precipitates after dispersal in a volume of Tris-
HCl buffer that made the suspension equivalent to the original 20%
homogenate were assayed also.  The next fractions were prepared by
centrifuging  the crude homogenates at 15,000g for 30 minutes and analy-
zing both the supernatant (microsomes + solubles) and the precipitate
(mitochondria).  Two other fractions were prepared by centrifuging the
15,000g supernatant at 105,000g for 1 hour.  The supernatant obtained
after centrifugation at 105,000g was used as an enzyme source, as was
the particulate fraction (microsomes).  All procedures were conducted
at 0-4°C to avoid loss of enzyme activity.

A typical incubation mixture contained 1.3 x 10"  micromoles of
                                 200

-------
radiolabeled 1-naphthol (44,000 dpm per incubation); 100 mg equivalents
of insect tissues in 0.5 ml buffer; 2.9 ml of 0.5 M Tris-HCl buffer
(pH 8); 0.1 ml (2 mg) of MgCl2; and 0.5 ml (1 mg) of UDPG.   The molar
concentrations for MgCl2 and UDPG in the final 4 ml incubation volume were
2.5 x 10"3 M and 4.12 x 10~4, respectively.

The 1-naphthol, in organic solvent, was transferred to a 25 ml Erlenmeyer
glask, and the solvent evaporated with care to deposit 1-naphthol uni-
formly over the bottom of the flask.  The other constituents were added
and the flasks were transferred to a metabolic shaker with the water
temperature at 37°C.  The cofactor was added and the incubation time,
15 minutes, was recorded from that point.  Reactions were terminated
by the addition of 2.0 ml of diethyl ether to the flask.

Unreacted 1-naphthol-1-  C from the enzyme preparations was extracted by
transferring the reaction mixture to a 15 ml glass-stoppered centrifuge
tube and extracting once with 10 ml of diethyl ether.  Further extraction
was not required since over 95% of the free 1-naphthol was recovered.
Volumes of both the ether and the water layers were recorded and aliquots,
1 ml ether and 0.2 ml water, were radioassayed.

In vitro glucuronide conjugation of 1-naphthol - Animal tissues were
weighed and sufficient Tris-HCl buffer (pH 7.0) to yield a 25% (w/v)
tissue homogenate was added, then the tissue was cut into small pieces
and homogenized in a Virtis homogenizer for 4 minutes.  The homogenate
was further homogenized until the cells were completely broken.

Various subcellular fractions were  isolated from the homogenate by
differential centrifugation.  The crude homogenate was first centrifuged
at 15,000g for 30 minutes and the supernatant decanted into clean
centrifuge tubes and spun at 105,000g for 60 minutes.  The microsomal
pellet was homogenized  in Tris-HCl  buffer of  sufficient volume to
achieve reconstitution  to the original 25% homogenate.  Therefore,  1 ml
                                  201

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 of  the microsomal  suspension  contained the microsomes from 250 mg of
 tissue.  All  the  above  operations were carried out at 0-4°C.

 For incubation, 125 mg  equivalents of rat tissue in 0.5 ml buffer; 2.9
 ml  of 0.05 M  Tris-HCl buffer  (pH 7); 0.1 ml  (2 mg) of MgCK; and 0.5 ml
 (1  mg) of UDPGA were added to a 25 ml Erlenmeyer flask containing
         _o
 1.3 x 10  micromoles of  the  labeled 1-naphthol.  The molar concentrations
                                                               o
 for MgClp and UDPGA in  a  final incubation volume were 2.5 x 10  M and
 3.9 x 10 M respectively.  The preparations were incubated at 37°C for
 15  minutes.

 Metabolite identification - The conversion of 1-naphthol to water soluble
 products was  used  as a  general indication of the conjugative enzyme
 activity in both  in vivo and  in vitro studies.  Therefore, the water
 phase of the  enzyme preparations and of the extract of houseflies and
 excreta were  radioassayed after extraction with organic solvent.  The
 percentage conjugation  of the applied 1-naphthol was calculated from
 these data.

 From more critical evaluation of the nature of the water soluble pro-
 ducts, these  materials were analyzed by tic.  Approximately 50 ug of
 1-naphthol glucoside, 1-naphthol  sulfate and 1-naphthol  standards were
 added to the  water soluble conjugates which were dissolved in methanol.
 The mixture was applied to Chromar tic sheets and developed first in
 hexane; this  step did not move any radioactivity from the origin but did
 remove fats and other interferring materials.  The tic was then develop-
 ed  in a 5:1 mixture of chloroform  and methanol  containing 10% acetic
 acid.  A 25%  aqueous solution of sulfuric acid was sprayed on the
 plates to detect the nonlabeled standards, and the radioactive areas
were located  by radioautography.   The radioactive conjugates were ex-
 tracted from  the tic plates and quantitated by liquid scintillation
counting.

Stability of  the enzymes during storage - Glucosyltransferase activity

                                 202

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in the housefly and cockroach were assayed after storage at various
temperatures.  The form of storage was either as the whole insects at
-20°C after which the 5,000g supernatant was prepared, or the 5,000g
supernatant stored at -20°, 0°, and 25°C.  The frozen samples were
stored as small individual preparations to avoid loss of activity during
thawing and refreezing.  For each analysis 100 mg equivalent of tissue
were used.

The rat liver, kidney and intestine were stored at -20°, 0° and 25°C
either as whole tissues in Tris-HCl buffer and the microsomal enzymes
then prepared, or the microsomal pellet was prepared from livers of
freshly killed rats and stored.  Individual, rather than pooled samples
were stored to avoid loss of activity during thawing and refreezing.
The effect of storage at -20°C on the 15,000g supernatant enzyme pre-
paration of rat kidney, lung, small intestine, caceum, large intestine,
heart, stomach, fat and brain also was determined.

Oxidative metabolism - The method used by Hart and Fouts (1965) to
measure the rate of TPNH oxidation by the rat liver microsomal enzymes
was employed.  The cuvet used as.a blank contained 40 mg equivalents of
tissue in a total volume of 3 ml of 0.05 M Tris-HCl buffer, pH 7.4.
The  cuvet used for oxidase assay contained 0.36 umole TPNH, and 40  mg
equivalents of tissue in a total volume of 3 ml of 0.05 M of Tris-HCl.
The optical density was measured at 340 mu in a model DV Beckman
spectrophotometer.

Sensitivity of glucuronyltransferase to various insecticides - DDT,
carbaryl, parathion, tedion, dipterex and disulfoton were evaluated  for
their effects on rat liver microsomal glucuronyltransferase activity.
The insecticides were introduced into a reaction flask as an acetone
solution just after the substrate was added.  The acetone was evaporated
using a gentle jet of air and other constituents of the reaction mixture
were added and the reactions carried to completion.  Sufficient quantities
                                  203

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of each insecticide were added so that their concentrations in the incu-
                       -5    -4       -3
nation mixtures were 10  , 10   and 10   molar.
Results and Discussion

Stability of glucosyltransferase during storage - The effects of storage
on glucosyltransferase activity in the housefly showed that the enzymes
stored as a 5,000g supernatant at -20° and 0°C were stable throughout
10 days storage period.  When the enzyme was stored at 25°C, the
glucosyltransferase enzymes retained 90% of its initial activity after
24 hrs but then the activity declined rapidly.  The preparation from
cockroaches which were allowed to feed until the time the enzyme was
prepared lost 53% of its activity in 4 days and 100% after 7 days,  whereas
in cockroaches not fed for 24 hrs before preparing the enzyme, there was
only 12% loss after 4 days and 27% loss of activity after 7 days.

The stability of the housefly TPNH oxidase activity was examined using
the 5,000g supernatant prepared from a whole body homogenate after  the
insects were stored for various periods of time.  In this case, about
25% of the initial activity was lost after 10 days of storage.  However,
an increase in the enzyme activity, a maximum of 45% during the first
24 hrs of storage was observed.

In vitro glucosylation by different insect species - The enzyme activity
was assayed using a 5,000g supernatant of tobacco hornworm larvae,
Manduca sexta (Lepidoptera), housefly, Musca domestica (Diptera),
Indian meal moth, Plodia interpunctella (Lepidoptera), alfalfa weevil,
Hypera postica (Cleoptera) and American cockroach, Periplaneta americana
(Orthoptera).   The results outlined in Table 64 indicate that: - (1) Each
of these insects were capable of conjugating about 77% of 1-naphthol to
form 1-naphthol  glucoside.  No other metabolites were detected.  (2) The
5,000g supernatant of the head, thorax and abdomen of houseflies were
individually as  effective in conjugating 1-naphthol  as the whole body
                                 204

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homogenate.  (3) The conjugative activity of various tissue preparations
from the tobacco hornworm larvae was similar to that of a whole body
homogenate.

Examination of glucosyltransferase activity in subcellular fractions of
housefly homogenates showed that crude homogenates of the whole body,
the 5,000g and 15,000g supernatants as well as the 5,000g, 15,000g and
105,000g precipitates were all equally active in conjugating 1-naphthol
(Table 65).  With these preparations, from 67 to 75% of the 1-naphthol
was conjugated.  The corresponding value for the 105,000g supernatant
was 44.6%.

Influence of age, sex and insecticide resistance - The glucosyltransferase
enzyme was assayed using the 5,000g supernatant prepared from the resistant
and susceptible houseflies at various stages of development.  Eggs of
both strains were high in glucosyltransferase activity while the enzyme
activity was lower in 3- and 5-day-old housefly larvae (Table 66).  The
level of glucosyltransferase activity in the pupal stage was comparable
to that in the eggs and in the adult flies.  Neither age, sex or
insecticide resistance had any influence on the conjugation of 1-
naphthol by the adult insects.

The fate of the radiocarbon after topical application of 1-naphthol-l-
  C to susceptible and resistant houseflies is shown in Table 67.  The
data show that both strains of flies excreted the 1-naphthol very
rapidly, with 50 to 60% of the dose eliminated by 4 hours.  Although
the individual metabolites of 1-naphthol in the excreta and flies
differed quantitatively, it does not appear that insecticide resistance
vastly effected overall conjugative metabolism in the housefly.  It was
established that glucosylation is an important in vivo metabolic
reaction as was indicated by the earlier in vitro studies.  After 4 hours,
for example, approximately 13% of the applied 1-naphthol had been
converted to the glucoside derivative.

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Conjugation by different animal species - The conjugative enzyme was
assayed using liver microsomes of various animal species (Table 68).
The results showed that the guinea pig, chicken and hamster livers were
more efficient in conjugating 1-naphthol than other livers.  With these
animals, over 85% of the added 1-naphthol was conjugated.  The enzymes
from the other animals conjugated less of the 1-naphthol but all
showed very active enzyme activity.  The distribution of glucuronyl-
transferase in several rat tissues other than the liver was examined.
Data in Table 69 show that the liver and kidney had the greatest
glucuronyltransferase activity per unit weight.  The lung IS.OOOg super-
natant formed the glucuronide in quantities almost equal to those from
the liver and kidney.  The activity of the glucuronide transferase in
the alimentary tract was less active than that in the liver, kidney and
lungs.

The conjugative enzyme activity in the alimentary tract seemed to be
dependent on the presence or absence of the mucosa in tissues used for
enzyme preparation.  For example, small intestine, caceum and large
intestine, which have large amounts of mucosa, gave higher activity
than those found in the stomach, which had less mucosa.  Washing the
inside of the intestine until most of the mucosa was removed, reduced
the enzyme activity by about 20%.  This indicated that most of the
enzyme activity was located in the mucosa and not in the muscular wall.

Stability of the glucuronyltransferase enzymes during storage - The
effects of storage on the conjugative enzyme activity in rat liver,
kidney and intestine stored as whole tissues and as microsomal pellets
showed that the enzyme activity of rat liver and kidney was very stable
during storage at -20°C.   None of the activity of the enzymes was lost
after 14 days of storage, and less than 10% of the original enzyme
activity was lost after 90 days of storage.

Enzymes from rat intestine were not so stable and, depended to a certain

                                 206

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extent on the type of preparation used for storage.   For example,  the
most stable preparation was the microsomal enzymes  from the  stored
intact intestine, as only 10% of the enzyme activity was lost  after  14
days of storage at -20°C.  Almost no activity remained  after 90  days.
On the other hand, enzymes stored as microsomal  pellets were consider-
ably less stable, with 90% of the enzyme activity lost  after 14  days
of storage.

The stability of the glucuronyltransferase enzyme prepared as  a  15,000g
supernatant from lungs, caceum, large intestine  were considerably  less
stable than that in the liver and kidney (Table  69)  with 30  to 40%
of the enzyme activity lost after 10 days of storage.  The small in-
testine, heart, stomach, fat and brain enzyme activity  dropped 10  to
30% of the initial activity after 5 days and to  5 to 20% after 10  days
of storage at -20°C.

The stability of enzymes in a 15,000g supernatant of rat liver when
stored at various temperatures showed that none  of the  enzyme  activity
was lost after a week of storage at -20°C, and that only about 5%  of
the activity was lost after 10 days of storage at 0°C.   When the
enzymes were stored at 25°C, 89% of the initial  activity was retained
after 12 hours, 50% after 24 hours and only 8% after 2  days.

Sensitivity of rat liver glucuronyltransferase enzymes  to insecticides  •
Several common insecticides were evaluated for their effect  on the
glucuronide transferase enzyme using the rat liver microsomal  enzyme.
The results of these were entirely negative.  None of the insecticides
inhibited the conjugating enzymes regardless of  the level of pest-
icide added to the system.
                                  207

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TABLE 64.  GLUCOSYLATION OF 1-NAPHTHOL-1-14C BY THE 5,000g SUPERNATANT
                  OF DIFFERENT INSECTS AND INSECT TISSUES3
Enzyme preparation	Percent conjugation of 1-naphthol
Housefly
  Whole body                                     77.1
  Head                                           77.3
  Thorax                                         80.6
  Abdomen                                        74.5
Tobacco Hornworm Larvae
  Whole body                                     79.4
  Whole body, less midgut                        78.1
  Midgut                                         72.6
  Malpigian tubules                              79.5
  Fat body                                       78.0
  Haemolymph                                      5.3
American Cockroaches
  Whole body                                     75.9
  Whole body, less midgut                        77.9
  Midgut                        .                 77.8
Indian Meal Moth
  Whole body                                     77.4
Alfalfa Weevil Larvae
  Whole body                                     76.1

  Enzyme from 100 mg of tissue was used for each analysis.
                                  208

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TABLE 65.  GLUCOSYLATION OF 1-NAPHTHOL-1-14C BY SUBCELLULAR FRACTIONS
                        OF A  HOUSEFLY HOMOGENATE3
Centrifugal fraction	Percent conjugation
Crude homogenate                                       68.7
5,OOOg supernatant                                     71.0
5,000g precipitate                                     66.7
15,OOOg supernatant                                    70.4
15,OOOg precipitate                                    72.0
105,OOOg supernatant                                   44.6
105,OOOg precipitate                                   74.6

a Enzyme from 100 mg of tissue was used for each analysis.
                                  209

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TABLE 66.  THE EFFECT OF AGE AND SEX ON THE CONJUGATION OF 1-NAPHTHOL BY
                     SUSCEPTIBLE AND RESISTANT HOUSEFLY
                      HOMOGENATES, 5,000g SUPERNATANT3

Enzyme
source
Eggs
Larvae,
Larvae,
Pupae
Adults
1 day
3 days
5 days
7 days
Percent
Susceptible

3 days old
5 days old









77.4
61.3
41.6
78.1
Female
old
old
old
old
11 days old
77
77
74
79
77
.9
.8
.0
.8
.9





flies








Male
81
77
79
79
78
.6
.2
.2
.6
.4
conjugation

Resistant




77.7
71.8
58.3
79.2
Fema 1 e
78.
78.
77.
81.
78.
0
7
5
6
8


flies








Male
76
77
77
78
76
.6
.1
.7
.7
.6
  Enzyme from 100 mg of tissue was used for each analysis.
                                  210

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TABLE 67.  FATE OF TOPICALLY APPLIED 1-NAPHTHOL-1-14C IN SUSCEPTIBLE AND
                     RESISTANT HOUSEFLIES AFTER 4 HOURS

Metabolites
Excreta
1-naphthol
1-naphthylglucoside
Unknown I
Unknown II
Unknown III
Total
Houseflies
1-naphthol
1 -naphthyl gl ucoside
Unknown I
Unextractables
Total
Total recovery
Percent
Susceptible

24.0
7.4
5.8
10.0
6.2
61.6

3.4
5.7
1.4
5.2
15.7
77.3
of applied dose
Resistant

15.5
10.7
TO.l
3.7
4.6
53.8

1.1
2.6
2.4
6.9
13.0
66.8
                                  211

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TABLE 68.  CONJUGATION OF 1-NAPHTHOL BY THE MICROSOMAL FRACTIONS OF
                      LIVERS FROM DIFFERENT ANIMALS3

Source of
liver enzyme
Guinea Pig
Hamster
Chicken
Rat
Sheep
Pig
Quail
Cow
Rabbit
Dog
Fetal Pig
Percent
conjugation
90.4
85.7
84.9
76.6
76.9
78.8
74.5
73.4
70.4
62.4
57.4
umoles 1-naphthol
conjugated/g tissue/
hr. x 10"2
3.8
3.6
3.5
3.2
3.2
3.3
3.1
3.0
2,9
2.6
2.4
a Enzyme from 125 mg of tissue was used for each analysis.
                                  212

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TABLE 69.  COMPARATIVE GLUCURONYLTRANSFERASE ACTIVITY IN THE 15,000g
              SUPERNATANT OF VARIOUS RAT TISSUES AND EFFECT OF
                    STORAGE AT -20°C ON THE ENZYME ACTIVITY3


Source
of
Enzyme
Kidney
Liver
Lung
Small intestine
Caceum
Large intestine
Heart
Stomach
Fat
Brain
Percent conjugation
of
1-naphthol
0 time
87.6
84.4
80.5
70.1
67.5
66.0
55.0
54.5
56.0
54.5
Percent of
initial activity
after
5 days
97.5
97.9
69.2
14.6
69.2
62.5
14.2
18.4
33.9
23.3
storage for:
10 days
97.7
94.9
61.1
13.4
65.6
68.0
10.4
8.1
26.1
18.2
  Enzyme from 125 mg of fresh tissue used for each analysis.
                                  213

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Synthesis  of Glycosi'des

Chemical syntheses  and acute  toxicity  of  the beta-D-O-glucosides of
carbaryl metabolites, 4-(N-methylcarbamoy1-oxy)-1-naphthy1-beta-D-
glucopyranoside  and 5-(N-methy1carbamoyloxy)-l-beta-D-g1ucopyranoside
are  reported.  The  preparation  of  their respective decarbamylated
products,  4-hydroxy-l-naphthyl-beta-D-glucopyranoside and 5-hydroxy-l-
naphthyl-beta-D-glucopyranos'ide are also  reported.  These syntheses can
provide material for toxicological evaluation and will enable identifi-
cation of  the  intact plant conjugates  without resorting to hydrolysis
of the glycones.  The synthetic conjugates may serve as standards to
aid  in the determination of the sugar  moiety of carbaryl plant conju-
gates, an  important consideration  which up to this time has been lacking.

Methods and  Materials

Chemicals  -  1,4-Naphthalenediol and 1,5-naphthalenediol were purchased
from Eastman Kodak  Company.  4- and 5-Hydroxycarbaryl were synethesized
by the reaction of  the corresponding naphthalenediol with methyl iso-
cyanate (Knaak et al., 1965).   Beta-D  (+) Glucose pentaacetate was
purchased  from Sigma Chemical Company, and boron trifluoride ether
complex (98%) was purchased from Matheson Coleman and Bell.  Methyl
tetra-0-acetyl-beta-D-glucopyranuronate was synthesized by the reaction
of a mixture of glucuronolactone,  sodium methoxide and methanol with
acetic anhydride and perchloric acid (Bollenback et al., 1955).
Anhydrous methanol  was prepared by the distillation of pesticide
quality methanol over magnesium turnings  (Fieser, 1957).  Benzene used
in the syntheses was dried over sodium.

Chrpmatography - Thin-layer chromatography was used to follow the pro-
gress of all  reactions, and to determine the purity of reaction products.

Silica gel  F-254 precoated plates  (0.25 mm thickness, Brinkman Instruments,
Inc., Westbury, New York) developed in either benzene-ether (7:3) or
                                 214

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petroleum ether-chloroform-ethanol (7:2:1) were used to separate 4- and
5-hydroxycarbaryl glucoside tetraacetates from their respective start-
"ing materials and other reaction products.  The R-: values for the 4-
and 5-hydroxycarbaryl glucoside tetraacetates were identical in the
benzene-ether system (Rf = 0.16) and in the petroleum ether-chloroform-
ethanol mixture  (Rf = 0.46).  Spots were detected by visualization
under ultraviolet, and by spraying with 10% methanolic sulfuric acid
with subsequent  baking at 140° for three minutes.

The glucosides of 4- and 5-hydroxycarbaryl were chromatographed on
aluminum oxide F-254, Type T, precoated plates (0.25 mm thickness,
Brinkman Instruments, Inc., Westbury, New York) developed in chloroform-
methanol-acetic  acid (75:15:10).  The Rf values for the 4- and 5-hydroxy-
carbaryl glucosides in this system were also identical; Rf = 0.49.  How-
ever, upon spraying the chromatograms with 10% methanolic sulfuric acid
and baking them  at 140°, the 4-hydroxycarbaryl glucoside stained a dark
gray.  The glycosides of 4- and 5- hydroxycarbaryl isolated from bean
plants had identical R-: values on silica gel plates developed in
chloroform-methanol-water (65:25:4) (Mumma et al., 1971).

Instrumentation  - The infrared data were obtained using KBr pellets with
a Beckman IR5A infrared spectrophotometer.  the NMR spectra were
determined with  a Varian Model T-60 spectrometer using tetramethylsilane
as the internal  standard.  The mass spectral data were recorded with a
Finnigan Series  1015C mass spectrometer at 70 ev.  The high resolution
mass spectral data were obtained on a Hitachi RMU-7 mass spectrometer.
Specific rotations were determined with a Bendix ETL-NPL Automatic
Polarimeter, Type 143A.  Melting points were determined with an
Electrothermal capillary melting point apparatus and are uncorrected.
Elemental analyses were performed by Galbraith Laboratories, Inc.,
Knoxville, Tenn.

Syntheses

                                 215

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Preparation of 4-hydroxycarbaryl glucoside tetraacetate (I) and 5-
hydroxycarbaryl glucoside tetraacetate  (II)-  (1).  4-(N-Methylcarbamoyl-
oxy)-l-naphthyl-tetra-0-acetyl-beta-D-glucopyranoside (I).  A mixture
of 7.2 g  (0.033 mol) of 4-hydroxycarbaryl, 11.7 g (0.03 mol) of beta-D
(+) glucose pentaacetate, 0.38 ml  (0.003 mol) of boron trifluoride ether
complex (98%), and 350 ml of anhydrous  benzene was refluxed with stirring
for 2 hr.  The dark red solution was cooled to 4° and the excess 4-
hydroxycarbaryl was removed by filtration.

The cooled filtrate was deacidified with a cold solution of 0.5 N sodium
hydroxide (2 x 75 ml).  The organic layer was separated, washed with
ice water (4 x 100 ml), dried over magnesium  sulfate, and concentrated.
The dark red syrupy residue was dissolved in  40 ml of hot ethanol.  The
solid which formed on standing was filtered to give 8.70 g of a mixture
of (I) and glucose pentaacetate, mp 95-125°.  The mixture was stirred
for 2 hr with 400 ml of diethyl ether in order to extract the unreacted
glucose pentaacetate.  Filtration gave  3.14 g (19%) of (I), mp 165^169°.
A single recrystallization from ethanol gave  2.94 g (18%) of (I) as
tiny, off-white needles, mp 169-171°.   (2).   5-(N-Methylcarbamoyloxy)-
1-naphthyl-tetra-O-acetyl-beta-D-glucopyranoside (II).  A mixture of
7.2 g (0.033 mol) of 5-hydroxycarbaryl, 11.7  g (0.03 mol) of beta-D (+)
glucose pentaacetate, 0.38 ml (0.003 mol) of  boron trifluoride ether
complex (98%), and 350 ml of anhydrous  benzene was refluxed with
stirring for 1 hr.  The dark red mixture was  allowed to cool to room
temperature and filtered to give 2.0 g  (28%)  of crude unreacted 5-
hydroxycarbaryl, mp 155-165°.  Recrystallizaiton from ethyl acetate/
hexane gave 1.51 g (21%) of 5-hydroxycarbaryl, mp 166-168° [lit. mp
166-167° (Knaak et al., 1965)].

The filtrate from the reaction mixture was cooled, washed successively
with a cold solution of 0.5 N sodium hydroxide (2 x 75 ml) and ice water
(4 x 100 ml), then dried over magnesium sulfate, and concentrated.  The
yellow syrupy residue crystallized upon addition of 1  1.  of diethyl
ether.  Filtration afforded 4.20 g of a colorless solid which was

                                 216

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recrystallized from ethanol  to give 3.84 g (23%) of (II)  as tiny,  color-
less needles, mp 165-168°.

Evaporation of the diethyl ether filtrate gave 7.55 g (65%) of crude
unreacted glucose pentaacetate, mp 115-122°C.   Recrystallization from
ethanol gave 5.60 g (48%) of glucose pentaacetate, mp 129-130° [lit.
mp 134° (Weast, 1968)].

Preparation of 4-hydroxycarbaryl glucoside (III) and 5-hydroxycarbaryl
glucoside (IV) - (1).   4-(N-Methylcarbamoyloxy)-l-naphthyl-beta-D-
glucopyranoside (III).  A mixture of 2.62 g (0.0048 mol)  of 4-hydroxy-
carbaryl glucoside tetraacetate (I) and 300 ml of anhydrous methanol
was cooled to 4°, and 1.04 ml (0.00048 mol) of a 0.463 N  barium
methyl ate solution (Mitchell, 1941) was added.  The reaction temperature
was maintained at 4°,  and after 7 hr of intermittent shaking a yellow
solution was obtained.  The solution was neutralized by the addition of
an exact equivalent of standard IN sulfuric acid (0.48 ml).  Removal
of the almost colloidal barium sulfate which formed was facilitated by
the addition of charcoal followed by filtration through Celite.

Evaporation of the filtrate left a syrupy residue which solidified upon
being stirred with diethyl ether (350 ml).  Filtration gave 1.45 g  (80%)
of (III), mp 204-208°.  Two recrystallizations from methanol/diethyl
ether (1:2) afforded 0.63 g (35%) of (III) as off-white crystals,  mp
216-218°.  (2).  5-(N-Methylcarbamoyloxy)-l-naphthy1-beta-D-
glucopyranoside (IV).   A mixture of 1.12 g (0.00205 mol)  of 5-hydroxy-
carbaryl glucoside tetraacetate (II) and 70 ml of anhydrous methanol
was cooled to 4°, and 0.44 ml (0.000205 mol) of a 0.463 N barium
methylate solution was added.  The reaction temperature was maintained
at 4°, and after 3 hr of intermittent shaking a light pink solution
was obtained.

The reaction mixture was processed in a manner similar to that described
above for 4-hydroxycarbaryl glucoside (III); 0.57 g &73%) of  (IV)  as
                                 217

-------
 a colorless solid, mp 178-182°, was obtained.   A single recrystallization
 from methanol/diethyl ether (1:3)  gave 0.31  g  (40%)  of (IV)  as  off-
 white crystals,  mp 197-200°.

 Preparation of 4-hydroxynaphthyl glucoside (V)  and 5-hydroxynaphthyl
 glucoside (VI) - (1)  4-Hydroxy-l-naphthyl-beta-O-glucopyranoside  (V).
.A solution of 0.50 g (0.0013  mol)  of 4-hydroxycarbaryl  glucoside (III),
 20 ml  of methanol, and 4.8 ml  (0.0013 mol) of a 0.277  N barium  hydroxide
 solution was kept at room temperature for 1/2 hr.  The solution was
 neutralized with. 0.71 ml  (0.0014 mol) of a 2.02 N  oxalic acid solution.
 The mixture was  cooled to 4°  to allow for complete precipitation of  the
 barium oxalate.   Charcoal  was  added and the  cooled mixture was  filtered
 through Celite.   The filtrate  was  evaporated to give 0.42 g  of  a brown
 solid, mp 235-240°.   A single  recrystallization from methanol gave 0.13 g.
 (2).   5-Hydroxy-l-naphthyl-beta-D-glucopyranoside  (VI).   A solution  of
 0.50 g (0.0013 mol)  of 5-hydroxycarbaryl  glucoside (IV),  20  ml  of
 methanol, and  4.8 ml  (0.0013 mol)  of a 0.277 N  barium  hydroxide sol-
 ution  was kept at room temperature for 1/2 hr.   The  reaction mixture was
 processed in the same manner as described above for  4-hydroxynaphthyl
 glucoside (V); 0.45  g of  a tan solid, mp 224-228°, was  obtained.
 Recrystallization from methanol  gave 0.23 g  (53%)  of (VI) as tan
 crystals, mp 232-234°.  A second recrystallization from isopropanol  gave
 the analytical sample,  mp 236-239°.

 Preparation of methyl  [5-(N-methylcarbamoyloxy)-l-naphthyl-tri-0-acetyl-
 beta-D-glucopyranosid]  uronate (VII)- A mixture of 5.20  g (0.024 mol) of
 5-hydroxycarbaryl, 7.52 g (0.020 mol) of methyl  tetra-0-acetyl-beta-D-
 glucopyranuronate, 0.28 ml  (0.0022 mol)  of boron trifluoride ether
 complex (98%), and 250  ml  of anhydrous benzene  was refluxed  with
 stirring for 16  hr.   The  dark  red  mixture was cooled to 4° and  the un-
 reacted 5-hydroxycarbaryl  was  filtered.

 The filtrate was washed successively with a  cold solution of 0.5 N
 sodium hydroxide (2  x 60  ml) and ice water (4 x  100 ml),  then dired over
                                 218

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magnesium sulfate, and concentrated.  The residue crystallized upon
addition of 50 ml of diethyl ether and was filtered to give 47. g of
an off-white solid.  Tic (silica gel; 7:3 benzene-ether) showed this
solid to a mixture of methyl tetra-0-acetyl-beta-D-glucopyranuronate
and (VII).  The solid was dissolved in 50 ml of acetone, and 6 g of
Florisil (60/100 mesh) was added.  The mixture was concentrated to
dryness by rotary evaporation and placed in a glass column containing
155 g of Florisil.  The column was eluted with 2 1. of diethyl ether.
The ether eluate was evaporated to give 2.5 g (33%) of unreacted methyl
tetra-0-acetyl-beta-D-glucopyranuronate, mp 171-175° [lit. mp 176.5-
178° (Bollenback et al., 1955)].  Further elution of the column with
1.2 1.  of a solution of 10% acetone in benzene, followed by evaporation
of the eluate gave 1.77g (16%) of VII, mp 167-172°.  Recrystallization
of a small amount of this solid from isopropanol gave the analytical
sample, as off-white crystals, mp 174-177°.

Toxicity studies - The acute toxicity of 4- and 5-hydroxycarbaryl and
their beta-D-0-glucosides to male white mice (Swiss-Webster, 20 g) was
evaluated by intraperitoneal injections in 0.1 ml dimethylsulfoxide.
The animals were observed for 3 weeks after treatment.

Results and Discussion

To synthesize beta-D-0-glucosides of 4- and 5-hydroxycarbaryl, the N-
methylcarbamoyl group in 4- and 5-hydroxycarbaryl was conveniently used
as a protecting group.  This was necessary since the condensation of
1,5-naphthalenediol with beta-D (+) glucose pentaacetate and catalytic
amounts of boron trifluoride ether complex in anhydrous benzene at
room temperature for 4 days gave the diglucoside of 1,5-naphthalenediol
in low yield.  The diglucoside was identified by its ir and nmr spectra.

The boron trifluoride method was evaluated since it had been used in
trace amounts as a catalyst for the condensation of beta-D (+) glucose
pentaacetate with various phenols (Bretschneider and Beran, 1949).  For

                                  219

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example,  1-naphthyl-beta-D-glucoside tetraacetate was obtained in 58%
yield when  the reaction was carried out  in benzene at room temperature
for  two days.  In the current study the.  reaction of 5-hydroxycarbaryl
with beta-D (+)  glucose pentaacetate and catalytic amounts of boron
trifluoride ether complex  in anhydrous benzene at room temperature for
5 days gave an 8% yield of 5-hydroxycarbaryl glucoside tetraacetate
(II).  Since the yield was low, the separation of (II) from the starting
material, glucose pentaacetate, was difficult.  It was subsequently
found that  by refluxing the reaction mixture for 1 hr a 23% yield of
(II) could  be obtained; refluxing for longer periods of time, up to
24 hr, did  not improve the yield.  This modification, besides almost
tripling  the yield, significantly reduced the reaction time, and also
permitted the facile removal of glucose pentaacetate from (II) by its
extraction  with diethyl ether.  4-Hydroxycarbaryl glucoside tetraacetate
(I)  was obtained in a similar manner from 4-hydroxycarbaryl in 19% yield.
(I)  and (II) were identified by their elemental analyses, and by their
ir,  nmr, and mass spectra.

The  ir spectra of (I) and  (II) were consistent with the proposed
structures.  Although these spectra were similar, they were distinguished
by the out-of-plane CH bending vibrations of the naphthyl ring.  The
frequency of the CH out-of-plane vibration is determined by the number
of adjacent  hydrogens on the ring (Williams and Fleming, 1966).  There-
fore, the ir spectra of (II) contained a single strong absorption at
791  cm, while the ir spectra of (I) gave several absorptions at 839,
778  and 766 cm"  due to this vibration.   The nmr spectra of (I) and
(II) were also consistent with the proposed structures.   These spectra
were taken  in acetone-d5, and upon shaking with deuterium oxide the NH
resonance dissappeared and the N-methyl  group resonated as a singlet.

The mass spectra of (I) and (II) both contained a weak molecular ion
at m/e 547, and a low intensity ion at m/e 490 due to the "parent" ion
minus CH^NCO.  An intense peak corresponding to the CH3NCO fragment
        O
at m/e 57 was also present in both spectra.   The base peak in each

                                  220

-------
spectra,  which occurred at m/e 43, was due to the acylium ion
The peaks in the mass spectrum of (II) that arose from the aglycone
moiety were similar to those obtained in the mass spectrum of (I).

The acetylated glucosides (I) and (II) were deacetylated with catalytic
amounts of barium methoxide in anhydrous methanol at 4°.  These reaction
conditions permitted the removal  of the acetate groups without causing
significant hydrolysis of the base labile carbamate group.  Therefore,
4-hydroxycarbaryl glucoside (III) was obtained in 80% yield by the
reaction of (I) with barium methoxide in anhydrous methanol for 7 hr
at 4°.  5-Hydroxycarbaryl glucoside (IV) was obtained in 73% yield  by
the similar hydrolysis of (II).  (Ill) and (IV) were identified by
their elemental analyses, and by their ir, nmr, and mass spectra.

The ir spectra of (III) and (IV)  were similar, but as with the acetyl-
ated glucosides (I) and (II), they could be distinguished by their
respective aromatic protons out-of-plane deformation.  The nmr spectra
of (III) and (IV) were also similar, but again they were differentiated
by the 2 proton singlet in the aromatic region of the nmr spectrum of
(III).

The mass spectra of (III) and (IV) proved to be very interesting.  Even
though glucosides are very polar organic compounds, a weak molecular
ion was obtained for (III) and (IV) at m/e 379.  A weak ion at m/e 322,
due to the "parent" ion minus CH3NCO, was also present in (III) and
(IV).  The OLNCO fragment was detected as a relatively intense fragment
at m/e 57 in both spectra.  The base peak in the mass spectra of
(III) and (IV) occurred at m/e 160 and was due to 1,4-naphthalenediol
and 1,5-naphthalenediol respectively.  The corresponding sugar moiety
was obtained at m/e 163 (CsH^Og©).  The latter peak was of low
intensity, but evidence was obtained for an intermolecular rearrangement.

It was shown that transcarbamylation had occurred between the phenolic
oxygen of the naphthalene ring and one of the hydroxyl oxygens on the
                                  221

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 sugar moiety.   A peak,  which  was  approximately  5  times as intense as
 the  163  peak,  was obtained  at m/e 220  in  the  spectra of  (III) and (IV).
 High resolution mass  spectral analysis  determined the elemental
 composition  of this peak  to be  either  C8H14^6N1 or C16H12^T  The latter
 compoistion  was discarded on  the  basis  of the chemical strucutre of the
 glucosides,  and therefore the peak at  220 was of  elemental composition
 C8H14°6N1  (tneoretica1  mass 220.0821;  actual  mass, (III) 220.089+
 0.010,  (IV)  220.080 + 0.010).   This elemental composition was consis-
 tent with  carbamylated  glucose.   It should be noted that the N-methyl-
 carbamoyl  group could be  on any one of  the four hydroxyl oxygens of the
 sugar.   Further evidence  for  this structure was obtained when the mass
 spectrum of  a  mixture of  1-naphthyl-beta-D-glucoside and 5-hydroxy-
 carbaryl were  taken separately, neither spectrum  contained an ion at
 this mass.

 Samples  of (III),  mp  204-208°,  and (IV),  mp 178-182°, prepared by the
 barium methoxide  procedure, were  shown  by aluminum oxide tic to con-
 tain an  impurity  which  traveled just below (III)  and (IV).  These
 impurities were identified  as their respective decarbamylated products
 4-hydroxy-l-naphthyl-beta-D-glucopyranoside (V) and 5-hydroxy-l-
 naphthyl-beta-D-glucopyranoside (VI).   The latter compounds were in-
 dependently  synthesized by  the  hydrolysis of  (III) and (IV) with a
 barium hydroxide  solution in methanol at  room temperature.  (V) and
 (VI) were  identified  by their elemental analyses, tic, and their ir
 spectra, which  was similar  to the  ir spectra  of (III) and (IV), but
 did  not  contain a  carbonyl  streching frequency at approximately 1720
 cm"  .  The instability of the carbamate ester in basic solutions was
 demonstrated when  approximately 50% hydrolysis was obtained with
 catalytic amounts  of  sodium methoxide in methanol  at room temperature.

 The  reaction of 5-hydroxycarbaryl  with methyl  tetra-0-acetyl-beta-
 D-glucopyranouronate and catalytic amounts of boron trifluoride ether
 complex  in refluxing anhydrous benzene for 16 hr gave a  15% yield of
methyl [5-(N-methylcarbamoyloxy)-1-naphthyl-tri-0-acetyl-beta-D-
                                  222

-------
glucopyranosid] uronate  (VII).  This reaction was significant since
aryl-beta-D-glucuronides are more commonly prepared by the condensation
of methyl  (tri-0-acetyl-alpha-D-glucopyranosyl bromide) uronate with a
phenol in  the presence of an acidic metal catalyst (Bollenback et al.,
1955; Coffey, 1967; Conrow and Bernstein, 1971), or by the fusion of
the acetylated methyl ester sugar with a phenol in the presence of an
acidic catalyst at reduced pressures (Bollenback et al., 1955).  The
structural assignment for (VII) is supported by its elemental analysis,
and by its ir, nmr, and  mass spectra.
                                                             *
(VII) was  deacetylated with catalytic amounts of barium methoxide in
methanol at 4°.  However, attempts at hydrolyzing the methyl ester
without  hydrolyzing the  carbamate ester were not successful.  The use
of barium  hydroxide at 4° hydrolyzed the carbamate group along with
the methyl ester.  The mass spectrum of the methyl ester of  the beta-
D-glucuronide of 5-hydroxycarbaryl had a weak molecular ion  at m/e 407.
The base peak was at m/e 160  (1,5-naphthalenediol).  The mass spectrum
also showed a peak at m/e 248, which corresponded to the thermal rearrange-
ment peak  at m/e 220 in  the mass spectra of (III) and  (IV).  Thermal
intermolecular rearrangements of this type may prove quite common in
the mass spectra of carbamate glucosides, and therefore may  be useful
in their identification.

.The attempted preparation of the beta-D-0-glucoside of N-hydroxymethyl-
carbaryl by a procedure  similar to that used for the preparation of
4- and 5-hydroxycarbaryl glucoside was unsuccessful.  Acetylation was
not accomplished since N-hydroxymethylcarbaryl was not stable in benzene
solutions  containing catalytic amounts of boron trifluoride.  Silica
gel tic  showed that it decomposed almost immediately in the  acidic mix-
ture to  at least four products.

Preliminary experiments  showed that the beta-D-0-glucoside of 3-hydroxy-
carbofuran (2,3-dihydro-2,2-dimethylbenzofuranyl-7 N-methylcarbamate)
could not  be synthesized using the procedure described herein.
                                   223

-------
3-Hydroxycarbofuran, beta-D  (+) glucose pentaacetate, and catalytic
amounts of boron trifluoride ether complex were refluxed in anhydrous
benzene for 16 hr.  The reaction mixture was processed in a manner
similar to that described for 4- and 5-hydroxycarbaryl glucoside
tetraacetates.  The major product isoalted, mp 235-240° dec., did not
contain the acetylated sugar moiety.  Its elemental analysis, and its
ir and nmr spectra indicated that it was a self-condensation product
of 3-hydroxycarbofuran.

The toxicity of 4- and 5-hydroxycarbaryl and their respective beta-D-
0-glucosides (III) and (IV) to mice are reported in Table 70.  In all
cases mortalities occurred within 2 hr after administration of the
test compounds, with no further mortalities occurring during an obser-
vation period of 3 weeks.  No mortalities occurred in the control mice.
As expected, glucoside formation greatly decreased the toxicity of
4- and 5-hydroxycarbaryl.  However, even though the aglycones differed
only slightly in toxicity, their glucosides showed a relatively greater
difference.  This could possibly indicate that some of the toxicity of
(IV) was due to the intact glucoside, and not entirely the result of
cleavage of the conjugated form to yield the toxic aglycone.  However,
it is possible that 5-hydroxycarbaryl glucoside is cleaved faster than
its 4-hydroxy analog in mice, resulting in an effectively higher con-
centration of 5-hydroxycarbaryl.  Further studies using the synthetic
conjugates will enhance the elucidation of this point and allow the
general toxicological  nature of the carbaryl  glucosides to be more
completely defined.
                                  224

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TABLE 70.  THE TOXICITY OF 4- AND 5-HYDROXYCARBARYL AND THEIR RESPECTIVE
              BETA-D-0-GLUCOSIDES (III) AND (IV) TO MICE3

Compound                                             LD5Q,mg/kgb
4-hydroxycarbaryl                                         50
4-hydroxycarbaryl glucoside (III)                       1550
5-hydroxycarbaryl                                         55
5-hydroxycarbaryl glucoside (IV)                         950

a Compounds were administered by intraperitoneal injection using
  dimethylsulfoxide as carrier.
  Mortality was recorded after 24 hours.
                                  225

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                            IV   REFERENCES

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                               14
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26.  Dorough, H. W., H.  M.  Mehendale and T. Lin.   Modification of carbaryl
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                                  228

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27.  Dorough, H. W. and 0. G. Wiggins.   Nature of the water-soluble
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33.  Gyrisco, F. G., D. J. Lisk, S. N.  Fertig, E. W.  Huddleston, F. N.
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41.  Kuhr, R. J.  Metabolism of carbamate insecticide chemicals in
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43.  Liu, S. Y., and J. M.  Bollag.  Carbaryl decomposition to 1-
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                                  230

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51.  Oonnithan, E. S. and J. E. Casida.  Oxidation of methyl- and
     dimethylcarbamate insecticide by microsomal enzymes and anti-
     cholinesterase activity of the metabolites.  J. Agr. Food Chem.
     16: 28 (1968).
                                    /
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     technique for the estimation of cholinesterase activity in boll
     weevils.  Annals. Entomol. Soc. Am. 57: 367-71 (1964).

                                  231

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55.  Stanier, R. Y.  Simultaneous adaptation.  A new technique for the
     study of metabolic pathways.  J. Bacteriol. 54: 339-48  (1947).

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     5,6-Dihydro-5,6-dihydroxycarbaryl glucuronide as a significant
     metabolite of carbaryl in the rat.  J. Agr. Food Chem. 20: 980
     (1972).

58.  Tewfik, M. S., and Y. A. Hamdi.  Decomposition of Sevin by a soil
     bacterium.  Acta. Microbiol. Polonica Ser B. 19: 133-5 (1970).

59.  Timmerman, J. A., H. W.  Dorough, J. R. Buttram, and B. W. Arthur.
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60.  Wasserman, M.  Effects of organochlorine insecticides on body de-
     fense systems.  Ann. Rev. N. Y. Acad. Sci.  160: 393 (1969).

61.  Weast, R. C.  "Handbook of Chemistry and Physics".   49th ed.,
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64.  Williams, S., and P.  A.  Mills.  Residues in milk of cows fed
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     (1964).
                                  232

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65.  Whitehurst, W. E., E. T. Bishop, F.'.E. Critchfield, F. G. Gyrisco,
     E. W. Huddleston, H. Arnold, and D. J. Lisk.  The metabolism of
     Sevin in dairy cows.  J. Agr; Food Chem. 11: 167 (1963).
                                   233

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                             V  PUBLICATIONS

1.    Dorough, H. W., R. B. Davis and 6. W. Ivie, 1970.  Fate of Tenrik-
           14
          C   in lactating cows during a 14-day feeding period.  J.
          Agr. and Food Chem. 18 (1): 135-42.
2.    Hicks, B. W., H. W. Dorough, and R. B. Davis. 1970.  Fate of
          carbofuran in laying hens.  J. Econ. Entomol. 63 (4): 1108-11.
3.    Culver, D. J., T. Lin and H. W. Dorough. 1970.  The effect of
          monoamine oxidase inhibitors on carbaryl metabolism.  J.
          Econ. Entomol. 63 (4): 1369-70.
4.    Dorough, H. W. 1970.  Metabolism of insecticidal methylcarbamates
          in animals.   J. Agr. Food Chem. 18 (6): 1015-22.
5.    Dorough, H. W. 1971.  Carbaryl residues in milk and meat of dairy
          animals.  Proceedings of the "International Symposium on
          Pesticide Terminal Residues".  Tel-Aviv, Israel, Feb. 17-19, 1971
6.    Mehendale, H. M. and H. W. Dorough. 1971.  Conjugative metabolism
          and action of carbamate insecticides.  Proceedings of the,
          "Second International Congress of Pesticide Chemistry" Tel-Aviv,
          Israel, Feb. 21-27, 1971.
7.    Mehendale, H. M. and H. W. Dorough. 1972.  In vitro glucosylation
          of 1-naphthol by insects.  J. Insect Physio!. 18: 981-90.
8.    Mehendale, H. M. and H. W. Dorough. 1972.  Glucuronidation
          mechanisms in the rat and their significance in metabolism of
          insecticides.  Pest. Biochem. and Physiol. 1: 307-18.
9.    Hicks, B. W., H. W. Dorough and H. M. Mehendale. 1972.  Metabolism
          of aldicarb pesticide in laying hens.  J. Agr. Food Chem.
          20 (1): 151-56.
10.   Mehendale, H. M., R. F. Skrentny and H. W.  Dorough. 1972.  Oxidative
          metabolism of aldrin by subcellular root fractions of several
          plant species.  J. Agr. Food Chem. 20:  398-402.
11.   Dorough, H. W.,  H. M.  Mehendale and T. Lin. 1972.  Modification of
          carbaryl metabolism in rats with monoamine oxidase inhibitors.
          J.  Econ. Entomol.  65 (4): 958-62.
                                   234

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 12.    Cardona,  R.  A.  and H.  W.  Dorough.  1973.   Syntheses  of  the  3-D-O-
           glucosides  of 4-  and  5-hydroxy-l-naphthyl  N-methylcarbamate.
           J.  Agr.  Food Chem.  (In Press).
 13.    Lin, T.  H.  and  H.  W.  Dorough.   Influence of selected insecticides
           on  carbaryl  metabolism in  rats  (In Preparation).
 14.    Lin, T.  H.,  El-Shourbagy, N. A.,  and H.  W.  Dorough.  Fate  of
           3-hydroxycarbofuran and its glucoside and  glucuronide  in rats.
           (In Preparation).
 15.    El-Shourbagy,  N. A. and H. W.  Dorough.  Factors  influencing  gly-
           coside  conjugation in insects  and mammals.   (In Preparation).
 16.    Dorough,  H.  W.,  J. P.  McManus  and  S.  S.  Kumar.   Chemical and
           metabolic  characteristics  of 1-naphthyl  glucoside.   (In
           Preparation).
THESIS
1.     Hicks, B. W.  "Fate of Furadan and Temik in Laying Hens".   Ph.D.
           1970.  Texas A&M University, College Station, Texas.
2.    :Lin, T. H.  "Influence of Selected Biologically Active Chemicals
           on the Mammalian Metabolism of Carbamate Insecticides."
           Ph.D. 1972.   University of Kentucky, Lexington, Kentucky.
3."    El-Shourbagy, N. A.  "Glycoside Conjugation in Insects and
           Mammals."  M.S. 1972.  University of Kentucky, Lexington,
           Kentucky.            -
4.     Rodriguez, L. D.  "Studies in Degradation of Carbaryl  by Soil
           Microorganisms."  Ph.D. 1973.  University of Kentucky,
           Lexington, Kentucky.
                                 235

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                            VI  SUMMARY

The current project "Metabolism of Carbamate Insecticides" was initiated
in 1965 and was originally funded by the National Institute of Health.
Detailed progress reports and requests for renewal were submitted in
April 1967 and in December 1969.  The present report covers research
conducted under EPA Grant Number R-802005 during the period of September
1, 1970 through August 31, 1973.

METABOLISM

Aldicarb

Increasing the number of days, up to 14, that cows were administered
aldicarb [2-methyl-2(methylthio-C  ) propionaldehyde 0-(methylcarbam-
oyl) oxime] at levels of 0.12, 0.6, and 1.2 ppm in the diet did not
alter the magnitude and nature of residues eliminated daily in the milk,
urine, and feces.  Parts per million total aldicarb equivalents in the
milk were approximately 1/100 that level of insecticide in the feed.
About 15% of the radioactive residues in the milk was aldicarb sulfone
and about 4% was aldicarb sulfoxide.  The remaining was hydrolytic
products and compounds of unknown identity.  Percentages of the doses
eliminated in the milk, urine, and feces were 1, 92, and 3 respectively.
Total aldicarb equivalents in the liver were 29, 123, and 164 ppb for
the three treatment rates, respectively, when the animals were
slaughtered 18 hours after the last treatment.   Twenty-six other tissue
samples contained either much lower quantities   of residues or none
at all.

Aldicarb metabolism in laying hens was invetigated and the nature and
levels of residues in the eggs and tissues were determined.  Single
oral doses of aldicarb and/or aldicarb sulfone  at 0.7 mg/kg were
excreted rapidly, with 75% of the doses in the  feces by 24 hr.  A large
portion of the feces metabolites (50-60%) was as water-soluble materials,
                                 236

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10% as unextractables, and the remainder primarily as known hydrolytic
products of aldicarb.  Only minute quantities were as toxic carbamate
compounds.  Aldicarb equivalents in eggs reached a maximum of 0.18 ppm
on the day after treatment but had declined to 0.01 ppm by 10 days.   In
muscle tissues, residues of 0.2 to 0.3 ppm 6 hr after treatment declin-
ed to 0.01 ppm or less by 10 days.  Residue levels in the liver and
kidney were about twice those in the muscle tissue.  The nature of the
aldicarb metabolites in the eggs and tissues was similar to that in the
feces.  Aldicarb in the diet of hens for 21 days did not appear to alter
the fate of the carbamate in the birds when compared to that when single
oral doses were administered.

Forty and 80% of a topically applied dose of aldicarb was absorbed by
boll weevils and houseflies during the first hour after treatment.
Complete disappearance from the surface of flies and weevils occurred
6 and 24 hours post-treatment, respectively.  The major metabolic path-
way in these species appeared to be one of oxidation with both the
sulfoxide and sulfone derivatives of aldicarb detected in the insects.
Aldicarb sulfoxide is a potent cholinesterase inhibitor and, as such,
probably contributes significantly to the insecticidal activity of the
parent carbamate compound.

Carbaryl

Cows fed on a diet containing 10, 30 or 100 ppm carbaryl-naphthyl-  C
for 14 days eliminated daily about 0.2 percent of the consumed doses
in the milk, 5-10 percent in the feces and 70-85 percent in the urine.
                                 14
Average daily levels of carbaryl-  C equivalents in the milk for the
three feeding levels were 0.02, 0.07 and 0.28 ppm respectively.  Residue
levels in the tissues after 14 days of feeding the three levels of car-
baryl were as follows:  kidney, 0.10, 0.53, 1.00; liver, 0.03, 0.10,
0.41; lung, 0.02, 0.06, 0.21; muscle, 0.01, 0.03, 0.10; heart, 0.01,
0.04, 0.10; blood 0.01, 0.04, 0.14; fat, 0.0, 0.02, 0.02.  The chemical
nature of the residues in the milk were determined.  Extraction with
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acetonitrile-water-chloroform was the desired solvents for extracting
the tissues.  Homogenation in water or phosphate buffer and centrifugal
fractionation showed that 15 to 20% of the radiocarbon in the liver and
kidney remained in the 15,000g pellet.  Centrifugation of the 15,000g
supernatant at 105,000g resulted in only trace amounts of radiocarbon
in the precipitant fraction.  After extraction of the precipitants
with acetonitrile and water, the solids from the liver contained from
                      14
7 to 10% of the total   C in the whole tissue; the solids from the
kidney contained about 5%.  Treatment of the precipitants with acid
(3N HC1 and reflux for 3 hrs) prior to extraction did not significantly
improve the extractability of the   C-residues.  The supernatant
fractions formed a precipitant fraction when acetonitrile-chloroform was
                           14
added.  Distribution, % of   C in tissue, in the solids, water phase,
and organic solvent phase of the supernatant extracts was as follows:
Liver - 40, 20, and 21; Kidney - 13, 35, and 31.  Comparable values
when the supernatant were treated with acid were:  Liver - 36, 23, and
26; Kidney - 6, 39 and 37.
The degradation of carbaryl was studied with three samples of Maury
soil:  Untreated soil with no recorded pesticide treatment; Carbaryl-
treated, which had received 4 Ibs/A carbaryl granules six months prior
to sampling; and Mixed Pesticide-treated, which had been subjected to
various pesticides for 15 years.  These soils, non-sterile and auto-
claved, were incubated under controlled conditions for periods up to
120 days with added carbaryl-l-naphthyl-l-14C, l-naphthyl-l-14C and
polar water-soluble metabolites of carbaryl produced in bean plants.
Total radioactivity in soil preparations to which carbaryl or polar
water-soluble metabolites had been added disappeared much faster from
non-sterile than from autoclaved soil, indicating biological attack.
Of the non-sterile soils, dissipation was fastest in Carbaryl-treated,
where 44 percent of the radiocarbon was lost after 48 hours incubation
at 27°C, 85 percent relative humidity.  Organosoluble radioactivity in
soils incubated with carbaryl was composed entirely of the parent
compound.  Water-soluble metabolites were present in small amounts.
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The loss in total radioactivity suggested the evolution of volatile
metabolites, of which only carbon dioxide could be demonstrated.   After
a loss of as much as 50 percent during the first 14 days of incubation,
radioactivity added as polar water-soluble metabolites persisted  in the
non-sterile soils thereafter.  Tic analysis indicated that organosoluble
fractions of the soil extracts contained aglycones different from those
originally present in enzymatic hydrolysates of the polar water-soluble
metabolites.  Fifty-eight bacterial and fungal isolations were obtained
from Mixed Pesticide-treated soil on silica gel with carbaryl  as  the
sole carbon source and 30 such isolations from Carbaryl-treated soil
on carbaryl agar.'  Those capable of attacking carbaryl in culture medium
were identified.

Carbofuran

Carbamate-resistant houseflies absorbed carbofuran slower and excreted
it faster than a susceptible strain of the insects.  This appears to be
an important factor in the added tolerance of the resistant flies to
carbofuran since both strains metabolized the compounds in an almost
identical manner.  Susceptible and carbamate-resistant housefly
cholinesterase recovered from inhibition with carbaryl and Baygon at
the same rate.  The degree of inhibition also was the same in both
strains, and no difference could be detected in the sensitivity of the
enzyme to the carbamate insecticide.  Resistant flies, however, did
appear to have greater cholinesterase activity than the susceptible
insects.

FATE OF CARBAMATE METABOLITES

1-Naphthyl Glucoside

When 1-naphthyl glucoside was administered orally to rats, 67% of the
dose was eliminated in the 0-24 hr urine.  About 10% of the dose was
as 1-naphthol, 24% as 1-naphthyl glucuronide and 10% as 1-naphthyl
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sulfate.  Urine  (0-24 hr) of rats treated with 1-naphthol contained
90% of the dose.  Only 1% of the dose was as free 1-naphthol, 73% as
the glucuronide  and 15% as the sulfate.  These studies demonstrated
that glucoside conjugates are not metabolized in the same manner as the
aglycones.

3-Hydroxy Carbofuran and its Glycosides

Approximately 60% of a single oral dose of 3-hydroxy carbofuran to rats
was eliminated in the urine within 32 hrs.  When 3-hydroxy carbofuran
glucuronide was  administered, 82% of the dose was in the 0-32 hr urine;
91% of a dose of 3-hydroxy carbofuran glucoside was eliminated in the
urine during the same time.  The percentage of the radiocarbon in the
urine which extracted into chloroform were 39, 18 and 10, respectively.
3-Hydroxy carbofuran was the only component of the chloroform extracts.
In bean plants,  3-hydroxy carbofuran was rapidly converted to the
glucoside form.

INTERACTIONS

Effect of Aldicarb on Methyl Parathion Toxicity

Aldicarb showed  only additive toxic effects to mice when administered
along with methyl parathion, an organophosphorus insecticide.  The
oxime hydrolytic product of aldicarb, which is nontoxic, did not alter
the toxicity of methyl parathion in any way, nor was the protective
action of the antidotes, atropine and 2-PAM, influenced by the
addition of the oxime derivative of aldicarb.

Modification of  Carbaryl Metabolism with MAPI's

The rate of carbaryl metabolism by rats was decreased by simultaneously
administering drugs commonly referred to as monaomine oxidase inhib-
itors and by prolonged exposure of the animals to one of the drugs in
                                 240

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i-n the drinking water.   Conjugative mechanisms of metabolism were affect-
ed most by the drugs although oxidative and  hydroxylative metabolism
were reduced to some degree.  Decreasing the rate of metabolism result-
ed in a slower rate of excretion of the carbamate from rats treated
with the drugs.  Tranylcypromine at 50 mg/kg, e.g., reduced excretion
of carbaryl-  C equivalents from rats by 50% during the 1st 48 hours
after treatment.  A rat intestine enzyme system, fortified with UDPGA,
conjugated 1-naphthol but did not metabolize carbaryl.  This in vitro
conjugating system was inhibited by the monoamine oxidase inhibitors.
The effect of these drugs on conjugation was not sufficient to suggest
that they might enhance the action of pesticides under practical condi-
tions.  However, the data do demonstrate that more effective inhibitors
of conjugation could be important in the safety and efficacy of chemicals
used to control insect pests.

Influence of Insecticides on Carbaryl Metabolism

Effects of simultaneous and/or pre-exposure of carbofuran, Ruelene and
coumaphos or DDT on the metabolism of carbaryl in rats and the enzymes
involved therein were investigated.  In addition, the urea and glucoside
content, and pH of the rat urine were considered.  Radioactive carbaryl
when given orally as a single dose (50 mg/kg) was rapidly eliminated
in the urine and feces of rats.  The excretion was complete by 72 hours
after the treatment with approximately 80% in the urine and 10% in the
feces of the rats.  Simultaneous doses of carbaryl and carbofuran
reduced the excretion of carbaryl equivalents by 12%.  Ruelene and
coumaphos caused a slightly faster excretion of carbaryl equivalents
when administered with the carbamate.  None of the compounds changed
the nature of carbaryl metabolites or changed the amount of carbaryl
residues in the body.  Similar results were obtained when these insect-
icides were administered in the diets of rats.  Protein content of the
liver, kidney and spleen were increased by DDT and to a lesser degree
by carbaryl.  DDT also enhanced mixed function oxidase enzyme activities
in the liver and the kidney, whereas carbaryl did not.  DDT increased
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the  in vivo glucurom'de and sulfate conjugation of 1-naphthol in rats
but  not the in vitro conjugation of 1-naphthol by rat liver microsomes.
Carbaryl did not affect the conjugating enzyme activity in the rat
liver.  Exposure of rats to the carbamate and/or organophosphates
resulted in increased urea and glucose in the urine, reduced the rate
of body weight gains, but had little effect on the pH of the urine.

CONJUGATION

Mechanisms

Optimum conditions for the in vitro glucuronidation of 1-naphthol were
established using rat liver and small intestine as enzyme sources.  The
liver enzyme system was used to study conjugative metabolism, per se,
and  in conjunction with oxidative metabolism while the intestine enzyme
was used to study conjugative metabolism exclusively.  Both carbaryl
and Banol required oxidative metabolism before conjugation could take
place.  However, when conjugative metabolism was reduced by limiting
the UDPGA concentration, the overall rate of carbamate metabolism was
decreased.   There was no accumulation of the nonconjugate metabolites
although excess NADPHp was present in the microsome system.  Inhibition
of the conjugating enzymes was demonstrated using several insecticide
synergists, including sulfoxide, piperonyl butoxide, and MGK-264, which
are established mixed-function oxidase inhibitors.  A number of
                  _3
insecticides at 10  M concentration had no effect on the in vitro
conjugating enzyme systems.   Glucuronides of several hydroxylated
carbaryl  metabolites were synthesized by the enzymes from the two sources
but the 5,6-dihydro-dihydroxy analog of carbaryl could not be conjugated.

Glucosylation in insects was investigated using tobacco hornworms
(Manduca sexta) as the primary test insect and 1-naphthol-  C as the
substrate.   Of 6 common co-factors tested, only UDPG was utilized by
the conjugating enzyme system.  Neither the hornworm nor housefly enzymes
could form the glucuronic acid derivative of 1-naphthol using UDPGA.
                                  242

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Centrifugal fractionation of the hornworm homogenates showed that the
glucosyltransferase activity was in the 105,000g soluble fraction.   In
the housefly, the enzyme activity was associated with the 15,000g pellet
and to a lesser extent with the 105,000g pellet.  In vitro inhibition
of the glucosyltransferase by sulphoxide, piperonyl  butoxide, and other
insecticide synergists were demonstrated.

Factors Influencing Conjugation

The occurrence and comparative activity of glycoside conjugating enzymes
were investigated in insect and vertebrate species.   All species readily
conjugated the naphthol, although there were quantitative differences.
Glucosyltransferase activity was greatest in the fat bodies, malphigian
tubules and midgut of the tobacco hornworm larvae.   In the rat,  the
comparable enzyme, glucuronlytransferase was most active in the  liver,
kidney and lungs.  Glycoside conjugating enzymes maintained almost 100%
of their activity when stored in the intact tissue or in various sub-
cellular fractions at -20° and 0°C for 90 days.   TPNH oxidase enzymes
lost most of their activity after a few hours of storage.  In vitro
glucosylation of 1-naphthol by houseflies was not greatly influenced
by developmental stage, age of the adult, sex or by acquired resistance
to insecticides.  The enzymes were not affected  by the addition  of
various insecticides to the reaction mixtures.   Glycoside conjugation,
like oxidation and hydrolytic metabolism, plays  a major role in  the
detoxication of insecticides and other foreign compounds.  The enzymes
responsible for glycosylation, however, are unlike these other enzyme
systems in that they are virtually unaffected by factors such as age,
sex, resistance and other toxicants.

Synthesis of Glycosides

4- and 5-(N-Methylcarbamoyloxy)-l-naphthyl-tetra-0-acetyl-beta-D-
glucopyranoside were synthesized by the condensation of the appropriate
hydroxy-1-naphthyl-N-methylcarbamate with beta-D (+) glucose
                                 243

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pentaacetate in the presence of catalytic amounts of boron trifluoride
ether complex.  Reaction of the acetylated beta-D-glucosides with
barium methoxide gave the corresponding beta-D-glucosides.  The ir,
nmr, and mass spectra of the beta-D-glucosides and their acetylated
analogs are reported and compared.  When administered ip to mice 4-
hydroxy-1-naphthyl-N-methylcarbamate was 31 times more toxic than its
beta-D-glucoside, 5-hydroxy-l-naphthyl-N-methylcarbamate was 17 times
more toxic than its beta-D-glucoside.  Methyl [5-(N-methylcarbamoyloxy)-
1-naphthyl-tri-O-acetyl-beta-D-glucopyranosid] uronate was prepared in
a manner similar to the acetylated beta-D-glucosides.  The deacetylated
methyl ester was prepared by hydrolysis with barium methoxide, however,
attempts to demethylate the product while leaving the carbamate ester
intact were unsuccessful.

The work conducted on this project has resulted in 11 publications in
print,  1  in press and 4 in preparation.
                                 244

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. R
      -o%l -74-002
                             2.
                                                           3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
   Metabolism  of Carbamate Insecticides
                                                           5. REPORT DATE
                                                               September 1973
                                                           6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
                                                           8. PERFORMING ORGANIZATION REPORT NO.
   H. W.  Dorough
9. PERFORMING ORGANIZATION NAME AND ADDRESS

   Department  of Entomology
   University  of Kentucky
   Lexington,  Kentucky  40506
                                                           10. PROGRAM ELEMENT NO.
             11. CONTRACT/GRANT NO.


                R-802005
12. SPONSORING AGENCY NAME AND ADDRESS
   EPA, Office  of Pesticides Program
   Research  Triangle Park, North  Carolina  27711
                                                           13. TYPE OF REPORT AND PERIOD COVERED
                                                              Final  Report
             14. SPONSORING AGENCY CODE
II . SUPPLEMENTARY NOTES
16. ABSTRACT
   The metabolic fate of aldicarb,  carbaryl, and carbofuran  was investigated  in  a
   variety  of biological systems.   In addition, the effects  of other insecticides  and
   certain  monoamine oxidase  inhibitors on carbaryl metabolism in rats was  studied.
   The fate of 3-hydroxy carbofuran,  its glucoside and  glucuronide, and naphthyl
   glucoside in rats was determined.   Using 1-naphthol  as  a  model compound, in vitro
   methods  were developed to  study  mechanisms of glycosylation in insects and mammals.
   The glucosides of 4- and 5-hydroxy carbaryl were prepared chemically and their
   acute  toxicity to mice compared  to the aglycones.  Results of these studies showed
   that carbamate insecticides  are  metabolized initially  by  hydrolytic- and oxidative-
   type reactions and the resulting products are then almost totally conjugated.   These
   conjugated products constitute  the majority of the terminal residues of  carbamates
   in both  animals and plants.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS  C. COSATI Field/Group
   Metabolism            Synthesis of
   Carbamate insecticides        glucosides
   Aldicarb
   Carbaryl
   Carbofuran
   Conjugation	
  Insecticide metabolism
  Fate  of carbamate in-
     secticides
18. DISTRIBUTION STATEMENT

    Unlimited
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    246
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