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Title: Pesticide methodology and fate in the environment
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Permanent Link: http://ufdc.ufl.edu/UF00094967/00001
 Material Information
Title: Pesticide methodology and fate in the environment summary progress report, January 1, 1967 to June 30, 1969
Physical Description: 60 leaves : ill. ; 28 cm.
Language: English
Creator: Pesticide Research Laboratory, University of Florida
Donor: unknown ( endowment ) ( endowment )
Publisher: University of Florida, Department of Food Science
Place of Publication: Gainesville, Fla.
Publication Date: 1969
Copyright Date: 1969
 Subjects
Subject: Pesticides -- Environmental aspects -- Florida   ( lcsh )
Genre: non-fiction   ( marcgt )
 Notes
General Note: Cover title.
General Note: "July 1, 1969."
General Note: "ES 00208."
Statement of Responsibility: Pesticide Research Laboratory, Department of Food Science.
 Record Information
Bibliographic ID: UF00094967
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 436169027

Table of Contents
    Front Cover
        Page i
    Table of Contents
        Page ii
    Introduction
        Page 1
        Page 4
        Page 5
        Page 6
    Progress reports by research projects
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
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        Page 30
        Page 31
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Full Text











TABLE OF CONTENTS


INTRODUCTION
Pages

Facilities and General Operation 1

Co-investigators on ES-00208 2


SUMMARY OF RESEARCH PROGRESS 2-6


PROGRESS REPORTS BY RESEARCH PROJECTS

1. Determination of N-methyl and N,N-
dimethylcarbamate Insecticides as
their Amide Derivative by Electron
Capture Gas Chromatography (Research
Team No. 1) 7-15

2. New and Improved Techniques for
Pesticide Residue Analyses (Research
Team No. 2) 16-34

3. Uptake, Translocation and Metabolism
of Pesticides in Higher Plants
(Research Team No. 3) 35-42

4 Interactions among Selected Insecticides
and Plant Systems (Research Team No. 4) 43-49

5 Methods of Increasing the Elimination
of Persistent Chlorinated Hydrocarbon
Pesticides from the Mammalian System
(Research Team No. 5) 50-54

6 Interrelationship between Pesticides
and the Effect of other Xenobiotic
Agents on the Metabolism of Mammals
(Research Team No. 6) 55-60









PESTICIDE METHODOLOGY AND FATE IN THE ENVIRONMENT
P.H.S. Grant ES-00208
Progress Report for period January 1, 1967 June 30, 1969

Introduction
The Pesticide Research Laboratory was occupied in June, 1965
and staffed with four faculty members by July 1, 1966. The ori-
ginal responsibilities of this laboratory were three-fold: (1) to
conduct cooperative research involving possible pesticide pro-
blems involving agricultural commodities of common interest to the
Southern Region of the United States, (2) to conduct research on
pesticides and their metabolites remaining on food commodities
originating in Florida, (3) to augment the above programs with
fundamental investigations involving pesticide methodology as a
basis for studies on the fate of pesticides in the environment.

The four co-investigators on ES-00208 that are physically
located in the Pesticide Research Laboratory are also involved to
some extent in several other programs involving pesticide research
pertinent to the state or region. A list of these allied pesti-
cide programs are as follows: State Project 1106: "Pesticide
Residues and Toxic Metabolites on and in Plant and Animal Products,"
State Project FS-01469: "Pesticide Residues in Agricultural Com-
modities and Environments" (contributing to regional research pro-
ject S-73), Interregional Project IR-4: "Evaluation of Current
Data and needed Research to Determine Tolerance Limits of Chemicals
for Minor Uses on Agricultural Products."

Facilities and General Operation:
The Pesticide Research Laboratory was constructed through the
pooling of regional research funds by all 14 of the states in the
Southern Region. Permanent staff and annual operating expenses
are borne by the Florida Agricultural Experiment Station and,
therefore, the laboratory is part of this organization. Most of
the major equipment items in current use in the research labora-
tories of this facility were obtained in 1966 through P.H.S. grant
ES-00100.

The areas of this facility which are devoted to or are avail-
able for the research conducted under ES-00208 are as follows:
five (5) research laboratories, two (2) instrument rooms, a radio-
logical laboratory, a 350F laboratory, and a thin-layer chromato-
graphy laboratory. The general analytical laboratory of this
facility is staffed and operated with state funds exclusively.
However, there is considerable "spin-off" of research findings from
the research laboratories to the pesticide investigations conducted
in the general analytical laboratory. There are two separate
buildings adjacent to the Pesticide Research Laboratory; the plant
growth chamber building and the solvent distillation and storage
building. The growth chamber building includes three large en-
vironmental control chambers, a tissue culture laboratory and a


-1-








Root entry is apparently the primary means of plant uptake
of certain chlorinated hydrocarbon pesticides when grown in
pesticide-contaminated soils. C14-labeled Azodrin, 2,4-D and
dieldrin were added to the roots of corn, okra, peanuts and cab-
bage. As indicated by radioautographs, Azodrin was found to move
in varying degree into the aerial portions of these plants. All
of the plants treated with 2,4-D were retarded in development when
compared with those treated with dieldrin. Extracts of roots and
shoots of all plants contained activity and the radioactive spots
that appeared on TLC plates were at the Rf of 2,4-D or dieldrin.
Corn plants were grown under controlled conditions to determine
the rate of uptake, persistence, direction of movement and degree
of metabolism of labeled Azodrin. Autoradiographs indicated that
the direction of movement of Azodrin was primarily from the point
of application toward the leaf tip. Unaltered labeled Azodrin
was recovered from the plant parts toward which the pesticide had
moved.

Green algae has been used as a model system for the study of
certain interactions of pesticides and green plants. The phenome-
non of co-distillation of organochlorine insecticides with water
had not been investigated under conditions used for the mainte-
nance of sterile algae cultures. It was found that co-distilling
dieldrin can be effectively trapped on a common gas chromatographic
column and easily eluted for quantitation. Dieldrin was found to
penetrate algal cells rapidly and a maximum per cell level was
reached within 6 to 24 hours following introduction. Between 1
and 4 days, the algae cellular portion contained increasing amounts
of labeled dieldrin and more exhaustive extraction was required
for its removal. No metabolites were detected. Azodrin did not
penetrate chlorella cells over a period of 4 weeks.

Dieldrin and trans-aldrindiol were found to rapidly penetrate
the particulate fraction of alfalfa, corn and rye tissue homoge-
nates; whereas Azodrin was found to remain completely in the incu-
bation or soluble cellular fraction. After 6 hours exposure to
the three plant homogenates, no metabolites of any of the compounds
were detected.

The root absorption of dieldrin by rye grown in sand appears
to be affected by environmental conditions. Low relative humidity
resulted in higher dieldrin residues in plants than when grown
under higher relative humidity. Lower temperatures also led to
greater dieldrin uptake in rye plants compared to plants grown
under higher temperatures. The effects of photoperiod on dieldrin
accumulations in rye plants is currently under investigation.

Metabolism in Mammalian Systems
On the premise that the degradation of DDT in vivo may be a
function of hepatic enzymes and that these enzymes might be sub-
ject to stimulation by the administration of various drugs (enzyme
inducers), efforts have been concentrated on the development of an
in vitro system to measure the effect of pretreatment with various
compounds on the degradation of DDT. Substances that accelerate
the in vitro metabolism of DDT in such a system might be expected







to have a stimulatory effect on the in vivo degradation of DDT
and thus hasten tissue depletion of t-Fis pesticide following high
level exposure. Pretreatment with several inducers or inhibitors
of rat liver hepatic microsomal enzymes appeared to produce no
effect on degradation of DDT to DDD in the system. From the ex-
perimentation to date on rats, it was concluded that the in vitro
degradation of DDT to DDD was due primarily to bacterial action.
Some liver tissues apparently provided better media for the growth
of anaerobic bacteria capable of degrading DDT. The preheating of
homogenate mixtures and the addition of antibiotics apparently was
effective only in inhibiting bacterial growth to the extent that
smaller amounts of DDD were formed over a 48 hour incubation period.

Aldrin is epoxidized to dieldrin in mammalian liver micro-
somes, and it previously was thought that no further metabolism
occurred. It has been shown, however, that rabbits fed dieldrin
excrete a number of metabolites in the urine, but primarily trans-
aldrindiol. The excretion of this metabolite increases during
chronic aldrin feeding, suggesting that the enzyme activity for
hydration of dieldrin is induced. Therefore, it appears likely
that dieldrin is hydrated in liver microsomes in a reaction mediated
by inducible enzymes.

Experiments with model cyclic epoxides in this laboratory have
shown that epoxide hydrase activity occurs in mammalian liver micro-
somes. Therefore, both indene oxide and cyclohexene oxide are con-
verted to their respective trans-glycols by rat and rabbit liver
microsomes. No cis-glycols were detected as products of these
reactions. Hydriaton of these epoxides does not require any
pyridine nucleotide coenzyme, and the reaction apparently does not
involve an oxidase enzyme. Since one of the principal products of
the in vivo metabolism of dieldrin is of the same stereochemical
configuration as are those produced in in vitro microsomal hydra-
tion of the simpler epoxycycloalkanes, it appears likely that the
hydration of dieldrin to trans-aldrindiol would be mediated by a
microsomal enzyme. Experiments to test this hypothesis were con-
ducted.

It was concluded that dieldrin is converted to trans-aldrindiol
in mammalian liver microsomes. The reaction requires TPHN and
oxygen and thus appears to be of the mixed-function oxidase class.
At least some of the other products of dieldrin metabolism in liver
microsomes would appear to be derived by further metabolism of
trans-aldrindiol.

Parathion is metabolized in mammalian systems by two main
pathways, both mediated by oxidative enzymes in liver microsomes.
Halogenated hydrocarbon insecticides have been known for some time
to induce the microsomal mixed-function oxidases of mammalian
livers in a manner similar to that of phenobarbital. Both of the
oxidative reactions of parathion are inducible with phenobarbital.
Therefore, it was decided to study the effects of induction with
chlorinated insecticides upon the two different pathways of micro-
somal parathion metabolism.








It was expected initially that the paraoxon formed in liver
microsomes could be assayed by measuring the inhibition of
cholinesterase in a Warburg respirometer. Difficulties were en-
countered using this procedure since apparently extensive tissue
binding of paraoxon occurs at low concentrations. Ethyl C14-
labeled parathion was used as a substrate. Thin layer chromato-
graphic techniques were developed for the separation and visuali-
zation of parathion, paraoxon, diethylthiophosphate and diethylphos-
phate. The amount of diethylthiophosphate formed was taken as a
measure of the detoxifying pathway, while the sum of the amounts
of paraoxon and its hydrolysis product (diethylphosphate) were
used as a measure of the paraoxon-producing pathway. Pilot ex-
periments with other induction systems are in progress. It would
appear, however, that the induction of the two pathways by DDT
is quantitatively similar.








Research Team No. 1


Title: Determination of N-methyl and N,N-dimethylcarbamate In-
secticides as their Amide Derivative by Electron Capture
Gas Chromatography.

Co-principal Investigator: C. H. Van Middelem

Because of the increasing commercial application of N-methyl
and N,N-dimethylcarbamate insecticides, there is a definite need
for developing rapid, sensitive gas chromatographic procedures for
the detection of residues of these compounds remaining on treated
crops. Unfortunately, the parent molecules of these particular
compounds cannot be satisfactorily chromatographed without deriva-
tion. Chemical alteration of the carbamate molecule is necessary
in order to render it susceptible to electron capture detection.

A number of derivatives for N-methyl and N,N-dimethylcarbam-
ate insecticides were synthesized and evaluated as to their poten-
tial electron capturing properties in a gas chromatograph. One
of the most promising derivatives, 4-bromo-N-methylbenzamide, was
used to quantitate, by electron capture gas chromatography, car-
baryl residues extracted from field-treated spinach and chicory.
Sulfuric acid hydrolysis was used to simultaneously convert car-
baryl to a methylamine salt and the crop extractives to water-
soluble products. Effective separation of the methylamine from
the water-soluble extractives was accomplished by making the
aqueous phase strongly alkaline which also catalyzed the coupling
of the free amine with the 4-bromobenzoyl chloride. In order to
verify the gas chromatographic results, all field samples were
also analyzed by the conventional colorimetric procedure for car-
baryl. The lower limits of detection, by employing the proposed
gas chromatographic procedure, were found to be 20 picograms for
pure standards and 0.2 ppm for fortified crop extracts.

The object of this investigation was to develop a practical
means of analyzing residues of carbaryl and other methylcarbamate
pesticides by electron capture GLC. It was found that the amine
portion of the carbamate molecule produced derivatives that were
not only quite sensitive to electron capture detection, but which
also exhibited a high degree of chemical and thermal stability.
Subsequently described are procedures developed to separate the
volatile amine from substrate extractives and conversion to EC-
sensitive benzamide derivatives of carbaryl. Spinach and chicory,
treated in the field with carbaryl, were successfully analyzed
by electron capture gas chromatography. It is expected that these
procedures will also have satisfactory application to other N-
methyl and N,N-dimethylcarbamates.

1-1. Development of Electron-Capturing Derivatives

Considerable effort was made to synthesize a derivative whose
molecular structure exhibited exceptional electron affinity. Four


-7-









basic groups of compounds were synthesized from methyl- and
dimethylamine, as illustrated in figure 1. The nitrated anilines
which were synthesized initially, exhibited considerably less EC
detector response than the benzamide group. Although the aliphatic
amides were found to be as sensitive as the benzamides, these
compounds were less easily prepared and were susceptible to hydro-
lysis. The fourth group synthesized were the sulfonamides which
were found to be relatively insensitive to electron capture and
lacked sufficient volatility for effective chromatography at
practical column temperatures. Therefore, the benzamide deriva-
tives were found to combine most of the required properties such
as high sensitivity to EC detection, ease of preparation, plus
thermal and chemical stability under appropriate column conditions.
The 4-bromomethylbenzamide, although not the most sensitive of
the benzamides tested, was the most satisfactory because the acid
chloride from which it was derived contained the least impurities.

Three methods of synthesizing the benzamides were evaluated
by reacting the amine with acetates, anhydrides and acid chlorides.
The latter procedure was found not only to be the most rapid, but
also resulted in the highest yields. After a suitable synthesis
procedure had been determined, consideration was given to substi-
tution of particular groupings on the ring and an evaluation of
which positions were optimum for maximum sensitivity to electron
capture.

The N-methylbenzamide without substitution was found to be
ineffective as an electron capture. The effect of substitution
was investigated by comparing the 4-nitro-N-methylbenzamide res-
ponse to that of 4-bromo-N-methylbenzamide. It was determined that
halogen substitution was the more sensitive to electron capture
detection. Para-iodo-N-methylbenzamide was found to be difficult
to prepare because of the very low solubility of 4-iodobenzoyl
chloride.

It soon became apparent that it would be necessary to consider
the physical consequences of substitution such as solubility and
volatility, in addition to the influence of substitution on EC
response. Although the 2,4-dichloro substitution produced approxi-
mately twice the EC response as compared to the 4-bromo, impurities
in the 2,4-dichlorobenzoyl chloride resulted in such large inter-
fering peaks that this derivative had to be eliminated from further
consideration. Unlike the 2,4-dichloro homolog, 4-bromobenzoyl
chloride was found to be relatively pure in a crystalline state
at room temperatures. Because of the essential requirement for
high acid chloride purity, further investigation into other possible
substitution patterns were discontinued. The EC sensitivity of
the 4-bromo-N-methylbenzamide derivative was determined to be
more than adequate to meet the analytical requirements for detec-
tion of microquantities of N-methyl and N,N-dimethylcarbamates.





O
CH NHC


0
O
CH NHC
3


SCI
CI


0


NO2


O
CH NHC CI
CI


S O
CH NHC (D- Br
L 3 -


IT BENZAMIDES


0
(CH3)N S


CI


0
CH NHS F


I ANILINES


IV SULFONAMIDES


O CI
CH3NHC-C-CI
31


OF F
CH NHC-C-C
3 F i
F F


F
-C-F
F


III AMIDES


Figure 1 Compounds synthesized and evaluated for
response to electron capture detection


N02




NO2


(CH32N
N


CH3NH









1-2. Conversion of Carbamates to Benzamides
The conversion of carbaryl to 4-bromo-N-methylbenzamide,
as conducted in this experiment, is illustrated in figure 2.

O
II H++
3 hydrolysis 3 3

OO
CARBARYL (neutralization) OH-


CI-C- -Br + CH3NH2

ACID CHLORIDE METHYLAMINE


(catalyst) OH-
0
O
CH NH-C-O -Br <
3- coupling
BENZAMIDE



Figure 2. Acid hydrolysis of carbaryl to methylamine and subse-
quent coupling with acid chloride to derive 4-bromo-
N-methylbenzamide.

Acid hydrolysis is preferred to basic hydrolysis since its use
results in the quantitative retention of methylamine as a salt.
The most suitable acid for this reaction was concentrated sulfuric
heated to 1250C. The acid-containing methylamine salt was diluted
in water and then covered with a known volume of benzene containing
an excess of the 4-bromo-benzoyl chloride. Sufficient sodium hydrox-
ide was added to neutralize the acid and alkalize the aqueous
phase. This permitted the released of the methylamine from its
water soluble salt, resulting in its being dissolved immediately
in the benzene layer. In a modified Schotten-Baumann synthesis,
the methylamine reacted with the acid chloride while being contin-
uously agitated in the water-benzene mixture. Conveniently,
excess sodium hydroxide was found to act as a catalyst for the
coupling reaction. The overall hydrolysis and coupling was quan-
titative on the micromole scale necessary for pesticide residue
analysis. Quantitative coupling was achieved in approximately
10 minutes and the resulting benzene phase was found to contain
no plant extractives detectable by EC gas chromatography. The


-10-









benzene solution containing the derivative in a predetermined vol-
ume was found to be sufficiently stable for satisfactory chroma-
tography, even after several days of refrigeration.

No separate treatment for cleanup of the crop extracts was
found to be necessary since it was incorporated in the hydrolysis
and coupling procedure. During hydrolysis of carbaryl, sulfuric
acid was found to simultaneously oxidize the plant extractives,
converting them to water soluble products and thereby effecting
the first cleanup step. Final extract cleanup took place during
the coupling reaction in which the methylamine was extracted into
the benzene layer while the acid-treated plant extractives remained
in the aqueous solution. Consequently, the benzene layer, con-
taining the derivative, was found to be relatively free of crop
extractives which may be manifested as interfering peaks in gas
chromatographic analysis.

Purified 4-bromo-N-methylbenzamide was used as a standard to
determine the yields for hydrolysis, coupling and recovery. Table
1 illustrates the detector response in the range of 0.2 to 10.0
nanograms of derivative.

Table 1. Electron capture detector peak height response to low
levels of 4-bromo-N-methylbenzamide.
9
4-bromo-N-methylbenzamide (1 x 10 amps full scale)
(Nanograms) Peak Height Response (mm)

0.0 0
0.2 8
0.4 16
0.6 24
0.8 32
1.0 36

(3 x 109amps full scale)
2.0 20
4.0 35
6.0 48
8.0 55
10.0 60


Complete coupling was achieved in 10 minutes as indicated in figure
3 by using EC-GLC to monitor the reaction of microgram quantities
of methylamine hydrochloride and an excess of acid chloride. By
following the described coupling procedure, it was found possible
to quantitate the acid hydrolysis of known quantities of carbaryl.
Efficient hydrolysis was obtained by using concentrated sulfuric
acid at an elevated temperature, thereby retaining all the basic
amine as a salt. Moreover, the oxidizing properties of sulfuric
acid were utilized to convert the plant extractives to water-soluble


-11







compounds, thereby facilitating a liquid-liquid extraction of methyl-
amine with water and benzene. Excessive background attributable
to plant extractives was eliminated by this procedure and recoveries
of carbaryl residues from crop extracts were found to be over 90%.


100-




z 80-
6O-
0
F-m
W



0

40-
V%


20-




0'


6 9 12
MINUTES


15


Figure 3.


Percentage completion of methylamine coupling with 4-
bromobenzoyl chloride to form benzamide derivative.


The interfering and non-interfering peaks illustrated in
figure 4 are due to hydrolyzed impurities from the acid chloride
reagent during the coupling reaction. This reagent-induced inter-
fering peak (high blank) was found to be of equal size in all
samples, regardless of the amount of pesticide or crop extractives
present.


-12-





80-


70-


60-


50-


40-


30-.


20-


3ppm Carbaryl
(50 gm Crop Equivalent)


N Reagent Blank


I
12


Minutes
'igure 4 Carbaryl peak interference attributable to acid chloride
impurities (no detectable interference from crop extrac-
tives equivalent to 50 grams).


-13-









It was found that extracts from as much as 50 grams spinach
or chicory resulted in no detectable interfering peaks. Efforts
to remove the interfering peak included "GC preparation" purifi-
cation of the 2,4-dichlorobenzoyl chloride and atmospheric control
to eliminate the possibility of methylamine contamination. In
addition, an assortment of columns and temperature programming
was used in an effort to displace the high reagent blank. The
problem was also apparent with the "4-bromo" compound but to a
lesser extent.

Evaluation of the data presented in Table 2 would indicate
that there is generally satisfactory comparative data obtained by
the two methods of analysis.

Table 2. Comparison of carbaryl residue data obtained by two
analytical procedures on samples of field-treated spinach
and chicory (expressed in ppm).

Spinach
Int Fld 1.0 Ib active/acre 2.0 Ib active/acre
Days* Rep. EC-GLC Colorimetric EC-GLC Colorimetric
1 A 28 28 80 94
3 20 18 46 33
8 4.5 4.5 23 18

1 B 38 33 100 120
3 4.0 3.0 11 11
8 3.0 0.8 10 5.0

Chicory
1 A 29 25 130 130
3 27 24 76 76
7.2 7.5 29 37

1 B 66 47 80 83
3 35 41 37 50
8 7.0 4.9 7.0 11
*Time interval since last of 4 field applications of carbaryl.


It is felt that the described gas chromatographic procedure has
several distinct advantages over the colorimetric method. Since
all processes following extraction are carried out in one flask,
the GLC procedure utilized considerably less glassware. The
derivative in the GLC procedure is stable for days whereas the
chromogenic agent in the colorimetric method is very unstable.
The colorimetric method is applicable only to carbaryl which forms
l-naphthol following alkaline hydrolysis. On the other hand, the
gas chromatographic procedure should be applicable to most N-
methyl and N,N-dimethylcarbamates. Preliminary work indicated that
the proposed electron capture GLC procedure can be successfully


-14-








applied to the analysis of residues of Matacil (4-dimethylamino-
m-tolyl methylcarbamate). The primary derivative used in this
study is relatively simple to synthesize and the usual time-
consuming cleanup is eliminated by the hydrolysis procedure des-
cribed. The limit of sensitivity of the EC-GLC procedure is
approximately 0.2 ppm which compares favorably to the colorimetric
method. Extreme sensitivity is not necessary for carbaryl analyses
because of the relatively high tolerance established for this
pesticide on agricultural commodities.

One of the disadvantages of the proposed gas chromatographic
procedure is the interfering high blank which is of no consequence
when dealing with substrates containing containing carbaryl resi-
dues of 1.0 ppm or greater. Another possible disadvantage of the
EC-GLC procedure may be the necessity for establishing a daily
standard curve when analyzing a group of samples.

Publications:
R. L. Tilden and C. H. Van Middelem. Determination of carbaryl
as an amide derivative by electron-capture gas chromatography.
J. Agr. Food Chem. (Submitted June 1969).


-15-








Research Team No. 2


Title: New and Improved Techniques for Pesticide Residue Analysis
Co-principal Investigator: H. A. Moye

2-1. Phosphorescence and fluorescence characteristics of newly
developed pesticides.

Nine promising new pesticides were investigated to determine
their fluorescent and phosphorescent characteristics with the in-
tention of uncovering potentialities for the analysis by fluores-
cence or phosphorescence of residues on agricultural commodities.
Excitation and emission maximum analytical curves and limits of
detection were recorded.

All compounds were found to be soluble in ethyl alcohol.
Since this solvent is easy to purify and freezes to a clear glass
at liquid nitrogen temperatures it was chosen for both the fluo-
rescence and phosphorescence measurements. Reagent grade anhy-
drous ethyl alcohol was distilled through a four foot vacuum
jacketed fractionating column. This was sufficient to produce a
product exhibiting neglible fluorescent and phosphorescent back-
ground.

An Aminco-Bowman spectrophotofluorometer was used for the
measurements with the phosphorescope installed for the phospho-
rescence measurements. Limits of detection were set at those
concentrations which would give an intensity reading of 1 per
cent full scale over the ethanol background reading. The instru-
ment was standardized with toluene in ethanol and the results are
shown in Table 1.

Table 1. Luminescent limits of detection and spectra.
Fluorescence Phosphorescence
Pesticide exc. em. Lim. of Det. exc. em. Lim. of Det.
Azodrin 290mu 407mu 2.3 x 10-8M
Bayer 25141 370 437 8.3 x 10-6
0,0'-diethyl-0-
3,5,6-trichloro- w
2-pyridyl phos- No Fluores- U
phate --- --- cence.
Dursban 354 417 4.7 x 10-6
Dyfonate 397 472 1.3 x 10-7
Dylox 352 414 1 x 107
Gardona --- --- No fluores-
cence.
Trifluralin --- --- No Fluores- 0
cence.
-7
Turbacil 382 481 1 x 10


-16-







It was felt that none of these compounds exhibited sufficient
fluorescence to warrant additional investigations for residue ap-
plication; in addition the emission maximum were all close to the
450 mu maximum most commonly found in crop extracts. None of these
compounds showed significant phosphorescence over that of the sol-
vent background.

2-2. Fluorescent dyes as indicators for methylamine, a hydrolysis
product of N-methylcarbamates.

An easily obtainable hydrolysis product of most N-methyl-
carbamate pesticides is methylamine. This compound was seen to
give litmus paper reactions in quantities as small as 50 ug. The
fact that many pH sensitive organic dyes fluoresce strongly in
certain forms offered a promise of using them for fluorescent
detectors for gaseous methylamine. By observing the fluorescence
of either the basic or acidic forms, and then creating a very small
pH change with the methylamine it was expected to see a corres-
ponding change in fluorescence.

The following dyes were studied in acidic and basic form for
fluorescence: 2,4-dinitrophenol, tetrabromophenolphthalein, 2-
nitro-l-naphthol, malachite green, 2,7-dichlorofluorescein, Sudan
Black B, Rhodamine B, Alizarin red, phenolphthalein, bromthymol
blue, N, N-dimethyl-p-phenylazoaniline, phenylazoaniline, and
azobenzene.

Of these only dichlorofluorescein and fluorescein appeared to
fluoresce strongly enough in both acidic and basic forms to be
suitable. Getting consistent fluorescence changes with uniform
amounts of methylamine added was impossible however, due to random
fluctuations (probably due to C02 absorption) of fluorescence of
the dye reagent. Rather than titrate in an inert atmosphere the
project was abandoned as impractical for use as a gas chromato-
graphic detector.

2-3. Phosphorimetric analysis of parathion

Parathion as p-nitrophenol had been shown to be highly sensi-
tive to phosphorescence measurement. It was analyzed in urine;
however, a thin-layer chromatographic cleanup was necessary to
eliminate interfering compounds that were co-extracted from the
urine. Preliminary work on celery extracts, that were hydrolyzed
according to the procedure to be described for parathion, showed
that they emitted phosphorescence at 400 mu as opposed to the 125
mu for the hydrolyzed parathion (p-nitrophenol). This wavelength
differential appeared promising for the analysis of parathion in
celery extract without preliminary cleanup.

The American Instrument Company spectrophotofluorometer was
used for the experiment. Two mm. slits were used throughout except
when 0.5 mm slits were used for recording of spectra, to provide
better resolution. The phosphoroscope attachment was installed to
allow measurement of the phosphorescence without the interfering
fluorescence.


-17-






The following procedure was ultimately arrived at: chopped
celery was extracted by shaking with hexane-isopropanol. The ex-
tract was concentrated by steam in a Kuderna-Danish flask and a
small aliquot added to the hydrolysis solution. Hydrogen peroxide-
sodium hydroxide was used for the hydrolysis of the parathion to
the strongly phosphorescent p-nitrophenol. The hydrolysis solu-
tion was acidified and the p-nitrophenol extracted into ethyl
ether, which was diluted with ethanol made basic with 2-amino-
ethanol for phosphorimetric measurement. Fortification levels of
0.08 to 60.0 ppm yielded recoveries between 79 and 112% (Table 1).

Table 1. Parathion recoveries from fortified celery; parathion
measured phosphorimetrically as p-nitrophenol.

Final Concentration Added Recovery
(x 10-8M) PPM PPM %

0.69 0.08 0.09 112
1.38 0.16 0.13 79
2.75 0.32 0.26 82
5.50b 0.64 0.56 87
7.5 0.87 0.74 85
5.0 x 10-6M 60.0 55.0 92

a Averages of four samples at each concentration.
b
Eight analyses gave a relative standard deviation of 4.2%.


A number of celery samples that had been analyzed colori-
metrically by the Florida Department of Agriculture mobile pesti-
cide laboratory at Belle Glade were obtained. These were in the
form of extracts that were capped and shipped without refrigera-
tion. Analyses were performed at our laboratory on these unclean
extracts by electron capture gas chromatography and phosphorimetry.
The results are summarized in Table 2.

In all cases the colorimetric data showed lesser amounts than
did the other two methods. The gas chromatography values in Table
2 contain an enhancement factor of 0.65. This is an average of in-
dividual values ranging from 0.6 to 0.7 and hence is somewhat in-
determinate.

The procedure will not distinguish among parathion, paraoxon
and p-nitro-phenol, the latter two naturally occurring on field
weathered crops. EPN which gives p-nitrophenol upon hydrolysis will
also be determined as parathion. Those compounds which were inves-
tigated for possible interference and which gave none were: 2-
chlorophenol, 4 nitrophenol, 2,4-dinitrophenol, pip' DDT, p,p'-DDD,
p,p'-DDE, dicofol (Kelthane), methoxychlor, toxaphene, Kepone,
Sulphenone, tetradifon (Tedion), Orthotran, ronnel, Co-Ral, Diazinon,
Guthion, Carbophenothion (Trithion), Aramite, isolan,carbaryl
(Sevin),Zectran, Bayer 44646, Bayer 37344, NIA 10242, UC 10854,
Imidan, 2,4,5-T, 2,4-D, p-chlorophenol, 2,4,5-trichlorophenol and
a-naphthol.


-18-









Analysis of parathion in celery by electron capture gas
chromatography, colorimetry, and phosphorimetry.


Sample Electron Capture ppm Colorime
214 4.6 2.0
215 5.8 2.5
216 3.9 2.5
217 5.6 4.6
218 5.1 3.6
219 5.3 3.6
220 5.6 3.6
221 4.7b 2.7
222 2.3
223 4.3 2.5
224 4.5 2.9
225 2.5
226
227 6.1 4.7
228 3.9
229 4.1 3.0
230 3.6 1.6
231 2.1
232 2.0
233 4.2 2.3
234 5.3 3.1
235 6.1 4.5
236 5.6 2.1
237 4.4 3.0
238 5.1 2.3
239 6.6 4.3
240 5.5 4.0
a. corrected for celery peak enhancement
b indicated lost in handling


try ppm


of parathion.


The method is not completely specific; neither is the Averell-
Norris procedure. The phosphorimetric method follows the same
sample-to-sample trends as both the Averell-Norris and the electron
capture methods, and it is at least as reliable as the electron
capture method.

Publications:
H. A. Moye. 1968. Comparison of a phosphorimetric procedure for
parathion on celery with the Averell-Norris and electron capture
methods, J. Assoc. Offic. Anal. Chem., 51, 1260.

2-4. Construction, operation and improvement of a microwave powered
discharge atomic emission gas chromatograph.
A microwave powered discharge atomic emission gas chroma-
tography detector was first reported by McCormack, Tong and Cooke.
This detector was used by Bache and Lisk for organic phosphate


19-


Table 2.


Phosphorimetry ppm
3.1
7.5
2.9
5.2
3.8
4.9
5.3
3.5
2.9
6.1
4.1
6.1

5.2
4.1
3.5
2.3
3.8
2.6
4.4
4.7
6.7
5.8
5.3
7.0
7.3
5.9









residues and organic iodine residues utilizing argon as the carrier
and discharge gas at atmospheric pressure. Subsequently they
reported a sensitivity enhancement by operation at reduced pressure.

A somewhat similar instrument was constructed and used to
study the emission characteristics of parathion, as a representa-
tive organophosphate, with respect to a number of parameters.
Consequently it was discovered that an enhancement of the atomic
phosphorous emission from parathion could be obtained over that
with either argon or helium by using a mixture of argon and helium.
This enhancement was also apparent for the chlorine emission of
p,p'-DDT and lindane, and also for the iodine emission of 2-iodo-
butane.

A block diagram of the chromatograph is shown in Fig. 1.
Warner-Chilcott Models 601-1 oven, 660 injection and output housing
and a 607-4 proportional temperature controller were used.Fig. 2
and 3 are photographs of the completed chromatograph. A 1P28
photomultiplier tube was used in conjunction with an Eldorado
model 211 photometer. The microwave power was generated by a Ray-
theon PGM10X1 generator. An American Instrument Co. scanning
monochromator, with 1200 lines/mm grating and bilaterally adjust-
able slits, Model 4-8401, was used. A second monochromator of the
same type was used in the scattered light tests. A tapered
cavity was used.

The phosphorous line at 253.57 mu was chosen to optimize
the instrument for discharge tube size, microwave power, discharge
tube ,pressure, slit width, carrier gas type and carrier gas flow
rate.

Of the tubes tested, 5-, 2-, 1- and 0.5 mm i.d., the 0.5 mm
thick walled (capillary) tube gave the highest sensitivity, in
terms of signal to noise ratios for parathion.

For all compounds tested microwave power did not affect
signal to noise ratios.

The monochromator was adjusted for optimum slit width. Fig. 4
shows the effect of slit width on the parathion signal to noise
ratio while fig. 5 shows the effect of slit width on the parathion/
lindane selectivity ratio.

A number of discharge gases were tested individually and
also mixed with argon. These were N2, CO2, H 2 and He. All
but He gave very high continuum radiation wit unstable and noisy
discharges when present even in trace quantities. Fig. 6 shows
the effect of He mixed with A on parathion signal to noise ratio.
Fig. 7 shows the effect of He mixed with A on parathion/lindane
selectivity ratio.

The limits of detection for a number of organophosphate
pesticides were determined and are shown in Table 1.


-20-








vac. pump


monochromator


photometer


ity


generator




recorder oven
A He


Figure 1 Block diagram of microwave powered emission gas chromatograph.


-,,,
--








































































Figure 2 Microwave emission gas chromatograph.


-22-









































Figure 3 Microwave emission gas chromatograph.


-23-


T~I







60 50/o SE 30 80/100 G.C.Q
4'x 1/8" GLASS
TOTAL FLOW: 27 cc/min.
COL.: 180 C
50- INJ.: 2100 C
GAS: 85%/ He-15%/0 A
POWER: 50%/
L \ PRESSURE: 25 m.m. Hg
)O- TUBE: 0.5 mm.
z 10 Ng Parathion

z
0 30-



20



10



I I I I I I I
10 20 30 40 50 60 70 80
SLIT WIDTH, p


Figure 4 Monochromator slit width versus parathion signal/noise.





425

400

375

350

325

300

275

250

225

200

175

150

125

100


*




50/o SE 30 80/100 G
4'x 1/8" GLASS
COL.: 1500 C
INJ.: 215 C
TOTAL FLOW: 27cc/n
GAS: 85/0oHe-15%/o A
POWER: 50%/o
PRESSURE: 25m.m.
\1 pg.9
TUBE: 0.5m.m. 10



*


.C .O.



nin.


Hg
Lind
Ng F


a | a


10 20 30 40 50 60
SLIT WIDTH,


70 80 90 100
p


re 5 Monochromator slit width versus parathion/lindane selectivity.


-25-


ane
'arathion


75





50-

25-


a a A







160
5/o SE 30 80/100 G.C.Q.
150- 4'x 1/8" GLASS
TOTAL FLOW: 27 cc/min.
140- COL.: 1800 C
INJ.: 220 C 10 Ng Parathion
130- SLIT: 6 p
POWER: 50%/o
120- PRESSURE: 25 m.m. Hg
TUBE:O.5mm
110

100

0 90-

80-

z 70
(D
60

50

40

30 -

20-

10

10 20 30 40 50 60 70 80 90 100
/o He

gure 6 Discharge gas composition versus parathion signal/noise.


-26-






680

640

600

560

520

480


5%/o SE 30 80/10C
4'x 1/8" GL
TOTAL FLOW: 27cc
COL: 1500CL 1800
INJ.: 2150C
SLIT: 6p
POWER: 50%/o
PRESSURE: 25m.m
TUBE:0.5 mm
10 Ng Parathion
1 pg Lindane


* a...


10 20 30 40 50
/0


60
He


70 80 90 100


ire 7 Discharge gas composition versus parathion/lindane selectivity.


-27-


)G.C.Q.
ASS
:/min
P



. Hg


0
S440

>400

> 360

w 320
w
)lJ


20ou

240

200

160

120

80


40F


I/ I N I1 o


I








Table 1. Limits of detection (based on peak heights) calculated
on signal/noise = 2/1.

Pesticide Limit of Detection (ng) Emission (mu)
Parathion 0.15 253.57
Trithion 0.07 253.57
Ronnel 0.19 253.57
Methyl trithion 0.34 253.57
VC-13 0.43 253.57
Aspon 0.48 253.57
Dicapthon 0.65 253.57
Ethion 0.48 253.57
Thimet 0.11 253.57
pp'-DDT 11.5 221.00
Lindane 7.2 221.00
2-Iodobutane 0.05 206.20

A new emission line was discovered for chlorine containing
molecules at 221.00 mu. This did not produce sensitivities as
high as for the iodine or phosphorous containing pesticides. The
206.20 mu line was used for the one iodine containing compound
measured, iodobutane.

Compared to electron capture detection the microwave detector
gave a remarkable degree of selectivity. Fig. 8 shows the responses
of the two detectors to a celery extract containing parathion.
The microwave detector gas chromatograph has been used in this
laboratory on a routine basis, giving progressively less operating
difficulties than those experienced with a variety of electron
capture instruments.

Publications:
H. A. Moye. 1967. An improved microwave emission gas chromatography
detector for pesticide residue analysis. Anal. Chem., 39, 1441.

2-5. A direct current powered discharge emission detector for
organophosphate analysis.
Using the basic gas chromatographic components described in
section IV that were used for the microwave detector studies the
possibility of using a direct current powered discharge for creating
atomic line emission from fragmented organic pesticides was inves-
tigated.

A wide variety of discharge tubes with a wide variety of dis-
charge energies were investigated. Also investigated were the
electrode material, electrode spacing, discharge gas composition,
discharge gas pressure, discharge gas flow rate, viewing segment
of discharge. The 253.57 mu phosphorous line of parathion was
again used for optimization. Fig. 1 shows the optimum discharge
tube configuration arrived at. The electrodes were of platinum
wire. This tube was operated at 255 V. across the electrodes with


-28-






M.W.


01




I I I I
2 3 4 5
10 /L


Figure 8 Response to extract containing parathion by E.C. (left) and microwave (right) detectors.


14l1


E.C.


;C'





















































































Figure 1 D. C. powered discharge tube.



-30-


1


//5. 5m?








25 milliamperes of current. Table 1 shows the limits of detection
determined for a number of organophosphates at 253.57 mu.


Table 1. Limits of detection.

Pesticide Limit of detection (Ng)

Parathion 0.7
VC-1-13 0.8
Ronnel 8.0
Ethion 1.0
Aspon 8.7
Methyl trithion 4.0
Dicapthon 6.5

These are not as good as those obtained with the microwave de-
tector, however, the instrument is still in the process of being
optimized for most of its parameters and should be capable of doing
considerably better.

2-6. A non-extinguishing flame photometric burner and housing for
gas chromatography.

The extremely useful flame photometric detector of Brody and
Chaney has two serious drawbacks, the flame goes out after each in-
jection and it is extremely expensive due to the special machining
required of the parts. This project was intended to design and
build a burner and housing which would eliminate these deficiencies.

It was noticed that a distinct similarity existed between an
acetylene torch cutting tip and the Brody-Chaney burner. Consequently,
a number of acetylene torch tips were purchased and assembled with
other brass parts by silver soldering (Fig. 1 & 2). The acetylene
tips that were tested were: K.G.M. S6-4, K.G.M. S6-5, Airco 144-12,
Goss 2272-1, Victor 1-111-3, Victor 6-1-115-6, Victor 1-101-0, and
Victor 4-1-101-4.

Wide ranges of H2, 02, Air and N2 were tried for each to assure
that the optimum operating regions would not be overlooked. The hy-
drogen flames were inspected visually in a darkened room while 1 ug.
quantities of parathion were repeatedly injected into the Varian 1200
gas chromatograph. The green chemiluminescence of phosphorous was
readily apparent with a few of the burners. The K.G.M. S6-4 was far
and away the superior, however, for luminescent intensity.

The relative responses of 0.4 ng of a methyl trithion and para-
thion mixture are shown in A for the Brody-Chaney burner and in B
for the acetylene torch tip burner of Fig. 3. This was with the 526
mu phosphorous filter in place. The response with the sulfur filter
in place was about one tenth of that of the phosphorous, as is the
case with the Brody-Chaney burner.

Publications:
H. A. Moye, A non-extinguishing flame photometric detector burner
and housing for gas chromatography. (Submitted to Anal. Chem.)


-31-












3.1"


0.38"


4.0"


S 6


C
D


El


Figure 1 Acetylene torch tip burner in assembled and disassembled forms.


QTP]







O.D.


I.D. 0.40"








O.D. 0.93"


040"


O.D. 063"








I.D. 0.68"


O.D. 0.70"


0 D 0.35 "


Figure 2 Individual parts of burner, with dimensions in inches.










































INJ.

4 ,O INJ.





igure 3 Response to 4NG of parathion and methyl trithion by Micro-Tek
burner (left) and acetylene torch tip burner (right).


-34-






Research Team No. 3

Title: Uptake, Translocation and Metabolism of Pesticides in
Higher Plants

Co-principal Investigator: N. P. Thompson

3-1. Transport of Auxins in isolated stems and leaves.

Separating an organ such as a leaf or an internode section
from the remainder of a plant makes possible a study of the be-
havior of the isolated system apart from other influences. It had
been shown in isolated Coleus internodes (Thompson, 1965) that
indole-acetic acid (IAA) had an effect on regenerating vascular
tissue very similar to that occurring in an intact plant. 2,4-D
had a similar but more pronounced effect at similar concentrations.
The purpose of this work was to investigate several plant species
in an effort to determine whether this was a general morphological
response to these auxins. Since vascular tissue in leaves bears
resemblance to that in stems, isolated leaves were also included
for study.

Peanut, okra, and pea plants were grown under controlled con-
ditions and internodes which had just ceased elongating were iso-
lated and treated with radiolabeled auxin at the morphological
apical or basal end. A horizontal wound was made severing several
conducting bundles in these isolated internodes as well as in in-
ternodes which were left on intact plants. After seven days inter-
nodes were divided into top, middle and bottom sections, the middle
section containing the wound. These sections were extracted with
ethanol for determination of radioactivity and then middle sections
were'stained and mounted on microscope slides for anatomical obser-
vation. Leaves also were isolated, treated at apical and basal ends
with radiolabeled IAA and 2,4-D and wounded in the midrib. After
seven days they were freeze-dried and radioautographs were made.
Leaves were stained and mounted for anatomical observation.

In each of the isolated stem sections and leaves, the movement
of applied auxin was basipolar. This can be seen in Fig. 1 which
illustrates the movement of 2,4-DC14 in peanut internodes. IAA-C14,
moves in a similar fashion when applied but it is less basipolar
and more mobile than 2,4-D (Fig. 2). The counts of regenerated
xylem cells in Table I, show that 2,4-D is morphologically more
active at a given concentration than IAA. It is evident that C14
applied as IAA reaches the wound area from basal application (Fig.
2). Since there is no stimulation of vascular tissue formation
around the wound as a result of basal application, it is suggested
that the mode of presentation of auxin to cells in the wound area
is important. What ever the criteria should be, basipetally moving
auxins meet them and acropetally moving auxins do not. There is
another alternative, though not presently considered likely, that
there is a differential metabolism of auxins moving in opposite
directions. The auxin-vascular regeneration relationship has now
been demonstrated in 3 additional species of higher plants and
appears to be a general phenomenon.


-35-









FIGURE 1


0.1% I.A.A.

66-XYLEM-15


FIGURE 2


-500


-200



S 102

BASE


r i1
I 0.01 / 2,4-D
I I


I I


S II
0.1-%. 2,4-D


I I 270-
9I I
I I 94-
I I


I ri

I I I
I API
I I


S I I I I
I I I I I I
I i I I
I I I
- I I I I I
I I I I
II I
I I I
S I I I I I

APEX


0.1 1


-.XYLEM--0


Figure 1 & 2 Log plot counts per minute 14C in isolated internodes. Solid area indicates point
of application. Central numerals are counts of regenerated xylem cells.
Figure 1 Peanut, solid line indicates 0.1% 2,4-D treatment, dashed line indicates
0.01% 2,4-D at apical end. Figure 2 Peanut, 0.1% IAA treatment


A I I
APEX


SI I -BA
BASE







Table 1.


Treatment


Regeneration of xylem cells 7 days after
intact plants and isolated internodes.a


Intact

Isolated
Apical Treatment
0.1% IAAb
0.1% 2,4-D
0.1% 2,4-D
1.5 mg/l 2,4-Dc
7.8 mg/l 2,4-D


Lanolin


Basal Treatment
0.1% IAAb
0.1% 2,4-D
1.5 mg/l 2,4-Dc
7.8 mg/l 2,4-D


Pea

38 (1)




259 + 45d
289 7 109e



11 + 5 (3)


16 + 9


Peanut

88 + 23



66 + 10
270 7 140
94 + 40


18 + 8


15 + 12
U


wounding


Okra

151 + 16



186 + 113


216 + 61 (3)e
722 7 263e


7+4


16 + 7

5 + 5 (3)
7


a. n = 4; numbers in parentheses denote different n value.
b. lanolin used as carrier
c. gel used as carrier
d. significantly different from control
e. significantly different from corresponding basal treatment


Publications: 14C 14C
Thompson, N. P., 1968. Polarity of IAA- and 2,4-D- transport
and vascular regeneration in isolated internodes of peanut. In:
Biochemistry and Physiology of Plant Growth Substances ed. Wightman
and Setterfield, Academic Press, pp. 1205-1213.

Thompson, N. P. (in prep.). The transport of auxin and regeneration
of xylem in okra, pea and peanut stems. American Journal of Botany.
(pre-print)

3-2. Transport of 2,4-D vs. Dieldrin.

The basipolar transport of auxins in plant tissue is well
known. This experiment compares the uptake and movement of the
auxin 2,4-D-C14 with Dieldrin-C14. Recent experimental work has
shown that Dieldrin is taken up by plant roots and moves into aerial
plant parts.

Internodes were excised from peanut plants grown under con-
trolled conditions. C14 labeled 2,4-D or dieldrin incorporated in
Dow 2645 gel was placed on the apical or basal end of the stem sec-
tions with plain gel placed on the other end. The internodes were


-37-








placed on glass microscope slides on moist filter paper in petri
dishes for seven days. The gels were carefully wiped off and
internodes were divided into 3 equal sections, morphological top,
middle and bottom. Gels and internode sections were extracted and
radioactivity was assayed by Liquid Scintillation Spectrometry.

Table 2 illustrates the acropetal and basipetal movement of
2,4-D-C14 and dieldrin-C-14, respectively. Analyses of the amount
of movement shows that 21% and 33% of the applied 2,4-D was taken
up by apex and base, respectively. Forty percent of that taken up
at the apical end moved through the tissue but only 6% moved through
the tissue after being taken up at the basal end. This indicates
a definite basipetal polarity of transport.

Fourteen percent of applied dieldrin was taken up following
apical application and 22% following basal application. The per-
centages dieldrin which moved through internodal tissue were 33%
apically applied, 30% basally applied. These results show a lack
of polarity of movement, in sharp contrast to the basipolar trans-
port of the growth regulator 2,4-D. The movement of dieldrin in
intact plants is under the influence of transpiratory forces and
other factors and could likely be different from that exhibited in
isolated internodes. Diffusion could be responsible for dieldrin
movement but there appears to be a higher concentration in the
receiver gels in each case indicating a lack of equilibrium between
final third of the internode and the receiver gel. A similar con-
centration against the gradient, across the internode-gel interface
is apparent in the transport of 2,4-D.

Table 2. The movement of 2,4-D-C14 and Dieldrin-C14 through isolated
internodes of peanut. a C14

7.8 mg / liter 2,4-D-C14 0 Plain
APEX BASE

44,846 190
3,939 44
4,073 639
217 3,262
2,739 8,530

35 mg / liter Dieldrin-C14

APEX BASE

\ 14,621 418
1,399 53
199 84
104 1,000
680 5,570


-38-







3-3. Uptake of Azodrin, 2-4-D and Dieldrin through the roots of
higher plants.

The initial entry of many pesticidal chemicals into plants is
through the roots. Root entry is apparently the primary means of
uptake of persistent pesticides following a previous soil contami-
nation. An artificial system is described which allows the quan-
titation of pesticide uptake into plant tissue.

Seeds of peanut, corn, soybean, cabbage and okra were germina-
ted on moist filter paper in petri dishes in the dark. When axial
polarity became apparent, that is root and shoot just began to be
visible, the seeds were placed on a sheet of wet filter paper
(about 12" x 3") such that the shoot ends were 1/2" from one long
edge of the paper. Germinating seeds were spaced about one inch
apart. A second piece of wet filter paper was placed on top of
the seeds and both pieces of paper with seeds in between were rolled
and placed root ends down in a beaker. Radiolabeled pesticides were
incorporated into water which was added to the beakers; additional
water was added as needed. After periods ranging from 4-14 days in
the beakers some plants were divided into root and shoot portions,
extracted and assayed for radioactivity by Liquid Scintillation
Spectrometry. A portion of this extract was placed on TLC plates
and the chromatograms formed were checked for metabolites. Other
plants were autoradiographed.

Azodrin-C14 which was added to the roots of corn, okra, peanut
and cabbage moved into aerial portions of these plants as shown by
extractions and radioautographs. In corn, extracts of washed roots
and unwashed shoots showed counts of 240 and 275 cpm respectively.
There is no implied significance in the number of counts, except
that a goodly amount moved into the shoots.

In okra, a portion of shoot extract contained 730 cpm and a
similar portion of root extract contained 475 cpm again showing
transport to the shoot.

In extracts of peanut the shoot contained 2,435 cpm, the root
205 cpm. It is doubtful that these counts express any relative
shoot-root value but they do indicate movement of Azodrin-Cl4 into
the shoot. Pre-coated Cellulose TLC plates spotted with these ex-
tracts and eluted with 40:9:1, acetonitrile:water:ammonium hydroxide
indicated only one radioactive spot at Rf 0.7-0.9. This is the Rf
of Azodrin in this solvent system.

Cabbage plant extracts developed similarly on TLC plates
showed also only one spot at the Rf of Azodrin in one experiment.
In a second experiment possible metabolites in the form of more
than one radioactive spot appeared. This aspect is being checked
further.

The movement of 2,4-D-C14 and dieldrin-C14 into peanut, corn
and soybean plants was investigated using the same system. All the
plants treated with 2,4-D in this way, including corn, were retarded


-39-







in development when compared with those treated with dieldrin. Ex-
tracts of roots and shoots contained radioactivity. Any radioactive
spots that appeared on TLC plates were at the Rf of 2,4-D or dieldrin,
respectively using silica gel plates developed in isopropanol:
ammonium hydroxide, 7:3 (2,4-D) and aluminum oxide plates developed
in 2% acetone in n-heptane (Dieldrin).

3-4. The Behavior of Azodrin in Corn.

Previous work with Azodrin on dicotyledonous plants showed
various degrees of metabolism and movement of the insecticide. The
purpose of this investigation was to determine the rate of uptake,
the persistence, the direction of movement and the degree of metabo-
lism of Azodrin by a monocot, corn.

Corn plants grown under controlled conditions of 16 hr. light
(23-26)-8 hr. dark (17-19) were treated with Azodrin C14 on the
leaf blade. C14 located on the 0-methyl and N-methyl position were
both utilized and results with each were compared. Autoradiographs
were made of some treated leaves 2, 7, 14 and 21 days following
treatment. Various plant parts were extracted with chloroform and
a portion of extracts were assayed for radioactivity by Liquid
Scintillation Spectrometry. Other portions were spotted on Cellu-
lose TLC plates and developed with Acetonitrile: Water: Ammonium
Hydroxide, 40:9:1. Partition coefficients of extracts in chloro-
form-water were also determined.

Autoradiographs indicate that the direction of movement of
Azodrin was primarily from the point of application towards the
leaf tip. No radioactivity was detected in untreated leaves on
the dame plant. 75-100% of the applied radioactivity could be re-
covered after 1, 2 or 3 weeks. Table 3 illustrates the location of
Azodrin in the leaf at various times after application. Of the
radioactivity recovered about 96% was in the form of Azodrin. The
remaining 4% could not be characterized by the procedures and levels
of radioactivity used.

When Azodrin is applied to corn leaves under the conditions re-
ported, a) it is taken up by the leaf, b) it moves primarily toward
the leaf tip and c) unaltered radiolabeled Azodrin can be recovered
from the parts to which it has moved.

Publications:
Whitesell, J. W., 1968. The Behavior of Azodrin in Corn. M. A.
Thesis, University of Florida, Gainesville.

Whitesell, J. W. and N. P. Thompson (in prep.). The Behavior of
Azodrin in Corn, J. Agr. Food Chem. (pre-print).


-40-








Table 3.


Distribution of 1C-labeled Azodrin in treated corn
leaves.


Location
of label


Rinse
prior to
analysis


Activity in Leafc


Azodrin in
leaf tip
me


Base Tip


N-methyl
N-methyl
N-methyl
0-methyl
N-methyl
N-methyl
0-methyl
N-methyl


N-methyl
N-methyl


No
Yes
No
No
Yes
No
No
Yes


No
Yes


One week after treatment
61 3 29 0.38
4 1 31 0.40
71 1 3 0.06
99 1 2 0.04
2 1 2 0.04
85 1 12 0.24
70 4 20 0.40
12 0.24

Two weeks after treatment


48 6
10 4


37
39


S.48
0.51


a specific activity approximately 1 uc/ml
specific activity approximately 2 uc/ml
C represents percentage of amount applied


3-5. Uptake of dieldrin from soil into field grown peanut plants.

Seeds of three varieties of commercial lines of peanut gene-
rally grown in the U. S., Starr Spanish, Florunner and Florigiant,
were sown in the field in soil which had been fortified with 0, 1#
and 4# per acre dieldrin according to a 3 replication randomized
block design. Plants were grown to maturity. Samples of soil
taken at planting and at harvest and plant hay, peanut meats and
shells were extracted and analyzed for dieldrin content. Samples
were analyzed by GLC using an F & M 700 gas chromatograph equipped
with an EC detector and a 3% QF1 column. Some extracts were spotted
an Aluminum Oxide TLC plates for confirmation of dieldrin.

The field in which plants were grown had been treated with
Aldrin two years prior to the test and dieldrin was present in the
0#/acre treatment as indicated in Table 4. All soil levels of
insecticide caused detectable residues in all plant parts. The
dieldrin content of each plant part, hay, meats and shells, was
proportional to the level of dieldrin present in the soil in which
the plant grew. There was no varietal difference in the dieldrin
content of the hay or meats but Florunner shells contained signifi-
cantly greater quantities of dieldrin than Starr Spanish and Flo-
rigiant shells. Both of these latter varieties contain more


-41-


Azodrin
applied
mg


Treated
Area


1.3a
1.3
2.0b
2.0
2.0
2.0
2.0
2.0


1.3
1.3


l- I







epidermal hairs than Florunner, and on this basis, the opposite
result would have been expected, that is, that the hairs would have
facilitated dieldrin uptake. It appears possible to predict levels
of dieldrin which will appear in plants grown in dieldrin contamina-
ted soil. Soil could be analyzed prior to planting and crop planting
plans adjusted to avoid over tolerances. As shown in Table 4, even
low levels of dieldrin present appear in peanut plant tissue.


Table 4.




(#/acre)
0
1
4


0
1
4


0
1
4


Dieldrin levels in soil and three peanut varieties after
soil fortification. (Expressed in p.p.m.)


Starr Spanish
Soil
Planting Preharvest
0.7 0.9
2.1 1.4
5.6 3.5


0.7
1.5
3.9


0.7
2.0
4.5


Florunner
0.8
1.7
3.4

Florigiant
0.9
2.1
3.1


Hay
0.06
0.14
0.24


0.06
0.15
0.27


0.07
0.10
0.28


Meat

1.33
2.50


0.65
1.35
3.00


0.80
1.13
3.53


Shells
1.15
4.16
9.48


1.56
4.55
11.21


1.56
3.10
9.64


-42-







Research Team No. 4


Title: Interactions Among Selected Insecticides and Plant Systems

Co-principal Investigator: W. B. Wheeler

4-1. Fate of Pesticides in Aqueous Chlorella Cultures.

Green algae, Chlorella pyrenoidosa Chick, have been used as
a model system for the study of certain interactions of pesticides
and green plants. Algae possess the same energy trapping systems
and biochemical capabilities as do higher plants.

The study of algae per se is also of value. Interactions,
especially among the rather persistent chlorinated insecticides
and micro-organisms, are potentially of ecological significance.
Both these organisms and certain insecticides are present together
for long periods of time; any effects of one upon another should
be studied.

A. Fate of Dieldrin
Two primary areas of investigation have been studied under
this title: 1) the trapping of dieldrin lost by co-distillation
with water from aqueous algae cultures; and 2) the absorption of
dieldrin by Chlorella.

1. Co-Distillation
The phenomenon of co-distillation of chloro-organic insecti-
cides with water has been recognized for some time, but it had not
been investigated critically under conditions commonly used for
the maintenance of sterile algae culture. Nor had any effective
trap been devised or adapted for the collection of co-distilling
insecticide.

The losses of dieldrin from an aqueous, cell-free medium
during various stages of processing are shown in Table 1. In this
case, it is assumed that the insecticide is added to the flasks in
hexane, the solvent evaporated, aqueous medium added, the flask
fitted with cotton stoppers, autoclaved and finally the sterile
flask incubated. This sequence of events led to very great losses
of dieldrin and this method was subsequently discontinued. In all
future work described, the insecticide was added to an established
culture in a small volume of acetone or ethanol. The problem of
loss by co-distillation during incubation still existed, however.

To solve this, the cotton plugs, normally used in the flasks
were replaced with two hole rubber stoppers. Compressed air was
piped into the flask through one hole and the other hole was fitted
with a gas chromatographic column packing trap which is illustrated
in Figure 1. The efficiency of the trapping material, held a dis-
posable Pasteur pipette, is illustrated in Table 2. In this case
two traps were placed in series so that all the insecticide which
passed through the first trap would be collected in the second.
The insecticide may be recovered quantitatively by eluting the
column packing with a few ml. of hexane or acetone.


-43-




















GC COLUMN


PACKING


GLASS WOOL


Figure 1 Gas Chromatographic Column Packing Trap







Table 1. Losses of dieldrin from Bristol's Medium at various
stages of processing

Process Micrograms of Dieldrin

Added Recovered* % Recovered
Dieldrin added
(Flasks No. 1,2) 10.0 10.0 100

Dieldrin added,
medium added,
stand 60 min.
(Flasks No. 3,4) 10.0 8.9 89

Dieldrin added,
medium added,
autoclaved
(Flasks No. 5,6) 10.0 5.1 51

Dieldrin added,
medium added,,
autoclaved, Incu-
bated 24 hours**
(Flasks No. 7,8) 10.0 2.9 29


average of duplicates
**
incubated at 300C

Table 2. Trapping efficiency of GC column packing traps*

Length of Trapping Time DPM

First 24 hours Trap 1 66,200
Trap 2 0
Second 24 hours Trap 1 40,800
Trap 2 0
Next 88 hours Trap 1 236,800
Trap 2 500

Air movement through the system was approximately 30cc/minute.


While the trap is very effective, significant quantities of
insecticide will be absorbed by the rubber stopper. This can be
conveniently measured and largely prevented by coating the stopper
with a 1-2 mm layer of paraffin and then analyzing the paraffin.
Quantitative recovery of all pesticide placed in an aerated aqueous
system may be obtained by utilization of the trapping device de-
scribed.

The treatment given to aqueous solutions containing dieldrin
governed the quantity of insecticide lost by co-distillation with
water. Co-distilling dieldrin may be effectively trapped on a common
gas chromatographic column packing and easily eluted for quantitation.


-45-








2. Absorption
It has often been implied that chloro-organic insecticides
penetrate algal cells, but no quantitative data from controlled
experimental conditions have been published. With this end in
mind, the following investigations were undertaken.

Chlorella pyrenoidosa Chick were grown in an aqueous medium
(the set up included GC column packing traps as discussed above)
to which 14C-dieldrin was added. Samples of the cellular suspen-
sion were taken at various time intervals and were extracted. All
extracts, trapped material, insoluble tissue residue and growth
media were subjected to liquid scintillation counting; and to thin-
layer chromatography and autoradiography. The results of the
liquid scintillation counting are presented in Table 3. The sam-
pling times, the percentage of the labeled material in each frac-
tion and the total counts per sample are listed. The ethanol
extract, the chloroform-methanol extract and the hyamine hydroxide
portions are indicative of the labeled dieldrin which was found
within the cells. It may be seen that as time progressed, the
amount and percentage of the labeled material within the cells
increased.

Table 3. Distribution of radioactivity of each sampling period*
(expressed as a percentage of the total dpm)

Time Medium** Cells** Traps Total
(hours) Acetone Ethanol C-MI H-HZ Radioactivity***
Rinse Extract (dpm)
0 64.5 33.5 2.0 0 0 313,300
6 44.8 44.1 10.9 9 9 0.51 330,700
12 40.5 30.6 25.2 03 0 3.65 301,000
24 33.0 33.6 29.8 tr tr 3.29 315,400
36 28.1 26.1 39.5 tr tr 6.07 291,300
48 23.9 25.6 45.1 tr tr 4.71 296,900
72 18.00 22.7 45.4 2.6 0.8 10.5 225,500

These illustrative data are from experiment B.
Averages of duplicate samples.
Total radioactivity in each 10 ml sample.
C-M Chloroform-methanol extract.
2H-H Tissue residue solubilized in hyamine hydroxide.
3tr Measurable radioactivity but less than 0.5%.

The absolute quantities of dieldrin, on a per-cell basis, were
also calculated. Table 4 shows data from two different experiments.
There is a general upward trend reaching a maximum value which is
approximately the same for the two experiments. Although initially
there was a two-fold difference in the quantity of dieldrin per cell
between the two experiments, the same maximum per-cell level would
indicate this value is in fact maximum for these conditions.


-46-







Table 4. Dieldrin in algae at each time period*
8
Time ug Dieldrin (x 10- ) per cell
(hours) (Expt. A)I (Expt. B)Z

0 0.20 0.097
6 0.98 0.49
12 1.14 1.02
24 1.11 1.30
36 1.20 1.26
48 1.34 1.44
72 1.50 1.08

* Calculated from total radioactivity found in the
ethanol, chloroform-methanol and hyamine hydroxide
extracts.
146 ug dieldrin added or 12.8 x 10-6 ug/cell
240 ug dieldrin added or 7 x 10-6 ug/cell


No metabolites of dieldrin were detected in any of the frac-
tions, although all were subjected to thin-layer chromatography
and autoradiography.

The absorption of 1C-dieldrin from aqueous medium by actively
growing Chlorella has been demonstrated. Dieldrin penetrated algal
cells rapidly and a maximum per-cell level was reached within 6 to
24 hours after introduction of the insecticide. The changes in
distribution of radioactivity in various extracts of the experi-
mental system with time are presented. As time passed, the cell-
ular'portion contained increasing amounts of labeled insecticide
and more exhaustive extraction was required for its removal from
these tissues; no metabolites were detected.

B. Fate of Azodrin
14 Chlorella grown in aqueous medium was exposed to N-methyl
C -Azodrin for periods up to four weeks. Table 5 presents the
distribution of the radioactivity between the cells and the nu-
trient medium in the experimental flasks which contained radio-
labeled insecticide (i.e. live algae and autoclaved algae) at each
sampling period. Essentially no label was detected in extracts of
cells or in the tissue residues which remained after extraction.

Table 5. Distribution of C-Azodrin between medium and cells at
each sampling period (expressed as a percentage of
total counts).

Time Live Algae Dead Algae
(days) Medium Cells* Medium Cells-
7 98 2 99.5 0.5
14 99.5 0.5 99.8 0.2
28 99.2 0.8 99.9 0.1

Derived from all extracts of cells and the insoluble cellular
tissue which remained after extraction.


-47-







The labeled material remaining in the medium broke down to
more polar labeled compounds with increasing time. No qualita-
tive or quantitative differences in breakdown were apparent among
the three experimental flasks (i.e. algae, dead algae, no ;-gae).
After 7 days, 30% and after 4 weeks, 22% of the total radio-
activity in the medium was intact Azodrin. One major breakdown
product was formed in increasing amounts with time.

Azodrin did not penetrate Chlorella cells over a period of
4 weeks. The insecticide did break down, however, in the aqueous
nutrient medium; the rate of breakdown was unrelated to the pre-
sence or absence of cells.

Publications:
Wheeler, W. B., Trapping of dieldrin lost from aqueous algae
cultures. J. Assoc. Offic. Anal. Chem., July, 1969 (In Press).

Wheeler, W. B., Experimental absorption of dieldrin by Chlorella
(Submitted to J. Agr. Food Chem.)

4-2. Interactions of Dieldrin, Trans-Aldrin Diol and Azodrin with
Tissue Homogenates of Corn, Rye and Alfalfa.

Corn, alfalfa and rye tissue homogenates were prepared by
blending (40C) aerial portions of each crop in sucrose-Tris buffer
and then filtering the slurry through cheese cloth. The homo-
genates were exposed in the light to each radioactive pesticide
individually for periods up to 6 hours. Samples were centrifuged
to give a "particulate" fraction and a "soluble" or supernatant
fraction. The "particulate" moiety contained all but the smallest
organelles and the "soluble" fractions contained any particles,
which did not sediment, as well as the soluble cellular components.

The results are only preliminary in nature and, therefore,
will be presented very generally.

Throughout the 6 hour period at least 90% of the dieldrin is
found in the particulate fractions of all three species. The par-
ticulate portion of a boiled alfalfa control possessed essentially
100% of the dieldrin found in the sample in contrast to the other
species which mimicked the biochemically active samples.

Essentially the same that was said regarding dieldrin may be
said for trans-aldrin diol (TAD). Perhaps more TAD remained in
the soluble fraction but the same behavior in the boiled controls
did occur.

The behavior of Azodrin was very similar to that reported
above with respect to the interactions with algae. Essentially no
Azodrin was detected in the "particulate" fraction; all remained
in the soluble portion.

The radioactive portions were subjected to thin-layer chroma-
tography and autoradiography in an attempt to detect metabolic
products. No metabolites were found.


-48-







Dieldrin and trans-aldrin diol rapidly penetrate the par-
ticulate fraction of alfalfa, corn and rye tissue homogenates,
whereas Azodrin remains completely in the incubation medium or
soluble cellular fraction. After 6 hours of exposure to the
homogenates of three plant species, no metabolites of any of the
compounds were detected.

4-3. Environmental Factors Affecting the Uptake of Dieldrin by
Rye.

Dieldrin is apparently absorbed and translocated by a large
number of crops. The mechanism of this phenomenon is unknown,
however. The purpose of this research is to determine what en-
vironmental factors affect uptake and to gain some insight as to
the mechanism of uptake.

Rye was grown from seed in sand containing 10 ppm dieldrin
with appropriate precautions to prevent surface contamination of
the plants. The effects of relative humidity, temperature and
photoperiod are being studied.

Presently, it may be stated that relative humidity does
affect plant levels of dieldrin. Rye grown under conditions of
low relative humidity does contain significantly more absorbed
dieldrin and possesses higher ppm levels.

The effects of temperature and photoperiod are not as pro-
nounced. The lower growth temperature (650F) did cause statis-
tically higher crop levels than the higher temperature (750F) but
the data concerning the effect of photoperiod (8 hours light vs.
16 hours light) are as yet inconclusive.

Greater uptake by plants grown under conditions of low rela-
tive humidity is expected if one assumes that the quantity of
dieldrin in the plants is related to the amount of water which
passes through the plants. Under conditions of low humidity more
water is transpired and the results obtained would be expected.

The low temperature effect, however, indicates that some active
process may be occurring. One would expect more water to pass
through the plants at 750F than at 650F and thus higher accumula-
tions would be expected at the higher temperature. Since this is
not the case, it appears that some other factors might be controlling
uptake, at least partially, and further investigations will be made.

The root absorption of dieldrin by rye grown in sand appears
to be effected by some environmental factors. Low relative humidity
(54%) causes higher plant residues than conditions of higher rela-
tive humidity (75%). Lower temperature (650F) also led to greater
plant accumulations than did higher temperatures (750F). Effects of
photoperiod (8 hours light vs. 16 hours light) are under investigation.

Publications:
Wheeler, W. B.,"Environmental Factors Effecting Dieldrin Uptake by
Rye" (Submitted to J. Agr. Food Chem.)


-49-







Research Team No. 5


Title: Methods of Increasing the Elimination of Persistent
Chlorinated Hydrocarbon Pesticides from the Mammalian
System

Co-principal Investigator: J. A. Himes

In Vitro Degradation of DDT in Mammalian Tissue Homogenates:
On the premise that degradation of DDT in vivo may be a func-
tion of hepatic enzymes and that these enzymes might be subject to
stimulation by the administration of various drugs (enzyme inducers),
research efforts to date have been concentrated on the development
of an in vitro system to measure the effect of pretreatment with
various compounds on the degradation of DDT. Substances that ac-
celerate the in vitro metabolism of DDT in such a system might be
expected to have a stimulatory effect on the in vivo degradation
of DDT and thus hasten tissue depletion of this pesticide after
high level exposure. To establish optimum conditions for such a
system, rat tissue homogenates were incubated with DDT for varying
periods of time at 370C and at room temperature. Once optimum
conditions were established, it was possible to subject rats to
various forms of pretreatment and the effects of treatment could
be determined. After incubation control and treatment, homogenates
were analyzed for DDT, DDD, and DDE.

Preliminary studies on rat tissue homogenates:
The preliminary work on the in vitro degradation of DDT in
tissue homogenates revealed that significant amounts of DDD were
present after incubation of rat liver homogenate with DDT for 48
hours at 370C. (See Table 1). No DDE was present in the incubated
homogenate mixture. Since a similar degradation of DDT to DDD did
not occur in muscle tissue homogenates, and since there was
apparently a slight difference in the amounts of DDT degraded in
male and female rats, it was felt that there was a possibility that
the degradation might be dependent upon cellular enzymes charac-
teristic of liver tissue rather than upon bacterial decomposition.

Effects of inducers and inhibitors of hepatic microsomal enzymes:
In order to assess the effects of hepatic enzyme inducers
and inhibitors, female rats were pretreated with microsomal en-
zyme inducers (sodium phenobarbital, sodium barbital and phenyl-
butazone) or an enzyme inhibitor (SKF 525-A) for several days
prior to sacrifice, and the in vitro degradation of DDT to DDD was
observed in liver homogenates from these rats as well as from con-
trol rats. Since percentages of degradation of DDT to DDD were
28, 36, and 37 respectively for the group pretreated with the
enzyme inducers, the group pretreated with the enzyme inhibitor
and the control group (see Table 2), no effect could be attributed
to enzyme inducers or inhibitors. Similarly no effect on DDT de-
gradation to DDD was observed when SKF 525-A (an enzyme inhibitor)
was mixed directly with the liver homogenate DDT mixture. (See
Table 3).


-50-







Table 1.


Percentage recovery
DDT mixtures.


of DDT, DDD, DDE from homogenate-


Rat Rat
No. Sex

III M


Rat
Tissue1

Liver
Liver
Control


IV F Liver
Liver
Control

Muscle
Muscle
Control

V M Liver
Liver
Control

Muscle
Muscle
Control

VI F Liver
Liver
Control

Muscle
Muscle
Control


DDT had been added
100 ppm.


Incu-
bation
(hours)

24
none
24


48
none
48

48
none
48

48
none
48

48
none
48

48
none
48

48
none
48


DDT Recov-
ered (% of
DDT added)

40.32
100.52
03

92.44
95.34
04

93.94
101.54
O3

17.34
101.94
03
4
91.0
87.94
03

44.64
100.04
03

104.84
104.34
03


DDD Recov-
ered (% of
DDT added)

46.42
02
03
4
4.44
4.44
04

0.54
0.24
03

47.84
04
03


DDE Recov-
ered (% of
DDT added)
trace2
trace2
03


4
39.0
04
03


to the homogenate for a final cone. of


2 Average determinations of 4 aliquots of tissue homogenate.

3 Duplicate determinations of 2 aliquots of tissue homogenate.
4 Average determinations of 5 aliquots of tissue homogenates.


-51-







Table 2. Effect of pretreatment on recoveries of DDT
liver homogenate-DDT mixtures.1


and DDD from


Enzyme inducer
or inhibitor
Pretreatment
Sodium Pheno-
barbital3


Incubated
DDT -DDD

41 30


Non-incubated
DDT DDD


Control
DDT DDD

0 0


2 Sodium Pheno-
barbital3
1 Sodium Barbi-
tal4
2 Phenylbuta-
zone

1 Saline
control
2 Saline
control
3 Saline
control

1 SKF 525-A7
2 SKF 525-A7
3 SKF 525-A7


53 21 86

38 32 92

56 30 105


38 42 96

33 35 90

37 34 96

35 36 95
37 36 104
39 37 96


1 DDT added to liver homogenates for final conc. of
2 Nd DDT added to liver homogenates.
Rats injected intraperitoneally with 75 mg sodium
kg/day in divided doses for 5 days.
Rats injected intraperitoneally with 75 mg sodium
in divided doses for 3 days.


100 ppm.


phenobarbital/

barbital/kg/day


5Rats injected intraperitoneally with 125 mg phenylbutazone/kg/day
in divided doses for 5 days.
6Rats injected intraperitoneally with 0.9% sodium chloride, volume
in same proportion to body weight as in treated animals.
7 Rats injected intraperitoneally with 20 mg SKF 525-A/kg, 2 hrs.
before sacrifice.


Table 3. Effect of addition of SKF 525-A on % recoveries of DDT
and DDD from liver homogenate-DDT mixtures.


Enzyme
Inhibitor
SKF 525-A, 10-4M
SKF 525-A, 10-5M
Saline Control


Incubated
DDT DDD

38 32
42 30


Non-incubated
DDT DDD
94 0
93 0


Controlz
DDT DDD
-0 0
0 0
0 0


1 DDT added to liver homogenates for final conc. of 100 ppm.
2
No DDT added to tissue homogenate.


-52-


Rat
No.
--


0 0

0 0

0 0


0 0

0 0

0 0

0 0
0 0
0 0







Studies of mechanisms involved:
These results indicated the likelihood that in vitro degra-
dation of DDT to DDD in liver homogenate was not an enzymatic
reaction and further experiments were designed to elucidate this
point. Expanded studies with rat tissue homogenates revealed the
following: (1) In a mixture of rat kidney and spleen homogenized
and incubated with DDT there was a definite degradation to DDD,
although not to the same degree as in liver homogenates (a mean
of 25.4% DDD in kidney-spleen vs. 56.6% in liver homogenate).
In this series, no sex differences were seen in either homogenate.
(See Table 4). (2) Although the amounts of DDD formed were con-
siderably smaller (25.4% vs. 56.6% for controls), significant de-
gradation of DDT to DDD did occur in liver homogenates to which
antibiotics (penicillin and streptomycin) had been added. Small
amounts of DDD (2.6%) were found in the kidney spleen homogenates
to which the same antibiotics had been added. (See Table 5).
(3) Pretreatment of liver and kidney-spleen homogenates by heating
for 30 minutes to 620C did not prevent significant degradation of
DDT to DDD (42.4% DDD for liver and 10.1% DDD for kidney-spleen
homogenate). (See Table 5). (4) Pretreatment by autoclaving did
not prevent the degradation of DDT to DDD in liver homogenates
(18.4% DDD) but did so in kidney-spleen homogenates. (See Table 5).


Table 4.


Recovery of DDT and DDE from rat tissue homogenate-DDT
mixtures.


Kidney-


1


2


Rat Rat Liver Spleen1 Muscle Liver
No. Sex DDT DDD DDT_ DDD DDT DD DDT D
T F .7 73.9 57.8 20.0 81.5 0 0
B M 8.8 50.4 43.5 25.7 76.5 0 0
C M 15.8 52.5 40.4 29.2 -
D F 19.0 49.5 44.9 26.8 0
Mean 13.6 56.6 46.7 25.4 79.0 0 0
DDT added to tissue homogenate for final conc. of 100 ppm.
No DDT added to tissue homogenate.


0T
0
0
0
0


Since these results were somewhat equivocable in regard to the
autoclaved homogenate mixtures, similar studies were done with
canine tissue homogenates. In these studies it was found that
comparable amounts of DDD were formed in liver, spleen and kidney
homogenates (38.3, 30.4 and 39.2% respectively) and that smaller
amounts of DDD (5.1%) were present in muscle homogenates incubated
with DDT. In contrast to the rat studies (where no DDE was found),
small amounts of DDE (1.5, 1.7, 1.2, and 2.4%) were found in in-
cubated liver, spleen, kidney and muscle homogenate mixtures. Of
the four homogenate mixtures pretreated by autoclaving, only the
kidney homogenate showed small (5.8%) amounts of DDD. No DDE was
found in any of the four autoclaved homogenate mixtures. (See
Table 6).


-53-


1







Table 5. Effect of various treatments on percentage recoveries
of DDT and DDD from rat tissue homogenate-DDT mixtures.

In Vitro Liver2 Kidney-Spleen2 Liver3
Treatment DDT DDD DDT DDD DDT DDD
None 4-6 0 76 T 6 54 7-
4
Antibiotics
added 45.6 25.4 77.8 2.6 0 0
Preheating 6 28.1 42.4 69.0 10.1 0 0
Autoclaving 43.5 18.4 74.3 0 0 0
1Percentages presented represent the mean for determinations on
4 rats (2 males and 2 females).
2
DDT added to tissue homogenate for final conc. of 100 ppm.
3
No DDT added to tissue homogenate.
4
25,000 Units of buffered potassium penicillin G and 50 mg of
crystalline dihydrostreptomycin sulfate added to each ml of
homogenate.
Homogenates placed in a water bath at 620C for 30 minutes.
6Homogenates autoclaved at 15 psi and 1210C for 20 minutes.
1
Table 6. Effect of autoclaving on percentage recoveries of DDT,
DDD and DDE from dog tissue homogenate-DDT mixtures.2

Rat Non-autoclaved Autoclaved
Tissue DDT DDD DDE DDT DDD DDE
Liver T7 3773 7T5 9T 7 -"T
Spleen 33.8 30.4 1.7 92.8 0 0
Kidney 23.4 39.2 1.2 80.0 5.8 0
Muscle 82.6 5.1 2.4 90.0 0 0
Controls 0 0 0 0 0 0
1
Mean values of determinations on 3 aliquots from each tissue.
2
DDT added to each homogenate for final conc. of 100 ppm.
3
Tissue homogenates to which no DDT had been added.

From the foregoing experiments the conclusion reached is that
under the experimental conditions present in this study the in
vitro degradation of DDT to DDD in liver and other tissue homo-
genates was due primarily to bacterial action. Some tissues (liver,
for example) apparently provided better media for the growth of
anaerobic bacteria capable of degradating DDT. The preheating of
homogenate mixtures and the addition of antibiotics were apparently
effective only in inhibiting bacterial growth to the extent that
smaller amounts of DDD were formed over a 48 hour incubation period.
The small amounts of DDD found after autoclaving the homogenate
mixtures was possibly due to incomplete autoclaving, or more likely
to breaks in sterile technique subsequent to the autoclaving.


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Research Team No. 6


Title: Interrelationship Between Pesticides and the Effect of
Other Xenobiotic Agents on the Metabolism of Mammals

Co-principal Investigator: K. C. Leibman

The work described in this section was engaged in for less
than two years, partially because of the unavailability of techni-
cal assistance for a part of the time and partially because the
investigator was away from the University for six months.

This work has centered around two projects: 1) The hydration
of dieldrin in liver microsomes, and 2) the effect of pretreatment
with various chlorinated insecticides upon the metabolism of
phosphorothionates.

6-1. The hydration of dieldrin in liver microsomes.

The purpose of this project is to study the fate of dieldrin
in liver microsomes. Aldrin is epoxidized to dieldrin in mammalian
liver microsomes, and it is often stated that no further metabolism
of dieldrin occurs in these organelles. It has been shown, however,
that rabbits fed dieldrin excrete a number of metabolites in the
urine, chief among them being 6,7-trans-dihydroxy-dihydroaldrin
(trans-aldrindiol). The amount of thTs metabolite excreted in-
creases during chronic aldrin feeding, suggesting that the enzyme
activity for hydration of dieldrin is induced. It thus appeared
likely that dieldrin is hydrated in liver microsomes in a reaction
mediated by inducible enzymes.

Experiments with model cyclic epoxides in this laboratory
have shown that epoxide hydrase activity occurs in mammalian liver
microsomes. Thus, both indene oxide and cyclohexene oxide are con-
verted into their respective trans-glycols by rat and rabbit liver
microsomes. No cis-glycols can be detected as products of these
reactions. Hydration of these epoxides does not require any
pyridine nucleotide coenzyme, and the reaction apparently does not
involve an oxidase enzyme.

Since one of the chief products of the in vivo metabolism of
dieldrin is of the same stereochemical configuration as are those
produced in in vitro microsomal hydration of the simpler epoxy-
cycloalkanes, it appeared likely that the hydration of dieldrin
to trans-aldrindiol would be mediated by a microsomal enzyme. Ex-
periments to test this were performed with the lyophilized 9000g
supernatant fractions of liver of rabbits which had been pretreated
with phenobarbital. Trans- and cis-aldrindiols were synthezed for
use as reference compounds and carriers; the former by acid treat-
ment of dieldrin, and the latter by osmium tetroxide oxidation of
aldrin. Procedures for the extraction and thin-layer chromatography
of the diols were worked out in preliminary experiments with these
synthetic diols.


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Incubation mixtures contained, except as noted, 0.2 M Tris
buffer, pH 7.5, 60 pM TPN, 10 mM MgS04, 1.2 mM EDTA, 0.6 mM ATP,
5 mM glucose-6-phosphate, 20 mM nicotinamide, 2 pc dieldrin-C14,
and enzyme preparation from 400 mg (wet wt.) of liver. The total
volume was 5 ml, and the dieldrin was introduced in 0.1 ml of
methylcellosolve. After 2 hour incubation at 370, the reaction
mixtures were cooled in ice, and about 0.1 mg of carrier trans-
aldrindiol was added. The mixture was immediately deproteinized
with HC104, neutralized with KOH, and after removal of precipitate,
was extracted three times with 5 ml portions of chloroform. The
combined extracts were evaporated overnight at room temperature;
the residues were taken up in ethanol and spotted on the thin-
layer chromatography plates coated with silica gel G; development
was with chloroform-ethanol (9:1). Reference spots of aldrindiols
were visualized by spraying with AgNO3/2-phenylethanol/H202 followed
by ultraviolet irradiation. Radioactive spots were localized by
radioautography. Areas of silica gel containing radioactivity
were scraped from the plates, and counted by liquid scintillation.

Radioautograms of the thin-layer chromatograms usually showed
five spots besides that for dieldrin, although in some cases as
many as nine metabolites could be detected. The spot which was
identical in Rf to trans-aldrindiol usually contained more radio-
activity than any other except that for dieldrin. This spot of
radioactivity was not separated from that of trans-aldrindiol,
visualized with the color reagent, in three different chromato-
graphic systems. No such identification of a radioactive spot
could be made with cis-aldrindiol. In different enzyme prepara-
tions, conversions of 0.5% to 4.0% of the dieldrin to trans-
aldrindiol were observed.

Under these conditions, increasing amounts of trans-aldrindiol
were formed during incubations of up to 2 hour duration (table 1).

Table 1. Formation of trans-aldrindiol from dieldrin at different
incubation times.
-4
Time (min) trans-aldrindiol (cpm x 10 )
15 1.65
30 2.27
60 2.41
120 2.92

Characteristics of the reaction were studied in lyophilized
preparations from rabbit liver as well as in fresh preparations
from rat liver. (Table 2). When the enzyme preparation was im-
mersed in boiling water before adding the rest of the components,
the extent of reaction was sharply curtailed. When the TPNH-
generating system was omitted from reaction mixtures containing
the lyophilized rabbit liver preparation, only 40% as much trans-
diol was formed as in its presence. Fresh 9000g supernatant frac-
tion from rat liver had a higher activity than did the lyophilized
rabbit liver preparation, but again removal of the TPNH-generating
system led to loss of about 60% of the activity.


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Table 2. Enzyme and coenzyme requirement for production of
trans-aldrindiol.


System

Complete
Boiled enzyme
No TPN of G6P


-4
trans-aldrindiol (cpm x 10 in experi-
ments with preparations from
Rabbit Rat
1.31 3
0.17


0.53


1.66


To test the oxygen requirements of the reaction, as well as
the effect of carbon monoxide, reaction mixtures were prepared in
Thunberg tubes, with the enzyme preparation in the sidearm. The
tubes were evacuated several times and filled with the appropriate
gas phase, after which the contents of the tube were mixed and
incubation started. The results (Table 3) show that the reaction
requires oxygen and is greatly inhibited by carbon monoxide.

Table 3. Effects of anaerobiosis and of carbon monoxide on trans-
diol production.
-4
Gas phase trans-aldrindiol (cpm x 10 )
02 1.30
N 0.17
5 02, 95% N2 1.18
5% 02, 95% CO 0.15


The effects of two compounds which inhibit many microsomal
oxidative reactions were tested. SKF 525A has for years been con-
sidered the classic inhibitor of these reactions. Metyrapone has
recently been shown in this laboratory to inhibit a number of such
oxidations. As shown in Table 4, both of these compounds exten-
sively inhibit the production of trans-aldrindiol from dieldrin.

Table 4. Effect of microsomal inhibitors on trans-aldrindiol
production.
-4
Inhibitor (1 mM) trans-aldrindiol (cpm x 10 )
None 4.73
SKF 525A 0.09
Metyrapone 0.16

A small amount of C14-labeled trans-aldrindiol became avail-
able from another project. When this was incubated with the same
system used for study of dieldrin metabolism, at least three other
spots containing radioactivity were found in the thin-layer chroma-
tograms (Table 5). The spot at Rf 0.34 is due to trans-aldrindiol.

Table 5. Metabolism of trans-aldrindiol.


Incubation time (min)


120


0.0
13
651


cpm in
0.34
2050
317


spot of Rf
0.60 0.75
0 0
341 174


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Some of the findings reported here, and the conclusions de-
rived therefrom, are different from those mentioned briefly in
previous progress summaries. This is because, after using a number
of preparations of C14-dieldrin which were quite free of radio-
chemical contamination in the chromatographic areas of interest,
we received two batches which contained radioactive materials
which chromatographed similarly to trans-aldrindiol. Before we
realized this, we drew a number of conclusions from our experi-
ments which later experiments, performed with purified C14-dieldrin,
showed to be in error. The above experiments were all performed
with labeled substrate shown to contain no radioactive contamina-
tion which would appear in chromatograms in the area of trans-
aldrindiol.

We conclude, therefore, that dieldrin is converted to trans-
6,7-dihydroxy-dihydroaldrin in mammalian liver microsomes. The
reaction requires TPHN and oxygen, and thus appears to be of the
mixed-function oxidase class. This is further borne out by the
fact that the diol production is inhibited by carbon monoxide,
which complexes with cytochrome P-450, the terminal oxidase-
oxygenase of microsomal mixed-function oxidation, and also by
SKF 525A and metyrapone, two potent inhibitors of mixed-function
oxidations. The activity appears to be different from the cyclo-
hexene oxide hydrase of liver microsomes, which has no requirement
for reduced pyridine nucleotide, and which is not inhibited by
SKF 525A or metyrapone.

Although it seems surprising on first glance that a reaction
which appears to consist merely of addition of the elements of
water should be mediated by an oxidase, other such cases are known.
Thus, the degradation of parathion to diethylthiophosphate and
p-nitrophenol, which would appear to be a hydrolysis, is mediated
by a TPNH- and oxygen-requiring enzyme of liver microsomes. Fur-
ther studies are required to establish the mechanism of such re-
actions.

At least some of the other products of metabolism of dieldrin
in liver microsomes would appear to be derived by further metabolism
of trans-aldrindiol.

6-2. Metabolic interactions between chlorinated hydrocarbon
insecticides and phosphorothionates.

The purpose of this project is to study the nature of the
effect of pretreatment of animals with chlorinated hydrocarbon in-
secticides upon the metabolism of phosphorothionates in liver mi-
crosomes, and the effect of such metabolic alteration upon the
toxicity of the latter. Parathion is metabolized by two main
pathways, both mediated by oxidative enzymes in liver microsomes.
One of these involves a sulfur-oxygen exchange with the production
of the active anticholinesterase paraoxon, which results in a
"toxification" of the phosphorothionate. The other results in the
breakdown of parathion to diethylthiophosphate and p-nitrophenol,
and is, therefore, a "detoxication." The toxicity of parathion
under any circumstance would depend upon the balance between these
two reactions.


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Halogenated hydrocarbon insecticides have been known for some
time to induce the microsomal mixed-function oxidases of mammalian
liver in a manner similar to that of phenobarbital. Both of the
oxidative reactions of parathion are inducible with phenobarbital.
It was, therefore, decided to study the effects of induction with
chlorinated insecticides upon the two different pathways of micro-
somal parathion metabolism.

It was initially expected that the paraoxon formed in liver
microsomes would be assayed by inhibition of cholinesterase in the
Warburg apparatus. Great difficulties were encountered with this
method, and no consistent results could be obtained in control ex-
periments. Apparently extensive tissue binding of paraoxon occurs
at low concentrations, thus vitiating the results of such methods.
Therefore, ethyl-Cl4-labeled parathion was used as a substrate;
since the label on the ethyl group is quite stable, all phosphate
products of metabolism would be labeled. Thin-layer chromatogra-
phic techniques were worked out for the separation and visualiza-
tion of parathion, paraoxon, diethylthiophosphate, and diethyl-
phosphate.

Reaction mixtures contained 0.6 mM TPN, 2.5 mM glucose-6-
phosphate 0.18 mM parathion containing 1.5 c of ethyl-C14-labeled
parathion, freshly-prepared 9000g supernatant fraction from 125 mg
(wet wt.) of rat liver, and phosphate buffer, pH 8, in 2 ml. total
volume. After 1 hour incubation at 370, cold acetone was added,
the mixture was placed in the freezer for 1 hours, and was then
centrifuged. The supernatant fraction was acidified and aliquots
were spotted on thin-layer chromatography plates coated with
silica gel G. Development was effected with two different sy-
stem's in order to achieve complete separation of the parathion,
paraoxon, diethylphosphate, and diethylthiophosphate. The amount
of diethylthiophosphate formed was taken as a measure of the de-
toxifying pathway, while the sum of the amounts of paraoxon and
of its hydrolysis product diethylphosphate were used as a measure
of the paraoxon-producing pathway.

A number of preliminary experiments have been done to standard-
ize the procedure, so that reproducible recoveries of metabolites
and unchanged substrate could be made. Pilot experiments are now
being performed to determine optimum conditions for induction of
rats with DDT. Data are now available for two such pilot experi-
ments (Table 6).

Pilot experiments with other induction regimen are in progress.
It would appear, however, that the induction of the two pathways
by DDT is quantitatively similar. Whether this is indeed the case
will be determined only after statistical numbers of animals are
tested in a common protocol.


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Table 6. Induction of parathion metabolism by DDT.


Pretreatment
Corn oil
DDT, 200 mg/kg
Corn oil
DDT, 200 mg/kg
Corn oil x 3 days
DDT, 25 mg/kg
/day x 3 days


Days
Before
3
3
5
5


Diethylthio-
phosphate
(nmoles/hr/g.
liver
122
315 (158%)*
186
531 (194%)
97


299 (208%)


Paraoxon + diethyl-
phosphate
(nmoles/hr/g. liver)
146
333 (128%)*
208
612 (185%)
103

350 (240%)


Figures in parentheses represent per cent stimulation.


Publications:
Leibman, K. C., 1968. Actions of insecticides on drug activity.
Internat. Anesthesiol. Clinics. 6:251.

Leibman, K. C. and E. Ortiz., 1968. Microsomal hydration of
epoxides. Federation Proc. 27:302.

Leibman, K. C. 1969. Effects of metyraphone on liver microsomal
drug oxidations. Mol. Pharmacol. 5:1.


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