THE METABOLISM OF O - ( 4 - B R O M O - 2 , 5 -

DICHLOROPHENYL) O-METHYL
PHENYLPHOSPHONOTHIOATE (LEPTOPHOS) IN WHITE
MICE AND ON COTTON PLANTS
R. L. HOLMSTEAD, T. R. FUKUTO, and R. B. MARCH
Department of Entomology, Division of Toxicology and Physiology
University of California, Riverside, Calif. 92502

The metabolism of 14C-labeled leptophos ( O - m e t h y l O - 4 - b r o m o - 2 , 5-

dichtorophenyl phenylphosphonothioate), a new phosphonothioate insecticide
chemical, was examined in the white mouse and the cotton plant. Leptophos, ad-
ministered orally to the mouse, is rapidly metabolized and excreted as degradation
products, principally in the urine. The major degradation products are 4 - b r o m o -
2, 5 - d i c h l o r o p h e n o l (in the form of conjugates), O - m e t h y l phenylphosphono-
thioic acid, methyl phenylphosphonic acid, and phenylphosphonic acid. Small
amounts of unchanged leptophos and leptophos oxon occur in the mouse feces.
In general, the same alteration products are also obtained when leptophos is applied
to the leaf surface of cotton plants. However, leptophos is quite stable on the cotton
plant and the major portion of the radioactive material recovered from the plant is
unchanged leptophos. The results indicate that leptophos is useful as a l o n g - t e r m
residual insecticide without the problem of biomagnification in the food chain.

Leptophos [ O - ( 4 - B r o m o - 2 , 5 - d i c h l o r o p h e n y l ) O - m e t h y l phenylphosphonothio-
ate, (trademarked Phosvel) is a new phosphonate insecticide chemical currently under de-
velopment by Velsicol Chemical Corporation (Chicago Ill.). Leptophos has shown promise
for the control of a broad spectrum of insect pests and as a fungicide against rice stem blast
(Velsicol Chemical Corp. 1971). Although phosphonothioate insecticide chemicals (e.g.,
EPN) were introduced over two decades ago, relatively little information is available on
their metabolism in animals and plants (Menn 1971). In view o f the general deficiency o f
information on the metabolism o f phosphonothioate insecticides and the imminent de-
velopment o f leptophos as a viable commercial insecticide, a study o f the metabolism of

S CI 0 CI

--0 Br ~ 0 Br

OCH3 CI OCH3 Cl
Leptophos (1) Leptophos oxon (2)

133
Archives of Environmental Contamination and Toxicology
Vol. 1, No. 2, 1973, © 1973 by
Springer-Verlag New York Inc.
134 R.L. Holmstead et al.

this material in the white mouse and on cotton plants was undertaken. This paper presents
results from this study made with 14C_labele d leptophos.

Materials a n d m e t h o d s

Solvents. All solvents used were AR grade. Solvents used for the estimation of radio-
activity in the counting vials were of scintillation quality.

Radiolabeled materials and model metabolites. Leptophos preparations labeled with

carbon- 14 at two different positions were provided by the Velsicol Chemical Corporation,
Chicago, Ill. but had been prepared by the New England Nuclear Corporation, Boston,
Mass. They are labeled as follows: O - ( 4 - - b r o m o - 2 , 5-dichlorophenyl-I 4 C ) O - m e t h y l
phenylphosphonothioate, specific activity 7.34 mCi/mmole and > 99% purity; 0 - ( 4 -
b r o m o - 2 , 5-dichlorophenyl) O-methyl phenyl-14C-phosphonothioate, specific ac-
tivity 6.23 mCi/mmole and > 99% purity; for convenience, these compounds are desig-
nated as phenoxy- and phenyl-labeled leptophos, respectively. Model metabolites were
synthesized in this laboratory or provided by Velsicol Chemical Corporation. The various
model compounds are listed in Table I along with relevant mass spectral data used for
structure verification. Mass spectra were obtained on a Finnigan Model 1015 mass spec-
trometer, using a direct-insertion probe and ionization voltage of 70 eV.

Thin-layer chromatography (TLC). TLC analyses were made with three different sys-
tems: System A, Silica H F - 2 5 4 (Merck) with a developing solvent of benzene-chloro-
form mixture (1 : 1 by volume); System B, microcellulose (J. T. Baker) with a developing
solvent of methanol-formic acid-water mixture (8:1.5:0.5 by volume); System C, Silica
H F - 2 5 4 (Merck) with a developing solvent of acetonitrile-water-ammonium hydroxide
mixture (8:1.8:0.2 by volume). The gel layer on all TLC plates was 0.75 mm thick. The
Rf values found for leptophos and its metabolites, for the three systems, are given in Table
II.

The location of the ~4C-labeled metabolites on the developed TLC plates was accom-
plished by means of a Berthold (Varian-Aerograph) Model LB 2723 thin-layer radio scan-
ner equipped with a dot printer. Quantitative determination of radioactivity was accom-
plished with a Packard Model 3003 Tri-Carb scintillation spectrometer.

Metabolism in mice. Female Swiss mice (Curd's Caviary, La Puente, Calif.), weighing
3 5 - 4 0 g, were treated orally with either the phenyl- or phenoxy- 14C_labeled leptophos
in olive oil at a dose of 25 mg/kg. The treated mice were held in individual metabolism
chambers which allowed the separate collection of urine and feces, and the provision of
food and water. Within 15 minutes after treatment, the mice developed muscular spasms
and convulsions which continued for 5 - 8 hours, after which all toxic signs and symptoms
disappeared. Urine and feces were collected at periodic intervals, immediately frozen, and
stored in a frozen state until analyzed.
 Metabolism of Leptophos in Mice and Cotton Plants 135

Typically, the urine samples were analyzed by the following procedure. Aliquots of a
known volume of the melted urine sample were first examined for radioactivity by scintil-
lation counting. The remaining urine was then extracted with three 5-ml portions of
benzene. The combined benzene extracts were examined for radioactivity, concentrated
under a gentle stream of nitrogen, and subjected to TLC analysis. The extracted aqueous
phase was concentrated by freeze-drying and the residue was subjected to TLC analysis,
using systems B and C.

In experiments carried out with phenoxy-labeled leptophos, the principal product

remaining in the aqueous phase, based on its TLC behavior, appeared to be a conjugate.
Accordingly, the residual product was subjected separately to enzymatic hydrolysis, using

Table I. Mass Spectral Dataa for the Various Model Compounds Used in
Identification of the Metabolites of Leptophos
Identification no.
and compound Mass spectral data: m/e (relative intensity)

1. leptophos 416 (0.2), 414 (1.2), 412 (2.3), 410 (1.4), 379 (3.6), 377 (17.6),
375 (13.3), 171 (100), 124 (13.0), 77 (47.6), 51 (18.2), 47 (14.2),
28 (18.5)
2. leptophos oxon 400 (0.5), 398 (2.8), 396 (5.8), 394 (3.5), 363 (1.5), 361 (4.8),
359 (3.6), 155 (100), 51 (25.0), 47 (6.8), 28 (17.4)
S
II
3. C6H 5 - P - O H 188 (95.6), 158 (43.5), 155 (18.1), 141 (47.1), 125 (100),
I 111 (60.8), 110 (40.6),91 (15.9), 77 (75.4), 65 (26.0), 63 (17.4),
OCH 3 51 (64.5), 47 (68.1)
O
II
4. C 6 H s - ~ - O H 173 (13.2), 172 (37.5), 171 (34.2), 159 (9.2), 158 (22.4),
94 (39.5), 91 (65.78), 77 (100), 51 (51.3), 47 (10.5)
OCH 3
O
II
5. C6H 5 P-OH 158 (39.5), 94 (100), 77 (21.8), 66 (14.3), 65 (11.8), 51 (24.8),
1 47 (6.3)
OH
C1
6. Br-~OH 246 (2.4), 244 (t4.7), 242 (30.8), 240 (20.8), 181 (2.t),
179 (6.0), t 77 (4.7), 149 ( t 1.6), 135 (6.3), 133 (9.2), 99 (7.9),
C1 97 (20.8), 35 (20.3), 32 (34.5), 28 (100)

aOnly values given are those which are important for identification purposes.
136 R.L. Holmstead et al.

~3-glucuronidase/aryl sulfatase (Calbiochem), at pH 5.0 and 35°C, and to acidic hydrolysis

at elevated temperatures. The radioactive hydrolysate was extracted into ether, concen-
trated, and analyzed by TLC.

The individual feces samples were extracted with five 10-ml portions of acetone, and
the extracts were combined, centrifuged, and examined for radioactivity and by TLC, in

Table II. Rf Values of Leptophos and Its Metabolites for the Three TLC Systems Used

I d e n t i f i c a t i o n no.
TLC system
and c o m p o u n d A C

t. leptophos 0.65 0.95 0.91

2. leptophos oxon 0.21 0.91 0.75
S
tl 0.0 0.75 0.48
3. C6tt s - ~ - O t t
O
t
CH 3
O
Jl
4. C6Hs- ~ OH 0.0 0.51 0.29

O
I
CH 3
O
11 0.0 0.64 0.03
5. C6Hs- ~ OH
OH
C1

6. Br-~OH 0.37 0.72

CI
C1

7. a Br-~O-M + 0.03
Cl

aThis salt,when extracted, acidified and rechromatographed, gave Rf values on systems

A and C identical to those of compound no. 6.
 Metabolism of Leptophos in Mice and Cotton Plants 137

the usual manner. Combustion of the feces, after extraction, showed very little, if any,
radioactivity remaining.

For mass spectral analysis, each metabolite was purified by repeated TLC. The meta-
bolites, which were isolated in the uncontaminated state and subjected to mass spectral
analysis, are: 1. leptophos, 2. leptophos oxon, 3. O - m e t h y l phenylphosphonothioic acid,
4. O - m e t h y l phenylphosphonic acid, 5. phenylphosphonic acid, and 6 . 3 - b r o m o - 2 , 5 -
dichlorophenol. The mass spectra of the recovered metabolites gave fragmentation patterns
virtually identical to those of the respective model compounds listed in Table I.

Metabolism in the cotton plant. The upper surfaces of the leaves of each of 4 or 5
young cotton plants (ca. 9 weeks old) were treated, by brush application, with the
following mixture: 3.'75 mg 14C-leptophos (equivalent to approximately 1 lb/acre), 20
mg Triton X - 1 0 0 , 1 ml water, and 0.5 ml acetone. The plants were maintained in the
greenhouse and provided water daily. The greenhouse temperature ranged from 70 to
85°C with a relative humidity range of 40 to 60%. At predetermined intervals, a plant was
removed from the greenhouse and the leaves were analyzed by the following procedure.
The leaves were excised at the base of the stem and rinsed with benzene to remove surface
residue. The rinsed leaves and stems were, in turn, homogenized with three separate 7 5 -
ml portions of benzene and three 7 5 - m l portions of methanol. The residual colorless
pulp was dried in a vacuum oven and retained for combustion analysis (see next section).

The combined benzene- and methanol extracts were each dried over magnesium sul-
fate, concentrated to a known volume ( 4 - 5 ml), and appropriate aliquots were taken for
combustion analysis preparatory to radioactivity determination. The remaining portions
were concentrated to near dryness, and 5 ml of acetone was added to the dry residues, and
the mixtures were centrifuged to separate extraneous plant material. This procedure was
repeated twice with separate portions of acetone and the combined extracts were con-
centrated and subjected to TLC analysis. Metabolites for mass spectral analysis were iso-
lated by extracting the relevant spots with an acetone-methanol mixture (1 : 1 by volume),
after repeated TLC purification involving all three systems.

Combustion analysis. Estimation of carbon-14 in cotton-leaf extracts and pulp was

accomplished by the method similar to that described by Oliverio et al. (1962). A Thomas
Ogg Model 11 oxygen-flask igniter was utilized for combustions. For a typical combus-
tion analysis, about 2 0 - 6 0 mg of dried plant pulp was weighed in a "combustion
envelope." The envelope was placed in a 2-liter suction flask and burned in an atmosphere
of oxygen in the flask igniter. After cooling, 15 ml of a mixture, consisting of 43 percent
phenylethylamine, 21 percent methanol, and 36 percent toluene (by volume) was injected
into the flask through a rubber septum for absorption of 14C-carbon dioxide. After
about one hour, an aliquot was withdrawn, mixed with scintillation fluid, and counted.
For the combustion of plant extracts, 0.20 ml of the extract was placed in the envelope,
dried in a vacuum desiccator overnight, and the envelope was burned and analyzed in the
usual manner.
138 R . L . Holmstead et al.

Results

Mouse metabolism. The results of metabolism studies performed in the white mouse
using phenoxy- and phenyl 14C_labele d leptophos are summarized in Tables III, IV, and
V.

In the case o f phenoxy-labeled leptophos, radioactivity is rapidly eliminated from

the mouse and elimination is virtually complete 48 hrs. after treatment, the bulk of the
excreted material being present in the urine (Table III). In comparison, elimination of
p h e n y l - l a b e l e d radioactivity is notably slower and radioactivity is detected in the urine
as long as 144 hours following treatment. It is estimated that the error in the administered
dose was approximately +10% and, therefore, that the total amounts recovered are close
to complete recovery of radioactivity. The rate in which the two labels in leptophos are
eliminated, in urine and feces, is presented graphically in Fig. 1. In spite of the possible
error in administration o f the radioactive compound, there is little doubt that the c a r b o n -
14 in the phenyl group is eliminated at a slower rate than that in the phenoxy group. A
possible explanation for the difference in elimination rates is that the conjugate of the
p h e n o x y - l a b e l e d phenol (no. 6) is eliminated more rapidly than the p h e n y l - l a b e l e d
phenylphosphonic acids (no. 3, 4, and 5).

Table III. Effect of Position of Radiolabel and Time on the Distribution o f

Radioactivity in Urine and Feces of a White Mouse Receiving a Single
Oral Dose of Leptophos Containing Carbon-14 Either in the Phenoxy
Group or in the Phenyl Group

Group in leptophos Time, Percentage of administered radioactivity

containing radiocarbon hr In urine In feces Total a

phenoxy 7 58.1 1.0 59.1

phenoxy 24 29.9 2.2 32.1
phenoxy 48 18.1 1.1 19.2
110.4 total recovery

phenyl 7 43.8 1.1 44.9

phenyl 24 23.9 7.7 31.6
phenyl 48 6.9 trace b 6.9
phenyl 72 2.7 trace b 2.7
phenyl 144 1.8 0 1.8
87.9 total recovery

aThe total recovery shown is based on the amount administered. The error in administered
dose is estimated to be + 10 percent.
b"Trace" refers to less than 0.1 percent of radioactivity administered.
 Metabolism of Leptophos in Mice and Cotton Plants 139

The major portion of the radioactivity eliminated from the mouse is in the form of
degraded products present primarily in the urine (Table IV and Table V). Leptophos
(no. 1) and the oxon (no. 2) are present in small amounts but essentially all in the feces.
With phenoxy-labeled leptophos, more than 90% of the urinary metabolite is in the form
of conjugated 4 - b r o m o - 2 , 5-dichlorophenol, although a small amount of free phenol
also is present. The exact nature of the conjugate (no. 8) is not established but both en-
zymatic 03-glucuronidase/aryl sulfatase) and acid hydrolysis yield only the phenol
(no. 6).

The phenyl-labeled leptophos gives O-methyl phenylphosphonothioic acid (no. 3),

O-methyl phenylphosphonic acid (no. 4), and phenylphosphonic acid (no. 5) as the
principal degradative products (Table V) present entirely in the urine. Compound no. 3
is the major component present in the early collection periods but its amount decreases
rapidly with time, eventually reaching a level about equal to that of no. 4 and no. 5. As in
the phenoxy label, both leptophos and leptophos oxon (no. 2) are found in the feces,
although unexplainably larger amounts of unchanged leptophos are detected with the
phenyl label.

The unknown metabolite(s) listed in Table IV and V cochromatograph with an

authentic sample of O ( 4 - b r o m o - 2 , 5-dichlorophenyl) phenylphosphonic acid on TLC

Table IV. Amount of Radioactive Metabolites Found at Various Times in the Urine
of a White Mouse Receiving a Single Dose of 14 C-Phenoxy-Labeled Leptophos

Percentage of recovered radioactivity

Identification no. at indicated time after dosing
and compound 7hr 24 hr 48 hr Total

1. Leptophos 0.7 0.8 0.2 1.7

2. Leptophos oxon 0.7 0.2 0 0.9
Cl
6. HO-~Br 4.5 1.2 1.5 7.2

Cl C1

8. C o n j - O ~ > - Br 47.0 26.5 15.6 89.1

El
Unknown a 0.6 0.5 trace b 1.1
100.0

aThe unknown material was not conclusively identified but it cochromatographs with an
authentic sample of O-(4-bromo--2, 5-dichlorophenyl) phenylphosphonic acid,
b,,Trace,, refers to less than O. 1 percent of recovered radioactivity.
140 R . L . Holmstead et al.

system A (Table ll). In view o f the minor extent to which this metabolite was formed, it
was not possible to isolate sufficient sample for mass spectral analysis.

Cotton plant metabolism. The data for the metabolism of leptophos after application
on the surface of cotton leaves, at a dosage equivalent to one pound per acre, are pre-
sented in Tables VI, VII, and VIII. F r o m the data in Table VI, it is evident that
leptophos does not penetrate readily into the leaf but remains on the surface or volatizes.
The primary mechanism b y which leptophos is lost appears to be by volatilization be-
cause there is a steady decrease in total measurable radioactivity with time. For example,
73 to 75 percent o f the applied leptophos is recovered one week after treatment compared
to 19 to 21 percent after five weeks. Relatively small amounts o f radioactivity are found
inside the leaf either as benzene or methanol extracts, or bound to the pulp. It is impor-
tant to point out that control experiments were made to show that radioactivity was not

Table V. Amount o f Radioactive Metabolites Found at Various Times in the Urine

o f a White Mouse Receiving a Single Dose o f 14 C-Phenyl-Labeled Leptophos

Percentage of recovered radioactivity

Identification no. at indicated time after dosing
and compound 7 hr 24 hr 48 hr 72 hr 144 hr Total

1. Leptophos 1.2 8.2 trace a trace a trace a 9.4

2. Leptophos o x o n 0 0.4 trace a trace a trace a 0.4
S

3. C 6 H s - P - O H 35.4 15.0 4.6 1.0 0.6 56.6

I
OCH 3
O
II
4. C6H 5 P - O H 8.9 5.7 1.5 1.1 0.7 17.9
1
OCH 3
O
II
5. C 6 H s - P - O H 5.5 6.3 1.8 1.1 0.8 15.5
J
OH
Unknown b 0.2 0.2
100.0

a"Trace" refers to less than 0.1 percent of recovered radioactivity.

bThe unknown material was not conclusively identified but it cochromatographs with an
authentic sample of O - ( 4 - b r o m o - 2 , 5 - d i c h l o r o p h e n y l ) phenylphosphonic acid.
 Metabolism of Leptophos in Mice and Cotton Plants 141

being lost in the w o r k - u p procedure,that is, to show that greater than 90 percent of the
applied radioactivity was recovered.

Qualitatively, the degradation of teptophos (no. 1) in or on the cotton leaf is similar to

that occurring in the white mouse (Table VII and Table VIII). The major component
present in the recoverable radioactivity one week after application is unchanged leptophos
(91 to 96 percent) and the amount of this substance gradually diminishes with time to ap-
proximately 29 percent in nine weeks. Most of the leptophos remains on the leaf surface
although significant but small amounts evidently are absorbed into the leaf. Little, if any,
leptophos oxon (no. 2) is present at any sampling period although other alteration
products [such as, the phenol (no. 6), in the form of an unknown salt, and the phenyl-
phosphonic acid derivatives (no. 3, 4, and 5), are present in varying amounts. No free- or
conjugated phenol is detected and essentially all of the phenol is isolatable as a salt (Table
VII). The latter substance is the principal degradation product present in the leaf but sub-
stantial amounts also occur on the leaf surface. The unknown material(s) from the
phenoxy-labeled leptophos treatment is present almost entirely in the pulp and, because
it is not extractable with methanol, it is considered bound to the plant pulp.

1 O0

A
v

8O
LU
tJ3
0
a
a
r" r' 60
IJ.I
I-
03
Z

a 4O

20

0 20 40 60 80 100' 120 t40

T I M E (hours)

Fig. 1. Rate of elimination of radioactivity from the white mouse, in urine and feces, after
oral administration of phenoxy-labeled ( O ) and phenyl-labeled ( • ) leptophos
142 R.L. ttolmstead et at.

Results obtained with the phenyl-labeled leptophos show that all three phenyl-
phosphonic acid derivatives (no. 3, 4, and 5) are present both in and on the cotton leaf.
The largest component among these acids is phenylphosphonic acid, particularly at the
later sampling periods. As in the case of the data obtained with the phenoxy-labeled
leptophos, a significant amount of the measurable radioactivity from phenyl-labeled
leptophos is inseparable from the pulp by methanol extraction and is considered to be
bound to the pulp. Unfortunately, the nature of this bound material is unknown but it
undoubtedly is not the same as the bound material obtained from plants treated with
the phenoxy-labeled leptophos.

Discussion

The proposed pathways, based on this study, for the metabolism of leptophos in the
white mouse are given in Fig. 2. The pathways are similar to those proposed for other
organophosphorus esters such as bromophos (Stiasni et al. 1967) and ronnel (Plapp and
Casida 1958).

Table Vl. Effect of Position of Radiolabel and Time on the Distribution of

Radioaetivity in the Wash, Solvent Extracts, and Pulp of Leaves of
Cotton Plants Receiving Topical Application of Leptophos Containing
Carbon-14 Either in the Phenoxy Group or in the Phenyl Group

Percentage of applied radioactivity

Group in leptophos recovered after indicated time
containing radiocarbon 1 wk 3 wk 5 wk 9 wk

Phenoxy
Wash 64.3 16.5 9.0 1.8
Benzene extract 5.6 6.2 4.5 2.4
Methanol extract 4.9 9.9 4.2 3.7
Pulp (bound) trace a 1.8 2.9 3.1
Total 74.8 34.4 20.6 ll.0

Phenyl
Wash 62.1 18,9 7.8
Benzene extract 7.0 8.1 4.8
Methanol extract 4.3 10.0 3.7
Pulp (bound) trace a 1.1 2.2
Total 73.4 38.1 18.5
a"Trace" refers to less than 0.1 percent of radioactivity applied.
 Metabolism of Leptophos in Mice and Cotton Plants 143

t•!--O•
s
Br , Lo.
OCH3 Cl ~ ; C H 3
leptophos (no. 1) (no. 3)
1 Cl
I
I
I
H O @ B r
I
CI
o (no. 6)

leptophos oxon
(no. 2)
OCH3
(no. 4)
cl

conj-O@Br
t
CI
conjugate (no. 8)
°"

(no. 5)

Fig. 2. Proposed metabolic pathways for leptophos in the white mouse

The formation of O-methyl phenylphosphonothioic acid (no, 3) probably occurs by

oxidative dearylation or by esterase catalyzed hydrolysis of leptophos, or a combination
of reactions similar to those demonstrated with other phosphorothionate insecticides (Yang
et al. 1971; Matsumura and Hogendijk 1964; Nakatsugawa et al. 1969). The desulfurated
acid (no. 4) can be formed by several possible mechanisms: 1) by direct oxidative hydrolysis
of teptophos (no. 1), 2) oxidative desulfuration of leptophos (no, 1) to the oxon (no. 2)
followed by hydrolysis, or 3) by desulfuration of the thionic acid (no, 3). The oxidative
hydrolysis of phosphorothionate esters by microsomal oxidase enzymes to give directly
the dialkyl phosphoric acid has been demonstrated by }SO studies with parathion
(Ptashne et al. 1971) and Dyfonate® (McBain et al. 1971). In vivo studies with O-ethyl
ethylphosphonothioic acid in the rat (McBain et aL 1972) and O, O-diethyl phosphoro-
thioic acid in the white mouse (Vinopal and Fukuto 1971) have shown that these thioic
acids are not desutfurated to their respective oxygen analogs and, therefore, mechanism
1) and 2) are the most reasonable pathways for the formation of no. 4. Phenylphosphonic
acid (no. 6) assumedly is produced by the hydrolysis of no. 4.
144 R . L . Holmstead et al.

The results obtained with the phenoxy-labeled leptophos show that 4 - b r o m o - 2 , 5 -

dichtorophenol (no. 6) or its conjugated form (no. 8) is the sole degradation product o f
this part of the leptophos molecule. There is not any evidence of the production o f any
hydroxylated or dehalogenated products. Thus, the overall metabolic fate o f leptophos in
mice is similar to that of parathion and related insecticides, and not any unexpected
products are formed. In general, leptophos is metabolized rapidly in the mouse, yielding
relatively nontoxic excretion products.

Leptophos evidently penetrates very slowly into the cotton leaf and the term "altera-
tion" is perhaps a better expression than "metabolism" for its behavior on cotton plants.

Table VII. Amount of Radioactive Metabolites Found at Various Times in the

Wash, Solvent Extract, and Pulp of Leaves of Cotton Plants Receiving
Topical Application of 14C_Phenoxy_Labeled Leptophos
Percentage of recovered radioactivity
Compound found after indicated time
in indicated material 1 wk 3 wk 5 wk 9 wk

Leptophos (no. 1)
surface wash 80.7 37.8 35.1 29.3
solvent extract 10.7 23.6 22.8 13.6
pulp 0 0 0 0

Leptophos oxon (no. 2)

surface wash trace c 0 0
solvent extract 0 trace c 0
pulp 0 0

Phenol (no. 6) plus salt

of phenol (no. 7) a
surface wash 5.3 9.9 8.7 7.2
solvent extract 3.3 23.2 19.4 21.8
pulp 0 0 0

Unknown b
surface wash 0 trace c 0 0
solvent extract 0 0.3 0 0
pulp 0 5.2 t4.0 28.1

aThese values are a sum of the free phenol (no. 6) and the salt of phenol (no. 7).
bThese values reflect the amount of unextractable radioactive material bound in the pulp.
C"Trace" refers to less than 0.1 percent of radioactivity recovered.
 Metabolism of Leptophos in Mice and Cotton Plants 145

Separate experiments in this laboratory have demonstrated that leptophos virtually has
not any systemic movement when applied to the stem o f cotton leaves, a finding which is
consistent with its poor penetration properties. It is difficult to ascertain whether the al-
teration of leptophos occurs primarily on the surface of the leaf, by photochemical
processes, or within the leaf, by plant processes; the evidence indicates that both altera-
tions occur. Nevertheless, the principal component recoverable at all time intervals is
leptophos itself, the majority of which is present on the leaf surface.

The overall evidence suggests that leptophos can be regarded as a persistent, bio-
degradable, organophosphorus insecticide chemical. Previous unpublished work in this
laboratory, using GLC as a means of analysis, has shown that leptophos is extremely stable
on glass and leaf surfaces in sunlight. The stability of ~4C-labeled leptophos to degrada-
tion on and in cotton leaves, together, with its rapid metabolism in the white mouse, sug-

Table VIII. Amount of Radioactive Metabolites Found at Various Times in the

Wash, Solvent Extract, and Pulp of Leaves of Cotton Plants Receiving
Topical Application of N C-Phenyl-Labeled Leptophos

% of recovered % o f recovered
c a r b o n - - 14 carbon--14
after i n d i c a t e d after i n d i c a t e d
Compound f o u n d Compound f o u n d
time time
in indicated in indicated
material 1 wk 3 w k 5 w k material 1 wk ~wk 5 wk

Leptophos (no. 1) Acid (no. 4)

surface wash 82.1 48.2 37.5 surface wash 0.8 0.3 0.5
solvent extract 14.3 30.1 31.3 solvent extract 0.5 0.3 I 1.6
pulp 0 0 0 pulp 0 0 0

Leptophos oxon Acid (no. 5)

(no. 2)
surface wash 0 0 0 surface wash 0 0 0
solvent extract 0 0 0 solvent extract trace t 6.2 10.3
pulp 0 0 0 pulp 0 0 0

Acid (no. 3) Unknown a

surface wash 1.6 1.3 4.3 surface wash 0 0 [ 0
I

solvent extract 0.7 0.8 2.7 solvent extract 0 0 I 0

pulp 0 0 0 pulp
i-
trace ~ I
2.8 11.8

aThese values reflect the amount of unextractable radioactive materiat bound in the pulp.
b"Trace" refers to less than 0.1 percent of radioactivity recovered.
t 46 R.L. Holmstead et al.

gests that it possibly is useful as a long-term, residual insecticide chemical without the
problem of biomagnification in the food chain.

Acknowledgment
This investigation was supported by Training Grant No. ES 47 from the National In-
stitute of Environmental Health Sciences, Research Triangle Park, N.C., Research Grant No.
EP 806 from the Environmental Protection Agency, Washington, D.C.,and a Research-
Training Grant from the Rockefeller Foundation, N.Y.

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Manuscript received August 7, 1972; accepted September 29, 1972.