Distribution and degradation of 14C-ethyl prothiofos in a potato plant and the effect of processing

Article abstract of DOI:10.1080/10426500801977970

Prothiofos and some of its degradation products have been synthesized in our laboratory for investigation purposes. The residual fate of 14C-ethyl prothiofos in different parts of potato plant was studied. The highest level of insecticide residues was detected in and on the leaves of potato plants. The residues of prothiofos insecticide were mainly located in the peels of potato tubers (peeling process removed 85% of the total residue after one month of the treatment), small amount penetrated into the pulp of potato tubers. The degradation products in the extracts of both peel and pulp of potato tubers were identified as prothiofos, prothiofos oxon, desethyl prothiofos, O-ethyl-S-propyl phosphorodithioate, O-ethyl phosphorothioate, O-ethyl-S-propyl phosphoric acid, O-ethyl phosphoric acid, despropylthio prothiofos, and prothiofos oxon sulfoxide. In addition 2,4-dichlorophenol was identified as such and in conjugated metabolites. Potatoes are processed in three ways: frying, boiling, and baking, which is simulated in home preparation. The amount of prothiofos residues was found to decrease on boiling (70%) and further on baking (82%) and frying (100%). The results indicated that frying process is the most effective method for reducing the amount of pesticide residues. Detectable residues were found in boiled potatoes (2.64 ppm), in the boiling water (0.24 ppm), and in the frying oil (0.71 ppm). From these results we concluded that the processed potatoes are safely used for human consumption. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426500801977970

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Phosphorus, Sulfur, and Silicon
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Distribution and Degradation
of 14C-Ethyl Prothiofos in a
Potato Plant and the Effect of
Processing
H. Abdel-Gawad a , L. M. Afifi b , R. M. Abdel-
Hameed a & B. Hegazi a
a Applied Organic Chemistry Department , National
Research Centre , Cairo, Egypt
b Radioisotopes Department, Atomic Energy
Authority , Cairo, Egypt
Published online: 10 Oct 2008.
To cite this article: H. Abdel-Gawad , L. M. Afifi , R. M. Abdel-Hameed & B. Hegazi
(2008) Distribution and Degradation of 14C-Ethyl Prothiofos in a Potato Plant and
the Effect of Processing, Phosphorus, Sulfur, and Silicon and the Related Elements,
183:11, 2734-2751
To link to this article: http://dx.doi.org/10.1080/10426500801977970
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Phosphorus, Sulfur, and Silicon, 183:2734–2751, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500801977970
Distribution and Degradation of 14C-Ethyl Prothiofos
in a Potato Plant and the Effect of Processing
H. Abdel-Gawad,1 L. M. Afifi,2 R. M. Abdel-Hameed,1
and B. Hegazi1
1Applied Organic Chemistry Department, National Research Centre,
Cairo, Egypt
2Radioisotopes Department, Atomic Energy Authority, Cairo, Egypt
Prothiofos and some of its degradation products have been synthesized in our labo-
14
ratory for investigation purposes. The residual fate of C-ethyl prothiofos in differ-
ent parts of potato plant was studied. The highest level of insecticide residues was
detected in and on the leaves of potato plants. The residues of prothiofos insecticide
were mainly located in the peels of potato tubers (peeling process removed 85% of the
total residue after one month of the treatment), small amount penetrated into the
pulp of potato tubers. The degradation products in the extracts of both peel and pulp
of potato tubers were identified as prothiofos, prothiofos oxon, desethyl prothiofos,
O-ethyl-S-propyl phosphorodithioate, O-ethyl phosphorothioate, O-ethyl-S-propyl
phosphoric acid, O-ethyl phosphoric acid, despropylthio prothiofos, and prothio-
fos oxon sulfoxide. In addition 2,4-dichlorophenol was identified as such and in
conjugated metabolites.
Potatoes are processed in three ways: frying, boiling, and baking, which is simu-
lated in home preparation. The amount of prothiofos residues was found to decrease
on boiling (70%) and further on baking (82%) and frying (100%). The results in-
dicated that frying process is the most effective method for reducing the amount of
pesticide residues. Detectable residues were found in boiled potatoes (2.64 ppm), in
the boiling water (0.24 ppm), and in the frying oil (0.71 ppm). From these results
we concluded that the processed potatoes are safely used for human consumption.
Keywords 14C-Ethyl prothiofos; degradation products; potato plants; processing and
residues
INTRODUCTION
Prothiofos (I), (Tokuthion, O-(2,4-dichlorophenyl) O-ethyl-S-propyl
phosphorodithioate) is an organophosphorus insecticide effective for the
Received 15 December 2007; accepted 6 February 2008.
We sincerely thank Prof. Dr. Issa Fakhr for his assistance in the synthesis of prothiofos
insecticide.
Address correspondence to L. M. Afifi, Radioisotopes Department, Atomic Energy
Authority, Cairo, Egypt. E-mail: abdelgawadhassan@yahoo.com
2734

14
C-Ethyl Prothiofos in a Potato Plant
2735
control of leaf—eating caterpillars, pseudoccus ssp., thrips, cockchafer
larvae, cutworms in a range of crops including vegetables, fruit, maize,
sugar cane, sugar beet, tea, tobacco, and ornamentals.1
It is selectively more toxic to insects than mammals due to the dif-
ference in metabolism of the thiopropyl group.2In spite of low toxicity
(LD50 950 mg/kg mouse and LD50 1700 mg/kg rats), fatal and nonfa-
tal poisoning cases have been reported in humans.3–5 Determinations
of its residues were carried out using several techniques such as GLC,
GC/MS and HPLC.6−10 Many researchers have studied how to minimize
pesticide residues in food products.11−13
The potato is tuber crop grown in the greatest quantities.14
Potatoes—members of the Solanaceae plant family—serve as major,
expensive low-fat food sources providing energy (starch), high-quality
proteins, fiber, and vitamins. Potatoes also produce biologically active
secondary metabolites, which may have both adverse and beneficial ef-
fects in the diet. These include glycoalkaloids, calystegine alkaloids,
protease inhibitors, lectins, phenolic compounds, and chlorophyll.15
Because organophosphorus insecticides are reported to affect cells,
animals, and humans, a need exists to develop a clearer understanding
of their roles both in the plant and in the diet. Since the typical use
pattern of (I) is leaves treatment, its metabolic pathways in plants after
foliar application have not been examined. Here we present data on
(a) the distribution of the organophosphorus insecticide prothiofos in
potato plant after its application in the field; (b) the identification and
determination of some of its degradation products; and (c) the removal
of the organophosphorus insecticide prothiofos and/or of its residues
from potato by processing. Each of these steps was studied separately
in order to determine which could account for residue changes.
EXPERIMENTAL
Spectral Analysis
1
The H NMR spectra were obtained on Jeol-EX (270 MHz) spectrom-
eter with tetramethylsilane (TMS) as internal standard for samples
dissolved in (CDCl3). The EI –MS (70 eV) spectra were run with a Jeol
JMS-AX500 mass spectrometer. The IR spectra were recorded as KBr
pellets with Nexus 670 FTIR spectrometer (Nicolet) at the National
Research Centre.
Chemicals
Chemical structures and abbreviations of the insecticide prothiofos and
of its supposed metabolites are shown in Figure 1.

2736
H. Abdel-Gawad et al.
FIGURE 1 Prothiofos and its supposed degradation products.
Prothiofos, O-(2,4-dichlorophenyl) O-ethyl-S-propyl phosphoroth-
ioate (prothiofos oxon) (II), O-(2,4-dichlorophenyl) S-propyl phos-
phorodithioate (III), O-(2,4-dichlorophenyl) O-ethyl phosphorothioate
(IV),O-ethyl-S-propyl phosphoro-dithioate (V) and 2,4-dichlorophenol
were synthesized as follows.
Synthesis of Prothiofos (Tokuthion) O-(2,4-dichlorophenyl)
O-ethyl-S-propyl Phosphorodithioate (I)
A mixture of 2,4-dichlorophenol (0.01 mol, 1.62 g) and triethylamine
(0.03 mol, 4.2 mL) in dry benzene (20 mL) was added dropwise to a
cooled (5–10◦C) solution of thiophosphoryl chloride (0.01 mol, 1.1 mL)
in dry benzene (20 mL) during 20 minutes. Stirring was continued for
4 h, followed by dropwise addition of propyl mercaptane (0.01 mol, 0.9
mL) in dry benzene (5 mL) at 5–10◦C, subsequently the temperature
was raised to 70–80◦C for 4 h, followed by dropwise addition of (0.6
mL, 9.25 MBq, Amersham, England) 14C-1-ethyl alcohol (0.01 mol) in
dry benzene (5 mL). The reaction mixture was stirred at 25◦C for 20 h,
filtered to remove the triethylammonium hydrochloride; and from the
filtrate, the solvent was evaporated under vacuum. The crude oil was
purified by silica gel column chromatography using hexane : diethyl
ether (9.5 : 0.5) for elution to give a colorless oil (Sp. Act. 0.02 mCi/g,
0.74 MBq/g), (yield 85%, 2.93 g). Thin layer chromatography on silica
gel plate showed one spot of Rf 0.56 in n-hexane : ethylacetate 99.5 : 0.5.
The radiochemical purity was >98% (Scheme 1). IR (KBr) νmax/cm−1
720 (P S), 1580, 1473 (C C ring stretch), 2966 (C-H aliphatic), 1160
:

14
C-Ethyl Prothiofos in a Potato Plant
2737
SCHEME 1 Route for preparation of 14C-ethyl prothiofos.
(P-Oaryl), 1022 (POC-). 1H NMR (CDCl3/TMS): δ = 0.95 (t, J = 7.0 Hz,
3H, CH CH2), 1.70 (m, 2H, C-CH -C), 2.95 (m, 2H, S-CH -), 1.37 (t, J =
3
2
2
7.2 Hz, 3H, CH -CH2), 4.28 (m, 2H, CH -CH3), 7.2–7.5 (m, 3H, arom-H).
3
2
EI-MS (m/z): 345 (M+), 347 (M++2), 349 (M++4), 183, 162, 164, 166,
155, 141, 98, 63.
Preparation of O-(2,4-Dichlorophenyl) O-ethyl-S-propyl
Phosphorothioate (Prothiofos Oxon) (II)
Nitric acid, 2.5 mL (0.059 mol, d = 1.49) was added dropwise to
252 µg of the cooled insecticide at 5◦C, and the mixture was stirred
at room temperature for 2 h. After removal of nitrogen dioxide gas in
vacuum, the mixture was poured into ice water (50 mL) and extracted
with ether (60 mL). The ether phase was washed with 10% cold sodium
bicarbonate solution and then with cold water till neutral. The ether
was evaporated under vacuum. The crude oil was purified on silica gel
column using hexane : diethyl ether (1:1) for elution. Yield: 64%, 162 µg.
Thin layer chromatography on silica gel plate showed one spot of Rf 0.01
in n-hexane : ethylacetate 99.5 : 0.5. IR (KBr) νmax/cm−1: 1220 (P=O),
1559, 1475 (C C ring stretch), 2922 (C-H aliphatic), 1037 (POC-).1H
NMR (CDCl3/TMS): δ = 0.99 (t, J = 7.0 Hz, 3H, CH CH2), 1.76 (m, 2H,
3
C-CH -C), 2.93 (m, 2H, S-CH -), 1.27 (t, J = 7.1 Hz, 3H, CH -CH2), 4.4
2
2
3
(m, 2H, CH -CH3) and 7.2–7.5 (m, 3H, arom-H). EI-MS (m/z): 329 (M+),
2
331 (M++2), 333 (M++4), 294, 252, 162, 164, 166, 98, 63.
Preparation of O-(2,4-Dichlorophenyl) S-propyl
Phosphorodithioate (III)
A solution of 2,4-dichlorophenol (0.005 mol, 0.81 g) and triethylamine
(0.01 mol, 1.4 mL) in dry benzene (20 mL) was added dropwise to a

2738
H. Abdel-Gawad et al.
cooled (5–10◦C) solution of thiophosphoryl chloride (0.005 mol, 0.507
mL) in dry benzene (10 mL) during 20 min. Stirring was continued for
4 h, followed by dropwise addition of propyl mercaptane (0.005 mol,
0.45 mL) in dry benzene (5 mL) at 5–10◦C. Subsequently the temper-
ature was raised to 70◦C for 4 h. The reaction mixture was filtered
and from the filtrate most of benzene was evaporated using a rotary
evaporator. The residue was dissolved in 50 mL of 0.1 N NaOH. After
extraction with diethyl ether (60 mL), the aqueous phase was acidified
to pH 1 with a few drops of concentrated HCl and extracted again with
ether (60 mL). The ether extract was dried over anhydrous sodium sul-
fate and the solvent was evaporated to leave colorless oil. The crude
oil was purified on silica gel column using hexane : diethyl ether (9.5 :
0.5) for elution (yield 50%, 0.8 g). Thin layer chromatography on silica
gel plate showed one spot of Rf (0.83) in n-hexane : ethylacetate 99.5 :
0.5 (Scheme 2). IR (KBr) νmax/cm−1: 3411 (OH), 720 (P S), 1580, 1475
(C C ring stretch), 2851 (C-H aliphatic), 2921 (CH arom-H), 692 (C-S).
1H NMR (CDCl3/TMS): δ = 0.99 (t, J = 7.4 Hz, 3H, CH CH2), 1.76 (m,
3
2H, C-CH -C), 2.97 (m, 2H, S-CH -), 7.2 (s, 1H, OH), 7.3–7.6 (m, 3H,
2
2
arom-H). EI-MS (m/z): 317 (M+), 319 (M++2), 321 (M++4), 282, 240,
162, 164, 166, 113, 63.
SCHEME 2 Preparations of prothiofos and its degradation products.

14
C-Ethyl Prothiofos in a Potato Plant
2739
Preparation of O-(2,4-Dichlorophenyl) O-ethyl
Phosphorothioate (IV)
Triethylamine (0.01 mol, 1.4 mL) and 2,4-dichlorophenol (0.005 mol,
0.81 g) in dry benzene (10 mL) were added dropwise to a cooled (5–
10◦C) solution of thiophosphoryl chloride (0.005 mol, 0.507 mL) in dry
benzene (10 mL) during 10 min. Stirring was continued for 4 h, followed
by dropwise addition of absolute ethyl alcohol (0.005 mol, 0.29 mL) in
dry benzene (5 mL). The reaction mixture was stirred at 25◦C for 18 h,
and filtered. From the filtrate the solvent was evaporated under reduced
pressure. The residue was dissolved in 50 mL of 0.1 N NaOH. After
extraction with diethyl ether (60 mL), the aqueous phase was acidified
to pH 1 with a few drops of concentrated HCl and extracted again with
ether (60 mL). The combined ether extract was dried over anhydrous
sodium sulfate and the solvent was evaporated to leave colorless oil. The
crude oil was purified on silica gel column using hexane : diethyl ether
(9.5 : 0.5) for elution (yield 34%, 0.48 g). Thin layer chromatography on
silica gel plate showed one spot of Rf (0.54) in n-hexane: ethylacetate
99.5 : 0.5 (Scheme 2). IR (KBr) νmax/cm−1: 3440 (OH), 722 (P S), 1580,
1474 (C C ring stretch), 2851 (C-H aliphatic), 2920 (CH aromatic), 670
(C S).1H NMR (CDCl3/TMS): δ = 1.24 (t, J = 7.1 Hz, 3H, CH CH2),
3
4.4 (m, 2H, C-CH -C), 7.4 (s, 1H, OH), 7.5–7.7 (m, 3H, arom-H). EI-
2
MS (m/z): 287 (M+), 289 (M++2), 291 (M++4), 252, 224, 162, 164, 166,
126.
Preparation of O-Ethyl S-propyl Phosphorodithioate (V)
A mixture of absolute ethanol (0.005 mol, 0.29 mL) and triethy-
lamine (0.01 mol, 1.4 mL) in dry benzene (15 mL) was added drop-
wise to a cooled (5–10◦C) solution of thiophosphoryl chloride (0.005 mol,
0.507 mL) in dry benzene (10 mL) during 15 min. The reaction mixture
was stirred at 25◦C for 18 h, followed by dropwise addition of propyl
mercaptane (0.005 mol, 0.45 mL) in dry benzene (5 mL) at 5–10◦C,
subsequently the temperature was raised to 70◦C for 3 h. The reaction
mixture was filtered, and from the filtrate most of the benzene was evap-
orated using a rotary evaporator. The residue was dissolved in 100 mL
of 0.1 N NaOH. After extraction with diethyl ether (60 mL), the aqueous
phase was acidified to pH 1 with a few drops of concentrated HCl and
extracted again with ether (60 mL). The combined ether extract was
dried over anhydrous sodium sulfate and the solvent was evaporated to
leave a colorless oil. The crude oil was purified on silica gel column using
hexane : diethyl ether (9.5 : 0.5) for elution to give a colorless oil (yield
68%, 0.68 g). Thin layer chromatography on silica gel plate showed one
spot of Rf (0.68) in n-hexane : ethylacetate 99.5:0.5 (Scheme 2). IR (KBr)

2740
H. Abdel-Gawad et al.
νmax/cm−1: 2962 (OH), 707 (P S), 2852 (C H aliphatic), 1485 (-CH2-),
1378 (-CH3), 692 (C S). 1H NMR (CDCl3/TMS): δ = 0.95 (t, J = 7.1 Hz,
3H, CH CH2), 1.8 (m, 2H, C-CH -C), 2.95 (m, 2H, S-CH -), 1.24 (t, J =
3
2
2
7.3 Hz, 3H, CH CH2), 4.2 (m, 2H, CH -CH3), 7.3 (s, 1H, OH). EI-MS
3
2
(m/z): 200 (M+), 198, 181, 172, 158, 139, 129, 75, 43.
Preparation of 2,4-Dichlorophenol (DCP (VI)
2,4-Dichlorophenol (DCP (VI) was prepared by hydrolysis of prothio-
fos as follows. Prothiofos (0.01 mol, 3.45 g) was mixed with 95% ethanol
containing 1% sodium hydroxide, and the reaction mixture was kept
at 60◦C overnight or stirred at room temperature for 5 h. The result-
ing precipitate was separated by filtration, dissolved in distilled water
(20 mL), and acidified with a few drops of 1N HCl. Crystallization from
aqueous ethanol gave (92%, 1.5 g) of 2,4-dichlorophenol, m.p. 43◦C.
Field Experiment
Valor potatoes (Solanum tuberosum L.) were planted in an isolated
area at the Experimental Station of the National Research Centre. The
complete randomized block design with four replicates was used. Each
plot had four 5 m rows. The complete tubers were planted in October
2005 and spaced 25 cm within the rows. Common cultural and fertil-
ization practices were followed. The 14C-ethyl prothiofos in very dilute
ethanol solution (to ensure that all radioactive material is transferred)
was topically applied manually with a micropipette on healthy leaves of
the plant after two months of agriculture following the Egyptian Min-
istry of Agricultureꢀ s recommended rate (12 mg/plant each time). The
pesticide was applied twice during the season with 15 days interval.
Sampling
Three potato plants were collected after 7, 14, 21, and 30 days from the
last application of the labeled prothiofos. Each plant was differentiated
into leaves, stems, roots, and tubers and they were weighed and stored
at −20◦C till analysis.
Three tubers were peeled with a knife in the way the peeling is usu-
ally carried out by consumers. The weights of the flesh (pulp) and of the
peel of each sample were recorded. The analysis was carried out sep-
arately. The concentration in whole unwashed tubers was estimated
taking into account the concentration in the flesh and the peel, and the
respective weights.

14
C-Ethyl Prothiofos in a Potato Plant
2741
Processing
Potatoes are processed in three phases: frying, boiling, and backing,
which is simulated in home preparation.
1. Three tubers were peeled, and cut into pieces for french fries. The
weights of the peel, and the flesh were recorded. Each subsample
was fried in soybean oil for 15 min. The weight of fries after cooking
was taken. The fries, as well as the oil, were analyzed.
2. Three tubers were boiled for 15 min in a pressure kettle, containing
1 l of hot water. The weight of each subsample was recorded before
and after boiling. The above methods of cooking were chosen since
they are among the most common cooking methods used around the
world. Also, the products are highly consumed by children.
3. Three tubers wrapped in aluminum foil were baked in an oven at
220◦C for 90 min.
Extraction and Isolation of Degradation Products
The potato samples (whole, peels, pulps, french fries, baked, and boiled)
were separately blended three times with methanol (100 mL each) for
three minutes and the mixture of each sample was filtered. The filtrate
was extracted three times each with 100 mL of chloroform and sodium
chloride solution (salting out, make separation easily) and was shaken
for 2 min. The combined chloroform extracts were dried over anhydrous
sodium sulfate, filtered, and from the filtrate the solvent was evaporated
under reduced pressure. Triplicates of organic and water layer samples
were radioassayed. The extract was analyzed by TLC for identification
of the metabolites.
The aqueous layer was divided into two parts: the first part was
evaporated to dryness in special dessicator over P2O5, extracted with
methanol (5 mL) and analyzed by thin-layer chromatography. The sec-
ond part was acidified by 50 mL 2N HCl, and heated for 12 h at 100◦C to
decompose conjugated phenolic compounds. After cooling the acidified
water phase was extracted with 50 mL chloroform three times and ana-
lyzed by TLC. The 14C distribution among the aqueous, organic soluble
fraction and the non-extractable residues was then calculated.
Chromatographic Analysis
Extracts of organic and aqueous layer of potato samples were achieved
by thin-layer chromatography (TLC) using silica gel 60 F254 thin-layer

2742
H. Abdel-Gawad et al.
chromatoplates (20–20 cm, 0.25 mm thickness, E. Merck). The following
solvent systems were used for development:
r
System 1: n-hexane : ethyl acetate 99.5 : 0.5 (v/v);
System 2: n-heptane : ethylacetate 99 : 1 (v/v);
System 3: n-heptane pure; and
System 4: n-heptane : chloroform : methanol 9 : 4 :1 (v/v).
r
r
r
Prepared degradation products were run alongside as references and
spots were detected under ultraviolet light at 254 nm and made visi-
ble by spraying the plates with a freshly prepared Hanes-Isherwood
reagent16 or by subjecting to I2 vapor after preliminary spray with
PdCl2 solution.17,18 To detected the phenolic compounds, the plate
were developed in the above systems and sprayed with ferric chlo-
ride/potassium ferricyanide solution, where blue spots on yellow back-
ground appeared.19
Radioassay
Radioactivity in the liquid extracts was determined by mixing each
aliquot with 10 mL of Packard Emulsifier Scintillator cocktail and an-
alyzed by liquid scintillation counting (LSC) with Packard Model TRI-
CARB 2300 TR. The background level of radioactivity in LSC averaged
30 dpm and was subtracted from the values for measured samples.
Radioactivity in the air dried unextractable residues from the treated
plants was combusted by using a Harvey Biological Oxidizer (model OX-
600). The librated 14CO2 was trapped in carbsorb scintillation cocktail
and assayed for radioactivity. The internal standard technique was used
for quench correction. The efficiency of combustion was determined to
be greater than 90%.
Thin-layer chromatography was used to trace and identify the degra-
dation products. After development, each plate was divided into 1 cm
zones, which were separately scrapped, extracted with 2 mL methanol,
and then counted for radioactivity in liquid scintillation counter.
GC/MS Analysis
GC/MS analysis was used for the identification of the parent com-
pound and its degradation products in this study. It was carried
out with GC/MS Finnigan Mat SSQ 7000, EI 70 eV. Operating
conditions: a capillary column DB-5, 30 m × 0.25 mm I.D. [(5%-
Phenyl)methylpolysiloxane]. The analysis was carried out at a pro-
grammed temperature: initial temperature 50◦C for 0 min, then

14
C-Ethyl Prothiofos in a Potato Plant
2743
increasing at rate of 5◦C /min, until it reaches 300◦C (kept for 5 min).
The injector temperature was set at 250◦C and the detector tempera-
ture at 280◦C. Helium was used as a carrier gas at 1 mL/min, injected
volume was 2 µL, and injection mode was splitless. The compounds
were identified by matching their MS with those recorded in the MS
library (Wiley) and comparison with those of reference compounds.
RESULTS AND DISCUSSION
Distribution of Radioactivity in Potato Tubers
The distribution of radioactivity of 14C-prothiofos was examined in
potato tubers samples. The use of a radiolabeled compound in this in-
vestigation was based on the assumption that the radiolabeled molecule
would be metabolized in the same manner as the unlabelled molecule.20
The topically applied radioactive material was subsequently translo-
cated through the plant. Some of this translocated radioactive material
was detected in the whole parts of the potato plants. Fourteen days
after the last treatment, the highest level of insecticide residues (49.5
ppm) was detected in and on the leaves of the potato plants. This high
level might be expected since the leaves are the most exposed parts of
the plant to the insecticide application. The accumulation of insecticide
detected in the roots after 14 days from the last treatment of insecticide
was 20 ppm, while the stem of the potato plant had 28.5 ppm. The level
of the residues was low on tubers and amounted to 4.8 ppm.
Table I shows the prothiofos residues in peels and pulps of potato
tubers collected at different time intervals from the last treatment with
TABLE I 14C-Residues in Potato Tubers (Peels and Pulps) After the
Last Application of 14C-Prothiofos
14C-residues (ppm)
Peels
Pulps
Time
Extract
Extract
Wt. of
tubers (g)
(Days)∗
Mean SD∗∗∗
Bound Mean SD∗∗∗
Bound
%**
7
14
21
30
0
0
0
0
0
0.22 0.04
0.54 0.04
8.2 2.02
0.64 0.19
0
0
0
0
118
175
161
196
0.00011
0.00314
0.01339
0.00350
3.76 1.96
11.7 0.35
3.6 0.23
∗Samples taken after the last application; ∗∗percentage of the applied dose; and
∗∗∗results are expressed as mean SD for three determinations of prothiofos level for
each sample.

2744
H. Abdel-Gawad et al.
14C-compound. The prothiofos residues in whole potato tubers increased
with time from 0.22 ppm to 19.9 ppm during the first three weeks,
then decreased rapidly at the end of 30 days of the last application and
amounted to 4.24 ppm. These results indicate that the highest level of
the insecticide residues was detected in and on the peels of potato tubers
and amounted to 3.6 ppm after 30 days of the last application. Peeling
process removed 85% of the total residues. Small amount of insecticide
residues (0.64 ppm) penetrated into the pulps. These results are in
agreement with those reported by many authors.21−23 In their studies
on distribution and degradation of some organophosphorus insecticides
on potato plants, Lentza-Rizos and Balokas24 found that the amounts of
chloropropham herbicide removed by peeling were 98% and 91% from
the first and second potato sample treated with this pesticide at 10
and 28 days, respectively. Mohamed25 found, that the loss was 49.6 and
52% for thin peeled, 57 and 68% for thick peeled, and 54 and 70% for
carbandum peeled in case of DDT and Lindan residues, respectively.
By extracting the potato tubers (peels and pulps) with methanol and
partitioning between chloroform and water, the activity in extractable
14C-residues in peels increased from 3.76 ppm to 11.7 ppm during the
first three weeks of the second application, then it decreased rapidly and
reached 3.6 ppm at the end of experimental period. The 14C-residues in
extractable layer of pulps also showed the same trends. There were no
bound residues detected in both peels and pulps of potato tuber and the
percent of recovery ranged from 90 to 97% as shown in Table II. Our
observations support the findings of other researchers,26,27 who found
that percentage of bound residues in the total 14C-labeled residues of
crop samples differs with the variety of pesticides and crops.
Identification and Characterization of Radioactive
Degradation Products in Potato Tubers
Because metabolic factors may be of importance in the risk assess-
ment of prothiofos and because there are very little data available on
prothiofos metabolism, we performed rather extensive series of exper-
iments to elucidate interactions and metabolic routes.28 Tables III and
IV represent the Rf values and the amounts (ppm) of the 14C insecticide
and some of its degradation products after three weeks and after one
month of the second application. In addition to the parent compound
(prothiofos) (I), prothiofos oxon (II), despropylthio prothiofos (IV),
O-ethyl-S-propyl phosphorodithioate (V), O-ethyl phosphorothioate,
O-ethyl-S-propyl phosphorothioic acid, O-ethyl phosphoric acid and
prothiofos oxon sulfoxide were detected. The phenolic compound

2745

2746
H. Abdel-Gawad et al.
TABLE III The Amount and Rf Values of 14C Prothiofos and Its Active
Degradation Products in Extracts of Different Parts of Potato Tubers
After 21 Days of Treatment
After three weeks (ppm)*
Rf values in various
solvent system
Pulp
Peel
Metabolites
1
2
3
4
Chloroform Aqueous Chloroform Aqueous
Prothiofos (I)
Prothiofos oxon (II)
Despropylthio
prothiofos (IV)
O-ethyl-S-propyl
phosphorodithiote
(V)
O-ethyl
phosphorothioate
(VII)
0.56 0.47 0.21 0.80
0.01 0.06 0.00 0.52
0.54 0.49 0.30 0.78
0.49
0.00
0.00
0.00
0.00
0.00
1.40
2.00
0.00
0.00
0.00
0.00
0.68 0.65 0.35 0.83
0.21 0.52 0.45 0.60
0.43 0.41 0.32 0.85
0.00
0.00
0.00
1.61
3.52
0.00
0.00
0.00
0.00
2.80
3.32
0.00
O-ethyl-S-propyl
phosphorothioate
(IX)
O-ethyl phosphoric
acid (VIII)
Prothiofos oxon
sulfoxide (X)
0.77 0.65 0.60 0.89
0.96 0.81 0.46 0.42
0.00
0.00
0.00
1.80
0.00
0.00
1.03
0.87
∗Data are mean of two replicates. System 1: n-hexane : ethyl acetate 99.5 : 0.5 (v/v).
System 2: n-heptane : ethylacetate 99 :1 (v/v). System 3: n-heptane pure. System 4:
n-heptane : chloroform : methanol 9 : 4 : 1 (v/v).
was found as the free phenol in the organic extract and as conju-
gate in the water extract, which was liberated by acid hydrolysis.
The conjugated metabolites are DCP–Glu [O-ß–D-glucopyranosyl-
2,4-dichlorophenol]
and
DCP–Glu–Mal
[(6-O-malonyl)-O-ß-D-
glucopyranosyl-2,4-dichlorophenol]. These findings are similar to
those obtained by GC/ MS for the extract of potato pulp after three
weeks of the last treatment as shown in Table V.
It is important to distinguish between the primary metabolic pro-
cess (triester to diester) and the secondary process involving the fur-
ther metabolism of the ‘leaving group’. In plants, pesticides generally
conjugate with sugars to form sugar conjugates as secondary metabo-
lites. The alcohol groups of primary metabolites are usually readily
conjugated.29 Laurent et al.30 found that 2,4-dichlorophenol was con-
jugated with sugar in edible plants (potato, sugar beet and tomatoes)
when treated with 2,4-dichlorophenoxy acetic acid (2,4-D).

14
C-Ethyl Prothiofos in a Potato Plant
2747
TABLE IV The Amount and Rf Values of14C Prothiofos and Its Active
Degradation Products in Extracts of Different Parts of Potato Tubers
After 30 Days of Treatment
After three weeks (ppm)*
Rf values in various
solvent system
Pulp
Peel
Metabolites
1
2
3
4
chloroform aqueous chloroform aqueous
Prothiofos (I)
0.56 0.47 0.21 0.80
0.01 0.06 0.00 0.52
0.54 0.49 0.30 0.78
0.00
0.00
0.00
0.00
0.00
0.00
0.167
0.162
0.00
0.00
0.00
0.87
Prothiofos oxon (II)
Despropylthio
prothiofos (IV)
O-ethyl-S-propyl
phosphorodithiote (V)
O-ethyl
0.68 0.65 0.35 0.83
0.21 0.52 0.45 0.60
0.00
0.00
0.00
0.15
0.00
0.00
0.00
1.10
phosphorothioate
(VII)
O-ethyl-S-propyl
phosphorothioate (IX)
O-ethyl phosphoric acid 0.77 0.65 0.60 0.89
(VIII)
Prothiofos oxon
0.43 0.41 0.32 0.85
0.00
0.00
0.00
0.23
0.25
0.00
0.00
0.00
0.00
0.00
0.52
0.60
0.96 0.81 0.46 0.42
sulfoxide (X)
*Data are mean of two replicates. System 1: n-hexane : ethyl acetate 99.5 : 0.5 (v/v).
System 2: n-heptane : ethylacetate 99 :1 (v/v). System 3: n-heptane pure. System 4:
n-heptane : chloroform : methanol 9 : 4 : 1 (v/v).
The proposed pathway for the degradation of 14C-prothiofos in potato
plant is shown in Scheme 3. Metabolic routes include oxidation to pro-
thiofos oxon, hydrolysis to 2,4-dichlorophenol, which is further conju-
gated with sugar in the plant, and cleavage of the propyl group leading
to the formation of despropylthio prothiofos as well as hydrolysis of the
TABLE V Structures and GC-MS Fragments of Extractable Potato
Pulp After Three Weeks from the Last Treatment
RT (min)
GC-MS (m/z)
Prothiofos (I)
O-ethyl-S-propyl
44.08
6.54
345, 347, 349, 183, 162, 164, 166, 155, 141
200, 198, 181, 172, 158, 139, 129
phosphorodithioate (V)
Prothiofos oxon sulfoxide (X)
O-ethyl phosphorothioate
(VII)
40.47
24.6
344, 308, 266, 207, 182, 161, 112
143, 115
2,4-dichlorophenol (VI)
3.79
163, 105

2748
H. Abdel-Gawad et al.
SCHEME 3 Proposed pathways for degradation of 14C-prothiofos in potato
tubers.
ethoxy group leading to the formation of desethyl prothiofos. Katagi
and Mikami31 noted that the metabolism of organophosphorus pesti-
cides in plants have revealed cleavage of the P-O-aryl linkage and O-
dealkylation to be among the most predominant metabolic pathways.
Enzymatic and acid hydrolysis released an aglycon and direct spectro-
scopic analyses of the metabolites implied that I primarily underwent
cleavage of the P-O-aryl linkage or hydroxylation of the aryl methyl
group similarly to other organophosphorus pesticides in plants. These
metabolites were also detected in rats5 and in human urine.2
Effect of Processing
Peeling process was the most effective way to remove the pesticide
residues from the crops.23,32−34 After three weeks of the last applica-
tion, peeling removed 59% of the 14C-residues, while 30 days after the
experiment it removed 85% of the total 14C-residues. The processing
methods lead to a high decrease in the total amount of 14C-residues

14
C-Ethyl Prothiofos in a Potato Plant
2749
TABLE VI Effect of Processing Methods on the Residues of 14
Prothiofos in Potato Tubers After Three Weeks of the Last
Application
C
Total 14C prothiofos (ppm)
Mean SD∗
Reduction (%)
Fresh whole potatoes (peeled)
Boiled whole potatoes (peeled)
Baking whole potatoes (peeled)
french fries from peeled potatoes
8.20 2.02
2.48 0.17
1.49 0.10
0.00
0.00
69.80
81.80
100.00
∗Results are expressed as mean SD for three determinations of prothiofos level for
each sample.
in peeled potato. Obvious reduction was detected after boiling (70%)
and was increased after baking (82%) and frying (100%) as shown in
Table VI. This decrease could be attributed to the time of exposure, the
effect of temperature and/or evolution of 14CO2 gas. Soliman35 found
that peeling, blanching, and frying are necessary to remove pesticide
residues from contaminated potato tubers. The residues detected in
boiling water were 0.24 ppm and in frying oil 0.71 ppm. Boiling is ef-
fective in reducing the level of water soluble pesticide residues and the
use of oil in cooking processes decreases the level of fat soluble ones.
The obtained data indicated that frying was more effective than the
other investigated potato processing methods in reducing the levels of
prothiofos residues. This finding is in accord with the results obtained
by many authors.12,21,25 Also these findings are in agreement with those
obtained by Lewis et al.36, who found that the residues of the pesticides
were significantly reduced to less than 2% and 10% of the maximum
theoretical residues level for potato crisp and jacket crisps, respectively.
The results demonstrate that the level of pesticide residues in potato tu-
bers is lowered, sometimes far below the recommended levels by wash-
ing or cooking.33
TLC and GC-MS revealed the presence of four degradation products
in addition to the parent compound in the extracts of both boiled and
baked potatoes, which were identified as prothiofos, prothiofos oxon,
O-ethyl phosphorothioate, prothiofos oxon sulfoxide and despropylthio
prothiofos, as shown in Table VII. The prothiofos residues level in the
different processing methods was found in the range of 0 to 0.04 ppm,
which was below the maximum residue limits (MRLs) of prothiofos in
potatoes (0.05 ppm).37 It can be concluded that the processed potatoes
can be safely used for human consumption.

2750
H. Abdel-Gawad et al.
TABLE VII The Amount, Rf Values and GC-MS Fragments of
14C-Prothiofos and Its Active Degradation Products in Extracts of
Pulp of Potato Tubers After Processing
Rf values
in systems
RT
(min)
Boiling Baked
GC/MS (m/z)
Sys 1 Sys 2 Sys 3 (ppm)∗ (ppm)∗
Prothiofos (I)
44.08 345, 347, 349, 183, 162, 0.56 0.47 0.21
164, 166, 155, 141
36.64 328, 294, 266, 224, 161, 0.01 0.06 0.00
142, 114
0.04
0.03
0.14
0.26
0.00
0.00
0.00
1.12
0.00
0.37
Prothiofos oxon
(II)
O-ethyl phospho- 24.6 143, 115
rothioate (VII)
Prothiofos oxon
sulfoxide (X)
Despropylthio
0.21 0.52 0.45
40.47 344, 308, 66, 207, 182,
161, 112
0.96 0.81 0.46
21.2 287, 289, 291, 252, 162, 0.54 0.49 0.30
164, 166 , 126
prothiofos (IV)
∗Data are mean of two replicates. System 1: n-hexane : ethyl acetate 99.5 : 0.5 (v/v).
System 2: n-heptane : ethylacetate 99 :1 (v/v). System 3: n-heptane pure.
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