Toxicological evaluation and bioavailability of 14C-fenitrothion bound residues on soybeans towards experimental animals

Article abstract of DOI:10.1016/j.fct.2008.06.015

Under local practice of Egyptian conditions, the application of ¹⁴C-fenitrothion on soybeans at a dose of 10 mg insecticide/kg grains, led to the formation of 21% of ¹⁴C-bound residues (non-extractable) after 24 weeks of storage. The

Full text of DOI:10.1016/j.fct.2008.06.015

Food and Chemical Toxicology 46 (2008) 3111–3115
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Food and Chemical Toxicology
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Toxicological evaluation and bioavailability of 14C-fenitrothion bound residues
on soybeans towards experimental animals
*
M. Farghaly, S. El-Maghraby
Department of Applied Organic Chemistry, National Research Centre, Dokki, Giza, Egypt
a r t i c l e i n f o
a b s t r a c t
Under local practice of Egyptian conditions, the application of 14C-fenitrothion on soybeans at a dose of
10 mg insecticide/kg grains, led to the formation of 21% of 14C-bound residues (non-extractable) after 24
weeks of storage. The external residues were 20% and the internal extracts were 55% of the applied dose.
Feeding studies on rats revealed that bound residues were bioavailable. After feeding rats for three days
with bound 14C-fenitrothion residues, the main portion of radioactivity was eliminated via expired air
(42%), urine (20%) and feces (11.5%). About 15% of the administered radioactivity was distributed among
various organs as, liver, kidney, lung, fat, intestine, blood, heart, and brain. Toxicity of bound residues of
14C-fenitrothion in stored soybeans was studied in mice through feeding experiments for three months at
a concentration of 1.9 mg/kg. The maximum inhibition in plasma and erythrocyte cholinesterase activity
was observed 22.5%, 18.9% and 8.6%, 9% after one and seven days, respectively. The obtained results
showed a slight significant elevation after three months in the activity of liver enzymes alanine amino
transferase, aspartate amino transferase and alkaline phosphatase. A moderate increase in blood urea
nitrogen and creatinine concentration was observed in the treated groups at the end of the experimental
period. The detected levels of albumin and total protein showed no significant compared to the control
values, of controlled animals, after three months.
Article history:
Received 3 December 2007
Accepted 23 June 2008
Keywords:
14C-fenitrothion
Stored soybeans
Bound residues
Bioavailability
Toxicity
Bioassay
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ase (ALT) activities, creatinine and urea which may play an impor-
tant role in the physiological functions and also accumulate in
Fenitrothion (O,O-dimethyl-O-(3-methyl-4-nitrophenyl phosp-
horothioate)), is a broad-spectrum organophosphate insecticide
that has been in use since 1959. It is employed in agriculture to
control insects on rice, cereals, fruits, vegetables, soybeans and cot-
ton (The Pesticide Manual, 1994). It is also applied in public health
measures to control flies, mosquitoes and cockroaches (WHO,
1992). Fenitrothion is widespread used for controlling insects in
stored grains. It is used only for treating the surface of storage ves-
sels, since it is moderately toxic to mammals LD50503 mg/kg
(Meaklin et al., 2002; Uygun et al., 2005). As with other organo-
phosphorus insecticides, fenitrothion is a contact-acting pesticide
which inhibits acetyl cholinesterase activity, thus disrupting the
nervous system (Sarikaya et al., 2004). The obtained results by
Okahashi et al. (2005) suggest that fenitrothion at in use levels in
the environment had no effects on the reproductive or endocrine
systems even at toxic dose that markedly suppressed brain cholin-
esterase activity in the experimental animals. Hayes and Laws
(1990) reported that fenitrothion has cytotoxic effects on the lungs
of rats. Treatment with fenitrothion caused a significant increase in
serum alanine amino transferase (AST), aspartate amino transfer-
different rat tissues (Kadry et al., 2001). Fenitrothion administra-
tion is associated with alteration of the ratio of various compo-
nents of phospholipids and neutral lipids in various organs of
rats up to 48 h (Roy et al., 2004a).
The aim of this present study was to evaluate the percentage of
the 14C-bound residues distributed through the grains after storage
for 24 weeks. This study also detects the amount of the insecticide
excreted and accumulated in rats and aimed to study the toxic
effect of fenitrothion residues focusing on the most important
biochemical parameters as acetyl cholinesterase, liver and kidney
enzyme activities.
2. Materials and methods
2.1. Chemicals
2.1.1. Synthesis of fenitrothion (I):
14C-fenitrothion-labelled at carbon atom of methyl groups-was synthesized by
a single vesseled reaction in our laboratory (Scheme 1).
This method involved the condensation of 0.28 ml (2 mol) of 14C-methanol in
toluene to 0.315 ml (1 mol) of thiophosphorylchloride in presence of triethylamine
0.81 ml (2 mol) and the mixture was stirred at 5–10 °C for 30 min, then filter the
product to remove the separated triethylamine hydrochloride and the filtrate was
evaporated under vacuum. The obtained (O,O-dimethylthiophosphoryl chloride)
in toluene was allowed to react with potassium salt of 3-methyl-4-nitrophenol
* Corresponding author.
E-mail address: somiaibrahem@yahoo.com (S. El-Maghraby).
0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2008.06.015

3112
M. Farghaly, S. El-Maghraby / Food and Chemical Toxicology 46 (2008) 3111–3115
determination of radioactive metabolites, 1 cm zones were removed from TLC
plates by scraping and then determined for radioactivity using a dioxane-based
scintillator.
*
S
P
Et3N
H3CO
*
2CH3OH
PSCl 3
+
Cl
*
H3CO
2.6. Feeding experiment and biochemical studies
CH3
+
S
*
In order to assess the toxicological potential of bound fenitrothion residues in
soybeans, a sub chronic feeding study on mice for 90 days was carried out. Standard
diet was mixed with extracted beans having bound fenitrothion residues and then
fed to a group of forty male Swiss mice of five weeks old (weighing 12 g 1) to en-
sure a daily dose of 1.9 ppm insecticide equivalent. The control group was fed the
same diet containing non-treated extracted soybeans. Throughout the time of the
experiment, mice were observed with regard to behavior, appetite, and body weight
gain. Feeding experiments lasted three months, where mice were examined at
intervals of 1, 7, 15, 30, 60, and 90 days for changes in plasma and erythrocyte cho-
linesterase activity.
H3CO
Reflux 2-3 hrs
60-80 OC
P
O
NO2
CH3
NO2
KO
*
H3-
Fenitrothion [I]
Scheme 1. Synthesis of 14C-fenitrothion.
For biochemical studies, animals were sacrificed under ether anaesthesia and
blood samples were collected in heparinized tubes from the orbital plexus of trea-
ted and control groups of mice for the assay of cholinesterase activity. There are at
least six animals in each control and treated group. Blood samples were centrifuged
to separate plasma and blood cells. Cholinesterase activity was determined accord-
ing to Ellman et al. (1961), as modified by Gorum et al. (1978).
In dry test tubes, samples of blood were taken and centrifuged to separate
serum that was used for assaying liver and kidney function enzymes. Alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) activities were
determined according to Reitman and Frankel (1957). Serum alkaline phospha-
tase activity was measured by Kind and King (1954), while blood urea nitrogen
concentration (BUN) was determined according to Urease-modified Bwrthelot
reaction by Charles and Crouch (1977). Serum creatinine determination was car-
ried out according to Jaffe reaction by Harry and Abraham (1968). The protein
concentration was determined by the Biuret method (Gornall et al., 1949). Serum
albumin concentration was determined using the Sigma Diagnostics albumin re-
agent (Sigma chemical), this method is based on the procedure of Doumas et al.
(1971).
while stirring for 30 min at room temperature. This mixture was refluxed for 2–3 h
at 60–80 °C, cool, added cold water and the organic layer was separated, dried over
anhydrous sodium sulphate. Under reduced pressure the solvent was evaporated to
give yellow-brown liquid (b.p 140–145 °C, 0.1 mm). The product was purified by
thin layer chromatography (TLC) plates Silica gel 60 F254 pre-coated plates
20 Â 20 cm layer thickness 0.5 mm Art.5744.The prepared 14C-fenitrothion had a
specific activity 2.96 MBq (0.08
et al., 1959).
lci/mg) and a radiometric purity 98% (Yoshihiko
2.2. Treatment and storage of seeds
Mature seeds of soybean (Var. Catler 71) with moisture content (12%) were
treated with 14C-fenitrothion at a dose of 10 mg insecticide/kg seeds according to
a standard procedure (Zayed and Farghaly, 1985). The seeds were stored under con-
ditions simulating those used locally. Samples were taken for analysis after 24
weeks of storage. The seeds used for the control experiment did not receive insec-
ticidal treatment and were stored under similar conditions.
2.3. Extraction and analysis of 14C-residues
2.7. Statistical analysis
Samples of treated seeds were washed with acetone: water (1:3) to remove the
external residues. The washed seeds were dried, crushed and extracted in a Soxhlet
apparatus for 24 h with methanol to obtain the internal 14C-extractable residues.
The radioactivity in both extracts was directly determined by liquid scintillation
counting (LSC) in a Packard model 3320 Scintillation Spectrometer. The extracted
soybean were analyzed for bound radioactive residues by combustion of a known
weight in a Harvey Biological Oxidizer (OX-600) followed by LSC of the produce
14CO2.
The obtained results were statistically analyzed by student’s t-test.
3. Results
3.1. Seed-bound residues
In a parallel experiment, bound residues were prepared using a non-labelled
fenitrothion to determine the adverse effects in mice fed extracted soybeans having
bound fenitrothion residues for 90 days.
The results obtained for the persistence of fenitrothion insecti-
cide on soybeans for 24 weeks of storage under controlled condi-
tions stimulating those used in practice (Table 1), indicate that,
fenitrothion readily penetrates the seed coat. The external 14C-res-
idues were 20% while the internal extract was about 55% of the
actual dose. The percentage of 14C-bound residues (non-extract-
able) of fenitrothion in treated beans was 21% of the applied dose.
A good recovery of radioactivity, which is generally over 95% of the
actual applied dose, could be achieved by the analytical procedures
used.
2.4. Bioavailability of bound residues in rats
Three months old, sexually mature white male rats (derived from Sprague–
Dawley strain) weighing 150 10 g were purchased from animal house colony,
Dokki, Giza Egypt. The animals were individually housed in glass metabolism cages
that allowed separate collection of feces, urine and expired air. The rats were con-
ditioned for two days to a daily diet consisting of standard feed mixed with ex-
tracted untreated soybeans for acclimation under the laboratory conditions
(27 3 °C). The animals were kept without food for 24 h and then fed the diet con-
taining bound 14C-fenitrothion residues for three days. To make the feed palatable,
the extracted beans were mixed thoroughly with an equal amount of white cheese
and the paste was left to dry.
3.2. Bioavailability to rats
Table 2 shows the elimination of radioactivity in expired air and
urine of rats following feeding with bound 14C-fenitrothion resi-
dues for 72 h. The maximum percentage of radioactivity was ex-
creted in the first 48 h. Distribution of release 14C-activity in rats
after feeding for three days is tabulated in Table 3. The major part
of radioactivity was eliminated via expired air (42%), while urine
and feces contained only 20% and 11.5% of the ingested 14C-activ-
ity, respectively.
The respiratory carbon dioxide was trapped in 10% NaOH solution. Urine, feces
and 14CO2 were collected separately for three days, and assayed for radioactivity.
After the end of three days each rat was lightly anaesthized with ether and blood
removed from the pumping heart. The animal was then killed with an overdose
of ether, and samples from liver, kidney, lung, fat, intestine, blood, heart and brain
were collected and kept frozen till analysis of radioactivity by combustion in Harvey
Biological Oxidizer (OX-600) and followed by LSC-counting.
2.5. 14C-Residues in urine and feces
Appreciable amounts of 14C-residues were also detected in
many organs of treated rats. Liver, kidney, fat and blood contain
10% of the administered dose of fenitrothion bound residues in
soybeans (Table 3). This indicates a high percentage of release of
the bound residues followed by distribution of these residues or
their degradation products among tissues. The relatively high per-
centage of liberated 14CO2 (42%) suggests that dealkylation is the
major route of degradation of bound fenitrothion residues in
The liberated aglycones were extracted with chloroform and then identified by
thin layer chromatography (radio-TLC) on silica gel plates using two solvent sys-
tems. System A: toluene: ethyl acetate: acetic acid (5:7:1; v/v). System B: toluene:
acetic acid (7:1; v/v).
Authentic samples were run alongside as reference substances. Fenitrothion
residues were made visible by spraying with vanillin–sulphuric acid reagent
(80 ml of ethanolic solution of vanillin 2.5% added to ice cold 400 ml of concen-
trated sulphuric acid). Fenitrothion and its metabolites gave pink, yellow, and
blue color spots upon heating the sprayed plates for 3–5 min at 110 °C. For

M. Farghaly, S. El-Maghraby / Food and Chemical Toxicology 46 (2008) 3111–3115
3113
Table 1
Residues of 14C-fenitrothion in soybeans stored for 24 weeks storage (expressed as mg insecticide/kg seeds)a
Actual dose (mg/kg)
14C-Residues
External extract
mg/kg SD
Recovery (%)
Internal extract
mg/kg SD
Bound residues
mg/kg SD
b
b
b
%
%
%
9
1.80 0.20
20
4.92 0.32
55
1.91 0.18
21
96
Data are mean of triplicate experiments standard deviation (SD).
a
Applied dose = 10 mg/kg.
Percentage of actual dose on soybeans.
b
3.3. Toxicological studies
Table 2
Elimination of radioactivity in expired air and urine of rats following feeding with
bound 14C-fenitrothion residues, during 72 h
Animal fed bound residues (resulting from treatment of seeds
with 10 mg insecticide/kg seeds) showed no statistically significant
reduction in body weight gain. None of the mice showed any signs
of toxicity during the course of the experiment as compared with
control mice.
Time (h)
% Of administrated dose
Carbon dioxide
Urine
l
g
%
lg
%
24
48
72
7.47
8.30
8.30
39.0
43.5
43.5
3.32
4.43
3.88
17.4
23.2
20.3
3.3.1. Effect on cholinesterase activities
Table 4 shows plasma and erythrocyte cholinesterase enzyme
activities in treated mice after feeding with extracted soybeans
containing bound fenitrothion residues for 90 days and control
mice. Both enzymes suffered only a slight inhibition during the
first seven days of the experiment.
Administrated dose = 100% (19.1
Data are means of three replicates.
l
g equivalent/rat/day).
Table 3
The percentage of plasma cholinesterase activity inhibition did
not exceed 22.5% through the first week while the percentage of
erythrocyte cholinesterase activity reached 9% inhibition during
the same time period. A complete recovery of these enzymes was
observed at the end of the feeding period.
Distribution of released 14C-activity in rats after feeding with 14C-bound fenitrothion
in soybeans after three days
Sample
Insecticide equivalent (
lg)
% Of administrated dose
CO2
24.07
11.63
6.59
1.75
1.05
0.80
1.25
0.65
1.05
0.15
0.65
42.0
20.3
11.5
3.5
2.1
1.6
2.5
1.3
2.1
0.3
Urine
Feces
Liver
Kidney
Lung
3.3.2. Biochemical studies in liver and kidney
The effects of feeding mice with extracted soybeans containing
1.9 mg/kg bound fenitrothion residues on certain biochemical
parameters in serum of mice for three months are illustrated in
Table 5. The activities of liver enzymes (AST, ALT, and ALP) showed
increase statistically significant after three months of feeding
experiments comparing with the control one, while, the concentra-
tion of total protein and albumin had no effects on treated mice
after the feeding period. The level of blood urea nitrogen and
creatinine clearance also showed significant increase in treated
mice as compared to the control animals (Table 5).
Fat
Intestine
Blood
Heart
Brain
1.3
Total released
49.64
88.5
– Administrated dose = 100% (57.3 mg equivalent/rat).
soybeans. The total recovery of 14C-activity amounted to more than
88% (Table 3).
4. Discussion
Chromatographic analysis of collected urine showed the pres-
ence of desmethyl fenitrothion (II) Rf = 0.72, 0.35 and 3-methyl-
4-nitrophenol (III) Rf = 0.80, 0.38, which represents the main resi-
dues in addition to traces of the parent compound fenitrothion
(I), Rf = 0.90, 0.75.
Under the storage condition used, bound fenitrothion residues
in stored soybeans amounted to about 20% of the actual applied
dose (Table 1). It should be noted that this value is relatively high
as compared with bound residues of some other organophosphates
in grains for the same time of storage such as, the non-extractable
Table 4
Effect of feeding mice with extracted soybeans containing 1.91 mg/kg bound fenitrothion residues on cholinesterase enzyme activities of mice for 90 days
Feeding period (day)
Plasma cholinesterase
Control mice Xa SD
Erythrocyte cholinesterase
Control mice Xa SD
Treated mice Xa SD
Inhibition (%)
Treated mice Xa SD
Inhibition (%)
1
7
15
30
60
90
0.481 0.051
0.439 0.062
0.476 0.043
0.479 0.041
0.490 0.081
0.494 0.068
0.373 0.049*
0.356 0.030*
0.406 0.028*
0.435 0.037*
0.466 0.054
0.468 0.049
22.5
18.9
14.7
9.0
4.9
5.0
0.908 0.092
0.925 0.116
1.067 0.128
1.070 0.080
1.116 0.133
1.148 0.130
0.830 0.085*
0.841 0.071*
0.980 0.093*
1.030 0.076
1.081 0.087
1.130 0.162
8.6
9.1
8.2
3.7
3.1
1.6
Recovery
0.474 0.063
0.463 0.070
2.3
0.918 0.072
0.920 0.010
–
a
Values are mean standard deviation for six animals.
* P < 0.05 compared to corresponding control.

3114
M. Farghaly, S. El-Maghraby / Food and Chemical Toxicology 46 (2008) 3111–3115
Table 5
ase agents for blood acetyl cholinesterase and other serine
esterase’s throughout the phosphorylation of the amino acid
(serine), present as a component of the active site of cholinester-
ase enzyme (Nigg and Knaak, 2000). It was found that, fenitro-
thion at in use levels in the environment had no effects on the
reproductive or endocrine systems of rats even at toxic doses
which markedly suppressed brain cholinesterase activity (Okah-
ashi et al., 2005).
Effect of feeding mice with extracted soybeans containing 1.91 mg/kg bound
fenitrothion residues on certain biochemical parameters in serum of mice for 90 days
Parameters
Control
Fenitrothion treated
AST activity (U/L)
ALT activity (U/L)
ALP activity (U/L)
TP (gm/dl)
ALB (gm/dl)
Urea (gm/dl)
28.25 2.50
19.00 1.82
12.70 0.49
6.81 0.19
3.36 0.11
62.50 3.42
0.82 0.02
43.75 3.40*
37.70 2.98*
14.58 0.38*
6.12 0.12
3.00 0.09
86.00 5.71*
0.97 0.02
Bain et al. (2004), suggested that peak cholinesterase enzyme
activities inhibition reached 19%, two hours after fenitrothion
ingestion in the low dose (2 mg/kg) and 68%, eight hours after
ingestion in high dose (20 mg/kg) to lizard animals. The slight sig-
nificance in blood urea nitrogen and serum creatinine (kidney
function parameter) had been generally considered as an indica-
tion for the toxicological significance of pesticide residues. Kadry
et al. (2001), reported that treatment with fenitrothion caused a
significant increase in serum AST and ALT activities and serum le-
vel of TB, creatinine and urea compared with of the control level.
Generally, results of AST and ALT may indicate degradation
changes and hypo function of liver as the effect of fenitrothion
on the hepatocytes was in the form of tissue damage in which cel-
lular enzymes are released from the cells into the blood stream.
The same finding was reported by Stevens and Gallo (1989) and
Paulino et al. (1996). The bioaccumulation of fenitrothion within
60 days during chronic treatment showed no metabolite but con-
tinuous reduction in fenitrothion concentration, indicating excre-
tion of pesticide and its products in urine and in feces (Roy et al.,
2004b).
Creatinine (gm/ dl)
(Values are mean SD, n = 6). AST: Aspartate transaminase, ALT: Alanine trans-
aminase, ALP: Alkaine phosphatase, TP: Total protine, ALB: Albumin.
*
P < 0.05 compared to corresponding control.
residues on 14C-chlorpyrifos was 11.3% (Zayed et al., 2003) and 14C-
pirimiphos-methyl was about 12% (Zayed et al., 2002) in soybeans.
14C-Fenitrothion was about 17% (Farghaly et al., 2007) and 14C-
malathion was 16% (Zayed et al., 1990) in faba beens.
Critical questions in connection with bound residues concern
the nature and identity of bound residues as well as their toxico-
logical significance with regard to bioavailability and biological
activity.
The results in Table 2 indicate that beans-bound 14C-fenitrothi-
on residues are highly bioavailability to rats. The data obtained are
in line with many other studies which indicate a moderate to high
bioavailability of grains bound 14C-pesticide residues in experi-
mental animals (Zayed et al., 1992; Mostafa et al., 1992; Khan
et al., 1992).
The metabolites identified in urine of rats suggest that the re-
leased bound residues of fenitrothion are further degraded through
secondary metabolic and ring hydroxylation before excretion
(Fig. 1).
Therefore, the present results obtained indicate that bean-
bound 14C-fenitrothon residues are highly bioavailable to rats. It
should be stressed that the presence of bound pesticide residues
can no longer be ignored in the evaluation of toxicological hazards.
Miyamoto et al. (1976) suggested that 14C-fenitrothion admin-
istered orally to rats was readily absorbed from the gastrointestinal
tract and distributed to various tissues and completely eliminated
in urine. 32P-Fenitrothion was also rapidly eliminated in the urine
and feces of white mice with a recovery of more than 90% after
three days of oral treatment (Hollingworth et al., 1967). Recently,
the highest concentration of fenitrothion residues in kidney and
lung was found after acute oral treatment than the detected resi-
dues in brain, liver, and heart (Kadry et al., 2001).
The inhibition of cholinesterase enzyme activity is considered
important for toxicological studies as supplementary to exposure
diagnosis. The inhibitory effect on acetyl cholinesterase was ex-
pected, as the organophosphate compounds are ant cholinester-
Conflict of interest
The authors declare that there are no conflicts of interest.
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