Environ Monit Assess (2010) 168:231–240
DOI 10.1007/s10661-009-1107-2
Monitoring health implications of pesticide exposure
in factory workers in Pakistan
Dilshad Ahmed Khan · Imran Hashmi ·
Wajiha Mahjabeen · Tatheer A. Naqvi
Received: 30 March 2009 / Accepted: 27 July 2009 / Published online: 8 August 2009
© Springer Science + Business Media B.V. 2009
Abstract The study aimed to determine the ha-
zardous health effects of pesticides exposure in
the factory workers by measuring plasma cho-
linesterase (PChE), pesticides residues, and renal
and hepatic biochemical markers. In addition,
we also assessed the knowledge, attitudes, and
safety practices adopted by the industrial wor-
kers. The study was conducted in three differ-
ent sizes of factories located in Lahore (large),
Multan (medium), and Karachi (small) in
Pakistan. Total 238 adult males consisting of 184
pesticide industrial workers (exposed group)
from large-sized (67), medium-sized (61), small-
sized (56) industrial formulation factories, and
54 controls (unexposed) were included in the
study. All the participants were male of aged
D. A. Khan · W. Mahjabeen · T. A. Naqvi
Department of Chemical Pathology,
Army Medical College,
National University of Sciences and Technology,
Rawalpindi, Pakistan
I. Hashmi (B)
School of Civil and Environmental Engineering,
National University of Sciences and Technology,
Rawalpindi, Pakistan
e-mail: hashmi71@gmail.com
18 to 58 years. PChE levels were estimated by
Ellmann’s method. Plasma pesticides residue ana-
lysis was performed by using reverse phase C-18
on high-performance liquid chromatograph and
GC with NPD detector. Plasma alanine amino-
transferase (ALT), aspartate aminotransferase
(AST), creatinine, urea, and gamma glutamyl-
transferase (GGT) were measured on Selectra E
auto analyzer. Plasma and C-reactive protein was
analyzed by Immulite 1000. The results revealed
a significant decrease in plasma post exposure
PChE levels (<30%) as compared to baseline in
the workers of small (29%) and medium (8%)
industrial units ( p < 0.001). Plasma cypermethrin,
endosulfan, imidacloprid, thiodicarb, carbofuran,
and methamidophos levels were found to be
higher than allowable daily intake. Serum AST,
ALT, creatinine GGT, malondialdehyde, total
antioxidant, and CRP were significantly raised
among the workers of small and medium pesticide
formulation factories as compared to large in-
dustrial unit and controls ( p < 0.001). The study
demonstrated that unsafe practices among small-
and medium-sized pesticides industrial workers
cause significant increase in pesticide exposure,
oxidative stress, and derangement of hepatic and
renal function.
Keywords Pesticides toxicity · Residues ·
Health effects · Industrial workers ·
Acetyl cholinesterase · Biochemical markers
232
Introduction
The health hazards of pesticides are a major
concern internationally especially in developing
countries, where pesticides are widely used in
agriculture. The economy of Pakistan is largely
based on agriculture. It contributes about 25% to
the national economy and provides employment
for over 44% of the labor force. Pesticide usage
has gradually increased in Pakistan, along with
the rest of the world. At present, more than 108
types of insecticides, 30 types of fungicides, and
six different types of rodenticides are being used
in Pakistan (Tariq et al. 2007).National and multi-
national companies are engaged in the lucrative
business of pesticides in Pakistan. According to
economic survey of Pakistan report (2008–2009),
the formulation capacities of the pesticide indus-
tries for granules, powder, and liquid products are
27,714 metric tons per annum.
Although pesticides furnish some benefits for
crop, they entail a number of risks and problems.
The unregulated packing of pesticides, careless
handling, improper disposal, and accidental spill
of these toxic compounds has led to the poten-
tially serious hazardous effects on human health
and environmental pollution. The residue of the
pesticides stays in the environment for variable
period of time, which poses serious threats to
environment and can lead to acute and chronic
effects on human life causing damage to health
or even death (Azmi et al. 2006). The three en-
try routes for pesticides are the skin, lungs, and
gut. The skin and respiratory tract provide a very
efficient surface for the absorption of pesticides.
High levels of pesticides residues are reported in
the houses and urine samples of factory workers
(Coronado et al. 2004).
Adverse effects of pesticide depend upon tox-
icity of the compound and duration of exposure.
Clinically, the workers may represent with symp-
toms like headaches, nausea, vomiting, skin irri-
tation, excessive sweating, respiratory problems,
and convulsions (Wesseling et al. 1997). Biomark-
ers may be used to detect the hazardous effects of
pesticides before adverse clinical health effects oc-
cur. Carbamate and organophosphorus (OP) com-
Environ Monit Assess (2010) 168:231–240
pounds inhibit the plasma cholinesterase (PChE)
levels and have inverse dose effects relationship
with it (El-Demerdash et al. 2001). Prolong ex-
posure to pesticides can affect multiple organs
including liver and kidney functions (Daniel et al.
2002; Peres et al. 2006). Many researchers also
tried to correlate various enzymes with the harm-
ful effects of pesticides on human liver especially
plasma alanine aminotransferase (ALT) and alka-
line phosphate (Misra et al. 1985; Goel et al. 2000).
It has also been seen that the exposure of more
than 20 years to the pesticides had resulted in fre-
quent infection and immunological abnormalities
(Daniel et al. 2002). Several studies have shown
that workers exposed to pesticides are more likely
to develop leukemia and brain, prostrate, and skin
cancers than the general population (Merhi et al.
2007; Cabello et al. 2001).
In Pakistan, little work has been done on bio-
chemical changes at cellular level in pesticide-
exposed occupational workers. The present study
aimed to determine the hazardous health effects
of pesticide exposure on the pesticide factory
workers, by measuring plasma PChE, pesticides
residues, and biochemical markers. The secondary
objectives were to evaluate the knowledge, atti-
tudes, and safety practices adopted by the indus-
trial workers. This study will serve as a step ahead
to create awareness and implementation of the
concept of safe occupational practice in workers
in the pesticide industries in our country.
Materials and methods
A cross-sectional comparative study was con-
ducted to investigate the effects of pesticide ex-
posure on pesticide formulation factories workers.
A preliminary survey of various industries was
performed to select sampling location and identify
target groups. On the basis of industrial size and
production of pesticides, three types of pesticide
industrial units, i.e., large-, medium-, and small-
sized, located in Lahore, Multan, and Karachi,
respectively, were randomly selected from the list
of factories in Pakistan.
Environ Monit Assess (2010) 168:231–240
Subjects
A total of 723 industrial workers consisting of 310
from large industry, 245 medium, and 168 small
industries were included by random draw from
the industrial employment record. Selection of the
industrial workers was done on basis of full-time
active involvement in preparation, storage, and
packaging of the pesticides in industry for at least
1 year. Total 238 adult males consisting of 184
pesticide industrial workers (exposed group) and
54 controls (unexposed group) finally recruited
in the study. The exposed groups of age range
between 18–58 years comprised of 67 workers
from large, 61 from medium, and 56 from small
formulation units were included in the study af-
ter taking informed consent. Medical history and
physical examination of the subjects were also car-
ried out. The workers who had diabetes mellitus,
hypertension, and viral hepatitis were excluded.
The unexposed groups composed of subjects with
similarity of age and socioeconomic status but
were not involved in pesticide production.
Blood sample collection
Five milliliters of blood was collected twice in
heparinized tubes. Pre-exposure baseline PChE
levels of industrial workers were collected in the
beginning of the season in March–April 2007, and
post-exposure PChE samples were collected in
July–August 2007. The sample was transported by
maintaining cold chain to the laboratory. Plasma
was separated by centrifugation at 1, 500 × g for
15 min. Samples were stored at −20◦ C in clean
plastic vials for enzymatic and pesticides analysis.
Analytical methods
Plasma enzymes and biochemical analysis was car-
ried out on Selectra E auto analyzer (Vita lab,
The Netherlands) following standards procedures
of Pioneer diagnostic kits (USA) at the medical
laboratory of Army Medical College, Rawalpindi.
233
Plasma PChE levels were estimated by Ellmann’s
method with butyrylthiocoline (Ellmann et al.
1961). Serum ALT was determined by the In-
ternational Federation of Clinical Chemistry and
Laboratory Medicine (IFCC) method (Bergmeyer
et al. 1998), serum total bilirubin by Jendrassik
and Grof method (Perry et al. 1983), plasma
albumin by bromocresol green (Doumas et al.
1997), and total protein by the Biurate method
(Kingsley 1942). Gamma glutamyltransferase was
determined by an IFCC reference measurement
procedure (Schumann et al. 2002). Serum creati-
nine was determined by Jaffe’s reaction (Spencer
1986) and urea by kinetic urease/glutamate de-
hydrogenase method (Sampson and Baird 1979).
Serum total antioxidant status was estimated by
kinetic colorimetric assay kit (Randox, Crumlin,
UK) on the automated clinical chemistry ana-
lyzer, Selectra E (Vita Lab, Netherland) by fol-
lowing the previously reported method (Miller
et al. 1993). Plasma malondialdehyde (MDA) was
assayed by using the method described by Gavino
et al. (1981). The extent of lipid per oxidation in
plasma was measured by the formation of thio-
barbituric acid reactive substance by using MDA
kit of Cayman (UK). Quantitative absorbance of
pink color was measured at 532 nm on photometer
(Shimadzu, Japan). Plasma and C-reactive protein
(hS-CRP) was analyzed by Immulite 1000 (DPC,
USA). The coefficient variation of the method was
found to be 3–5%.
Pesticide residue analysis
All the extraction and cleanup steps were stan-
dardized and checked for quantitative recove-
ries. Pesticides extraction was performed by
solid-phase extraction by LiChrolut RP-18
(Merck, Germany). The cartridge was condi-
tioned with 2 ml methanol (SPE) followed by 2 ml
water. Serum 1 ml was diluted with 1 ml water
and mixed vigorously to get homogenous mixture;
this was allowed to trickle slowly on the column
and rinsed with 2 ml ultra pure water in order to
elute the sample throughout the column under
234
the atmospheric pressure. Finally, the column was
dried under vacuum on SPE station for 30 s and
rinsed with 2 ml, n-hexane and eluted with 3 ml
n-hexane, dichloromethane (1:1). The solvent was
evaporated, and sample was resolved using 25 μl
toluene.
Cleanup was made by USEPA Method 3620B
(US-EPA 1996). Florisil was activated at 130◦ C
overnight and cooled in a dessicator before use.
Column was packed with 1 g of Florisil. Extract
was transferred to the column and eluted with
hexane and diethyl ether. Eluent was collected
and evaporated to dryness. Final sample were
prepared in 2 ml of acetonitrile.
Instrumental analysis of samples
The instruments used in this study was high-
performance liquid chromatograph (HPLC;
Shimadzu, Japan) consisting of binary LC pump
and UV/VIS detector. The residues of methomyl,
thiodicarb, imidacloprid, cypermethrin, and endo-
sulfan were determined by using reverse phase
C-18 column (VA-ODS 150 × 4.6 Shimadzu,
Japan) at wavelength of 254 nm with flow rate of
1 ml/min on HPLC. Analysis was performed using
60% acetonitrile, 20% methanol, and 20% water
as mobile phase. All the chemicals used during
analysis were HPLC grade (Merck, Germany).
Calibration of the instrument was done before
the sample analysis by using analytical pesticide
standards purchased from Riedal-de Haen AG
Seelze, (Germany). Every possible care was taken
to observe good laboratory practices to avoid
contamination and keep the precision with in 5%.
Analysis of methamidophos, chlorpyrifos, and
carbofuran were done by using Elite-5 column
(HP-5ms, 30 m × 0.32 mm × 0.25 μm) and
NPD detector on GC Model Clarus-500 (Perkin
Elmer, USA). The injector and detector temper-
atures were 225◦ C and 300◦ C, respectively. Ini-
tially, the oven was ramped to 100◦ C at a rate
of 20◦ C/min; Ramp 2:5◦ C/min to 150◦ C (5 min)
Ramp 3:10◦ C/min to 200◦ C. Helium gas with a
flow rate of 50 ml/min was used as a carrier. Three
microliters of the samples was injected through
10 μl. Hamilton syringe by the solvent flush injec-
tion technique. Calibration of the instrument was
done before the sample analysis by using analyti-
Environ Monit Assess (2010) 168:231–240
cal pesticides standards purchased from Riedal-de
Haen AG Seelze (Germany). Pesticide residues
in the samples were identified on the basis of
retention times and quantified on the basis of
peak area by using external standard method. The
retention time for methamidophos, chlorphyrifos,
and carbofuran were 8.2, 17.3, and 8 min. The
recoveries of the insecticides were found to be in
the range from 81% to 94%.
Statistical analysis
The data were entered into SPSS version-15
(SPSS Inc, Chicago) for statistical analysis. Me-
dian, percentile, and interquartile range (IQR)
of all biochemical markers were calculated. Post-
exposure PChE in the pesticides packing workers
as compared to baseline levels by Wilcoxon rank
sum test. Biochemical, oxidative, and inflamma-
tory markers in the workers of three pesticides
packing industries were analyzed by Kruskal–
Wallis test. Mann–Whitney U test was applied
for pairwise group comparisons. Association of
reduction cholinesterase (%) with change in bio-
chemical markers in pesticide industrial workers
was established by Spearman’s coefficient correla-
tions. P value of <0.05 was considered significant.
Results and discussion
Total 184 pesticides factory workers participated
from the three pesticides formulation industrial
units. All the workers were males with age ranging
from 18 to 58 years. The duration of pesticide ex-
posure among the workers ranged between 1 and
19 years. The pesticides workers of small-sized
industry experienced more symptoms of central
nervous (Fig. 1), gastrointestinal, and respiratory
systems than the other two factories. Headache
followed by dizziness, vomiting, and shortness of
breath and skin rash were quite high among these
workers. Few workers complained of fatigue and
tiredness.
Pesticide exposure is a global public health is-
sue. Pesticide industrial workers are constantly ex-
posed to a variety of toxic chemicals (Javed et al.
Environ Monit Assess (2010) 168:231–240
Fig. 1 Distribution of symptoms reported by the pesticide
industrial workers
2006). Extensive exposure of pesticides causes ad-
verse health effects in workers ranging from minor
respiratory problems and eye symptoms (Murphy
et al. 2002) up to cancerous, genotoxic, repro-
ductive, and neurotoxic diseases (Sanborn et al.
2007). In our study, headache, dizziness, vomit-
ing, shortness of breath, skin rash, fatigue, and
tiredness were quite high among exposed workers
Fig. 2 Comparison of
PChE levels in the
industrial workers as
compared to control
235
as reported by other researchers (Catharina et al.
2002).
Cholinesterase activity
The present study demonstrated a decrease in
plasma level of post exposure PChE in the pesti-
cides packing workers as compared to baseline.
Box plot of PChE levels in pesticides exposed
worker of three industries are illustrated in
Fig. 2. The workers of small and medium indus-
trial unites had significantly lower median (IQR)
post-pesticides exposure vs. baseline PChE 5,273
(4,781–6,479) vs. 7,117 (6,403–8,006) U/l and 5,453
(6,167–6,897) vs. 6,580 (6,580–8,399), respectively
( p < 0.001). The workers of large industrial unit
had similar trend of median (IQR) 6,735 (5,792–
8,091) vs. 6,905 (5,908–8,129), but change in PChE
was not significant ( p = 0.082).
236
Fig. 3 The number of workers of three pesticide formula-
tion units who had significant exposure to pesticides during
work (PChE > 30%)
Depression in PChE levels >30% as compared
to baseline was considered pesticides exposure
(Ohayo-Mitoko et al. 2000). The workers of three
pesticide-formulation industrial units had depres-
sion of PChE more than 30% in small- (n = 16),
medium- (n = 5), and one in large-sized industry
(Fig. 3).
Most of the pesticides inhibit the PChE, which
is helpful for detecting the early acute effects of
poisoning and monitoring the pesticide exposure.
In order to estimate the pesticide-induced toxicity,
Fig. 4 Pesticide residues
in the plasma of industrial
workers (n = 184)
Plasma pesticides levels (mg/L)
Environ Monit Assess (2010) 168:231–240
PChE activity has been used for several years in
pesticide-exposed workers. PChE levels are more
reliable indicators than the symptoms attributed
to exposure for risk assessment and monitoring of
pesticide intoxication in farm workers (Dasgupta
et al. 2007). As PChE is regenerated quickly in the
liver, follow-up testing to monitor recovery after a
depression >40% from baseline should be carried
out repeatedly until the level returns to within
20% of baseline. The measurement of erythrocyte
PChE activity in pesticide workers who formulate
OPs can be a good monitoring factor and is rec-
ommended to be performed in a regular manner
(Ranjbar et al. 2002).
The present study revealed that workers of
small- and medium-sized factories had signifi-
cant OP and other pesticide residues in their
blood (Fig. 4). The industrial workers had car-
bofuran, cholorpyrifos, cypermethrin, endosulfan,
imidacloprid, and methamidophos residue above
the acceptable daily intake (ADI) (Australian
Government 2008).
This result is also consistent with a study which
reported significantly decrease activity of PChE
in exposed group as compare with control group
(Javed et al. 2006). In a study by Hernández et al.
Environ Monit Assess (2010) 168:231–240 237

Table 1 Biochemical markers in pesticide exposed workers as compared to control

Parameters Small industry Medium industry Large industry Control
(n = 56) (n = 61) (n = 67) (n = 54)
Median (IQR) Median (IQR) Median (IQR) Median (IQR)
Hemoglobin (g/dl) 13.98 (13.02–14.90)* 14.70 (14.20–15.50) 14.86 (14–15.42) 14.92 (14–15.60)
Total bilirubin (μmol/l) 10 (6–13) 9 (7–12) 9 (8–12) 8 (6–11)
Alanine aminotransferase (IU/L) 34 (24–53)** 31 (23–41)** 26 (21–33) 25 (20–29)
Asparate aminotransferase (IU/L) 25 (20–41)** 27 (22–35)** 24 (20–32) 24 (21–27)
Alkaline phosphate (IU/L) 187 (158–217) 195 (168–232) 183 (166–212) 175 (161–213)
Albumin (g/L) 43 (39–44) 44 (42–45) 43 (41–46) 43 (42–47)
Urea (mmol/l) 4.50 (4.0–5.20) 4.20 (3.80–5.20) 4.20 (3.50–4.80) 4.10 (3.80–4.90)
Creatinine (μmol/l) 87.50 (77.0–94.75) 90.0 (78.50–97.50) 89.0 (77.0–98.0) 83.0 (78.0–91.0)
* p =< 0.05, ** p =< 0.01

(2004), both directly and indirectly exposed work-
ers presented significant decreased levels of PChE
at the time of maximum exposure to pesticides.
Biochemical markers
The industrial workers of small- and medium-
sized industries had significant hepatic injury
when compared to the large-sized industry and
control. The enzyme activity of ALT and aspar-
tate aminotransferase (AST) were significantly
raised ( p < 0.001) among the pesticide-exposed
industrial workers as compared to less-exposed
workers of large-sized industry Table 1. Other
markers such as plasma bilirubin, albumin, to-
tal proteins, urea, and creatinine were not sig-
nificantly changed among pesticides formulation
workers. Our studies also revealed inverse cor-
relation between the PChE with AST (r = 0.42;
p < 0.01) and ALT (r = 0.50; p < 0.01) in the
pesticide-exposed workers.
Oxidative stress markers
The pesticide-exposed industrial workers had in-
creased oxidative stress indicated by significantly
raised plasma total antioxidant, gamma glutamyl-
transferase (GGT), MDA, and CRP levels in
small-sized industrial workers as compared to
other two industries (Table 2). Plasma CRP levels
were also increased due to nonspecific inflamma-
tory changes in the exposed workers.
Our study had revealed significant association
between reduction PChE (%) with change in bio-
chemical markers of oxidative stress and inflam-
mation in pesticides small-sized industrial factory
workers. The workers also had negative correla-
tion between cholinesterase and MDA (r = 0.54;
p < 0.01), GGT (r = 0.21; p < 0.05), and CRP
(r = 0.55; p < 0.01). The post-exposure reduc-
tion PChE (%) with total antioxidant status (r =
−0.34; p < 0.01) is exhibited in Fig. 5.
The workers knowledge regarding the health
hazards of pesticide and safety were assessed on

Table 2 Comparison of oxidative stress and inflammatory biochemical markers among pesticide exposed workers of three
different industrial units (n = 184)
Parameters Small industry Medium industry Large industry
(n = 56) (n = 61) (n = 67)
Median (IQR) Median (IQR) Median (IQR)
Total antioxidants (mmol/L) 1.53 (1.37–1.66)* 1.56 (1.40–1.66)* 1.65 (1.47–1.70)
Gamma glutmyl transferase (U/L) 26 (22–30)** 21 (19–26) 20 (19–22)
Malonylialdehyde (μmol/L) 2.44 (1.72–4.00)** 1.62 (1.51–2.46) 1.85 (1.55–2.41)
C-reactive protein (mg/L) 1.55 (0.90–2.65)** 0.80 (0.30–1.40) 0.90 (0.30–1.50)
* p =< 0.05, ** p =< 0.01
238
Fig. 5 Correlation of
PChE with total
antioxidant level in the
industrial workers
Total antioxidant (mmol/L)
the pretested questionnaire by the health team.
Among 184 workers, 62% were not literate and a
few (10%) had completed their high-school level
education. Only the workers (95%) of large-sized
industry had formal training in safe pesticide han-
dling and packing. About 90% of the workers
had knowledge of the detrimental health effects
of pesticide but were not well aware of the safety
precautions regarding pesticide handling. How-
ever, many workers reported (85%) to wash hands
and bodies after work.
The participants had casual attitude in handling
of pesticide and personal protection. We observed
that 90% workers of small-sized industry did
not use any personal protective equipment dur-
ing pesticide handling while 35% medium-sized
industrial workers used gloves and ace masks.
Large-sized industry workers (95%) used proper
protective head cover, gloves, and masks during
work. Large-sized industry possessed a compe-
tent, knowledgeable, and experienced staff and
was meeting the standard of occupational health
and safety measures. They were fully equipped
to meet any emergency situation and workers’
pesticides exposure was regularly monitored by
measuring PChE levels. The workers of large in-
dustry were knowledgeable and experienced, and
Environ Monit Assess (2010) 168:231–240
they practice the standards of occupational health
and safety.
The study demonstrates that casual attitude
and unsafety practices among small industrial
workers causes significant decrease in PChE levels
and increased multiple pesticide residues. Pro-
longed exposure of pesticides causes derangement
of hepatic renal function. This is accompanied by
increased concentration of oxidative stress and in-
flammatory markers. On the basis of information
gathered, best management practices at work-
place were recommended to industrial units for
the safety of their workers. Baseline data regard-
ing health risks caused by the unsafe handling and
packing of pesticides thereby help government
and policy makers in reviewing pesticide-related
health and environmental policies.
Conclusions
In conclusion, unsafe practices among industrial
workers cause significant decrease in PChE lev-
els, increased oxidative stress, and derangement
of hepatic function in workers. Furthermore, the
workers of small and medium factories had little
knowledge about the safety practices and use of
Environ Monit Assess (2010) 168:231–240
basic protective equipment. Concerted efforts are
required to create awareness, change in attitude,
and safety practices among the industrial workers
for prevention of hazardous health effects.
Acknowledgements This study was a part of the Project
No. 20-770/R&D/2007/446 entitled, “The Effects of Envi-
ronmental Pesticides Residues on the Health of workers in
Pesticide Manufacturing Industries of Pakistan” supported
by Higher Education Commission (HEC) Pakistan. We
acknowledge the participation of pesticides formulation
industrial units from Karachi, Multan, and Lahore for
extending their cooperation in all aspects of the study and
continuous support.
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