Annual Report 2007-08

Health Hazards in Ship Breaking Workers of Alang

RR Tiwari, SK Dave, SR Tripathi, NG Sathwara, PK Majumdar,
BD Patel, RA Rathod and JB Vyas

Abstract

The ship breaking workers are exposed to various hazards such as accidents and injuries, exposure to
asbestos, heavy metals like lead and polychlorinated biphenyls and noise exposure. The present cross
sectional study was carried out to find the pattern and prevalence of health morbidities among ship breaking
workers and their associated epidemiological factors. The study included 405 ship-breaking workers. Data
collection on pre-designed proforma was followed by complete clinical examination, pulmonary function
testing and chest radiography (PA View) of each worker. Blood sample of gas cutters was collected for lead
estimation. The study also included audiometry of 187 workers and workplace monitoring for air lead levels.
The radiological evaluation showed 12(2.96%) subjects with 1/1 and higher opacities whereas the
spirometric evaluation revealed 65 (16%) subjects with obstructive, 6 (1.5%) with restrictive and 5 (1.2%)
with combined type of impairment. The workplace air lead level monitoring revealed normal lead values while
3
23(18.4%) subjects showed blood lead levels >30g/m . The audiometric evaluation of workers suggested
normal results for all the workers. The workers in ship breaking are exposed to dust and lead.
Keywords: Ship-breaking, lead, asbestos, pulmonary function test

Introduction Materials and Methods

Ship breaking is the process of dismantling an obsolete vessel's
structure for scrapping or disposal. The Alang-Sosiya Ship-Breaking
Yard (ASSBY) located in the Gulf of Cambay in the Bhavnagar District of
Gujarat State in India is the biggest ship-breaking yard, which employ
some 15,000 to 25,000 workers directly for ship-breaking activities1.
The complete dismantling of an obsolete ship is further divided into sub
processes which include removal of asbestos containing materials by
workers known as Gudadiwale in local language, pulling the ship with
the help of wires to the beaching point, sorting of the materials
according to different metals of which they are made of, cutting the iron
parts of the ship with the help of gas cutter and other miscellaneous
work like housekeeping of the plot on which these activities are carried
out, loading and unloading of the heavy parts on and off the trolley for
marketing purpose, driving the trolleys, vehicles and cranes, etc. All
these processes expose workers to a wide range of hazards or
workplace activities or conditions likely to cause injury or illness. These
include exposure of asbestos fibres, heavy and toxic metals such as
lead, mercury, cadmium, zinc and copper, PCB in paint, PVC and
welding fumes2,3. Thus, the present cross sectional study was carried
out to find the pattern and prevalence of health morbidities among ship
breaking workers and their associated epidemiological factors.
The present study was carried out among 405 ship breaking workers.
Interview technique was used as tool for data collection, which was
later on recorded on a pre-designed proforma. This was followed by
complete clinical examination, pulmonary function testing and chest
radiography (PA View) of each worker. 5ml blood sample was drawn
from cubital vein in a heparinised vacutainers in aseptic condition from
those workers working in the process of gas cutting to estimate the
blood lead levels as they were directly exposed to fumes generated
during the cutting process. Also the audiometry of about 187 workers
was carried out. The workplace monitoring was carried out for air lead
levels. The environmental monitoring for asbestos fibre levels could not
be done as during the study period in none of the ship the process of
removal of asbestos containing material was going on. For lead
estimation, blood samples were digested using HNO3 and H2O2 mixture
in Microwave digester (ETHOS 1600). The digested samples were
analyzed using AAS, Perkin Elmer, U.S.A. model no 3100.
Air samples were collected using personal sampler at the rate of
2-3 LPM for 2-3 hours and respirable high volume sampler (Envirotech
Ltd, New Delhi) at the rate of 0.92 to 1.1 m3/min for 8 hrs. Collected air
samples were digested using HNO3 and HClO4 (2:1) mixture.

7
 Annual Report 2007-08 Annual Report 2007-08

Table 1: Distribution of pulmonary function status according to Table 2: Findings on chest radiography in ship breaking workers
duration of exposure Opacities No % Right ear Left ear
Exposure N Pulmonary Function Status Normal 357 88.2
duration Normal Obstructive Restrictive Combined Pleural thickening 6 1.5
<5 101 87 (86.1) 11(10.9) 1 (1.0) 2(2.0) Old healed tuberculosis 2 0.5
5-10 135 104 (77) 25(18.4) 3 (2.2) 3 (2.2) Emphysema 2 0.5
10-15 119 100 (84) 18 (15.1) 1 (0.8) 1(2.3) Cardiomegaly 4 1.0
³15 50 38 (76) 11 (22) 1 (2) - 1/0 small regular opacities 3 0.7
Total 405 329 (81.3) 65 (16) 6 (1.5) 5 (1.2) 1/0 small irregular opacities 19 4.7
Numbers in parenthesis indicate the percentage 1/1 small regular opacities 1 0.2
The digested samples were analyzed using Atomic Absorption 1/1 small irregular opacities 7 1.7
Spectrophotometer (Perkin Elmer double beam model no. 3100) at the 1/2 small irregular opacities 1 0.2
wavelength of 283.3 nm. Calibration of the instrument and the samples Small regular and irregular opacities 2 0.5 Figure 2: Average hearing level (dB) according to duration of exposure
Figure 1: Average hearing level (dB) according to duration of exposure
were compared with the lead standard of National Bureau of Standards
Large opacities 1 0.2
(NBS), USA. Data analysis was carried out using Epi-Info5 software
(WHO). when radiographs were classified according to the ILO Classification of liberated in the fumes gets diluted by the ambient air. However as the Acknowledgment: The investigators are grateful to AR Shah, MH
Pneumoconiosis3, 12 (2.96%) were found to have findings suggestive workers work without any respiratory protective device, individual Vakharia, MR Varia, RC Patel, Raksha Agarwal, Divija Patel, MI Sheikh
of interstitial lung fibrosis. This can be attributed to lung parenchymal blood lead levels were more than 30mg/dL in 23 (18.4%) workers. and Shaswat Dodia for the technical help rendered during this study.
Results and Discussion
pathologies such as asbestosis. However, when the radiological Similar results were reported in earlier study8.
The demographic characteristics of study subjects showed that the abnormalities were analyzed according to different processes, it was References
majority of the workers were less than 35 years of age with mean age found that such cases were evenly distributed among all processes. Table 4 shows the air lead levels at the worksite and away from the
being 30.86±7.79 years. All the subjects were male. This is due to the This is because each workers works in multiple processes. worksite. The air lead levels were also below the normal levels 1. Rousmaniere P and Raj N. Ship breaking in the developing
3
fact that the work demands heavy labour. About 81% of the workers prescribed. The normal values for open area is 1.5mg/m while that for world: Problems and Prospects. Int J Occup Environ Hlth 2007;
were married while remaining were single. Though half of the workers The musculoskeletal complaints like low back pain and joint pains workplace area is 50mg/m3. The workplace and ambient lead levels at 13:359-368.2. International Labour Organization. Safety and
were educated up to middle and secondary levels, about one-third were more common being present in 21.1% and 10.5% respectively. working plots were within normal level. Health in Shipbreaking: Guidelines for Asian Countries and
were illiterate also. 57.4% of the workers were working in this This could be due to heavy lifting5,6 and continuous working in squatting Turkey,2004.http://www.ilo.org/public/english/standards/rel
occupation for more than 9 years. The mean duration of exposure was posture7. Any type of injury from trivial abrasions to major injury The result of hearing assessment for right and left ear is shown in m/gb/docs/gb289/pdf/meshs-1.pdf. Accessed February 25,09.
9.67±0.28 years. 125 (41%) workers were working in gas cutting resulted during work in ship breaking processes in last one year period Figures 1 & 2 respectively. Mean threshold of hearing of exposed group 3. Mattorano D, Harney J, Cook C, Roegner K. Metal exposure
followed by 49(16.1%) in maal paani and 31(10.2%) in Jodi. Only 10 was present in 17.1% subjects. Though some of the workers were was just above the normal level in the right ear but the left ear showed during ship repair and ship breaking procedures. Appl Occup
(3.2%) were working in miscellaneous process like as cook, removing using personal protective devices like helmets and gumboots but the mild hearing loss. The 20-25 years exposure group had little higher Environ Hyg. 2001; 16:339-349.
the cabins in the ships and machine repairer. The respiratory quality and maintenance of such PPEs were found to be sub-standard. levels at frequencies 4000-8000Hz. Audiometric evaluations showed 4. International Labour Organization. Encyclopaedia of
complaints such as cough and breathlessness were present only in that the hearing acuity of all the workers was within normal limits. Occupational Health and Safety, volume II.3rd revised edition,
3.2% and 3.9% workers respectively. Other complaints included Table 3 shows the mean lead levels according to the duration of Though the workers are exposed to noise, it is intermittent in nature and ILO, Geneva, 1983: 2102-2104.
anorexia in 28 (6.9%), bleeding from gums in 21(5.5%) and frequent exposure among the gas cutters. Though an increase in mean blood this might not affect the hearing much. 5. Miranda H, Viikari-Juntura E, Punnett L, Riihimäki H.
burning in micturition in 19 (5%) workers. lead levels is observed with increasing duration of exposure, the Occupational loading, health behavior and sleep disturbance as
difference was found to be statistically non-significant. Also the mean Thus to conclude, the ship breaking workers are exposed to predictors of low-back pain Scand J Work Environ Health. 2008;
Table 1 shows the distribution of pulmonary function
.
levels were below the permissible levels of 30mg/dL The reason being asbestos dust, heavy metals like lead and intermittent noise. 34:411-419.
abnormalities according to the duration of exposure. Table 2 shows the the process of gas cutting is carried out in open space, the lead Table 4: Air lead levels at and away from the worksite (mg/m3) 6. van der Burg JC, van Dieën JH, Toussaint HM. Lifting an
findings observed on the chest radiography of study subjects. 359 unexpectedly heavy object: the effects on low-back loading and
Table 3: Mean blood lead levels according to exposure duration Place Plot No. Open area Worksite balance loss. Clin Biomech. 2000; 15:469-477
(88.6%) had normal chest radiographs. In remaining subjects small
irregular opacities of category 1/0 of ILO classification of Exposure group N Blood Pb levels (mg/dL) Sosiya 3 0.772 4.42 7. Van Vuuren BJ, Becker PJ, van Heerden HJ, Zinzen E, Meeusen
Pneumoconiosis4 were present in 19 (4.7%), 6 (1.5%) had pleural <5 14 13.32 ± 9.79 127-128 1.19 14.65 R. Lower back problems and occupational risk factors in a South
thickening while other abnormalities included old tuberculosis in African steel industry. Am J Ind Med. 2005; 47:451-457.
5-10 41 20.33 ± 9.93 132 0.07 0.76
2(0.5%), emphysema in 2 (0.5%) and cardiomegaly in 4 (1%) subjects. 8. Nosal RM, Wilhelm WJ. Lead toxicity in the ship breaking
10-15 51 19.21 ± 9.65 Alang 73 0.19 2.56 industry: the Ontario experience. Can J Public Health. 1990;
In all 48 (11.4%) had radiological abnormalities. The spirometric
parameters revealed that only 6 (1.5%) had restrictive while 5 (1.2%) ³15 19 24.61 ± 9.04 7 1.68 7.08 81:259-262.
had combined restrictive and obstructive type of abnormality. However Total 125 19.74 ± 9.99

8 9
Annual Report 2007-08

Musculoskeletal Pain and Discomfort and Work Stressors Among Women

in Fish Processing Activities
A Nag, PK Nag, H Vyas and P Shah

Abstract

The objective of the study is to describe workers with musculoskeletal pain and discomfort (MSDs), to identify
work aspects associated with pain and discomfort and to correlate the stress symptoms to pain among
women engaged in fish processing activities. A descriptive study was conducted among women workers
(N=450) selected at random from fish processing units of the coastal regions of Gujarat in Western part of
India. The major activities performed by the workers were grading, sorting, peeling, and packing. About 72%
of workers reported pain and discomforts chiefly in upper back, knee and shoulders. Relative risk estimate
suggests that workers suffering from fatigue and those reported socio domestic disruption were at a higher
risk to pain and discomforts. Analysis of correlation coefficient between work aspects (work stressors) and
MSD suggest that physical work environment, physical activity, manual material handling, cold work
environment and poor task clarity were correlated. In fish processing industry work involves repetitive
movements and may be associated with a considerable risk of musculo-skeletal disorders. Physical and
psychosocial risk factors at work may potentially interact to further increase the risk of symptoms of MSDs.
Strategies aimed at the prevention of these work related musculo-skeletal disorders should reduce exposure
to physical and psychosocial workplace risk factors.

Keywords: Musculoskeletal discomfort, fish processing, work stressors

Introduction Materials and Methods

It has already been mentioned in our previous Annual Reports that due
to the nature of the job, women in fish processing activities are
exposed to cold environment, use improper hand tools and
implements and adopt awkward working postures to perform
occupational activities. Many studies in literature establish
relationship between work factors and MSDs. Personal, psychosocial
job stress and work organizational factors in sewing machine
operators1, work tasks and psychosocial job stress associated to MSD
in forestry2. Physically strenuous works, carrying heavy weights and
3
environmental factors and mental stress of German workforce have
been associated to pain and discomfort. Such correlation studies help
to down line the key causative factors that are associated to pain and
discomforts and thereby help to draw plans that intervene to make the
work and work factors less stressful. The objective of the study is to
find out the prevalence of musculo-skeletal pain and discomfort
among fish processing workers, to identify occupational aspects
associated with pain and discomfort and to correlate the psychosocial
stress symptoms with the pain and discomforts prevailing among
women engaged in fish processing activities.
The prevalence of musculo-skeletal pain and discomfort among the
women fish processing workers were evaluated using NIOSH
4
checklist , including checkpoints on the severity of the pain, the work-
day lost due to pain, the remedial measures adopted by the workers to
minimize the pain and the worker's perception on the causative
factors for pain. To identify the work aspects and stressors, the
workers were individually interviewed by using a multi-method
ergonomic checklist5. The physical health and well-being were
examined using the standard questionnaire. The checklist entries
were responded by a single digit on a five-point common severity
agreement/ disagreement scale - strong disagreement (1) to strong
agreement (5), suggesting that the low value is the positive indicator of
the perception of absence of the stress. The score for each section of
the checkpoints were arrived by adding the individual scores obtained
for each item and then dividing it by maximum cumulative score
possible under that section. In other words, for each of the work
stressors, the outcome value would range within 0 to 1. Required
ethical clearance for the study was obtained from the ethical
committee of the Institute.

11
 Annual Report 2007-08 Annual Report 2007-08

Results and Discussion Table 2: Responses of women to variables related to MSD (N=450)
0.9
The workgroup (N=450) was relatively young with a mean age and job Severity of pain (%) 60
0.8
involvement of 23 ± 6.4 and 2.8 ± 2.0 years respectively (Table 1). Mild 52
50 0.7
Work activities primarily performed by the workers in units were Moderate 35

Prevalence of MSD (%)

0.6
grading, peeling, ring cutting, sorting and packing. More than 70% of
Severe 13 40

Relative loading
workers were literate and approximately 70% were unmarried. About 0.5

72% of workers reported pain and discomfort (MSD) in different Worker's perception to the cause of pain (%) 0.4
30
regions of the body and there was high prevalence of pain and Posture 23 0.3

discomfort in upper back (54%) followed by lower back (33 %), knee Work environment 35 20
0.2
(35 %), shoulder (27 %), hand (17%), finger (9%) and calf (13%) 0.1
Work method 6
shown in Figure 1. The women workers less than 25 yrs of age (OR 2.6, 10 0
95% CI 1.3-5.2) showed higher risk of development of MSD in lower Work load 27

MMH
IIIumination
Job speicalization

Work place design

Mental overload

Training

Task situation

Work organization
Temperature

Work posture

Tools
Physical activity

Job autonomy

Task clarity

Machinery safety

Machinery
Auxillary support

Skill requirement

Seating arrangement
Job feed back
back than the women who were more than 25 yrs of age. Risk Exhaustion 9
0
estimates suggest that workers performing grading (OR 2.4, CI 1.3- Others (Non occupational) 2 Uppper Knee Lower Shoulder Neck Hand Elbow Calf Ankle Finger Thigh
back back
4.2) and peeling (OR 2.5, 95% CI 1.3-4.8) were at relatively higher risk
to develop pain in upper back and hands which is associated to Worker's selection of remedial measures (%) Figure 1. Prevalence of MSD in fish processing workers Figure 2. Relative weightage of work stressors
repetitive hand movements involved in the processing activities. Risk Self chosen pain killer 20
to overall pain and discomfort was found among women engaged in Seek advice from doctors 16 Workers' chosen perception to the cause of MSD like constrained to the health hazard can be mitigated by suitable intervention and make
ring cutting (OR 20.7, CI 9.8-43.8) and mixed tasks (OR 4.5, CI 1.9-10.5). Apply ointment 20 posture (23%), cold environment (35%) and work load (27%) were the work safe and less strenuous. Fish processing workers are at high
Table 2 shows that severe pain was experienced by 13% of the found to have correlation with the pain in lower back, upper arm ect., risk to pain and discomfort in different body regions, due to varied
women whose pain persisted, even in the night, long after the None 44
signify the necessity of the involvement of the workers representation nature of activities in inclement working conditions. The behavioral
cessation of work. Women had mixed responses when asked about while taking into account the intervention program to minimize the symptoms, job specialization, skill requirements, physical activity,
their perception to the cause of pain and discomfort. As perceived by treatment for prevention of pain and only 16% of the women consulted MSD among these women. Repeated handling of unergonomically psychosocial aspects, work environment and postural constraints have
the workers, working posture, work environment and workload were the doctors to minimize the pain. designed tools as knives and scissors, impose biomechanical strain association with the development of MSD. The study points out the
the factors contributed to the development of pain and discomfort. and increase their risk to MSD and need to be replaced by properly importance of suitable work designs and ergonomics intervention to
The selection of remedial measures differed among women and designed hand tools. Also women perform processing activities in cold prevent musculo-skeletal problems among the women.
nearly 44% of the women were indifferent towards adopting Work stressors and MSD: The descriptive values obtained gave
environment without any personal protective garment, might be one of
the response of the workers to the work aspect as a stressor. The References
the causative factors for the development of MSD in different body
response of the workers to these work stressors is shown in Figure 2.
Table 1: Physical and occupational characteristics of women fish regions. High prevalence of MSD among the women fish processing 1. Wang, PC, Rampel, DM, Harrison, RJ, Chan, J., Ritz, BR 2007. Work
Illumination, low temperature, dust, constrained work posture, high
processors (N=450) workers suggest that they are at risk and the intervention strategy must organizational, and personal factors associated with upper body
physical activity, psychosocial aspects as work scheduling, poor job
be developed and implemented immediately. MSD among fish musculoskeletal disorders among sewing machine operators.
Age (Mean ± SD) 23 ± 6.4 autonomy and poor job feedback had more weighttage among the
processors is a major health problem. Fish processing activities share Occu. Enviro. Med. 64(12): 806-813.
work stressors as perceived. The Pearson's correlation matrix showed
<25 years (%) 75.3 many similarities that are attributed to the poor work conditions, which
that high physical activity, job specialization, cold environment, poor 2. Hagen, K, Magnus, P, & Vetlesen, K, 1998. Neck /shoulder and low-
Work experience (Mean ± SD) 2.8 ± 2.0 pose varieties of health hazards including MSD. Women can face
job feedbacks and strenuous work postures were associated to pain back disorder in the forestry industry: Relationship to work tasks
significant risks from the processing activities they perform,
<3years (%) 47.6 and discomfort in the lower back. Psychosocial aspects as poor job and perceived psychosocial job stress. Ergonomics. 41-1510-
underlining the importance of adequate risk-assessment and
autonomy and mental overload had correlation to discomfort in knee. 1518.
Education -- Literate (%) 72 prevention measures for them. Musculo-skeletal pain and discomfort
Pain in hand was associated to work environment, absence of 3. Schneider S., Lipinski S, Schiltenwolf M., 2006 Occupations
Marital status -- Unmarried (%) 69 are the results of interaction of the factors associated with the job and
auxiliary support and manual material handling. High physical associated with a high risk of self-reported back pain:
work environment, environmental factors and characteristics of the
Work shift (hr) 12 activities, job specialization, task environment, cold environment, representative outcomes of a back pain prevalence study in the
individual factors. As observed in the study, working conditions and
poor job feedbacks and poor job autonomy and task clarity, strenuous Federal Republic of Germany. Eur Spine J. 15,821-833.
Tasks (Women performs more than one task) (%) methods, unergonomically designed work station and tools, cold work
work postures and high mental overload were associated to pain and 4. Cohen, AL, Gjessing, CC, Fine, LJ, Bernard, BP, McGlothlin, JD,
Grading 39 environment, constant standing posture, long working hours etc., were
discomfort in lower back. Physical activity and poor task clarity 1997. Elements of Ergonomics Program-A primer based on
the predominant factors contributing to the development of MSD.
Sorting 15 associated to discomfort in upper back. Pain in hand was associated workplace evaluation of musculo-skeletal disorders. DHHS
Perceptions of intensified workload, monotonous work, limited job
Packing 50 to work environment (cold), absence of auxiliary support, manual (NIOSH) Publ. No. 97-117.
control, low job clarity, and low social support were also associated
handling and poor task clarity. Discomforts in hand and finger 5. Nag PK. 1998. Work systems-checklists. In: ILO Encyclopedia of
Mixed tasks 15 with various work-related MSD.
associated to cold environment, high physical activity, manual Occupational health and Safety, Fourth ed. Geneva, ILO
Peeling 27 material handling. The double fold risk of development of MSD among Ergonomics pp. 29.14-29.24 (Chapter 29).
younger group of women workers, who were less than 25 years of Conclusion
Ring cutting 16
age, attracts special attention regarding implementation of ergonomic Work related pain and discomfort lead to sickness absence and to add
BMI (Mean ± SD) 21.4 ± 4.1 intervention in the fish in processing units with an immediate effect. to the burden of disease. The components at work that might attribute

12 13
 Split contour & Porous backrest

Annual Report 2007-08

Arm rest

Angle & Height Adjustment

Vibrators
Waterfall seatpan

Chair Height Adjustment

Tilt mechanisam of seatpan
Stabilometric Signal Analysis of Women Sitting Postures
A Nag, PK Nag, S Pal and H Vyas
Foldable Footrest with Castor
Abstract

The study examines the utility of the force platform measurements- Centre of pressure (CoP) displacement
parameters and force exertion in order to explore the influence of sitting modes governed by sitting behavior
and seat components. A test rig of two piezoelectric force platform, one platform was placed to serve as a
chair seat pan (P2) and second platform (P1) was placed on ground to serve as a footrest. Eleven women
participated and adopted eleven sitting modes arms right angled with upright back, arms on lap with upright
back, arms on lap with slouched back, one leg crossed over the other with upright back, back supported with
arms on lap, upright unsupported and supported back (95 recline) with variations in armrest height which
was adjusted at 17, 20 and 23 cm. Force exertion and CoP displacements parameters derived from force
platform were included in the study. In addition to descriptive, in order to understand the influence of sitting
modes on CoP displacement parameters, one-way (sitting modes) repeated measures analysis of variance
(ANOVA) was done with time blocks as covariate. The combined inclusion of back support and armrest in the
seat system reduced the load at seat pan; this is suggestive of increased dissipation of load from seat pan to
armrest, back support and marginally to feet. ANOVA results indicated that changes in the sitting modes had
discernable effects on the medio-lateral and anterio-posterior CoP displacement. The typical characteristics
of stabilometric dimension brings an insight into the influence of seat components and sitting behavior on the
postural balance characteristics therefore is important in human seat interface design.
Keywords: Stabilometric dimension, sitting postures, CoP displacement

Introduction
The sitting habits and behavior vary considerably with the designs of
chair-desk complex. Since prolonged sitting work is a potential risk to
the spinal, paraspinal and other musculo-skeletal structures,
researches have been focused on the postural load at the
intervertebral discs. This contribution describes the orthogonal force
components recorded from the ground reaction force and analysis of
body CoP displacements due to chair sitting modes and the
components of seat.
Materials and Methods
F
Eleven healthy, young, sedentary women (age - 31.7 ± 6.9 years,
body height - 150.4 ± 5.1 cms, body weight - 48.3 ± 5.7 kg, body
Tz mass index: 21.6 ± 3.2, spine length - 44.7 ± 4.7 cms) volunteered in
the study. A test rig of two piezoelectric force platform (sized 40 x 60
cm, Kistler, Switzerland, model 9268AA), one being placed to serve as
a chair seat pan (P2), stabled and vertically adjusted by a heavy duty
Force Platform signals mechanical jacking mechanism and the second platform placed at
Dynamic change in Kg Force ground (P1) as footrest (Figure 1). The sitting on a force platform
Force Componants
recorded three counteracting forces due to the body weight along the
three orthogonal axes Fx, Fy, and Fz, and the three moments around
the three axes Mx, My, and Mz. These signals were analyzed using
SMART analyzer software (BTS Bioengineering, Italy) to derive force
components and platform signals from P2 (seat pan) were used to
obtain the x and y coordinates of the CoP displacement corresponding
to medio-lateral (ML) and anterio-posterior (AP) time series
respectively with SWAY software (BTS Bioengineering, Italy). The
statistical analysis was accomplished by the software SPSS 16.0
package. In order to understand the influence of postures on CoP
displacement parameters, one-way (sitting modes) repeated
measures analysis of variance (ANOVA) was used with time blocks as
covariate. The least significant difference test was applied to produce
post hoc multiple comparison of the test measures, with respect to
variation in sitting modes, arms and back support.
Results
Force distribution (kgf) at seat pan and feet that was derived in eleven
chair-sitting modes is given in Table 1. The force distributed at the
seat pan (38.9 kgf) was recorded highest in Mode C (slouched back),
and the force at feet was 8.9 kgf (i.e., the combined force at P1 and P2
was 47.8 kgf, which was 99% of the body weight measured in

15

standing posture). With the back supported, the load at the seat pan
reduced by about 8%, as compared between Mode B (unsupported
back) and Mode E (supported back at 950 recline). With unsupported
back and increase in the height of armrest from 17 to 23 cm, the force
distributed at the seat pan reduced from 35.9 to 34.2 kgf. The
combined inclusion of back support and armrest in the seat system for
Modes F, G and H (arm rest height 17, 20 and 23 respectively) reduced
the load at seat pan to 32.9, 32.6 and 32.3 kgf respectively; this is
suggestive of increased dissipation of load from seat pan to armrest,
back support and marginally to feet. Directional ML and AP CoP
displacements of eleven sitting modes are shown in Figure 2. Repeated
measures of ANOVA indicated that the ML CoP displacement varied
across the sitting modes (p<0.01). The unsupported upright sitting
modes (Modes A and B) had highest AP CoP displacement (194 mm),
however ML CoP displacement (p<0.001) was significantly less in
Mode A compared to Mode B. Among the sitting modes, Mode E
(supported back) caused highest ML CoP displacement, when back
was supported at 950 recline. The post hoc analysis showed that the
legs being crossed one over the other (Mode D) caused relatively larger
ML CoP displacement (p<0.001), with reference to sitting mode B. The
unsupported upright sitting (Mode B) resulted in significantly greater
AP CoP displacement (p<0.001), compared to those in slouch sitting
(Mode C). The supported back (Mode E) caused reduction in AP CoP
displacement (p<0.001) and increased ML CoP displacement
(p<0.001) compared to those in upright unsupported sitting (Mode B).
For the pooled data, the scatter of the ML and AP CoP displacements
was negatively correlated (r = - 0.283, p<0.001). The linear regression
equation indicated that the unit increase in the ML CoP displacement
had a corresponding decrease in the AP CoP displacement.
Armrest heights caused significant change in the ML (p<0.001)
and AP CoP (p<0.001) displacements when the back was upright. The
ML CoP displacement in Mode J (armrest height 20 cm) was relatively
less compared to Mode I (armrest height 17 cm) (p<0.01) and Mode K
(armrest height 23 cm) and higher than Mode B (arms on lap)
(p<0.0001). Mode I (arm rest height 17 cm) varied from Mode K
(armrest height 23 cm) in AP CoP displacement (p<0.001). With back
unsupported, the increase in height of armrest from 20 cm (Mode J) to
23 cm (Mode K) significantly influenced the AP CoP displacement
(p<0.001). Mode G had considerably higher ML CoP displacement
(p<0.05) than Mode H. The study includes analysis of nearly eleven
hundred platform signals of women recorded for different sitting
modes and thereby identified seven distinct CoP patterns (Figure 3).
The anterior-posterior (Pattern 3) CoP displacement was predominant
in Mode A (upright sitting, arms at right angle), Mode C (slouched back),
Mode D (crossed legs) and Mode E (supported back). The medio- lateral
(Pattern 1) CoP displacement was predominant in Mode B (upright sitting
arms on lap) i.e. about 42% of the total distribution of patterns.
AP CoP displaced (mm) = 187.7 1.02 ML CoP displaced (mm)
16
Annual Report 2007-08 Annual Report 2007-08
Discussion Table 1. Force distribution (Kgf) at seat pan and feet in different chair sitting modes
Modes Posture configuration (P2- Seat pan) Kgf (P1- feet) Kgf
The present study emphasizes that seat components and sitting
modes implicate a change in body passive acceleration, CoP A Upright unsupported back, legs right-angled at knee and armsright-angled at elbow 38.8±5.4 8.9±1.2
displacement and CoP oscillation pattern2-4 The armrest and back- B Upright sitting unsupported back, legs right-angled at knee and arms rested on lap 38.6±5.6 9.0±1.3
support led to reduction in 16.3% of the net load exerted at the seat C Slouched back, legs right-angled at knee and arms rested on lap 38.9±5.7 8.9±1.3
pan. The armrest took about 8% of the net force exerted at the seat D Upright unsupported back, legs crossed one over the other, and arms rested on lap 38.5±4.5 9.0±1.0
pan. The extent of load dissipated by the components of seat indicated
E Supported back at a recline of 95o with arms rested on lap 36.0±5.3 9.6±1.2
likely mitigation of the compressive and shear stresses on the spinal
and other paraspinal structures. The present observation emphasizes F Supported back, armrest at the height of 17 cm 32.9±5.0 10.3±1.1
that the armrest, as a seat component, had greater contribution to G Supported back, armrest at the height of 20 cm 32.6±5.8 10.3±1.3
reduce load at seat. Load distribution outlined the overall reduction H Supported back, armrest at the height of 23 cm 32.3±5.3 10.4±1.2
and transfer of the load from seat pan to armrest, back support and I Upright unsupported back, armrest at the height of 17 cm 35.9±6.0 9.6±1.4
feet. The study defines amplitude of ML and AP CoP displacement to
J Upright unsupported back, armrest at the height of 20 cm 34.4±5.6 9.9±1.3
range narrowly within 6-14 mm and 160 to 195 mm respectively in
chair-seated modes. In chair seating modes oscillation patterns found K Upright unsupported back, armrest at the height of 23 cm 34.2±5.7 10.0±1.3
to be predominant in anterior-posterior and medio-lateral direction. Values are mean ± SD
References
1. SWAY software manual, release 1.4.4, BTS
Engineering, Milano, Italy.
Platform (P2) Height adjustment
2. Kanaya, T., Gresty, M.A., Bronstein, A.M.,
seat pan Buckwell, D., Day, B., 1995. Control of the head
in response to tilt of the body in normal and
labyrinthine-defective human subjects. Journal
of Physiology, 489, 895-910.
3. Keshner, E.A., 2000. Modulating active stiffness
Platform(P1)
affects head stabilizing strategies in young and
foot rest
elderly adults during trunk rotations in the
vertical plane. Gait and Posture, 11, 1-11.
4. Fenety, P.A., Putnam, C., Walker, J.M., 2000. In-
chair movement: validity, reliability and
Figure 1.. Force platform for stabilometric study
implications for measuring sitting discomfort.
Applied Ergonomics, 31, 383-393.
Men and women have subtle postural orientation due to Figure 2: CoP displacement in different chair seated modes
circumstances of sitting as well as seat used. Analysis of seat
dynamics bears significance to assess design and consequences for
sitter. The present stabilometric study quantifies relative share of
body weight distributed at seat pan and feet and low amplitude body
oscillation characteristics influenced by back unsupported or
supported or slouched sitting, back supported sitting, position of
arms (on lap, at right angle from elbow and on armrest) and position of
leg. The study finding strongly indicated that the backrest and
armrest shifted weight from the seat pan. The armrest influenced the
directional CoP displacements. With the backrest, medio-lateral CoP
displacement increased in comparison to back unsupported sitting. Figure 3
The typical characteristics of stabilometric dimensions bring an
17
Annual Report 2007-08

Assessment of Feasibility of Dust Control Devices for Small Silica

Flour Milling Units in India
LJ Bhagia, MI Shaikh, SL Dodia and P Sekhar

Abstract

Exposure to crystalline silica produces silicosis and silico-tuberculosis. There are two type of silica mills viz.
hammer type (product mesh size <100) and ball mill type (product mesh size 100-500). At Godhra all mills
(17) are hammer type whereas at Beawar most of the mills (about 300) are ball type. However, there are some
hammer types of mills at Beawar also. In ball mills, exposure occurs during feeding and bagging operations.
Exposure to silica dust in ball mills is reported. Total dust concentrations ranged from 9.88 to 13.14 and 11.78
to 25.36 mg/m3 in feeding and bagging operations respectively. The respirable dust varied from 2.19 to 2.48
3
in feeding and 2.89 to 4.39 mg/m bagging operations.

Keywords: Silica milling, quartz, dust control system

Introduction
Silicosis is the most ancient and common of all occupational diseases.
Even today, it continues to be among the most serious occupational
diseases. The crystalline free silica, the agent responsible for the
causation of silicosis, is one of the most powerful fibrogenic matter
found in nature. Silicosis occurs in occupations like silica milling1,
agate industry2-4, slate pencil industry5,6, ceramic and pottery7,
foundries, glass, mining of stones containing free silica, etc. Ambient
silica dust exposure in vicinity of agate and slate-pencil industry has
8,9
also been reported. There is no cure for silicosis at present but the
disease is preventable. For the prevention, exposure of workers to
crystalline silica needs to be reduced. Engineering control is the most
powerful tool for the reduction of dust exposure. This study was
conducted with the objectives to monitor dust levels in silica mills,
design a dust control system and test the efficacy of dust control
system.
Materials and Methods
Type of mills: There are two type of mills viz. hammer type (mesh size
<100) and ball mill type (mesh size 100-500). At Godhra all mills (17)
are hammer type whereas at Beawar most of the mills (about 300) are
ball type. About 25 hammer types of mills are also at Beawar.
Manufacturing process (Ball mills): The process includes
feeding of raw materials to ball mill. Different size of particles is
separated by mesh separator (100 - 500 mesh size). Normally 200
mesh size product is manufactured. Over size particles are fed back
to ball mill. Product is collected manually in bags. Exposure occurs at
feeding and bagging operations
Exposure assessment: Five ball mills were selected for
monitoring dust levels at Beawar. House keeping in general was very
poor in all the mills. The quality of bags used for product was also very
poor. The exposure of workers to silica dust occurs at feeding and
bagging operations. Workers are exposed continuously for 8 hours.
Total and respirable dust concentrations were evaluated at feeding
and bagging sites. Dust concentrations were evaluated by using SKC
samplers. Dust was collected on membrane filters.
Results and Discussion
Total and respirable dust concentrations are shown in Table 1. Total
dust concentrations ranged from 9.88 to 13.14 and 11.78 to 25.36
3
mg/m in feeding and bagging operations respectively. The respirable
3
dust varied from 2.19 to 2.48 and 2.89 to 4.39 mg/m in feeding and
bagging operations respectively. Dust levels need to be reduced by
proper engineering control. At a glance Table 1 show that dust
concentrations are higher than the permissible level of 0.1 mg/m3 for
respirable and 0.3 mg/m3 for total dust. Dust levels in bagging are
more than the same in feeding of raw materials. Hence more suction
is required at bagging site. Keeping this in view dust control system is
designed.

19

Table1: Dust concentrations (mg/m3) in ball mills
Mill Feeding
Total dust Respirable dust
A 10.74±6.42 (5) 2.45±0.71 (6)
B 9.88±2.68 (4) 2.37±0.69 (6)
C 10.03±2.97 (6) 2.31±1.76 (6)
D 12.88±2.88 (5) 2.19±1.62 (6)
E 13.14±4.29 (4) 2.48±1.57 (6)
Numbers in parenthesis indicate number of samples
Design for the dust control system: Dust control system is
designed by NIOH using ACGIH ventilation guidelines10. Dust control
system involves hood (one each at feeding and bagging points)
ducting, centrifugal fan and bag filters with reverse pulse jet system.
The reverse pulse jet systems are most commonly used. The pulse jet
cleaning mechanism uses a high pressure jet of air to remove dust
from the bag. Bags in the bag house compartment are supported
internally by rings or cages. Bags are held firmly in place at the top by
clasps and have an enclosed bottom. Dust laden-air is filtered through
the bag, depositing dust on the outside surface of the bag. The dust
cake is removed from the bag by blast of compressed air injected into
the top of the bag tube. The blast of high pressure air stops the normal
flow of the air through the filter. The air blast develops into a standing
wave that causes the bag to expand as the bubble travels down the bag
tube. As the bag flexes, the cake fractures and deposited particles are
discharged from the bag. Total flow of the system is 1200 Cubic feet
per minute (CFM). Horse-Power of the motor is 5 hp (1440 RPM).
Speed of the fan is increased to 2300 RPM by using pulleys. Owners
were encouraged to install dust control system for feeding and
bagging operations. One of the owners has agreed to install the system
on experimental basis. MoU has been signed with the mill at Beawar
for installation of dust control system. The work is in progress for
testing the efficacy of dust control system.
References
1. Bhagia LJ, Tiwari RR, Sharma YK, Saiyed HN. Silicosis and its
control in small-scale silica mills in India. WHO newsletter Gohnet
(Global Occupational Health Network), 12; 12-15, 2007.
2. NIOH Report. Prevention, control and treatment of silicosis and
silico tuberculosis in agate industry- report submitted by NIOH to
the Ministry of Health and family welfare, Govt. of India, 2004.
20
Annual Report 2007-08 Annual Report 2007-08
Environmental cum Epidemiological Survey in Asbestos Jointing
Bagging Material Manufacturing Industry
Total dust Respirable dust A Saha, LJ Bhagia, YK Sharma, PK Majumdar and JB Vyas
12.90±3.32 (5) 3.70±1.26(6)
11.78±1.02 (4) 3.44±0.97 (6)
Abstract
17.65±2.04 (6) 3.51±0.40 (6)
15.82±3.36 (5) 2.89±0.64 (6) A cross sectional study was carried out among the workers of asbestos jointing material industry with the aim
25.36±8.22 (4) 4.39±1.92 (6) of evaluating the working environment as well as the workers. Seventy workers participated in this study.
Every individual subject was interviewed with a pre-designed questionnaire to collect information in relation
to personal, occupational and morbidity details. Lung function test was carried out in all subjects. All the study
3. Bhagia LJ, Parikh DJ, Pandya GL, Vyas JB and Saiyed HN. subjects were evaluated by radiological examination as per ILO guidelines. Environmental monitoring of
Assessment and control of silica dust exposure in agate grinding asbestos fibers was also carried out. Pulmonary function test revealed that 11.4% workers were having
units, Khambhat, India. Indian journal of Occupational Hygiene obstructive and 2.9% were having restrictive abnormality. Fiber level in all the workplaces were below
and Safety. 1;2: 20-27, 2007. national and international standards. No worker was found to have radiographic features suggestive of
interstitial lung fibrosis.
4. Sadhu HG, Parikh DJ, Sharma YK, et al. A follow up study of health
status of Small-scale Agate Industry Workers. Indian J. of Ind.
Keywords: Asbestos, jointing material, pulmonary function
Med. 41;3: 101-105,1995.
5. Saiyed HN, Parikh, DJ, Ghodasara, NB, et al: Silicosis in slate
pencil workers. An environmental/medical study. Am. J. Ind.
Med. 8: 127-133,1985. Introduction accumulate and cause scarring and inflammation, which can
affect breathing and lead to serious health problems4. Asbestos has
6. Saiyed HN and Chatterjee, BB: Rapid progression of silicosis in Asbestos is the generic name given to a group of fibrous mineral been classified as a known human carcinogen (a substance that
slate pencil workers: A follow up study. Am. J. Ind. Med., 1985; 8: silicates found in nature. Exposures to asbestos fibres occur during causes cancer) by the U.S. Department of Health and Human Services,
135-142. mining, milling, processing, and transportation of asbestos and also the EPA, and the International Agency for Research on Cancer5-8. The
7. Saiyed HN, Ghodasara NB, Sathwara NG, Patel GC, Parikh DJ, during manufacturing of asbestos products. Known major health occurrence of health effects of asbestos exposure also depends upon
hazards of asbestos include cancer of lung, mesothelioma of pleura the type of asbestos used. While the occurrence of asbestosis, lung
Kashyap SK. Dustiness, Silicosis and Tuberculosis in Small Scale
and peritoneum and a specific fibrous disease of lung known as cancer and mesothelioma is widely reported with the use of
Pottery Workers. Indian J Med Res.; 102: 138-142,1995.
asbestosis. All types of asbestos fibres are responsible for human amphibole variety the target cells10. Murlidhar and Kanhere11 reported
8. Bhagia LJ. Non-Occupational exposure to silica dust in vicinity of mortality and morbidity. Chrysotile is thought to be less harmful than 23% asbestosis in a survey of 181workers of an asbestos industry of
slate pencil industry, India Published Online in Environmental other fibre types but it is difficult to substantiate after standardization India. The mean age of the workers was 54 years and all had a
Monitoring and assessment (Springer publication), April 2008. for exposure levels, type of exposure, duration of exposure, etc. minimum of 20 years of exposure to asbestos. Lung function test
Harmful effects of asbestos also depend on several factors like size revealed that 62% of workers had FVC values less than 80% of
9. Bhagia LJ, Parikh DJ, Saiyed HN. Ambient silica monitoring in
vicinity of agate industry, Khambhat, India. Indian journal of and type of fibres, duration of exposure, use of personal protective predicted standard. Radiological findings revealed that 92% and 17%
devices, etc. Chrysotile asbestos, the most common variety in use, of the subjects were having parenchymal fibrosis and pleural
Occupational Hygiene and Safety, 1; 6-10, 2007.
has been consumed in roofing, insulation, pipe and other moulded abnormalities respectively11. Such studies conducted in India are
10. ACGIH: Industrial ventilation, A manual of recommended practice, goods, gaskets, friction materials (brake linings, clutch facings) etc.1,2 . insufficient and considerably old. Hence, need of such studies is
25th edition, Signature Publications, 2004. Asbestos-cement industries manufacturing corrugated and flat desperately felt to assess the present day asbestos handling
sheets, moulded goods, pipes etc. are the major consumers of workplaces and workers. The Company manufactures Compressed
asbestos worldwide, accounting for about 85% of all use. These Asbestos Fibre Jointing Sheets as one of its major products. Asbestos
products contain 10-15% of asbestos3. fibre is mixed with binders solution and fillers and then passed through
the Calendar Machine to form Compressed Asbestos Fibre Jointing
Asbestos fibers when breathed in may get trapped in the lungs Sheets. These sheets are cut, as par required size, graphited, if
and may remain there for a long time. Over time, these fibers can required printed, bundled and packed.
21
Annual Report 2007-08
Materials and Methods
In India the chief use of asbestos is in roofing, pipeline and fireproofing.
Asbestos cement product manufacturing industries producing AC
sheets and pipes are the major user of asbestos in our country. Apart
from this a portion of asbestos use in our country is committed by
manufacturers of other asbestos containing materials (e.g. friction
materials, jointing materials etc.). This study was carried out among
the workers of asbestos jointing material industry with the aim of
evaluating the working environment as well as the workers. Seventy
workers participated in this study. The aim and purpose of the study
was explained to the participants and consent was obtained
accordingly. Every individual subject was interviewed with a pre-
designed questionnaire to collect information in relation to personal,
occupational and morbidity details of the workers. Lung function test
was carried out in all subjects. Forced vital capacity (FVC) was
recorded by Spirovit-sp-10 (Schiller Health Care Ltd, Switzerland).
Three successive recording were made in standing posture and the
nose clip was used. The best of the three performances was
considered for calculation purpose. Other different parameters like
FEV1, FEF25-75 were also calculated from the same tracings. All volumes
obtained were expressed in body temperature on atmospheric
pressure of air saturated with water vapour (BTPS). Body height and
body weight were measured in bare feet on a standard scale.
Pulmonary function test values were predicted from the standard
prediction equation. The instrument was calibrated every day before
starting the experiment. FVC and FEV1 were expressed in litres, FEF25-75
in litres/sec and FEV1% was presented as the ratio of FEV1 and FVC.
All the study subjects were evaluated by radiological examination
as per ILO guidelines. Chest X rays (PA view) were obtained with the
subject in inspiration. These X rays were examined using ILO
Classification of Pneumoconioses Radiographs. Data were analyzed
using Epi Info 5 and SPSS software.
Table 1: Distribution of pulmonary function test parameters according to age and experience
Age Group (yrs.) FEV1
Up to 30 3.57 ± 0.62
31-40 3.29 ± 0.55
41-60 2.87±0.75
Significance (One way ANOVA) F=5.98; p<0.01
Experience Group (yrs.)
Upto 5 3.19 ± 0.61
6-10 3.33 ± 0.76
11 and above 3.16 ± 0.7
Significance (One way ANOVA) NS
23
Fiber concentrations in the workplaces were evaluated using SKC
personal samplers with a flow rate of 1-2 ltr./min. The membrane filter
method using phase contrast microscopy was used. Samples were
collected on cellulose acetate membrane filters (diameter 25mm,
pore size 0.8mm) using personal samplers with flow rates of 1-2 LPM.
Samples were shifted to the laboratory and slides were prepared using
acetone-triacetin method. Fibres (length >5 m, width <3 m and
aspect ratio ³3:1) were counted using Walton-Beckett graticule at
magnification of 400X. Fibre levels were then compared with the
permissible levels mentioned in the Indian Factories Act, 1948 (1
fibre/ml) and the recommended exposure levels of OSHA (0.1
fibre/ml), NIOSH (0.1 fibre/ml).
Results and Discussion
Mean age of the study subjects was 37.8±9.02 years (range 22-60
years). Majority of the workers were more than 30 years of age.
Twenty-four workers (39.3%) were of more than forty years age.
Thirty workers (42.9%) were in 31-40 years age group. Sixteen
workers (22.9%) were up to thirty years of age. Among the total
number of workers 63 (90%) were married and the rest were not. Only
27.1% workers had more than secondary level education and about
14.3 % workers illiterate. Mean body mass index of the workers was
20.6±4.4. Majority (57.1%) had normal body mass index values
(18.5-24.99) whereas 30% and 12.9% workers had values lower and
higher than normal respectively. Among the total number of workers,
10 (14.3%) were smokers and 28 (40%) workers were chewers.
Majority of the workers (51.4%) were from Process Division followed
by Maintenance (24.3%) and Packing/Loading (8.6%). Other workers
participated in the study were from Stores and other departments
including persons employed for cleaning (15.7%). Mean job duration
of the workers was 11.77 ± 7.71 years. Minimum experience was 1
year and maximum experience was 30 years. Majority of workers
(45.7%) had experience of more than 10 years, whereas 25.7%
FVC FEF 25%- 75%
4.07 ± 0.75 4.22 ± 1.36
3.76 ± 0.65 3.94 ± 1.18
3.43 ± 0.97 3.15 ± 1.23
F=3.19; p<0.05 F=4.34; p<0.05
3.65 ± 0.71 3.98 ± 1.67
3.85 ± 0.84 3.67 ± 1.46
3.69 ± 0.89 3.61 ± 1.30
NS NS

workers had duration of work of 6-10 years. Almost twenty nine
percent of workers were having experience of up to 5 years of work.
So far as morbidity is concerned, 5.7 % of the workers were
suffering from frequent cough during work, 2.9% from backache and
2.9% from joint pain. Headache (5.7%), eye irritation (2.9%) and nasal
irritation (2.9%) during or immediately after work were the other
complaints of the workers. Pulmonary function test revealed that
11.4% workers were having obstructive disorder, 2.9% were having
restrictive abnormality and rests were normal. Significant effect of age
(Table 1) on pulmonary function test parameters' values was observed
but no such significant effect was observed in relation to work
experience. Radiological findings suggestive of Koch's infection (Right
Upper Zone) were observed in two workers. One of them had right-
sided pleural effusion also.
Environmental monitoring was carried out at four sampling
locations. Fiber concentrations are shown in Table 2. Permissible
Exposure level (PEL) in India is 1 fiber/ml whereas other agencies like
NIOSH, ACGIH and OSHA recommend 0.1 fiber/ml. It can be seen from
table 2 that fiber concentrations are lower than PELs.
Table 2: Mean Fiber Concentration (fibers/ml) at different locations
Location No. of Fiber levels
Samples (Mean±SD)
Fiber Feeding 12 0.0382 ± 0.0224
Calendermachine 12 0.0135 ± 0.0128
Cutting/finishing 6 0.0107 ± 0.0071
Near godown 6 0.0217 ± 0.0114
Conclusion
Fiber level in all the workplaces were below the national and
international standards. Only three percent workers were found to have
restrictive type of pulmonary function impairment. No worker was
found to have radiographic features suggestive of interstitial lung
fibrosis.
Acknowledgment: We are thankful to Idrish Shaikh, S Dodia and M
Joshi for the help rendered during the study.
References
1. ATSDR. 2001. Toxicological Profile for Asbestos. NTIS Accession
No. PB/2001/109101. Atlanta, GA: Agency for Toxic Substances
and Disease Registry. 146 pp.
24
Annual Report 2007-08
2. HSDB. 2003. Hazardous Substances Database. National Library of
Medicine. http://toxnet.nlm.nih.gov/cgibin/sis/htmlgen/HSDB.
3. Pigg BJ (1994). The uses of chrysotile. Ann Occup Hyg 38: 453-
458.
4. Agency for Toxic Substances and Disease Registry (June 2006).
Asbestos:Health Effects http://www.atsdr.cdc.gov/asbestos/
asbestos/health_effects/index.html.
5. Agency for Toxic Substances and Disease Registry (September
2001). Public Health Statement for Asbestos.
http://www.atsdr.cdc.gov/toxprofiles/phs61.html.
6. National Toxicology Program. Report on Carcinogens. Eleventh
Edition. U.S. Department of Health and Human Services, Public
Health Service, National Toxicology Program, 2005.
7. U.S. Environmental Protection Agency. Health Effects Assessment
for Asbestos. U.S. Environmental Protection Agency, 1984.
EPA/540/1-86/049 (NTIS PB86134608).
8. International Agency for Research on Cancer. Asbestos. IARC
Monographs on the Evaluation of Carcinogenic Risks to Humans,
vol. 14. Lyon, France.
http://monographs.iarc.fr/ENG/Monographs/vol14/volume14.pdf.
9. Harvey G, Page M, Dumas L. Binding of environmental
carcinogens to asbestos and mineral fibres. Br J Ind Med 1984,
41: 396-400.
10. Gerde P & Scholander P (1989). A model for the influence of
inhaled mineral fibers on the pulmonary uptake of polycyclic
aromatic hydrocarbons (PAH) from cigarette smoke. In: Wehner
AP & Felton DV ed. Biological interaction of inhaled fibers and
cigarette smoke. Richland, Washington, Battelle Pacific, pp 97-
120.
11. Murlidhar V, Kanhere V. Asbestosis in a composite mill at Mumbai:
a prevalence study. Environmental Health: A Global Access
Science Source 2005; 4:24.
Annual Report 2007-08

Respiratory Response to Tobacco Dust Exposure in the Bidi Binders

BP Chattopadhyay, PK Gangopadhyay, AK Mukherjee,
DS Munda, S Das and SM Hossain
Abstract
Bidi industry is one of the country's oldest home-based industries in the unorganised sector. Bidi making is a
skilled job. The occupational stress is associated with long hours of work in sitting posture, and exposure to
tobacco dusts. In the present study total 198 bidi binders (Male-109 and Female-89) were investigated. A
detailed history of illness was collected and clinical examination was performed. The pulmonary function
status was made by spirometric method. Slow Vital capacity (SVC) and forced vital capacity (FVC) was
recorded spirometrically and peak expiratory flow rates by Wright's peak flow meter. All other lung volumes
(SVC, FVC, FEV1) and flow rates (FEF0.2-1.2ml, FEF25-75% and FEF75-85%were calculated from the same tracings. Blood
samples were taken from 169 bidi binders' for IgE and total and differential count of leucocytes. Total dust
concentrations of the bidi manufacturing process are between 0.49 to 1.09 mg/m3, and respirable dust was
between 0.26 - 0.68 mg/m3, which are below the ACGIH TLV of nuisance dust. About 53.7% of the workers
complained of cough; chronic bronchitis in 41% and breathlessness by 39% binders. The lung volumes and flow
rates of the male bidi binders are significantly higher compared to the females. The PFT values are gradually
reduced as the duration of work exposure increased. The mean eosinophil count and IgE level were found higher
than normal range in both sexes. As a whole 25.7% male and 11.2% female bidi binders had respiratory
function impairments. The occurrence rate is much higher among the males compared to females. The
respiratory impairments among the bidi binders may be due to their exposure to tobacco dust during the work.

Keywords: Bidi binders, Tendu leaf, Pulmonary function tests, Spirometry

Introduction breathlessness were reported symptoms of the women bidi workers .

3

Bidi industry is one of the country's oldest home based industries

employing over 3 million workers consisting of males, females and
children. There are different processes involved in bidi making. About
85% of World's bidi production is coming from 290,000 bidi making
1
units in India . Bidi is manufactured in different states like West
Bengal, Kerala, Karnataka, Andhra Pradesh and Madhya Pradesh. On
an average, each worker can roll from 400 to 1000 bidis per day
depending on individual skill and time.
The air borne tobacco dust and microbes generated during bidi
processing is inhaled by the workers. These are the probable risk
factors of causing respiratory disorders. Studies carried out on
tobacco processing workers reported symptoms like headache,
2
nausea, giddiness, vomiting, tiredness, loss of appetite etc . It
revealed that the rate of urinary excretion of nicotine and cotinine was
high in exposed workers. Backache, headache, pain in the neck, pain
in the hands & legs, cough with or without expectoration,
The ventilatory capacities of tobacco factory workers in Libya
decreased in comparison with age matched unexposed workers4. The
respiratory symptoms and lung function impairment in workers in a
5
tobacco processing plant was evidenced in Croatia The health status
of the workers exposed to tobacco dust showed a fairly high frequency
6
of respiratory diseases .
In bidi binders the occupational stress is associated with long
hours of work in floor sitting posture, and exposure to tobacco dusts
7
and poor ill ventilated working conditions. Gopal reported the main
symptoms among the female bidi workers are backaches, neck
aches, headaches, burning senses on of eyes, pain in the legs and
numbness of the fingers, cough, giddiness, breathlessness, urinary
burning sensation, white discharge, joint pains and swelling etc.
Keeping in view the hazards of tobacco dust, the present study was
under taken to investigate respiratory health status among the bidi
binders.

25
Annual Report 2007-08
Materials and Methods
Bidi making is a skilled job. Bidi is an indigenous cigarette in which
tobacco is rolled in a processed tendu leaf and tied with a cotton thread.
The rolled bidis are bundled and then dried by using a Chula for 10 - 15
minutes. The tendu leaves are moistened and then cut into piece of
required size and shape by the workers with the help of scissor.
Processed tobacco is mixed up by hand and then it is wrapped and
rolled in the cut piece of tendu leaf in such a way that one end is broader
and the other end is narrower and that end is tied with thread so as to
maintain shape and tobacco should not come out.
Environmental Study: Work place environmental monitoring in five
bidi manufacturing units was carried out at Mograhat areas of West
Bengal. Assessment of personal exposure to respirable dust carried out
by attaching personal sampler with respirable sampling head to the
workers and sampling for 2-3 hours at a rate of 2 litres per minute
(LPM) during the work. Work place area was also monitored for total
dust levels by placing a high volume dust sampler in the processing and
bidi binding area with a 4.7 mm sampling head on the Glass fibre filter
paper at a rate of 28.36 LPM for 2-3 hours consecutively for two days.
During the course of sampling, thermal parameters like dry bulb (DB),
wet bulb (WB), natural wet bulb (WNB), globe temperature (GT) were
recorded and wet bulb globe temperature (WBGT) in the work areas
were calculated.
Medical Study: In the present study 198 bidi binders (Male-109
and Female-89) were investigated. Mograhat area was chosen
considering the approachability and assurance of participation of the
subjects voluntarily. A questionnaire, tested and validated in field
condition, was administered to each participant. Prior to administering
questionnaire written consent was obtained from each subject for his
or her voluntary participation that was required for clinical examination.
The data were analysed using Epi Info software (WHO).
Pulmonary Function Tests: The pulmonary function status of the
bidi binders was assessed by spirometric method. Slow Vital capacity
(SVC) and forced vital capacity (FVC) was recorded spirometrically
(Chest Graph-801, Tokyo, Japan) and peak expiratory flow rates by
Wright's peak flow meter (Clement & Clark, UK). Other lung volumes
and flow rates were measured from the same tracing. The smoking
history including frequency of smoking of the subjects were recorded.
Bidi binders are divided into non-smokers, smokers and left ex-
8
smokers . The personal history, present duration of work along with
their past and present work history was recorded9. The PFT
measurements were made in a comfortable standing position and body
height and body weight was measured by a standard scale without
footwear. All measured lung volumes obtained were expressed in body
temperature pressure saturated with water vapour (BTPS) 10. Body
27
surface area (BSA) was calculated from the Du-Bois and Du-Bois
11
formula . Pulmonary function test values were predicted from the
standard prediction equation of the Kolkata normal subjects12. The
criteria followed for categorization of the severity of restrictive
impairments is based upon the ratios between predicted and observed
values of SVC and obstructive impairments based on the observed
12
FEV1% .
Immunological Study and Leucocyte Count: Blood samples were
taken from 169 bidi binders for immunological study that is for IgE and
total and differential count of leucocytes. Immunoglobulin E (IgE)
enzyme immunoassay was done by enzyme linked Immunosorbent
assay (ELISA) method13 by taking the serum from the blood sample and
using the test kit of Biocheck Inc. Burlingame. Leucocytes count was
made by standard method using Eosin and Haematoxylin stain.
Results and Discussion
The samples collected during area monitoring within the bidi-
manufacturing units (Table-1) showed total dust concentrations are
3 3
0.49-1.09 mg/m , which are below the ACGIH TLV of 10 mg/m , the TLV
of total nuisance dust as there is no separate TLV for the tobacco dust.
The personal exposure of the workers engaged in the bidi processing
units to respirable dust was between 0.26 - 0.68 mg/m3. The exposure
level of respirable dust was also found below the permissible standard
of nuisance dust. In the manufacturing process there are heating
operations for drying and baking of the rolled bidis which release
pungent smell and fumes in the work areas.
Table 1: Static air and personal exposure of workers to respirable
dust in the bidi binding units
Bidi Static air concentration Personal exposure to
binding unit of total dust (mg/m3) † respirable dust (mg/m3) #
1 1.09 0.26 ± 0.19,
(0.08 - 0.56) (n=4)
2 0.80 0.39 ± 0.13,
(0.20 - 0.49) (n=5)
3 0.63 0.52 ± 0.69,
(0.05 - 1.89) (n=5)
4 0.62 0.33 ± 0.11,
(0.24 - 0.48) (n=5)
5 0.49 0.68 ± 0.30,
(0.32 - 1.13) (n=5)
†: High Volume Sampling. #: Low Volume Sampling,
Figures in the parenthesis are ranges, n = number of sampling
 Annual Report 2007-08 Annual Report 2007-08

Table 2: Meteorological parameters recorded in the different Table 4: Distribution of musculo-skeletal symptoms as reported Table 5: Pulmonary function test values of the male and female bidi binders according to BMI range (Mean ± SD)
bidi-binding units by the subjects BMI range SVC FVC FEV 1 FEV 1% FEF.2 -1.21 FEF25 -75% FEF 75-85% PEFR
Bidi binding No. of DB (ºC) % RH WBGT (ºC) Bidi Binders (kg/m2 ) (l) (l) (l) (l/sec) (l/sec) (l/sec) (l/min)
unit observation Symptoms Male Female Total
<18.5 2.70 2.67 1.17 80 4.07 2.38 0.83 350
1 3 23.7 ± 0.2 51.3 ± 14.6 21.7 ± 0.7 (n=109) (n=92) (n=201) (n=46) ±0.66 ±0.67 ±0.71 ±10.96 ±1.84 ±1.31 ±0.50 ±105.89
2 11 20.8 ± 7.2 48.9 ± 18.7 19.5 ± 6.5 Headache 19 (17.4) 18 (19.6) 37 (18.4)

(n=109)
18.5 to <25 3.02* 2.91 2.42*** 83 5.06** 2.74 0.89 393*

M a le
3 5 20.8 ± 7.1 48.4 ± 19.0 19.5 ± 6.5 Pain in Neck 72 (66.1) 72 (78.3) 144 (71.6) (n=58) ±0.65 ±0.71 ±0.66 ±8.21 ±1.88 ±1.32 ±0.48 ±91.01
4 5 20.6 ± 7.4 48.8 ± 20.2 19.3 ± 6.8 Joint Pain 67 (61.5) 47 (51.1) 114 (56.7)
### ##
5 12 25.2 ± 3.4 48.8 ± 8.5 23.4 ± 2.6 Low Back Pain 80 (73.4) 72 (78.3) 152 (75.6) >=25 3.18 3.13 2.63 84 6.46 3.02 0.85 446
(n=5) ±0.36 ±0.38 ±0.18 ±6.03 ±1.23 ±0.74 ±0.20 ±49.30
Table 2 shows the measurements of thermal parameters in these Pain in Hands and Legs 19 (17.4) 7 (7.6) 26 (12.9)
manufacturing units. The maximum value of WBGT temperature Neuropathy 87 (79.8) 58 (63.0) 145 (72.1) <18.5 2.40 2.35 2.14 91 3.70 2.91 1.33 324*
obtained was 23.4 ºC in the manufacturing unit 5 and the minimum was (n=26) ±0.41 ±0.41 ±0.41 ±9.12 ±1.02 ±0.78 ±0.64 ±63.57
Values in the parenthesis indicate percentage
19.3 ºC in the manufacturing unit 3. The WBGT temperatures in all

F e m a le
18.5 to <25 2.29 2.21 1.93* 87 3.21 2.49* 1.04* 329

(n=89)
these manufacturing units were below the WBGT TLV of 30 ºC for light Nutritional deficiency disorders like anaemia (17.9%), angular
stomatitis (57.2%) and glossitis (33.3%) were also prevalent. Glossitis (n=57) ±0.37 ±0.37 ±0.40 ±10.82 ±1.13 ±0.89 ±0.51 ±56.36
nature of continuous job as recommended by ACGIH. The dust levels
measured in the bidi processing units are within the limit for the and angular stomatitis prevalence was more in males whereas
nuisance dust. anaemia more in females. Other symptoms like loss of appetite, feeling >=25 2.39 2.19 1.81 87 3.22 2.39 1.04 303
of cramps, etc. were also present among the bidi binders. About 5.2% (n=6) ±0.65 ±0.86 ±0.78 ±11.77 ±1.97 ±1.07 ±0.43 ±51.64
Among the total workers studied the average age were 48.25 ±
12.82 years. The mean height and weight were 155.87 ± 7.81 cms and workers were hypertensive (>140/90 mm of Hg; male 6.4%, female *p<0.05, **p<0.01, ***p<0.001 when compared with corresponding earlier BMI range
##
<0.01, ###p<0.001BMI <18.5 Vs >=25
48.00 ± 8.95 Kgs respectively. Majority of them were literate more in 4.3%). Most of the symptoms and signs may be sequelae of handling
case of males. The general symptoms of the subjects are presented in tobacco, awkward posture and nutritional deficiency. Table 6: Pulmonary function test values of the male control subject and bidi binders according to smoking habits (Mean ± SD)
Table 3. Weakness, chronic diarrhoea and dyspepsia were more SVC FVC FEV1 FEV1% FEF.2 -1.21 FEF 25 -75% FEF 75 - 85% PEFR
prevalent in male, whereas giddiness showed higher prevalence in The lung volumes (SVC, FVC, FEV1, FEV1%) and flow rates (FEF0.2- Smoking category
(l) (l) (l) (l/sec) (l/sec) (l/sec) (l/min)
females. 1.21ml, FEF25-75%, FEF75-85%, PEFR) of the male bidi binders were significantly
Non -Sm 3.13 3.10 2.70 87 5.65 3.43 1. 19 429
higher compared to the females. The pulmonary function test (PFT)
About 53.7% workers complained of cough and chronic bronchitis (n=19) ±0.60 ±0.65 ±0.64 ±7.97 ±1.38 ±1.11 ±0.63 ±66.07

(n=109)
values were low in females compared to male bidi binders. In male bidi

M a le
was noted in 40.8% subjects. Breathlessness was complained by Sm 2.89 2.82 2.31 * 81 ** 4.61 * 2.48 ** 0.82 ** 376 *
binders there was a gradual decrement of PFT values as the age
substantial number of subjects (39.3%). Rhonchi were detected in (n=78) ±0.64 ±0.64 ±0.62 ±8.18 ±1.88 ±1.27 ±0.42 ±96.40
increases. Female bidi binders in the age range 20 yrs had low lung
10.4% of the subjects. These symptoms and signs were present in # @## @ ## # # ## ## ##
higher frequencies in male subjects. Incidentally a large number of male volumes and the flow rates compared to the age range (21-30 and 31- Ex -Sm 2.56 2.35 1.83 77 3.76 2.10 0.64 315
workers were smoker. Inhalation of tobacco dust and smoking might 40yrs). Male bidi binders had higher PFT values, compared to females. (n=12) ±0.80 ±0.85 ±0.82 ±15.11 ±2.55 ±1.34 ±0.35 ±120.93
be contributing in developing these complaints. The symptoms related Level of significance* p<0.05, **p<0.01 Non-smoker compared with the smokers. @P<0.05 @P<0.01 Smoker compared with the
The PFT values of the bidi binders (Table 5) were divided according # ##
Ex-smokers p<0.05, p<0.01 Non-smoker compared with the Ex-smokers
to musculoskeletal system is summarised in Table 4. Pain in neck, low
to their BMI into three categories, that is <18.5, 18.5 to <25 and ³ 25.
back pain and joint pain were highly prevalent. Typical posture of bidi the male bidi binders were less compared to the 11-20 years exposure obstructive) type (7.34%) among the male bidi binders. The
The PFT values of the male bidi binders are less in <18.5 category,
rolling might be a probable cause of these ailments. Presence of group, but in females, no such trend was observed. corresponding values in females are 3.37% and 7.87% but there was
compared to other two categories.
headache, Burning and itching of eye, conjunctival redness and no combined type of impairment. As a whole, 25.7% male bidi binders
dimness of vision were the other complaints of the workers. The Hb, total RBC and WBC count of the male and female bidi and 11.24% female bidi binders had respiratory function impairments.
Only male bidi binders are smokers, who were divided into non- binders were within normal range. The eosinophil count was higher
Table 3: Distribution of general symptoms as reported by the The pulmonary function impairments are shown in Figure 1.
smokers, smokers and ex-smokers, according to their smoking habit than the normal range in male and female bidi binders. The serum IgE
subjects (Table 6). The mean values of all pulmonary function parameters were level was found to be higher in both male and female bidi binders Among the bidi binders, 5.26% of the non-smokers had
Symptoms significantly higher in non-smokers followed by smokers and ex- compared to the normal range (0-200 IU/ml). restrictive and obstructive type of impairments (Table 7). Among the
Bidi Binders Male (n= 109) Female (n = 92) Total (n= 201) smokers. The bidi binders are divided into the three groups according to their smokers, the impairment was 24.4% and in ex-smokers 58.3%, but in
Weakness 64 (58.7) 49 (53.3) 113 (56.2) serum IgE level, 0-200 IU/ml, 200-400 IU/ml and >400 IU/ml. The non-smokers it was 10.5%.
The work exposure of the bidi binders of both sexes were divided maximum number of bidi binders had >400 IU/ml of serum IgE level.
Giddiness 40 (36.7) 41 (45.1) 81 (40.5) into <10 yrs, 11-20 yrs, 21-30 yrs, 31-40 yrs, 41-50 yrs and ³ 50 Mustajbegovic et.al18 reported that the ventilator capacity in
The PFT values in male bidi binders were found to be higher in the low
Chronic Diarrhoea 45 (41.3) 29 (31.5) 74 (36.8) years. No female subjects had working history more than 31-40 years IgE level compared to the high IgE level but in females, higher IgE level tobacco workers showed a reduction in FEV1, FEF50% and FEF25% in
Dyspepsia 60 (55.0) 46 (50.0) 106 (52.7) and above. The PFT values are gradually reduced as the duration of group had high PFT values. The pulmonary function impairments are of relation to predicted values. In the present study reduction of lung
Values in the parenthesis indicate percentage work exposure increased from 11-20 years onwards. The PFT values of restrictive (7.34%), obstructive (11.01%) and combined (restrictive and volumes i.e., SVC, FVC, FEV1, FEV1%, flow rates were noted in bidi

28 29
 Annual Report 2007-08 Annual Report 2007-08

were higher among the male bidi binders (25.7%) and the 11. Du-Bois D, Du-Bois EF. Clinical calorimetry. A formula to estimate
impairments were comparatively low among the female bidi binders the approximate surface area if height and weight be known.
(11.24%). Arch. Intern. Med 1916; 17:868-871.
12. Chatterjee S, Saha D, Chattopadhayay BP. Pulmonary function
Acknowledgment: The investigators thankfully acknowledge studies in healthy non-smoking men of Calcutta. Annals of
30 cooperation of the bidi binders and the technical assistance rendered Human Biology. 1988; 15: 865-874.
by SK Bhattacharya, J Alam, SK Roy, M Chatterjee, D Chakraborty, and
T Dasgupta. This project was partially supported by the Department of 13. Engvall E, Perlmann P. Quantitation of specific antibodies by
Science and Technology, Govt. of West Bengal. The authors thankfully enzyme linked anti immunoglobulin in antigen-coated tubes. J
25
acknowledge the co-operation rendered by Mondal of Mograhat gram Immnol 1973,109: 129-135.
Panchyet, Kalo Gazi, Jugal Baidya and other members of the gram rd
14. Morgan WKC, Seaton A. Occupational Lung Diseases. 3 Edition,
panchyet. Philadelphia, London, Toronto, Montreal, Sydney, Tokyo, W.B.
Saunders Company 1975 p79.
References
15. Ignacak A, Guizik TJ, Gorski L, Czerniawska-Mysik G, Adamek-
Guzik T: Influence of tobacco dust on the respiratory system and
1. Bidis more lethal than cigarettes: Health Ministry, The Statesman, selected immunological parameters. Przegl lek. 2002; 59(10):
Kolkata edition, 14th May 2008. 789-792.
2. National Institute of Occupational Health, Ahmedabad, - 16. Mahimkar MB, Bhisey RA: Occupational exposure to bidi tobacco
Occupational Health survey of Women Workers engaged in Bidi increases chromosomal aberrations in tobacco processors.
making (rolling). Annual Report 1980. Mutat Res. 1995; 334(2): 139-144.
3. National Institute of Occupational Health, Ahmedabad, - 17. Bagwe AN, Bhisey RA: Occupational exposure to tobacco and
Occupational Health survey of Women Workers engaged in Bidi resultant genotoxicity in bidi industry workers. Mutat Res. 1993;
making (rolling), pp 78-86. Annual Report 1983-84. 299(2): 103-109.
4. Mengesha YA and Bekele A: Relative chronic effects of 18. Mustajbegovic J, Zuskin E, Schachter EN, Kern J, Luburic- Milas
Figure 2 : Respiratory impariments of male and female bidi binders occupational dust on respiratory indices and health of workers in M, Pucarin J.: Respiratory findings in tobacco workers. Chest;
three Ethiopian factories. Am J Ind Med 1998; 34: 373-80. 123 (5): 1740- 1748.
5. Imbus HR: Clinical aspects of occupational medicine. In: 19. Osim EE, Musabayane CT, Mujamda J. Lung function of
Table 7: Respiratory impairments of male bidi binders according to smoking habit
Occupational medicine. Edited by Carl Zenz, O. Bruce. Dickerson, Zimbabwean farm workers exposed to flue caring and stacking of
Smoking Habit Restrictive Obstructive Combined Gross Total
_ Edward, P. Horvath, JR, Third edition, Mosby, London, 1994; 3. tobacco leaves. S Afr Med J. 1998; 88(9): 1127-1133.
Male Non-smoker (n=19) 1 (5.26) 1 (5.26) 2 (10.52)
Bidi
binders Smoker (n=78) 5 (6.41) 8 (10.26) 6 (7.69) 19 (24.36) 6. Umadevi B, Swarna M, Padmavathi P Jyothi A, Reddy PP. 20. Valic F, Beritic D, Butkovic D. Respiratory response to tobacco
(n=109) Ex-smoker (n=12) 2 (16.67) 3 (25.00) 2 (16.67) 7 (58.33) Cytogenetic effect in workers occupationally exposed to tobacco dust exposure. Am Rev Respir Dis 1976; 113(6): 751-755.
Values in parenthesis are percentages dust; Mutat. Res. 2003; 535(2): 147-154.
21. Kjaergaard SK, Pederson OF, Frydenberg M, Schanheyder H,
7. Gopal. M. Health of women workers in the bidi industry. Occu Anderson P, Bande GJ. Respiratory disease and lung function in a
binders. Smoking bidi or cigarettes got a profound effect on PFT tobacco dust might have negative health effects in female workers. India (2000) Medico Friends Circle Bulletin, Jan-Feb 2000. tobacco industry. Arch Environ Health. 1989; 44(3): 164-170.
values and smokers had much less PFT values, compared to the non- The respiratory symptoms reported by the present female bidi
smokers8. Osim et.al19 evidenced that the FEV1% of the tobacco farm binders are more compared to male, which corroborates the study of 8. Chattopadhayay BP, Alam J. Spirometric function of ventilatory 22. Viegi,G, Paggiaro PL, Begliomini E, Vaghetti E, Paoletti P, Giuntini,
workers was different from that of the control. The present study Viegi22. There was a gradual decrement of pulmonary functions as function of non-smokers and different graded of smokers of C. Respiratory effects of occupational exposure to tobacco dust.
showed that as the duration of exposure increases there was a their duration of work exposure increased. The PFT values are found Calcutta. Ind J Environ Protec. 1996 14: 274-279. Br J Ind Med. 1986; 43(12): 802-808.
decrement of lung volumes and flow rates in male and female bidi higher in the low IgE level bidi binders compared to the high IgE level.
9. Chattopadhayay BP, Saiyed HN, Alam J, Roy SK, Thakur S, 23. Mukhtar, MS, Rao GM, Gamra NS, Afan AM, Zendah MI.
binders. The respiratory symptoms were more in female bidi binders Dasgupta TK. Injury into occurrence of Byssinosis in Jute mill
23 Respiratory effects of occupational exposure to tobacco dust.
compared to males. The low prevalence of respiratory symptoms was Mukhtar et.al. stated that the small airways are affected by
workers. J Occup Hlth 1999; 41: 225-231. Respiration; 1991; 58(5-6): 271-276.
found in non-exposed persons20. Kjaergaard et.al21 stated that higher exposure to tobacco dust. Huuskonen et.al24 reported a tendency
prevalence of cough and shortness of breath was present among the towards restriction and obstruction especially in small airways of 10. Cotes JE. Lung function assessment and application in 24. Huuskonen, MS, Husman K, Janvisalo J, et al. Extrinsic
tobacco workers. Their results highly corroborated with the results of tobacco industry workers and the diffusion capacity also decreased in Medicine3rd Edition, Oxford, Blackwell Scientific Publication, allergic alveolitis in the tobacco industry. Br J Ind Med.
the present study. Viegi 22 established that prolonged exposure to 18% of the workers. In the present study the respiratory impairments 1976. 1984; 41(1): 77-83.

30 31
Annual Report 2007-08

Studies on Hydrocarbon Chain (C6-C9) Characteristics of Benzene and

Alteration of Heme-Metabolism in Lymphocytes of Human Exposed to
Benzene and Substituted Benzene
AK Mukherjee, SK Bhattacharya and SK Roy
Abstract

The study was undertaken for exposure assessment of eight C6 - C9 aromatic hydrocarbons in and around the
petrol pumps, biological monitoring of urinary metabolites among the exposed and to correlate the exposure
of benzene and substituted benzene with the early health effects. Environmental levels of aromatic
hydrocarbons were collected in charcoal sorbent tubes and analysed by GC, FID technique. The urinary
metabolites were estimated by reverse phase HPLC. The personal exposure to PM10 observed in the range,
174-960 µg/m3 and the levels at the immediate outdoor air around petrol pumps were between, 176-401
3
µg/m . The PM10 around the petrol pumps having particles, 18.3 to 53.3 % of < 4.7 µm size and 1.4 to 20.1%
particles, below 1.1 µm size. The exposures to toluene were highest (AM, 735.7 µg/m3; (GM, 500.2 µg/m3)
among all the hydrocarbons; followed by benzene (AM, 199.1 µg/m3 & GM, 145.7 µg/m3), ethyl benzene (AM,
152.7 µg/m3 & GM, 119.9 µg/m3), o-xylene (AM, 112.3 µg/m3 & GM, 109.6 µg/m3), m-, p-xylenes (AM, 122.1
3 3 3 3
µg/m & GM, 85.1 µg/m ) combined, propyl benzene (AM, 73.5 µg/m ; GM, 62.1 µg/m ) and mesitylene (AM,
51.2 µg/m3; GM, 44.1 µg/m3). The outdoor air hydrocarbons also followed the similar trend but the levels were
lower. The pre-shift and post-shift urinary tt-MA were 0.24 and 0.91 mg/g creatinine and for SPMA were 0.90 and
1.33 mg/g creatinine respectively. The urinary MA, HA and MHA in pre- and post-shift samples were 1.69 and
2.89 mg/g creatinine, 5.67 and 10.01 mg/g creatinine and 0.45 and 0.66 mg/g creatinine respectively. Air
benzene levels, both in personal exposure and immediate outdoors were observed to be higher than the
recommended levels of exposure. Air benzene level correlated significantly with pre- and post-shift urinary tt-MA
and SPMA and the air ethylbenzene, toluene and xylene also correlated significantly with pre- and post-shift
urinary MA, HA and MHA. The post-shift urinary metabolites were significantly higher than the pre-shift values
in case of the tt-MA and MA among the petrol pump workers. A better correlation was observed between the
personal exposure to benzene and relative excretion SPMA than tt-MA. A decreasing order in case of urinary
excretion of the metabolites, HA, MA and MHA was noticed similarly with the sequence of environmental
levels of hydrocarbons, toluene, ethylbenzene and xylene The post-shift urinary metabolites of workers were
significantly higher than that of the occupationally unexposed population, particularly for tt-MA and HA.
Keywords: Aromatic hydrocarbon, benzene, human exposure,particulate matter, tt-MA

Introduction used as exposure marker of benzene at different levels. tt-MA and

Petrol pumps are one of the major sources of VOCs pollution in ambient
air of the urban localities. The petrol pump workers are exposed to
these compounds, which might enter into the body by inhalation and
skin absorption. The inhaled compounds (about 20%) are excreted
through exhale air and more than 80% of the absorbed VOCs are
metabolised in the body and excreted as urinary metabolites. Benzene
produces a variety of urinary products like phenolic compounds, tt-
muconic acid (tt-MA) and S-phenyl mercapturic acid (SPMA), which are
SPMA have been proposed to be the two urinary metabolites for
biological monitoring of benzene exposure at levels below 5 ppm.
SPMA has been proved to be a more reliable biomarker at level below
1
1 ppm than tt-MA due to its longer elimination half-life .
Exposure to toluene occurs simultaneously with benzene and
hippuric acid (HA) is well known indicator of toluene exposure2.
Moreover, hippuric acid production by human showed significant
inter- and intra-individual variation influenced by factors like diet,

33

intake of medicines, alcohol consumption etc.3,4. Xylene with its three
isomeric forms (-o, -m and -p) is one of the major constituent of VOCs.
It is reported that about 95% of absorbed xylene is metabolised in liver
to methyl hippuric acid (MHA)5. Mandelic acid is a recognized
biomarker of ethyl benzene6. Jang et al.7, reported significant
correlation between urinary metabolites like mandelic acid and methyl
hippuric acid for exposure to ethyl benzene and xylene respectively,
along with the environmental concentrations of ethyl benzene and
xylene. At low exposure both methyl hippuric acid and mandelic acid
can be used as a indicator for commercial xylene exposure7.
Muzyka et al8, observed the alteration of heme metabolism in
lymphocytes of workers exposed to diesel fuel. Both ALA and PP levels
have significantly increased among the exposed workers with respect
to lymphocytes compared to the control. ALA synthase activity was 2.5
fold higher in lymphocyte of workers exposed whereas ferrochelatase
activities have been found decreased, and protoporphyrin level
elevated8.
9
Studies are limited in Indian cities. Mohan Rao et al , in their study
of non-methane hydrocarbons in industrial locations of Bombay,
observed benzene concentrations above 5 ppb at four of the five
chosen sites and its concentration was more than that of toluene9.
Samanta et. al10., reported the levels of VOCs in road side air of Kolkata
over a period of three years from 1992 to 1994 during winter10. A
similar study in Delhi11 reported total ambient non methane VOC in 13
sites and the levels were correlated with traffic volume. The diurnal and
12
seasonal variations were also observed. Mukherjee et al conducted
the exposure of drivers and conductors to benzene, toluene and xylene
(BTX) in state transport buses of Kolkata12. Studies have been reported
on behavioral pattern, pulmonary function status and biological
monitoring among the petrol pump workers in India13 -16. In the light of
the literature review the present report incorporates the environmental
assessment of C6 - C9 hydrocarbons around the petrol pumps, personal
exposure of the workers to C6 - C9 hydrocarbons (e.g. benzene, toluene,
ethyl benzene, o-, m-, -p-xylene and n-propyl benzene) at work and
biological monitoring of these compounds in urine of the workers. The
urinary metabolites, namely, tt-muconic acid, S-phenylmercapturic
acid (SPMA), mandelic acid (MA), hippuric Acid and methyl hippuric
acid were studied.
Materials and Methods
Ten petrol pumps, located in North and Central Kolkata were selected
for personal exposure assessment of workers in the petrol pumps with
respect to respirable dust (PM10) and mono aromatic C6 - C9
hydrocarbons namely, benzene, toluene, ethylbenzene, o-, m- and p-
xylenes, n-propanyl benzene, isopropanyl benzene. Environmental
34
Annual Report 2007-08
monitoring of these components at the immediate air around the
petrol pumps was undertaken. All the petrol pumps are located within
the busy traffic zones and are situated by the side of main roads.
Biological monitoring of the C6 - C9 hydrocarbons in urine were carried
out among the petrol pump workers.
The urinary metabolites, trans, trans- muconic acid (tt-MA), S-
phenylmercapturic acid (SPMA), hippuric acid (HA), methyl hippuric
acid (MHA) and mandelic acid (MA) were estimated.
Environmental study: Static air sampling: Air sampling for static
air PM10 was undertaken in the outdoor air at a height of 7 to 10 feet
from the ground level by a 8-stage cascade impactor (1 ACFM) for a
period of 6 8 hours at rate of 1 Cubic Feet per Minute (CFM). Three
consecutive days samples were collected on the same filter paper in
each site. The gravimetric weight differences of the 8-impactor stages
(9.0, 5.8, 4.7, 3.3, 2.1, 1.1, 0.7 and 0.4 µm.) were used for estimation
of dust concentration and particle size distribution.
Monitoring of the hydrocarbons in static air around were carried
out at the outdoor static air by using a Air Check low volume sampler,
model 224-PCXR4, SKC Inc. Air samples were collected in sorbent
tubes (size, o.d. 6mm x length 70 mm.), packed with activated
charcoal (60 80 mesh) in two compartments (100 / 50 mg) and kept in
holder connected to a constant flow pump, by drawing air at a rate of
0.1 LPM for a maximum period of 3 - 4 hours within the period of 9 AM
to 5 PM. The flow rate of air sampling pump was calibrated regularly
before and after sampling by using a primary airflow calibrator (Ultra
Flow, SKC, USA).
Personal Exposure: The study included both the, filler group and
non-filler group of workers for exposure monitoring in petrol pumps.
The filler group is engaged to fill the fuel (diesel / petrol) into the
vehicles and the non-filler categories in the petrol pumps donot fill
fuels into vehicles, e.g., manager, cashier and other supervisory staff.
Personal exposure monitoring of 38 petrol pump workers were done
for the aromatic hydrocarbons by attaching a personal sampler (Air
Check low volume sampler, model 224-PCXR4, SKC Inc.) to the lapel
of the worker fixed with an activated charcoal sorbent tube under
similar sampling conditions.
Gas chromatographic analysis of VOCs: The VOC samples
collected in activated charcoal tubes were desorbed in carbon
disulfide (CS2) for 1 - 2 hour and analysed for benzene, toluene,
ethylbenzene, o-,m- and p-xylene, n-propanyl benzene, isopropanyl
benzene. Estimations were done by using a Gas Chromatograph,
model, Auto System XL GC (Perkin Elmer) attached with a flame
ionization detector (FID) and a capillary column, length 30 m, i.d, 0.53
mm., PE 624 (Perkin Elmer).
Annual Report 2007-08
The quantification of individual hydrocarbons in the samples was
done by using a seven point calibration curve of different
concentrations of the standard compounds prepared in CS2.
Fluorobenzene was used as internal standard to avoid injection error
and trace amount of benzene contamination in the solvent. The
retention times (RT) obtained for benzene, fluoro benzene, toluene,
ethylbenzene, m- & p-xylene, o-xylene, n-propanyl benzene and
mesitylene were 7.16, 8.32, 13.46, 18.52, 19.31, 20.75, 23.78 and
24.50 minutes respectively. All the calibration curves showed the
value of co-efficient of determination (r2), 0.995 or more.
Biological monitoring of VOCs: Study subjects: The biological
monitoring included an exposed population of 31 subjects from the
petrol pumps. Besides, 22 subjects were included who belong to
occupations involved in official and other jobs mainly, not exposed to
the nature of conditions as the petrol pump workers. The details of the
occupational history, age, weight, smoking habit, food habit, and year
of exposure in the job were recorded. Spot urine samples were collected
from the exposed workers in the morning hours before starting the
work (Pre-shift) and also at the end of the shift (Post-shift). Spot urine
samples of occupationally unexposed population were also collected.
Analysis of urinary metabolites: The collected urine samples
were undergone sample pre-treatment using the strong anion
exchanger (SAX) column (500mg/3ml., Whatman) for solid phase
extraction (SPE), attached with Vac-Elute vacuum elution system.
Prior to extraction, the SAX cartridge was conditioned by washing
with methanol (2 to 3 times), followed by washing with phosphate
buffer (pH 6.4) and the elution rate was adjusted to 2 to 3 ml. 10ml
urine-phosphate buffer mixture (1:1) was passed through SAX column
adding 1ml. each time. The SAX column was cleaned by washing with
water, then with phosphate buffer and 1.0% aqueous acetic acid. The
column was dried. Finally, the analyte was eluted by 10% aqueous
acetic acid to a volume 4 - 5 ml.6 The analysis was performed by a
HPLC (Shimadzu, Japan) consisted of an HPLC system controller
(Model : SLC-10 A, VP-Series), connected with an automated liquid
sampler (Model: LC- 10AT, VP series), spectrophotometer (Model:
SPD M 10A, Diode Array Detector), and column oven. The column
used was ODS-2 Hypersil, 250mm x 4.6mm., particle size 5 mm.
(Thermo). The mobile phase used was 0.1% Trifluoro Acetic Acid (TFA)
in acetonitrile and 0.1% Trifluoro Acetic Acid (TFA) in water with a
programmable time range, 0-50 min. at a flow rate of 1 liter per
minute. Each metabolite was estimated by scanning the
chromatogram at specific λmax. The tt-MA was measured at λmax, 263 nm,
SPMA at 205 nm, MA at 202 nm, HA at 228nm and MHA at 202 nm.
The reverse phase high performance liquid chromatographic
(HPLC) technique was used for estimation of the metabolites (e.g. tt-
MA, SPMA, MA, HA and MHA). A control urine-phosphate buffer
mixture (1:1) was spiked with 6.25 mg/ml, 12.5 mg/ml and 18.75
mg/ml of standard mixture respectively and the each spiked urine was
injected to the HPLC after the clean up procedure by solid phase
35
extraction (SPE). The tt-MA, SPMA, MA, HA calibration curves showed
the value of co-efficient of determination (R2), 0.995 or more except
SPMA (R2 = 0.919). The percentage recovery of each metabolite was
studied in the spiked normal urine samples of occupationally non-
exposed group.
Results and Discussion
The petrol pump workers included in the study are mostly come
from the outskirts of Calcutta and belong to lower socio-economic
status and low educational background. This occupation was
classified as low-skilled but with high risk of benzene exposure along
with co-exposure to other aromatic volatiles25. Out of the total 31
exposed subjects, 9 subjects (25.8 %) were reported to be smoker in
the category, 1 - 10 cigarettes per day, 1 subject (3.2%) in the
category, >11 - 24 cigarettes per day and 22 subjects (71%) were
non-smokers. Most of the workers are non-vegetarian. The mean age
of these workers was 32.45 years, (range:17-71 years) The years of
exposure, < 5 years, 20 subjects, > 5 - 10 years, 4 subjects and > 10
years 7 subjects. The mean body weight of the 31 subjects was 54.7
kg. (range:45-66 kg). Non-occupationally exposed group of 22
persons were selected as positive control. 20 subjects were males
and only 2 were females. All the subjects were non-vegetarian with
only 4 smokers. The average age of 22 subjects was 40.5 years
(range:22-58 years). The mean weight of these subjects was 63.3 Kg.
(range:48-84 kg).
Table 1 shows the respirable dust (PM10) exposure of petrol pump
workers and the static air concentration at immediate outdoor around
the petrol pump. The mean PM10 level of the workers found to be very
high (481.1 mg/m3) and the concentrations depended on factors like
the location of the pump with respect to the traffic junction, incoming
of vehicles to the pump for re-fuelling and meteorological conditions.
The highest value of personal exposure (960 mg/m3) found to be
alarmingly high. The mean PM10 level in the ambient air around the
petrol pump was 248 mg/m3. The PM10 around the petrol pumps
consists 18.3 to 53.3 % particles, < 4.7 mm size and 1.4 to 20.1%
particles, below 1.1 mm. The particle size distribution found to vary
from pump to pump depending on the size of the pump, its location
and traffic volume near the pump.
Table 1 : Personal exposures of the workers to PM10 and static air
concentration of PM10 around the petrol pump
Parameters PM10 Concentrations (µg/m3)
Personal exposure of petrol 481.1 ± 237.8
pump workers* (n) 12 (174.5 - 960.4)
Static air around petrol 247.7 ± 90.9
pump† (n) 15 (176.0 - 401.2)
* †
Personal sampling technique., Static air sampling technique,
Values are mean ± SD, (range), n=number
 Annual Report 2007-08 Annual Report 2007-08

Table 2: Personal exposure and static air concentrations of Volatile organic compounds (VOCs) in and around different petrol pumps mg/m3) 17. But when personal exposure to benzene within the petrol and MHA reported as individual metabolite or combined with one or
pump region is compared to the Air Quality Standards prescribed in two. In the present study all the five metabolites are estimated
Type of Benzene Toluene Et. Benzene m-,p-Xylene o-Xylene Prop. Mesytylene different countries, the value is about 5 to 40 times higher. The air together by same method after the solid phase extraction (SPE) using
samples µg/m3 µg/m3 µg/m3 µg/m3 µg/m3 benzene µg/m3 benzene levels at the immediate outdoor static air around petrol pump strong anion exchange (SAX) cartridge, followed by elution in
199.1±181.4 735.7±702.7 was more than 22 times higher than the prescribed European minimum volume of 10% acetic acid 20. It was observed that the post-
Personal 152.7±119.0 122.1±110.1 112.3±107.0 73.5±60.8 51.2±30.1 3
Commission limit value for benzene (5 μg/m , annual average) and also shift values are significantly higher than the pre-shift values for the
Exposure 35 - 728 85 -2965 33 - 590 26 - 403 24 - 489 30-345 22-147
found several times higher than the recommended Air Quality Standard metabolites, tt-MA (p <0.001) and MA (p <0.05) among the petrol
145.7* 500.2* 119.9* 85.1* 109.6* 62.1* 44.1* of 5 ppb (16.25 μg/m3) as annual mean (Air Quality Standard, 2007) by pump workers. The estimations of metabolites in the normal urine of
37# 38# 34# 29# 34# 30# 28# the UK expert panel for benzene in air with a target to reduce to 1 ppb 18. occupationally unexposed subjects and the same urine spiked with
The benzene levels at the immediate outdoor static air around petrol standard metabolites (6.25 ppm) showed the recovery ranges, 82.0
Static air around 110.9±60.1 443.7±304.0 120.3±70.3 137.0±128.1 78.2±44.6 92.7±77.6 72.6±54.4 pump in Kolkata when compared to the reported values in different
petrol pump 40-238 86-980 22-268 22-419 28-153 23-241 23-187 urban environment like residential area, traffic intersections, petrol
96.5* 336.4* 98.5* 89.7* 66.3* 69.7* 57.6* pumps of Delhi, the values were comparable and particularly to that of
the petrol pumps. The highest value obtained in case of the personal
11# 11# 11# 11# 11# 11# 10# exposure of petrol pump workers of Kolkata, is much higher than the air
Values are expressed as AM±SD, Range; *GM = Geometric mean; number of samples #
benzene level reported at petrol pump of Delhi. The values obtained in
Table 2 shows the personal exposure of workers and static air
3
for benzene (1.6 mg/m or 0.5 ppm), toluene (50 ppm, 188.14 mg/m ),
3 this study are higher than the air benzene levels reported from the major
concentrations of VOCs around petrol pumps. The arithmetic and
3
ethylbenzene (100 ppm, 433.54 mg/m ), xylene (100 ppm, 433.54 cities of the world. Besides, the values of VOCs (e.g, benzene, toluene, Et-
geometric mean outdoor air (static air) concentrations found to follow mg/m3) 17. But when personal exposure to benzene within the petrol benzene, m-, p- xylene, and o- xylene) obtained in the petrol pump areas of
more or less the similar trend but with values lower than that of pump region is compared to the Air Quality Standards prescribed in Kolkata city are much higher than that of different areas in Delhi. The
personal exposure due to the aerial dilution. As the petrol pump different countries, the value is about 5 to 40 times higher. The air toluene levels were reported highest among all the VOCs in Delhi air as
workers perform their job in the open air, their exposure levels was benzene levels at the immediate(50 ppm, 188.14 mg/m3), observed in the present study19.
expected to be much lower than the prescribed workplace ACGIH TLVs ethylbenzene (100 ppm, 433.54 mg/m3), xylene (100 ppm, 433.54
Table 3 presents the levels of urinary metabolites of the different
Table 3: Levels of urinary metabolites of different aromatic hydrocarbons among the workers exposed in the petrol pumps aromatic hydrocarbons among the workers engaged in the petrol
pumps. The values of the metabolites are expressed, both in terms of
Pre - shift Post - shift milligram per liter of urine and milligram per gram of creatinine. Several
Metabolites studies on estimation of urinary metabolites, like tt-MA, SPMA, MA, HA
Urine Urine -Creatinine Urine Urine - Creatinine Figure 1: Correlation between personal exposure to benzene
(mg/ L ) (mg/g creatinine) (mg/ L ) (mg/g creatinine) Table 4: Levels of urinary metabolites of different aromatic (µg/m3) and relative excretion of tt-MA.
0.41 ± 0.58 0.24 ± 0.30 0.95 ± 0.39 * * 0.91 ± 0.51# # hydrocarbons among non-occupationally exposed
(0.04 – 1.84) (0.02 – 1.57) (0.05 – 1.68) (0.03– 2.56) population in and around Kolkata city
tt-MA
(n = 20) (n = 20) (n = 31) (n = 31)
Levels of urinary metabolites of
1 10 ± 1.05 0.90 ± 0.92 1.29 ± 1.03 1.33 ± 1.58 non - occupationally exposed group.
(0.09 – 3.37) (0.09 – 3.34) (0.11 – 4.14) (0.11 – 8.16)
SPMA Metabolites Urine (mg/ L ) Urine creatinine
(n = 26) (n = 26) (n = 33) (n = 33)
(mg/g creatinine)
#
1.85 ± 1.82 1.69 ± 1.86 2.98 ± 1.43 * 2.89 ± 1.77 tt-MA 0.31± 0.18 0.36 ± 0.20
MA (0.16 – 6.99) (0.10 – 6.72) (0.22 – 15.92) (0.28 – 12.55)
(n = 20) (0.02 - 1.71) (0.03 - 2.06)
(n = 21) (n = 21) (n = 30) (n = 30)
SPMA 0.73 ± 0.52 0.73 ± 0.56
8.08 ± 7.15 5.67 ± 5.80 12.44 ± 10.68 10.01 ± 8.76
(n = 21) (0.05 - 5.16) (0.10 - 6.22)
HA (0.11 – 25.72) (0.17 – 20.30) (0.67 – 44.07) (0.72 – 35.35)
(n = 21) (n = 21) (n = 26) (n = 26) MA 1.58 ± 1.34 1.95 ± 1.67
(n = 20) (0.11 - 6.36) (0.11 - 6.36)
0.52 ± 0 .49 0.45 ± 0.43 0.77 ± 0.67 0.66 ± 0.65
MHA (0.09 – 2.02) (0.06 – 1.56) (0.12 – 2.58) (0.16 – 2.96) HA 1.86 ± 1.91 3.00 ± 3.00
(n=18) (n=18) (n=20) (n=20) (n = 14 ) (0.12 - 4.39) (0.11 - 8.36)
Values are expressed as mean±SD; Range and number of samples; tt-MA = tt- Muconic Acid
MHA 0.70 ± 0.69 0.69 ± 0.65
SPMA = S-Phenylmercapturic acid; MA = Mandelic Acid; HA = Hippuric Acid and MHA = Methyl Hippuric Acid. (n=17) (0.08 - 2.85) (0.07 - 2.37) Figure 2: Correlaton between personal exposure to benzene
* p<0.05; ** p < 0.001; (When compared with urine metabolite results of Pre-& Post-shifts values of urine). (µg/m3) and relative excretion of SPMA
# p< 0.05; # #p<0.001 (compared with urine metabolite results of Pre-& Post-shifts values with respect to creatinine) tt-MA = tt- muconic acid; SPMA = s-phenylmercapturic acid; MA = Mandelic Acid;
HA = Hippuric Acid and MHA = Methyl Hippuric Acid

36 37

98.4%, 81.3 104.5%, 91.8 100.8%, 80.5 104.5%, 80.5 96.4%, 81.4
92.8% respectively for tt-MA, SPMA, MA, HA and MHA. Urinary benzene
metabolites, tt-MA and SPMA are well-established biomarkers of
benzene exposure at level below 5 ppm. It is also reported that at
exposure below 1 ppm (3.2 mg/m3) SPMA is very specific biomarker of
benzene than tt-MA20. Estimation of these metabolites, tt-MA and SPMA
simultaneously in this study revealed a better picture of benzene
exposure among the petrol pump workers in the range, <1ppm to 5
ppm. The benzene levels both, personal exposure and static outdoor air,
at the petrol pumps showed values much below 1 ppm. The lower
values of tt-MA compared to SPMA may be accounted due to the fact
that SPMA is comparatively more sensitive at levels < 1ppm (Table 3). It
may also be observed in Figures 1 and 2 showing a correlation between
personal exposure to benzene (g/m3) and relative excretion of tt-MA and
SPMA respectively. Table 4 shows the urinary metabolites of aromatic
hydrocarbons among the occupationally unexposed population. On
comparing the levels of urinary metabolites in post-shift samples of
exposed worker, engaged in petrol pumps to that of the occupationally
unexposed population, it was observed that the levels of tt-MA (p<
0.001), SPMA (p< 0.05), MA (p< 0.001) and HA (p< 0.001), expressed
in mg/L urine among exposed group were significantly higher.
However, in case of the values of urinary metabolites expressed in mg/g
creatinine, the levels of tt-MA (p< 0.001) and HA (p< 0.01) were found to
be significantly higher.
References
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Annual Report 2007-08
8. V. Muzyka, S. Bogovski, A. Viitak, T. Veidebaum, Alteration of
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Roychowdhury, A, S Sen “Occupational exposure of drivers and
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Dhawan. Lung function abnormalities among petrol pump
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(ACGIH). 1999 TLVs and BEIs. Threshold Limit Values for
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Annual Report 2007-08
Biomonitoring of Exposure to Urban Air pollutants in Traffic Policemen
HR Rajmohan and B Ravichandran
Abstract
The urinary excretion of 8-hydroxy deoxyguanosine has been used as a biomarker of oxidative DNA damage in
both the clinical and occupational setting. The urinary 8-hydroxydeoxyguanosine in traffic policemen posted
at busy traffic junctions were estimated along with the control population away from the busy traffic junctions
those doing administrative job. A total of 100 urinary samples (56 samples of traffic policemen and 44
samples of control population were collected for estimation of 8-OHdG and analysed using enzyme linked
immunosorbent assay (ELISA). The mean 8-OHdG significantly higher (14.24 ± 12.53 mg/g creatinine) than
level of those of control group (8.34 ± 6.24 mg/g creatinine) (p<0.01). The comparison made between the
study and control groups taking into consideration over all experience the results showed 13.88 ± 12.35 mg/g
creatinine 8-OHdG in traffic policemen and 8.34 ± 6.24 mg/g creatinine 8-OHdG in control subjects which was
statistically significant (p<0.01). The 8-OHdG levels were significantly higher in non smokers of study group
compared to the control group (p<0.01). The study showed that urinary 8-OHdG is also associated with
occupational and other lifestyle factors.
Keywords: Urinary 8-hydroxy deoxyguanosine, traffic policemen, ELISA
Introduction benzene concentration was in the range of 123.20 - 4048.00 mg/g
creatinine in traffic policemen and 30.30 - 2462.90 m/g creatinine in
Urban air contains a diversity of chemical compounds, some of which petrol filling station workers. It is recognized that these population are
are genotoxins. The urinary excretion of 8-hydroxy deoxyguanosine at high risk. Hence, biomonitoring of urinary 8-OHdG, an indicator of
has been used as a biomarker in both the clinical and occupational oxidative DNA damage might indicate the likely extent of harmful
setting.1-6. Exposure to various environmental pollutants such as
effect that may occur on these groups. Urinary 8-OHdG is easy and
polyaromatic hydrocarbons, fly ash and fine particulate matter 2.5
non-invasive to collecting compared to collect tissue or leukocyte
containing metal fumes of vanadium, chromium, manganese, nickel,
samples.
copper and lead increased the level of urinary 8-OHdG 7-9.
Occupational exposure to benzene is a known carcinogen. A dose
Materials and Methods
response relationship was found between personal exposure to
benzene and urinary 8-OHdG concentrations3. In order to expound the The urinary 8-hydroxydeoxyguanosine in traffic policemen posted at
health effects arising out of occupational exposure, monitoring of busy traffic junctions were estimated along with the control population
biomarkers of early genetic effect will be a useful tool. away from the busy traffic junctions those doing administrative job. A
total of 100 urinary samples (56 samples of traffic policemen and 44
Studies were carried out on air pollution revealed that urban samples of control population) were collected for estimation of 8
population is exposed to various inorganic and organic pollutants. An OHdG and analysed using enzyme linked immunosorbent assay
increased risk of cancer has been reported in occupations with heavy (ELISA). The collected urine samples were centrifuged at 2000 rpm for
exposure to traffic related pollution10. Recently study on health risk 10 minutes and the supernatants were used for the determination of 8
assessment of rural and urban population due to indoor / ambient air OHdG. Urinary 8 - OHdG levels and subsequently adjusted by urinary
pollution of Bangalore city showed that the concentration of benzene, creatinine levels.
toluene and m- xylene varied from 4.35-55.10, 8.73-103.61 and
1.53-135.67 mg/m3 respectively in traffic policemen and 1.200- The data pertaining to personal habits like smoking, alcohol
444.6, 0.5 - 130.8 and 0.50-195.6 mg/m3 in petrol filling station consumption, nutritional data and other potential confounding factors
workers. The trans, trans-muconic acid a known biomarker of were collected using a questionnaire survey.
39
Annual Report 2007-08

Results and Discussion Studies on urinary 8-OHdG as a biomarker of oxidative DNA

damage in workers exposed to fine particulates was carried out by Jee
The levels of 8-OHdG among the study and control subjects according Young Kim et al9 and concluded that urinary 8-OHdG increased after
to their age groups are given in Table 1. It was observed that the traffic occupational exposure to fine particulate matter. Chum-Yu Chaung et
policemen below the age group of <30 showed 10.06 ± 4.37 mg/g al.,10 studied the urinary 8- hydroxydeoxyguanosine (8-OHdG) on taxi
creatinine levels of 8-OHdG compared to the same age group of people drivers from traffic exhaust and/or smoking in exposed and non-
in control subjects (6.45 ± 2.11 mg/g creatinine). In the 31-40 age
exposed individuals, and found the level significantly higher in drivers
group of traffic policemen the levels were 18.50 ± 16.15 mg/g
than in community men (13.4 ± 4.7 vs. 11.5 ± 4.7 mg/g creatinine).
creatinine where as in the control group it was 10.42 ± 8.71 mg/g
creatinine. Similarly in the higher age group traffic policemen the Table 2: Distribution of study and control subjects according to work
levels were 11.18 ± 9.13 mg/g creatinine and 12.16 ± 9.39 mg/g duration of exposure
creatinine in 41-50 & ³51 age groups respectively. The 8-OHdG levels Duration of Study Control
in the control subjects of the same group were 9.02 ± 6.96 mg/g Exposure (yrs)
creatinine and 7.22 ± 4.97 mg/g creatinine respectively. The mean of <10 20 (36.4) 7 (15.9)
total study population the 8OhdG levels were significantly higher (14.24
11-20 22 (38.2) 12 (27.3)
± 12.53 mg/g creatinine) than the control group (8.34 ± 6.24 mg/g
creatinine of 8-OHdG) (p<0.01). 21-30 10 (18.2) 19 (43.2)
³31 4 (7.3) 6 (13.6)
The distribution of participants according to duration of exposure is Total 56 (100.0) 44 (100.0)
shown in Table 2. The 8-OHdG levels according to their years of
experience is given in Table-3. The traffic policemen those working Table 3: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine)
less than 10 years were shown the levels of 8-OHdG 17.61 ± 17.10 in study and control groups according to work experience
mg/g creatinine and those working more than 11 years and below 20 Experience (yrs) Study Control
years were shown 11.47 ± 7.56 mg/g creatinine of 8-OHdG levels.
<10 17.61±17.10 (20) 9.94±7.61 (7)
These values when compared to the control subjects not working in the
traffic zones were shown 9.94 ± 7.61 mg/g creatinine among those 11-20 11.47±7.56 (22) 9.73±5.06 (12)
below 10 years of experience. Even when we look into the control 21-30 13.48±9.93* (10) 7.20±6.41 (19)
group working between 11-20 and 21-30 years of experience the ³31 8.06±6.35 (4) 7.30±6.83 (6)
levels were 9.73 ± 5.06 mg/g creatinine, and 7.20 ± 6.41 mg/g
creatinine respectively. The levels were significantly (p<0.05) higher Total 13.88±12.35** (56) 8.34±6.24 (44)
than the control group in 21-30 years of experienced traffic policemen. *p<0.05 ; **p<0.01 ; Mean ± SD
Those working more than 31 years the levels were 8.06 ± 6.35 mg/g The 8-OHdG levels among study and control groups according to
creatinine in traffic policemen and 7.30 ± 6.83 mg/g creatinine in their smoking habits is shown in Table-4. The 8-OHdG levels
control subjects. The comparison made between the study and control compared to the smokers of traffic policemen and control group were
groups taking into consideration over all experience the results showed 7.61 ± 4.72 mg/g creatinine and 7.10 ± 4.83 mg/g creatinine,
13.88 ± 12.35 mg/g creatinine 8-OHdG in traffic policemen and 8.34 ± respectively. The data analysis among the smokers and non-smokers
6.24 mg/g creatinine 8-OHdG in control subjects which were of the present study contradicts the earlier studies, as regard to
statistically significant (p<0.01). excretion of 8-OHdG. The earlier studies showed that the smoking
Table 1: Levels of 8-hydroxy -2 deoxyguanosine (µg/g creatinine) in habit had effect on 8-OHdG excretion. Several studies showed an
study and control groups according to age group increase in urinary 8-OHdG concentrations in smokers compared with
12,4
Age group (yrs) Study Control nonsmokers . Smoking can have a considerable effect on the
7
concentration of 8-OHdG. However, studies by Nilsson et al failed to
<30 10.06±4.37 (7) 6.45±2.11 (3)
show the effect of smoking on the urinary excretion of 8-OHdG.
31-40 18.50±16.15 (23) 10.42±8.71 (6) Whereas studies by Jee Young Kim et al.9 reported that mean baseline
41-50 11.18±9.13 (15) 9.02±6.96 (18) 8-OHdG concentration were not significantly different between
smokers and nonsmokers.
³51 12.16±9.39 (11) 7.22±4.97 (17)
Total 14.24±12.53** (56) 8.34±6.24 (44) Previous occupational studies, in which workers were exposed to
Mean±SD ; (Number) ; **p<0.01 various carcinogens, including PAHs and asbestos, found that smoking

41
 Annual Report 2007-08 Annual Report 2007-08

Table 4: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and smoking habits Table 7: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and alcohol consumption
Age group Study Control Study Control
Smokers Non-Smokers Smokers Non-Smokers Age group (yrs)
(yrs) Alcohol consumption Alcohol consumption
³ 30 9.03 (2) 10.47± 2.79 (5) 8.44 (1) 5.46 (2) Yes No Yes No
31-40 10.02±6.50 (4) 18.13±15.59 (19) 10.62 (2) 10.31±11.24 (4) ≤30 10.77 9.77±5.08 6.68 6.34
41-50 6.44±2.82 (4) 12.90±10.11 (11) 1.41 (2) 9.98±6.80 (16) (2) (5) (1) (2)
³51 5.81±3.14 (3) 14.54±9.97 (8) 7.86±5.55 (4) 7.03±5.01 (13) 31-40 18.62±10.54 14.70±13.85 8.65±6.10 12.18±11.97
(12) (11) (3) (3)
Total 7.61±4.72 (13) 15.23±12.40 (43) 7.10±4.83 (9) 8.66±6.58 (35)
41-50 16.95±11.36 * 8.75±6.92 6.84±5.46 10.77±7.79
*p<0.05 ; **p<0.01 ; Numbers in parenthesis indicate sample size
(5) (10) (8) (10)
≥51 12.30±10.95 11.79±4.57 10.25±3.67 6.57±5.08
Table 5: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and smoking habits
(8) (3) (5) (12)
Study Control
Age group (yrs) Total 15.64±10.40** 11.60±9.89 7.88±4.92 8.58±6.89
Chewers Non Chewers Chewers Non chewers (27) (29) (17) (27)
≤30 - 9.78±5.08 (7) - 6.45±2.11 (3) *p<0.05; **p<0.01; Numbers in parenthesis indicate sample size

31-40 12.19 (2) 17.89±15.84 (21) - 10.42±8.71 (6) Table 8: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and food habit
41-50 33.98 (2) 9.03±5.31 (13) - 8.88±7.15 (18)
Age Group Study Control
≥51 6.13 (2) 13.47±10.56 (9) 12.35 (1) 6.90±4.95 (16) Vegetarian Mixed Vegetarian Mixed
Total 14.61±13.37 (6) 13.86±12.28 (50) 12.35 (1) 8.17±6.34 (43) ≤30 5.60 11.84±3.24 7.56 4.23
(2) (5) (2) (1)
*p<0.05 ; **p<0.01 ; Numbers in parenthesis indicate sample size
31-40 26.88 17.71±16.71 - 12.32±8.23
Table 6: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and chewing habits (2) (21) (6)
41-50 10.65±6.29 11.53±10.99 8.05±5.12 9.51±7.89
Age Group Study Control
(6) (9) (6) (12)
Coffee / Tea Coffee / Tea
≥51 7.98±6.44 13.73±10.19 5.51±3.41 9.15±5.94
Yes No Yes No (3) (8) (9) (8)
≤30 9.40±4.39 (6) 14.01 (2) 6.34 (2) 6.68 (1) 11.75±8.57* 14.99±13.49 6.65±3.96 9.74±7.12
Total
(13) (43) (17) (27)
31-40 18.50±16.15 (23) - 12.32±8.23 (5) 0.90 (1)
Mean ± S.D; *p<0.05; Numbers in parenthesis indicate sample size
41-50 11.18±9.13 (15) - 9.85±6.92 (16) 2.39 (2)
Table 9: Levels of 8-hydroxy-2-deoxyguanosine (µg/g creatinine) in study and control groups according to age and exercise habit
≥51 12.72±9.70 (10) - 7.20±5.14 (16) 7.59 (1)
Age group Study Control
Total 14.39±12.72* (54) 14.01 (2) 8.90±6.34 (39) 3.99±3.21 (5)
*p<0.05; Numbers in parenthesis indicate sample size Exercise No exercise Exercise No exercise
≤30 6.59±3.37 (3) 12.21±3.61 (4) 7.56 (2) 4.23 (1)
status was not a significant predictor of urinary 8-OHdG levels 5, 6, 13. smokers. They concluded that oxidative stress imposed by cigarette
14
However, Besarati Nia et al ., reported that 8-OHdG was consistently smoking has a low impact upon certain pathways involved in DNA 31-40 10.03±5.58 (10) 21.12±14.03 (13) 10.32±9.74 (5) 10.90 (1)
increased among smokers. Heavy exposure to air pollution in damage and the antioxidative defense system. Studies by Yun-Chul 41-50 6.95±3.09 (4) 12.72±10.21 (11) 7.25±4.89 (10) 11.25±8.76 (8)
occupational settings in terms of diesel exhaust, polyaromatic Hong et al.15 concluded that cigarette smoke contains numerous
≥51 12.26±10.94 (8) 11.90±4.64 (3) 6.99±5.47 (12) 7.77±4.03 (5)
hydrocarbons, and benzene has been associated with increased 8- carcinogens. It has been shown to generate reactive oxygen species
OHdG excretion, whereas non-occupational exposure to ambient air and to induce oxidative damage in isolated DNA's as well as to Total 9.83±7.38 (25) 16.06±11.65* (31) 7.69±5.88 (29) 9.60±6.92 (15)
pollution was not significantly lower in smokers as compared with non- produce 8-OHdG in cell cultures. It has been reported that smokers *p<0.05; Numbers in parenthesis indicate sample size

42 43

had a 50% higher concentration or urinary 8-OHdG than non-smokers,
which correlates with the study by Yun Chul, which showed that
smokers who smoke less that 20 cigarettes a day showed a 48%
increase in the excretion of 8-OHdG. The 8-OHdG levels among study
and control groups, according to their exercise habit is shown in Table 9.
The excretion of 8-OHdG levels were high in traffic policemen those not
doing exercise when compared to the administrative staff. Among study
group the levels were significantly higher (p< 0.05) in those not doing
exercise compared to those having habit of doing moderate exercise.
15
Hiroshi et al studied the effects of exercise, working conditions, meat
intake, body mass index and smoking habit and other life styles on 8-
OHdG excretion. It was found that moderate physical exercise, and high
body mass index reduced the 8-OHdG excretion, while physical labour,
smoking and low meat intake (less than once/week) increased the
excretion levels.
Conclusion
The study showed that the level of urinary 8-OHdG is associated with
occupational and other lifestyle factors. Differences between the results
of the present study and other investigation might explain variations in
sample size, sample composition, and methods of measurement of
urinary 8-OHdG. Inclusion of more number of participation from traffic
policemen might highlight the association of 8-OHdG excretion and their
exposure to air pollutants.
Acknowledgment: Technical assistance rendered by A Mala, Vijayendra,
MN Prakash, V Sehar, S Raghavendra Rao, N S Marula Siddiah, N Thara,
MR Subbamma, HC Rangaswamy, D Krishnamurthy, R Muniyappa, J
Revathy, N Hema are acknowledged. Assistance rendered by Police
Department, Bangalore City is gratefully acknowledged.
References
1. Erhola M, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H et al
(1997): Biomarker evidence of DNA oxidation in lung cancer
patients: association of urinary 8-hydroxy 2'-deoxygunosine
excretion with radiotherapy, chemotherapy and response to
treatment. FEBS Lett 409: 287-291
2. Honda M, Yamada Y, Tomonaga M, Ichinose H, Kamihira S (2000):
Correlation of urinary 8-Hydroxy-2'-Deoxyguanosine (8-OHdG) a
biomarker of oxidative DNA damage and clinical features of
hematological disorders: a pilot study. Leuk Res 24: 461-468.
3. Lagorio S, Tagesson C, Forastiere F, Iavarone I, Axelson O, Carere A
(1994): Exposure to benzene and urinary concentration of 8-
Hydroxy-2'-Deoxyguanosine, a biological marker of oxidative
damage to DNA. Occup Environ Med 51: 739-743.
4. Pilger A, Germadnik D, Schaffer A, Theiler A, Pils P, Sluka F at al.
(2000): 8-Hydroxydeoxyguanosine in leukocyte DNA and urine of
quartz exposed workers and patients with silicosis. Int Arch Occ.
Environ Health 73:305-310.
44
Annual Report 2007-08
5. Tagesson C, Chabiuk D, Axelson O, Baranski B, Palus J,
Wyszynska K (1993): Increased urinary excretion of the oxidative
DNA adduct, 8-Hydroxydeoxyguanosine as a possible early
indicator of occupational cancer hazards in the asbestos, rubber
and azo-dye industries. Pol J Occ Med Environ Health 6: 357-368
6. Toraason M, Hayden C, Mariow D, Rinehart R, Mathias P, Werren D
et al (2001): DNA strand breaks, oxidative damage, and 1-OH
pyrene in roofers with coal-tar pitch dust and/or asphalt fume
exposure. Int Arch Occup Environ Health 74:396-404.
7. Nilsson R, Nordlinder R, Moen BE, Ovrebo S, Bleie K, Skorve AH,
Hollund BE, Tagesson C (2004): Increased urinary excretion of 8-
hydroxy deoxyguanasine in engine room personnel exposed to
polycyclic aromatic hydrocarbons. Occup Environ Med 61, 692-6.
8. Yoshida R, Ogawa Y, Mori I, Nakata A, Wang R, Ueno S, Shioji I,
Hisanaga N (2003): Association between oxidative stress levels
and total duration of engagement in jobs with exposure to fly ash
among workers at municipal solid waste incinerators.
Mutagenesis 18, 533-537.
9. Jee Young Kim, Sutapa Mukherjee, Long Ngo and David C
Christiani (2004): Urinary 8-Hydroxy-2'-Deoxyguanosine as a
Biomarker of Oxidative DNA Damage in Workers Exposed to Fine
Particulates. Environmental Health Perspectives 112, 6, 666-671.
10. Chun-Yu Chuang, Chen-Chen Lee, Yu-Kang Chang and Fung-
Chang Sung (2003). Oxidative DNA damage estimated by urinary 8
hydroxydeoxyguanosine: influence of taxi driving, smoking and
areca chewing.Chemosphere. 52(7):1163-1171.
11. Burgaz S, Demircigil GC, Karahalil B, Karakaya AE (2002):
Chromosomal damage in peripheral blood lymphocytes of traffic
policemen and taxi drivers exposed to urban air pollution.
Chemosphere Apr, 47 (1): 57-64.
12. Loft S., Vistisen K, Ewertz M, et al. (1999) Oxidative DNA damage
estimated by 8 hydroxydeoxyguanosine excretion in humans:
influence of smoking, gender and body mass index.
Carcinogenesis. 13: 2241-2247.
13. Toraason M (1999) 8-hydroxyguanosine as a biomarker of
workplace exposure. Biomarkers 4:3-26.
14. A.Besarati Nia, F.J.Van Schooten, P.A.E.L.Schilderman,
T.M.C.M.De Kok, G.R.Haenen, M.H.M.Van Herwijnen, E.Van
Agen,D.Pachen,J.C.S.Kleinjans (2001). A Multi-biomarker
approach to study the effects of smoking on oxidative DNA
damage and repair and antioxidative defense mechansisms
Carcinogenesis 21( 2):395-401.
15. Yun-Chul Hong, Hye-sook and Eun-Hee Ha 2000 influence of genetic
susceptibility on the urinary excretion of 8-hydroxydeoxyguanosine
of firefighters, Occup. Environ. Med, 57:370-375.
16. Hiroshi Kasai, Noako Iwamoto-Tanaka, Toshiaki Miyamoto,
Kiyoshi Kawanami et al, (2001). Life style and urinary 8-
hydroxydeoxygaunosine, a marker of oxidative DNA damage,
effect of exercise, working conditions, meat intake, body mass
index, and smoking, Cancer Res, 92:9-15.
Annual Report 2007-08
Occupational Noise and Hearing Assessment of Workers
in a Refurbishment Unit
SR Tripathi and R Vishwakarma*
Abstract
A total of 38 workers, age (21-34 yrs. with normal ears, duration of employment 1-8 yrs), working in a
refurbishment unit, were subjected to hearing assessment. The data for each subject was obtained twice i.e.
once (Day 1) at the pre- exposure level and on the second occasion (Day 3) at the post exposure level. The
result showed that the mean threshold of the workers were well within normal threshold of hearing on low
frequencies as well as on high frequencies, before work. However, after work the threshold of hearing
increased a little which was also within the normal level of hearing. The sound pressure level (SPL) has been
also carried out near the workers' ear level, which was found to be in the range of 85-116 dBA at different
locations.
Keywords: Hearing assessment, threshold limit, frequency, sound pressure level
Introduction The adverse health effects of noise exposure can be prevented, or
at least minimized, by reducing noise exposure to safe levels. The best
Workers in certain industries experience high exposure to potentially method of noise reduction is to implement engineering controls such
hazardous noise levels. These industries include agriculture, mining, as modifications to the noise source and/or to the workplace
construction, manufacturing and utilities, transportation, machinery, environment. Where technology cannot adequately control the
aircraft, weapons, and industrial operations contribute to a potentially problem, personal hearing protection such as ear muffs or plugs are
noise hazardous environment. Man-made technology advances have
used. The use of personal protection is only an interim measure while
led to the installation of broad spectrum of machines and as a result
engineering controls for permanently reducing noise levels are being
the work environment as well as the community environment is now
explored and implemented.
threatened with undesired sounds resulting in noise pollution. Its
effects are insidious and often go undetected1. Problems arise when Assessment of the magnitude of the noise problem, the levels
sound damages the human hearing. and quality of noise generated vis-à-vis the risk to hearing impairment
of workers were assessed in a gas turbine with refurbishment facility.
Intensity of noise is not the only factor to determine the It was also intended to identify the noisy equipment that could assault
magnitude of hearing loss, there are other variables which is required on the health of the workforce at their place of works. The main
to understand the spectrum of noise characteristics, duration of occupational activity of the workers was grinding operation. Each
exposure, physical dimension of workroom, and other personal factor worker was working in a separate small cabin as a result they were
such as age, sex, temperament, susceptibility, personality etc2 Long- working very close to the machine. With this background it was
term occupational exposure to high level of noise carries an increased decided that for the promotion of well being of the work force following
3-5
risk of perceptive hearing loss . A number of studies have reported future course of action should be taken.
that noise causes serious hazards to hearing6-10 cognitive11,
neuromotor functions12 and psychological disturbances such as lower This study was conducted with the objective to quantify level of
level of work performance 1 3 increased annoyance 1 4 - 1 6 , exposure to noise, particularly at high frequencies (3, 4, 6 & 8 KHz), to
hypertension17,18, hearing interference and speech intelligibility19,20, determine the hearing ability through audiometric performance and to
industrial accident 21. recommend a hearing conservation programme to protect personnel
*Prof. ENT, Civil Hospital, Ahmedabad from noise higher than permissible levels.
45
 Annual Report 2007-08 Annual Report 2007-08

The criteria for acceptance as normal ears considered were: (a) analyzed for each ear of the subjects for all test frequencies. The
NDT 8 9 10 11121314 1516 17 Tympanic membrane intact, (b) Rinne test positive, (c) No history of schematic diagram of the noise survey area has been shown in Fig. 1.
Zyglo Facility
F G C congenital or acquired conditions associated with sensory neural
hearing loss. (d) Audiogram compatible with clinical findings Weber Procedure: As testing threshold of audibility with speech signals
test, nerve defects, absolute non- conduction, polypus, etc. were also involves problems that have not as yet been completely solved or identified,
taken in to account for categorizing ears as normal. pure tone audiometry was carried out for the present investigation. In the
individual experiment, in a quiet room (less than 10 dB) subjects were
Criteria for hearing impairment: Hearing impairment is considered briefed about the nature and purpose of the study. The hearing
Gangway E D to occur when the average of the hearing threshold levels at assessment was carried out (tone lasting for 2 sec. pulsing of the tone
audiometric frequencies of both ears exceed 25 dBA. at 0.5/ sec.) The intensity of the tone was raised by 5 dB until the
threshold of hearing was determined at each test frequency viz 125 Hz,
Design of the study: Pure tone air conduction hearing threshold 250Hz, 500Hz, 1KHz, 1.5KHz, 2KHz, 3KHz, 4KHz, 6KHz.and 8KHz. Care
Fixture was obtained in a quiet room. Threshold of hearing is defined as the was taken to ensure reliable reporting of the subjects' 'just audible
Storage minimum decibel level (dB) at which the subject responds at least two sound'. Misses (error of omission) and false alarms (error of
VL CNC
Supervisor 2 3 4 5 6 7
1 2
Area Cabin times on ascending trial. The data for each subject was obtained twice commission) were avoided.
A B (i) at the pre- exposure level before the work shift (Day1) and (ii) at the
post exposure level at the end of 8 hr. work shift (Day 3). Half of the Results
Figure 1. Schematic diagram of the noise survey area subjects were tested in the sequence before- after work shift, while for
the rest of the subjects the testing was in the reverse sequence of after- The sound pressure level (SPL) has been carried out near the
before work shift, so as to eliminate the effects of practice on the workers' ear level, which was found to be in the range of 85-116 dBA
Table 1: Sound pressure level (SPL) at different locations of noise survey area
performance of the test. Pure tone threshold was obtained using at different locations (Table 1) and octave band analysis was also
Noise Levels Monitoring Frequency Levels descending - ascending threshold crossing technique. The data was carried out at all locations (Figure 2 and 3). The noise level at the places
Location Component Abrasive Unit SLM 63 125 250 500 1K 2K 4K 8K
wheel
A Fr-6 Segment Abrasive cone dBA 96 60 62 67 71 75 81 82 85
B Fr-9 2nd Stage Mounted point dBA 92 58 70 75 81 82 82 85 89
B Fr-9 2nd Stage Abrasive cone dBA 99 62 65 72 83 97 100 105 107
C Fr-6 TP Abrasive cone dBA 115 62 69 79 87 92 104 113 114
D Gangway Gangway dBA 85 65 62 62 67 82 83 85 85
E Gangway Gangway dBA
st
F Fr-9 1 Stage Abrasive cone dBA 95 57 62 65 69 72 82 87 88
G Fr-6 TP CL Abrasive cone dBA 116 65 65 70 81 89 95 105 110 Figure 2: Frequency spectrum of noise originating from equipment Figure 3: Frequency spectrum of noise originating from equipment

Grit Blasting machine dBA 85 70 72 70 68 69 74 77 78

Materials and Methods
Subjects: A total of 38 workers with normal hearing ability (as
assessed by questionnaire) included in the study. They were in the age
range of 21-34 yrs. with the duration of employment 1-8 yrs and
working in the noisy work environment for 8 hours daily. Instruments
used for the study are sound level meter:- 2218, fitted with condenser
microphone (Bruel & Kjer, Denmark), Octave frequency analyzer-
1616, (Bruel & Kjer, Denmark), Audiometer: Model 700MK IV (M/s
Arphi India Ltd.).Tunning Fork: Vibrates at 512Hz.
Following criteria were maintained for non-inclusion of workers in
audiometry:
(a) The subjects' present noise level was not amenable to
quantitative description.
(b) Not served in the armed forces, or had been exposed to gunfire.
(c) Whose past noise exposure was different from that of their
present occupation.
(d) Who were known to have existing or previous ear disease or
abnormality. Figure 4: Mean hearing level (dB) for the right ear of exposed group Figure 5: Mean hearing level (dB) for the left ear of exposed group
(e) Head injury with history of unconsciousness or skull fracture.

46 47

Fr-6 Segment, Fr-9 2nd Stage, Fr-6 TP, Fr-9 1st Stage and Fr-6 TP CL
was 96, 99, 115, 95 and 116 respectively, which is very high than the
recommended safe limit value. The noise level at the places Fr-6
Segment, Fr-9 2nd Stage, Fr-6 TP, Fr-9 1st stage and Fr-6 TP CL was 96,
99, 115, 95 and 116 respectively, which is very high than the
recommended safe limit value.
The work force were subjected to hearing assessment, before
entering the noisy area (pre exposure) and followed by 8 hours
exposure (post exposure). The data for the workers who were found to
be free from ear pathology were analyzed. The geometrical mean
threshold of hearing for the right ear and left ear of the exposed group
was calculated. Hence, the mean of the value of both the ears are
given by duration of exposure (Figure 3 & 4). It may be noted that the
mean threshold of the workers were well within normal threshold of
hearing on low as well as on high frequencies, before work. However,
after the work threshold of hearing increased a little which was also
within the normal level of hearing.
Discussion
The sound pressure level shows that the noise level at many places
was higher but hearing impairment was not found because they were
using earmuffs during their work. But it is not a permanent solution.
Also the exposure is not for 8 hours daily and sound pressure level is
not always at high level. It may be concluded through the problem of
noise with regard to hearing impairment was not observed but the
management is interested to reduce the noise levels in working area
and hence consulted the engineers to prepare a sound proof-working
chamber for permanent solution.
Conclusion
The sound pressure level (SPL) ranged from 85-116 dBA. Octave
Band Analyses revealed that noise was high frequency
characteristics, which was at some places more than recommended
values. Hearing assessment shows that no noise induced hearing
loss was found among the workers because they are using ear
protectors. These ear protectors are temporary arrangement so
management is trying to make a sound proof-working chamber for
permanent solution.
References
1. Harris CM. Handbook of noise control. Second edition. McGraw
Hill Book Company, USA, Chapters 9 &15, 1997.
2. Webb JD. Noise control in industry, Sound Research Laboratories
Ltd., England, 19, 1976.
3. Bauer P, Korpert K, Neuberger M, et al. (1991) Risk factors for
hearing loss at different frequencies in a population of noise-
exposed workers. J Acoust Soc Am; 90:3086-98.
4. Prince MM, Stayner LT, Smith RJ, et al.(1997) A re-examination of
48
Annual Report 2007-08
risk estimates from the NIOSH Occupational Noise and Hearing
Survey (ONHS). J Acoust Soc Am; 101:950-63.
5. Ahmed HO, Dennis JH, Badran O, et al. Occupational noise
exposure and hearing loss of workers in two plants in eastern
Saudi Arabia. Ann Occup Hyg 2001; 45:371-80.
6. Holme RH, Steel KP. J Assoc Res Otolaryngol. 2003 Dec 5 [Epub
ahead of print]. Progressive Hearing Loss and Increased
Susceptibility to Noise-Induced Hearing Loss in Mice Carrying a
Cdh23 but not a Myo7a Mutation.
7. Tambs K. Hearing loss caused by noise, otitis and head injuries.
Tidsskr Nor Laegeforen. 2003 Nov 6; 123(21): 3047-49.
8. Moshi NH, Riwa P (2003) Noise induced hearing loss among
industrial workers in Dar es Salaam. East Afr Med. J; 80 (6) : 298 - 02.
9. Adera T, Gaydos JC. Identifying comparison groups for evaluating
occupational hearing loss: a statistical assessment of 22
industrial populations. Am J Ind Med. 1997 Feb; 31(2): 243-9.
10. Maisarah SZ, Said H. The noise exposed factory workers the
prevalence of sensori-neural hearing loss and their use of
personal hearing protection devices. Med J Malaysia. 1993 Sep;
48 (3): 280-85.
11. Bhattacharya SK, Tripathi SR and Chatterjee BB (1986) Influence
of exposures to low intensities of sound at different frequencies
on the cognitive abilities and sensori-motor coordination. Ind J
Med Res 84,430-436.
12. Bhattacharya, SK, Tripathi SR and Kashyap SK (1989) The
combined effects of noise and illumination on the performances
efficiency of visual search and neuromotor task components, J.
Human Ergol, Tokyo, 18,41-43.
13. Cohen A (1973). Extra-auditory effects of occupational noise. Part
II. Effects on work performance. Natl Saf News 108 (3):68-76.
14. Schultz TJ (1978). Synthesis of Social Surveys on Noise
Annoyance. Journal of the Acoustical Society of America, Vol. 64,
No. 2, , pp. 377-405.
15. Taylor SM (1984). A path model of aircraft noise annoyance.
Sound and Vibration 96(2): 243-260.
16. Öhrström E, Björkman M, Rylander R (1988). Noise annoyance
with regard to neuro-physiological sensitivity, subjective noise
sensitivity and personality variables. Psychol Med 18:605-613.
17. Wu TN, Ko YC, Chang PY (1987). Study of noise exposure and high
blood pressure in shipyard workers. Am J Ind Med 12:431-438.
18. Talbott E, Findlay R, Kuller L, et al (1990). Noise-induced hearing
loss: a possible marker for high blood pressure in older noise-
exposed populations. J Occup Med 32 (8): 685-689.
19. Lindeman HE (1976). Speech intelligibility and the use of hearing
protectors. Audiology, 15 348-356.
20. Webster JC. Effects of Noise on Speech Intelligibility. In
Proceedings of the Conference Noise as a Public Health Hazard,
(1969), p. 49-73.
21. Moll van Charante AW, Mulder PGH (1990). Perceptual acuity and
the risk of industrial accidents. Am J Epidemiol 131(4): 652-663.
Annual Report 2007-08
Pesticide Exposure Amongst Formulators and Health Effects
VK Bhatnagar, AB Patel, SSA Zaidi, SR Tripathi, SN Sinha, VK Shivgotra
Abstract
The study was conducted in a unit engaged in production and formulation of organophosphate insecticides
(Triazophos and Acephate). The findings indicated that the workers involved in production and formulation of
these insecticides had significantly low level of RBCs cholinesterase activity. Serum level of IgM reflected
significant increase, indicating impairment of immune system of subjects. The levels of thyroid functional
tests (T3, T4 and TSH) were within normal range. The findings pertaining to neurobehavioral test battery
indicated that maintenance and exposed group workers showed significant elevation in finger dexterity error
test. Further, a significant decrease in neurobehavioral tests like forward and back memory, total digit symbol
test, and increase in hand steadiness test was observed in maintenance group workers.
Keywords: Pesticide formulators, triaziphos, acephate, cholinesterase, neurobehavioural test
Introduction general physical (anthropometric data, blood pressure, conjuctiva and
sclera) and systemic examination of respiratory, cardiovascular and
Pesticides are toxic chemicals and pose risks to the user groups. These gastrointestinal systems; allergic respiratory, allergic skin and
are important factors in agricultural development and protection of chronic symptoms to peripheral neuropathy were noted. An
public health in our country, since the tropical climate is very conducive electrocardiogram (ECG) of each worker was also done.
for pest breeding. Their production and formulation in industrial settings
may involve exposure risk to the workers.
Blood samples of the subjects from cubital vein were obtained
The existing database on health of workers involved in the production with the help of vacutainer (BD Vacutainer, Plymouth, PL6 7BP, UK).
and formulation of pesticides in the country is scanty, therefore, the Serum was obtained by centrifuging blood sample at 5,000 rpm for 5
0
study is designed to address the magnitude of health risk among min and stored at 20 C till the time of analysis. The activities of serum
pesticide formulators with an objective to evaluate the magnitude of glutamate oxaloacetate transaminase (SGOT, EC 2.6.1.1), serum
health risk with special reference to neurobehavioural changes. glutamate pyruvate transaminase (SGPT, EC 2.6.1.2) were estimated
following the procedure of Bergmeyer et al 1. The activities of alkaline
Materials and Methods phosphatase (AlP, EC 3.1.3.1) and lactate dehydrogenase (LDH, EC
1.1.2.27) were estimated according to the method of Empfehlungen2
The unit belongs to organized sector and engaged in production and and Mathieu3. The absorbance of these parameters was recorded on
formulation of organophosphate insecticides (Triazophos and RA-50 Chemistry Analyzer. Plasma Cholinesterase (ChE, EC 3.1.1.8)
4
Acephate). A total of 161 workers were included as subjects in this activity was estimated according to method of Ellman et al and that of
study. These were divided into three groups; i) Control Group (n=40) RBCs cholinesterase (ChE, EC 3.1.1.7) was estimated following the
comprised of employees of administration department e.g. managers, 5
procedure of Worek et al . The absorbance of plasma and RBCs ChE
executives, clerks, typists, stenographers, peon, orderlies etc ii) activities were measured on Varian Cary-100 Spectrophotometer. In a
Maintenance Group (n=50) comprised of mechanics, electricians, subset of samples, the immunological profile IgG, IgM and IgA)
engineers, fitters and helpers etc and iii) Exposed Group (n=71) and thyroid functional tests (T3, T4 and TSH) were also estimated.
comprised of workers, supervisors involved in the production and
Neurobehavioral test battery involving (Tweezer Dexterity, Finger
formulation of organophosphate insecticides. There was specific work
Dexterity, Hand Steadiness, Card Sorting, Mental Control, Memory
schedule (8 hr shift duty) for exposed and maintenance group workers
test, and Digit Symbol) was administered to this study.
whereas the control group subjects had 8 hr general duties without any
shift. Each subject was interviewed for their demographics, smoking
habits, alcohol intake, medical history, past and present illness, work Data presented as Mean ± SD. All statistical analysis was
conditions and exposure to other chemicals. Their detailed performed using SPSS software. Analysis of variance (ANOVA) was used
occupational history (past and present) was recorded on pre-coded for comparison among groups followed by group wise comparisons by
proforma. They underwent detailed clinical examination including Dunnett's T3 test. Linear regression was used for studying various
49
 Annual Report 2007-08 Annual Report 2007-08

Table 1: Distribution of subjects according to the duration of their Job Table 4: Neuro-behavioural test performance of study population
Duration of Exposure Control Maintenance Exposed
Group N Age (Years) Neurobahavioural Test Battery
<1 1-2 2- 5 >5 (n=40) (n=49) (n=71)
Mean SD
Tweezer Dexterity No. of pins 42.13  1.238 41.71  0.988 39.58  1.086
Control 40 35.029.98 4 15 10 11
Error 2.78  0.378 2.04  0.240 2.87  0.276
Maintenance 50 31.389.08 18 5 15 12
% accuracy 93.01  0.988 94.54  0.766 91.30  1.163
Exposed 71 28.456.81 29 10 16 16
Finger Dexterity No. of pins 29.05  0.992 29.76  0.838 30.58  0.832
N= number of subjects
Table 3: Correlation between RBCs and Plasma ChE in study population error 1.73  0.245 2.92  0.318 2.61  0.244
relationships among continuous variables. Two tailed tests were used
Plasma ChE % accuracy 93.47  1.005 89.67  1.212* 90.99  0.871
for all comparisons with level of significances as 5% (p < 0.05).
Parameter Control Maintenance Exposed Hand Steadiness 5.19  0.161 5.89  0.143 5.58  0.121
Results and Discussion (n=40) (n=50) (n=71) 41.31  1.347 41.61  1.122 41.41  0.917
Card Sorting Design Error
RBCs ChE Coefficient 0.282 0.459 0.402 0.49  0.168 0.51  0.124 0.62  0.123
Data on distribution of subjects in relation to their duration of job have (Time in sec.)
% accuracy 98.93  0.351 98.95  0.244 98.42  0.392
been mentioned in Table 1. There were 40 subjects in control group
p value 0.078 0.001 0.001
(aged: 35.02 ± 9.98 years), 50 subjects in maintenance group (aged: 116.51  0.409 118.14  1.351 115.96  0.306
31.38 ± 9.08 years), and 71 subjects in exposed group (aged: 28.45 Card Sorting Face
also been evaluated (Table 3). A positive correlation was observed Error 0.72  0.183 1.41  0.252* 1.24  0.183
6.81 years), The levels of various biochemical indices estimated in the (Time in sec.)
blood samples of study population are given in Table 2. The serum between plasma ChE and RBCs ChE in maintenance group (r = 0.459; % accuracy 99.38  0.157 98.79  0.216 98.93  0.157
levels of GOT, GPT, alkaline phosphatase and lactate dehydrogenase p < 0.001) and in exposed group (r = 0.402; p < 0.001). Hand Dynamometer 39.65  1.179 39.29  1.129 40.08  0.769
were within normal limits in the maintenance and exposed groups Mental Control (30”) Time 8.95  0.576 9.35  0.398 9.08  0.286
Data pertaining to neurobehavioural test battery e.g. tweezer
subjects.
dexterity, finger dexterity, hand steadiness, card sorting (design and Error 0.10  0.061 0.38  0.102 0.27  0.08
face value), hand dynamometer, mental control, memory (forward and % accuracy 98.88  0.546 96.23  0.825* 97.32  0.643
However, the activities of RBCs cholinesterase were significantly
backward) and digit symbol test have been given in Table 4. While
inhibited (p < 0.05) in exposed group subjects, indicating appreciable Mental Control (45”) Time 16.23  0.998 20.43  1.547 18.73  1.040
analyzing the data, three factors (total score, error and percentage of
exposure to the pesticides from their working environment (Figure 1). Error 2.45  0.533 3.30  0.465
accuracy) have been considered to evaluate the effect of pesticides on 3.00  0.523
The depression in ChE activity is an effect biomarker of pesticide load
the neurobehavioral test performance. The result indicated that % accuracy 90.36  2.003 88.82  1.919 87.84  1.669
and in accordance to our previous published studies amongst
exposed group workers have shown significantly lower performance
pesticide formulators and with other studies conducted on sprayers of Memory Forward 6.08  0.176 4.96  0.227*** 5.79  0.104
6,7 in digit symbol test (% of accuracy) compared to control group.
pesticides in field conditions . The exposure of OP compounds Backward 4.73  0.164 3.47  0.216*** 4.37  0.128
Exposed workers did not reflect any deficit on memory test, mental
manifests as a cholinergic crisis and diagnosis is based on the clinical
control and also in their motor performance test (tweezer dexterity, Digit Symbol Total 66.15  1.816 58.0  1.731*** 65.24  1.238
signs and symptoms as well as the measurement of inhibition of
finger dexterity and hand steadiness). Similarly no significant deficit Test
erythrocyte (RBC) and/or plasma cholinesterase (ChE) activities. The Error 0.90  0.352 1.82  0.375 2.13  0.398
was found on card sorting and hand dynamometer performance of
correlation between plasma ChE and RBCs ChE in three groups have % accuracy 98.75  0.505 96.79  0.661 96.66  0.671*
maintenance and exposed group subjects. Quite a few studies of OP
Table 2: Levels of various biochemical indices in the study population exposed workers showed no change in their performance in some of Data represented as mean ± SE; *p < 0.05; **p < 0.01; ***p < 0.001
8
Indices Control Maintenance Exposed the neurobehavioural test . In one of the studies, tremor was related to Table 5: Levels of immunological profile in study population Table 6: Levels of thyroid hormones in study population
9 10,11
(n=40) (n=50) (n=71) exposure to combination of pesticides but not to OPs . Available
data indicated that grip strength was not related to the exposure of Tests Control Maintenance Exposed Tests Control Maintenance Exposed
Hb (gm/dl) 13.78  1.55 13.64  1.36 14.20  1.35 12 13 14
(n=12) (n=28) (n=11) (n=12) (n=28) (n=11)
subjects to fumigants , DDT , or multiple pesticides .
ALKPase (U/l) 136.57  4.29 141.56  3.38 136.59  2.68
IgA T3
LDH (U/l) 327.72  9.52 312.83  8.67 320.62  7.70 The maintenance group of workers showed poor performance on 281.88 ± 19.27 291.39 ± 11.69 272.42 ± 15.52 2.36 ± 0.14 2.41 ± 0.17 2.42 ± 0.10
(mg/dL) (nmol/L)
accuracy score of finger dexterity (% accuracy) and card sorting.
RBCs ChE (mU/umol Hb) 492.35  7.56 488.67  9.09 464.6  7.97* Though there is no significant difference on their total score and error IgG T4
 #
3421.3  1.25 3326.64  1.27 3144.47  1.28 score. Similarly the memory test, (both forward and backward) has 1455 ± 55.32 1473.86 ± 56.55 1374.10 ± 30.68 138.58 ± 7.29 131.46 ± 10.92 131.11 ± 5.61
Plasma ChE (U/l) (mg/dL) (nmol/L)
 #
22.72  1.39 22.26  1.52 23.48  1.50
shown deficiency in their performance scores than the control group
SGPT (U/l) IgM TSH
subjects. Similar results have been shown in workers exposed to OPs and 160.13 ± 10.13 191.19 ± 16.56 192.08 ± 9.18* 3.14 ± 0.55 4.08 ± 1.36 3.06 ± 0.41
 26.11 1.25 26.1 1.24 27.57  1.27 (mg/dL) (mIU/L)
applicators in field conditions15,16. Card sorting (face value) and mental
#
SGOT (U/l)
#
Data expressed as mean ± SE; GM ± GSD; *p < 0.05 control (accuracy 30 seconds time) have also shown poor accuracy. Data presented as mean ± SE *p < 0.05 Data presented as mean ± SE

50 51

Figure 1. Level of RBCs cholinesterase in study population
In a subset of samples, the serum level of IgM reflected significant
increase, indicating impairment of immune system of subjects at the
level of exposure studied by us (Table 5). The levels of thyroid
functional tests (T3, T4 and TSH) were within normal range (Table 6).
The findings on significantly low RBCs ChE activity in exposed group in
our study may indicate that the operational activities pertaining to
production and / or formulation of these chemicals in industrial
settings should be done with utmost care e.g. minimizing the risk by
using protective devices to avoid absorption of these chemicals into
the body and by instituting educational preventative modules on
hygienic measures at periodic interval in order to ensure the safety to
the workers. The analysis of data pertaining to clinical examination
along with ECG of each subject of study population is in progress.
Conclusion
The findings indicated that the workers involved in production of
technical grade material and their formulation had comparatively
significantly low level of RBCs cholinesterase activity. Serum level of
IgM reflected significant increase, indicating impairment of immune
system of subjects at the level of exposure studied by us. The levels of
thyroid functional tests (T3, T4 and TSH) were within normal range. The
neurobehavioral test battery indicated that maintenance and exposed
group workers showed significant elevation in finger dexterity error
test. Further, a significant decrease in neurobehavioral tests like
forward and back memory, total digit symbol test, and increase in
hand steadiness test was observed in maintenance group workers.
Acknowledgment: We are thankful to MR Variya, Yogesh Shah, SN
Yadav, KA Patel, Raksha Agarwal, Divija Patel, Safia M Arab, Mukesh
Vakharia, SV Upadhyay, Atul Shah, NJ Vaghela, Yamini Panchal for the
technical assiatance during the study.
References
1. Bergmeyer H.U., Bowes G.N. Jr., Horder M. Jr., Moss D.W.
Provisional recommendations on IFCC methods for the
measurement of catalytic concentrations of enzymes. Part 2.
52
Annual Report 2007-08
IFCC method for aspartate aminotransferase. Clin. Chem. Acta.
1976; 70: F19F42.
2. Empfehlungen der Deutschen Gesellschaft fur Klinische Chemie.
Z Klin Chem u Klin Biochem 10 Jg 1972, 182-192.
3. Mathieu MM, Artur Y ,Albury C et al. les members de la
commission Enzymologie de la Societe Francaise de Biologie
Clinique (1982): Recommandations pour la mesure de la
concentration catalytique de la lactate deshydrogenase dans le
serum humain d 30C. Ann.Biol.Clin.,40,123-125.
4. Ellman GL, Courtney KD, Andres V Jr, Featherstone RH. A new
and rapid colorimetric determination of acetylcholinesterase
activity. Biochem Pharmacol 1961; 7: 88-95
5. Worek F, Mast U, Kiderlen D, Diepold C, Eyer P. Improved
determination of acetylcholinesterase activity in human whole
blood. Clin Chim Acta 1999; 288:73-90
6. Srivastava AK, Gupta BN, Mathur AK, Mathur N, Mahendra PN,
Bharti RS. The clinical and biochemical study of pesticide
sprayers. Hum Exp Toxicol 1991; 10: 279-283.
7. Bhatnagar VK, Karnik AB, Suthar AM, Zaidi SSA, Kashyap R, Shah
MP, KulkarniPK, Saiyed HN. Biological indices in formulators
exposed to a combination of pesticides. Bull Environ Contam
Toxicol 2002; 68:22-28.
8. Ames R, Steenland K, Jenkins B, Chrislip D, Russo J. 1995.
Chronic neurologic sequelae to cholinesterase inhibition among
agricultural pesticide applicators. Arch Environ Health 50:440-444.
9. Davignon LF, St Pierre J, Charest G, Tourangeau FJ. 1965. A
study of the chronic effects of insecticides in man. Can Med
Assoc J 92:597-602.
10. London L, Myers JE. 1998. Use of a crop and job specific
exposure matrix for retrospective assessment of long-term
exposure in studies of chronic neurotoxic effects of
agrichemicals. Occup Environ Med 55:194-201.
11. Steenland K, Dick RB, Howell RJ, Chrislip DW, Hines CJ, Reid TM,
et al. 2000. Neurologic function among termiticide applicators
exposed to chlorpyrifos. Environ Health Perspect 108:293-300.
12. Anger W, Moody L, Burg J, Brightwell WS, Taylor BJ, Russo JM, et
al. 1986. Neurobehavioral evaluation of soil and structural
fumigators using methyl bromide and sulfuryl fluoride.
Neurotoxicology 7:137-156.
13. Van Wendel de Joode B, Wesseling C, Kromhout H, Monge P,
Garcia M, Mergler D. 2001. Chronic nervous-system effects of
long-term occupational exposure to DDT. Lancet 357:1014-1016.
14. Kamel F, Rowland A, Park L, Anger W, Baird D, Gladen B, et al.
2003. Neurobehavioral performance and work experience in
Florida farmworkers. Environ Health Perspect 111:1765-1772.
15. Korsak RJ, Sato MM. 1977. Effects of chronic organophosphate
pesticide exposure on the central nervous system. Clin Tox 11:83-95.
16. Cole DC, Carpio F, Julian J, Leon N, Carbotte R, De Almeida H.
1997. Neurobehavioral outcomes among farm and nonfarm rural
Ecuadorians. Neurotoxicol Teratol 19:277-286.
Annual Report 2007-08

Determination of Urinary t,t-MA and S-Phenyl-Mercapturic Acid by Liquid

Chromatography Tandem Mass Spectrometer in Footwear
and Diamond Industry Workers
VN Gokani, PB Doctor, HS Trivedi, RR Teli and PR Mansuri

Abstract

The urinary t,t-MA and S-PMA are the specific and sensitive biomarkers for the determination of low levels of
benzene exposure. A sensitive LC-MS/MS method was developed for the simultaneous determination of t,t-
MA and S-PMA from human urine samples in a single run using dual column of two different ID to reduce the
matrix effect. The recovery of spiked standard of t,t-MA (40ng/ml) in urine was 95.4±12.3% ranging from
79.4-114% and for SPMA (20ng/ml) was 69.7 ± 9.5% ranging from 60.1 - 89%. The limit of quantitation
(LOQ) for t,t-MA was 5 ng/ml and that of SPMA was 0.03 ng/ml at the S/N ratio of 10. This method was applied
on spot urine samples of 26 exposed subjects of footwear industry and 52 subjects of diamond industry, and
12 control subjects. The observed values of t,t MA and SPMA in these subjects are below the values of
biological exposure indices (BEI) described by ACGIH of benzene exposure . However, both t,t- MA and SPMA
values in one exposed subject, and SPMA in two subjects of the foot wear industry were found higher than BEI.

Keywords: Trans, trans muconic acid, S- phenylmercapturic acid, benzene, LC MS/MS

Introduction
There is strong evidence that benzene is hematotoxic and causes
various types of leukemia. It has been classified as a group - I
1
carcinogen by IARC . Two minor urinary metabolites, trans, trans
muconic acid (t,t-MA) and s-phenylmercapturic acid (SPMA) are
specific biomarkers of benzene exposure and can be used to monitor
the benzene exposure in working environment2-7.
SPMA is considered to be a more specific biomarker than t,t MA
for low level exposures to benzene, because excretion of t,t MA might
be influenced by the uptake of sorbic acid (a widely used food
preservative). While SPMA is formed after conjugation of the highly
reactive benzene oxide with glutathione. Dietary or no other sources for
excretion of SPMA than exposure to benzene have been reported so far.
In this study, we have used a liquid chromatography /
electrospray tandem mass spectrometry method for the
determination of t,t-MA and SPMA in the same analytical run from the
urine samples. This method was applied to monitor benzene exposure
among child laborers of footwear industry of Agra and diamond
industry workers of Surat. Thefootwear industry is a significant
segment of the leather industry in India and ranks second among
footwear producing countries next to china. According to estimate, as
many as 25000 children between the age of 10 and 15 yrs are mainly
employed in shoe making both for the domestic and international
markets. Children work on soling (fixing upper portion of shoe to
leather or rubber soles) with glue. The children working are exposed to
benzene that is used as a solvent in glues. Most children suffer from
respiratory problems, lung diseases and skin infections through
constant exposure to glue and fumes.
Materials and Methods
Chemicals: t,t-MA and SPMA standards were procured from Sigma-
Aldrich, Methanol (MeOH), Acetonitrile (ACN) and HPLC water of LC
grade were obtained from JT Baker ltd, USA, Iso propanol and
disodium hydrogen phosphate (Merck (I) Ltd), Ethyl acetate,
Creatinine, Formic acid from Fluka, Acetic acid and Hydrochloric acid
were obtained from Across Organic, USA. Durapore Membrane filter
from Milipore were used for the fitering of mobile phase. QSAX, 100
mg/3cc cartridge for sample clean up and extraction of analytes was
used from Analchem Ltd, India.
Sample collection: Spot urine samples from randomly selected
30 exposed child labourers working in footwear manufacturing at
Agra, 15 matched control groups from school children studying in
government school located in the neighboring area, 60 workers

53
Annual Report 2007-08
engaged in the different processes (20-Planners, 20-Grinders, 10-
Markers, 10-Laser operators) of Diamond Manufacturing industries
at Surat were collected in polyethylene container in cool condition.
Frozen samples were transported in dry ice and preserved at -20 oC until
analysis.
Sample clean up and extraction of analytes from urine samples:
One ml urine sample of each subject was passed through a QSAX
cartridge (100 mg/3cc) that was preconditioned with 1ml MeOH and 1
ml 2mM phosphate buffer (pH 6.8). Urine sample was applied at slow
flow rate without drying the cartridge. Cartridge was rinsed with 1ml
HPLC water followed by 1ml 2mM phosphate buffer (pH 6.8) and 1ml
0.1% aqueous acetic acid under full vacuum for 1 min. The analytes
were eluted with 2x0.5 ml of 10% aqueous acetic acid. Final eluate
was transferred to 10 ml centrifuge tube. One ml ethyl acetate was
added to the eluate and vortex for 2-3 minutes. Upper organic layer
(0.5 ml) was separated into a vacelute tube and evaporated it to
dryness under nitrogen stream at room temperature. Dry residues
were reconstituted in 0.5 ml of mobile phase and 5.0 µl was injected
for LC-MS/MS analysis.
Liquid chromatography: Perkin Elmer series 200 system
equipped with quarternary pump, autosampler and peltier column
oven was used for the analytes separation. HPLC separation was
performed with dual column in series to minimize the matrix
suppression on ionization. Altima C18- 50 x 2.1 mm id, 3µm particle
size (Altech) and C18- 50 x 4.6 mm id, particle size 5 µm (Analchem
ltd.) were used at the flow rate of 400 µl/min. A phenomenex universal
guard column 4.0 mm Lx 3.0 mm id was used to protect the columns.
Gradient profile of mobile phase, started with 40% aqueous formic
acid (0.1%) and 60% methanol with 0.1% formic acid was held for 3
mins. It was then increased from 40% to 80% in 4 min. and held for
further 2 min. It was then reduced from 80% to 40% in 2 mins, where it
was held for 3 min. to wash the column. The total run time was 14 min.
Mass spectrometry: An API 3000 triple quadrupole mass
spectrometer from Applied Biosystem was used for MS/MS analysis.
The mass spectrometer was equipped with a turbospray (ESI)
interface. Mass and product ion spectra were acquired in negative
ionization mode.MRM transitions were monitored for quantitative
analysis of t,t-MA and SPMA. Mass to charged ion ratio (m/z) of 141
and 238 were selected as precursor ions (Q1) for t,t-MA and SPMA
respectively, while m/z 97 and 109 were selected as product ions (Q3).
For each analyte the compound dependent parameters like
Declustering potential (DP), Focusing potential (FP), Enterance
potential (EP), Collision energy (CE), and Collision exited potential
55
(CXP) were optimized by continuous infusion mode and the source
dependent parameters like Nebulising gas (NEB), Auxilliary gas
(heater gas), Curtain gas (CUR), Ion spray voltage, and source
temperature were optimized by flow injection analysis (FIA) mode.
Calibration curve: Calibration curves were prepared by
processing spiked urine sample of healthy, non-smoker individual.
Calibration standards were prepared by spiking urine to final
concentrations of 5.0, 10.0, 20.0, 25.0, 40.0, 50.0 and 100.0 ng/ml of
t,t-MA and SPMA mix prepared from reference stock standards.
Calibration curves were prepared by plotting peak areas versus
concentrations of standards. The calibration curves were generated
using linear regression analysis.
Limit of detection (LOD) and limit of quantitation (LOQ) : The LOD
and the lower LOQ were define as three and ten times respectively the
standard deviation of the LC-MS/MS peak areas detected at the
retention times of the analytes of interest in blank urine samples.
Recovery: The recovery of t,t-MA and SPMA was calculated by
comparing the area responses of extracted and non extracted
standard solutions containing t,t-MA at 40 ng/ml and SPMA at 20
ng/ml, each analyzed for six times.
Precision and accuracy: The inter-day and intra-day accuracy
and precision were determined from the analysis on three
independent QC samples tested on the same day and the five days of
the study. Precision is expressed as the relative standard deviation
(RSD) of the values found over the mean for each concentration (%
CV). FDA acceptance criteria for precision are RSD < 20%.
Matrix effect on ionization: Pot column infusion experiments
were performed to assess ion suppression. By using an infusion
pump, a continuous posts column infusion of standard solution was
introduced into the analytical LC system through a T-connector,
during injection of an extract of urine. Ion suppression of analyte's
signal by the urinary matrix was examined as negative
chromatographic peak from the elevated baseline.
Creatinine measurement: 24 hours creatinine clearance is a
measure of kidney function. Creatinine of spot or random urine
sample is a measure of concentration or dilution of the urine. Urinary
creatinine provides a good adjustment for the variations in
concentration of spot urine samples due to differences in fluid intake
among workers. Therefore creatinine of all the spot urine samples
was measured by the alkaline picrate method8. All the results were
expressed as t,t-MA and SPMA µg/g creatinine.
 Annual Report 2007-08 Annual Report 2007-08

Table 1 Urinary concentrations of t,t-MA and SPMA (µg/g creatinine) in footwear industry workers

Metabolite Exposed Controls

N Mean± SEM Range N Mean ± SEM Range

t,t -MA 61.16±11.86 5.61 -259.84 40.2 ± 8.05 13.91 -118.41

26 12
SPMA 9.41 ± 0.31 0.07 -91.7 0.51 ± 0.15 0.04 -1.83
N= number of samples

141.9 96.9 Table 2 Urinary concentrations of t,t-MA and SPMA in diamond industry workers
Type of Operations N Concentration of t,t MA and SPMA (µg/g creatinine)

t,t MA SPMA
Planning 18 24.62 ± 2.57 0.21 ± 0.04
(3.22 – 70.69) (0.02 – 0.81)
Grinding 14 36.44 ± 6.56 0.08 ± 0.01
238.3 101.1 (7.71 – 101.97) (0.01 – 0.27)
Marking 10 13.15 ± 2.92 0.07 ± 0.02
(ND – 18.89) (ND – 0.17)
Laser operation 10 17.03 ±  4.38 0.16 ± 0.04
(5.61 – 64.84) (0.04 – 0.47)
Figure 1: Extracted Ion chromatogram of t,t-MA and SPMA
Values are Mean ± SEM; Figures in parentheses indicate the range; N= number of samples

Results
Total run time of analysis was 14 minutes. t,t-MA was eluted at 3.1 min.
and SPMA was eluted at 5.3 min. The product ions mass spectrum for
t,t-MA and SPMA were recorded in negative ionization mode. The most
intensive fragment (product ion) for t,t-MA was detected at m/z 97,
while for SPMA it was detected at m/z 109. These transitions were
selected as the quantitative transitions for t,t-MA and SPMA. The
extracted ion chromatogram of t,t-MA and SPMA is shown in Figure 1.
The linearity of the calibration curve was determined over the
ranges 10-100 ng/ml and 5-50 ng/ml for t,t-MA and SPMA
2
respectively. For each analyte, the coefficient determination (r ) was
greater than 0.99. The detection limit for t,t-MA was 1 ng/ml and that
of SPMA was 0.03 ng/ml, while Limit of Quantitation for tt-MA was 5
ng/ml and for SPMA was 0.1 ng/ml at the S/N ratio of 3 and 10,
respectively.
The recovery (Mean ± SD) of spiked standard of t,t-MA (40ng/ml)
in urine was 95.4 ± 12.3% (n=6), ranging from 79.4-114% and for
SPMA (20ng/ml) it was 69.7 ± 9.5% (n=6) ranging from 60.1-89%.
The intraday and interday coefficient variation (% CV) were below 20
% for both t,t-MA and SPMA, which met the FDA acceptance criteria.
During the experiment of ion suppression effect of urine, the negative
peaks revealed that the matrix effect was not the same for all retention
times. Therefore, dual column of different diameter were used to set
the retention time of analytes and reduced the matrix effect on elution.
Quantitation of Urinary t,t-MA and SPMA in Footwear Industry:
Urinary concentration of t,t-MA and SPMA of exposed and control
workers are given in Table 1. Out of total 30 urine samples of exposed
workers, the creatinine values of 4 urine samples were found below
the normal range. In 26 samples of exposed subjects the
concentrations (Mean ± SEM) for t,t MA and SPMA were found 61.16 ±
11.86, ranging from 5.61 - 259. 84 µg/g creatinine and 9.41 ± 0.31,
ranging from 0.07 ± 91.7 µg/g creatinine, respectively. In control
workers, out of 15 urine samples the creatinine values of 3 samples
were found below the normal range. In 12 samples, the
concentrations (Mean ± SEM) for t,t- MA and SPMA were found 40.2 ±
8.05 µg/g creatinine, ranging from 13.91 to 118.41 and 0.51 ± 0.15
µg/g creatinine, ranging from 0.04 to 1.83 µg/g creatinine,
respectively.
Quantitation of urinary t,t-MA and SPMA in diamond Industry: Out
of total 60 samples, two urine samples of planner and 6 samples of
grinder did not process because of the lower values of creatinine than
the normal range. Urinary concentrations of t,t-MA and SPMA of
workers are given in Table 2. The concentration of SPMA was found
higher in planner and laser operators compared to grinders and
markers. According to survey carried out by NIOH, all other groups
except grinders and laser operators use solvents. Usually markers and
planners sit in the same room.

56 57

Discussion
A sensitive and specific LC-MS/MS method has been standardized for
the simultaneous measurements of two urinary metabolites, t,t-MA
and SPMA, which are known to be good biomarkers of low levels of
benzene exposure in humans. Previous studies have indicated that the
exposure level could influence the ratio of benzene metabolites. At low
levels of exposure to benzene, excretion of t,t-MA can be relatively high
even when SPMA excretion is very low9,10. Urinary SPMA in workers
exposed to 0.5-ppm benzene has been reported in the range from 7.2to
25 µg/g creatinine. Similarly, the predicted excretion of t,t-MA ranged
from 0.39 to 1.1 mg/g creatinine for 0.5 ppm benzene exposure11. As
t,t-MA is a metabolite of sorbic acid12 the values of t,t-MA in urine are
strongly influenced by the consumption of food or drinks containing
sorbic acid which is used as a preservative, while SPMA is a specific
biomarker of benzene exposure at low level.
It is observed by Wiesel et al13 that at relatively low benzene
exposure levels, the complexity of the urine matrix suppressed the
ionization. The suppression of monitored analytes by co-eluting matrix
compound was also observed in our method. Therefore, in HPLC
separation dual columns of different dimensions were used to reduce
the matrix suppression on analytes at their elution times.
In a study, among Chinese footwear workers, it was reported that
these workers were exposed to high levels of benzene, toluene and
other toxic solvents contained in the adhesive used in the shoe making
process and many workers have been affected with aplastic anemia,
leukemia and other health problems14.
The observed values of t,t MA and SPMA in our study are below the
values of Biological Exposure Indices (BEI) i.e. 500 µg/g creatinine for
t,t-MA and 25 µg/g creatinine for SPMA, described by ACGIH15 of
exposure to benzene. Except in one exposed subject in the footwear
industry the values of t,t-MA and SPMA, and in two subjects the values
of SPMA were found higher than BEI described by ACGIH. These results
also support that the diamond manufacturers have stopped the use of
benzene in diamond processing, as exposure is very low. Observed
values might be due to exposure through other sources of
environmental pollution.
References
1 IARC Monograph on the evaluation of carcinogenic risk to humans:
Occupational exposure in petroleum refining, crude oil and major
petroleum fuels, IARC; Lyons, France 1989:45.
2 Inoue O, Seiji K , Nakatsuka H, et al. Urinary trans, trans muconic
acid as an indicator of exposure to benzene. Br. J. Ind. Med. 1989;
46:122-127.
58
Annual Report 2007-08
3 Ghittori S, Maestri L, Rolandi, L. Lodola L, Fiorentino ML, Imbriani
M. The determination of t,t-MA in urine as an indicator of
exposure to benzene. Appl. Occ. Env. Hyg., 1996; 11:187-191
4 Ruppert T, Scherer G, Tricker AR, Adlkofer T. trans, trans-
muconic acid as a biomarker of non-occupational environmental
exposure to benzene. Int. Arch. Occup. Environ Health, 1997;
69:247-251.
5 Van Sittert NJ, Boogard PJ, Beulink GD. Application of the urinary
S- phenylmercapturic acid test as a biomarker of low level of
exposure to benzene in industry. Br. J. Ind. Med. 1993; 50:460-469.
6 Ghittori S, Imbriani, M Mestri L, Capodaglio E, cavallen A.
Determination of s-Phenaymercapturic acid in urine as an
indicator of exposure to benzene. Tox. Lett. 1999; 108: 329-34.
7 Melikian AA, Qu Q, Shore R. et al. Personal Exposure to different
levels of benzene and its relationships to the urinary metabolites
s-Phenylmercapturic acid and trans, trans- muconic acid. J
chromatogr. B Analyst Technol. Biomed. Life Sci. 2002; 778:
211-221.
8 Harold Varley Determination of creatinine in urine. Practical
Clinical Biochemistry, Vol 1, Allan H Gowenlock and Maurice Bill
th
(Eds.), 5 Edition, 1980; 484-485.
9 Subourin PJ, Bechtold WE, Griffit WC Bimbaum LS, Lucier and
Henderson RF. Effect of exposure concemntration, exposure rate
and route of administration on metabolism of benzene by F344
rats and B6C3F1 mice. Tox. Appl. Pharm.1989; 99: 421.
10 Henderson RF, Subourin PJ, Bechtold WE, Griffith WC, Medinsky
MA, Bimbaum LS and Lucier GW. The effect of dose, dose rate,
route of administration and species on tissue and blood levels of
benzene metabolites. Environ. Health Perspect. 1989; 82:9.
11 Melikian A, O'Concor, R, Prahalad AK , Hu P, LI H, K agan M
Thompson S. Determination of the urinary benzene metabolites
S-phenylmercapturic acid and trans,trans-muconic acid by
liquid chromatography/electrospray tandem mass
spectrometry. Carcinogenesis, 1999; 20 (4): 719-726.
12 Barbieri A, Sabatini L, Accorsi A, Roda A & saverio Violante F.
Simultaneous determination of t,t-muconic,Sphenylmercapturic
and S-benzylmercapturic acids in urine by a rapid and sensitive
liquid chromatography /electrospray tandem mass spectrometry
method. Rapid Communication in Mass spectrometry. 2004; 18:
1983-1988.
13 Weisel c, Y u R, Roy A, Georgopoulos P. Env. Hlth Persp. 1996;
104:1141.
14 Chen MS, Chan A China's “ Market economics in Command”
Footwear workers Health in Jeopardy. Int. J. Health Serv., 1999;
29 (4) : 793-811.
15 American conference of Governmental Industrial Hygienists.
Threshold Limit Values and Biological Exposure Indices for 2001,
American Conference of Govt Industrial Hygienists, Cincinnati,
OH, 2001.
Annual Report 2007-08
Occupational Work Stress Among Cycle Rickshaw Pullers
CK Pradhan, B Dalal, K Nayak and S Thakur
Abstract
The study was undertaken to evaluate the effect of rickshaw pulling on physiological and biochemical
parameters of cycle rickshaw pullers. Fifteen cycle rickshaw pullers and eight control subjects, in the age
group of 20-39 years were selected randomly from K olkata. Each subject participated for two consecutive
days for determination of maximal oxygen uptake (VO2max) and energy expenditure study for doing work in
three phases. Pre and post exercise blood samples were collected and analysed for biochemical parameters.
VO2max of the rickshaw pullers was significantly higher than those of the control subjects. Average working
heart rate, peak heart rate and energy expenditure of the cycle rickshaw pullers and the control subjects
showed that the workloads of both the groups are heavy to very heavy. The energy requirement was same for
both the groups while doing heavy work. Significant difference in glucose level was observed between
rickshaw pullers and control subjects at pre-exercise condition. The Lactate Dehydrogenase (LDH) in pre-
exercise condition of cycle rickshaw pullers was higher than normal range. Increasing trend of Creatine
K inase (CK ) was noted after exercise in pullers. Cycle rickshaw pulling is categorised as heavy to very heavy job.
Keywords: Rickshaw pullers, maximal heart rate, energy expenditure, creatine kinase
Introduction Approval of the ethical committee of the institution was obtained.
Written consent was obtained from all subjects prior to study.
A large population is engaged in pulling cycle rickshaw as occupation
in India and other South Asian countries. Strenuous physical activities Experimental Design: Each rickshaw puller participated for two
influence their physiological and metabolic responses. The workload consecutive days. On the first day, body height, weight and resting
1
of the cycle rickshaw pullers has been categorized as heavy . It is heart rate were recorded. Body surface area (BSA)6 and body mass
reported that significant fall in blood glucose takes place after heavy index (BMI) were calculated from the height and weight of the
2
exercise . Earlier study indicated that serum creatine kinase (CK ) subjects. Subsequently, the subjects were asked to pull rickshaw with
increased during first hours after exercise3. Heavy exercise increases two passengers for a period of 15 minutes according to their own habit
lactate production. Lactate dehydrogenase (LDH) activities enhances of pedalling frequency on the same route in order to follow a standard
after heavy physical exercise or submaximal exercise for a long road condition. After completion of this work, they are allowed to take
duration4,5. However, data on difference in physiological responses rest for 15 minutes. This phase of 30 minutes (work plus rest) was
considered as Phase I. Again the cycling was continued and
during various work phases and biochemical characteristics induced
designated as Phase II and Phase III. During pulling cycle rickshaw,
by cycle rickshaw pulling were not available. The objective of the
heart rates were monitored every minute by heart rate monitored
study is to evaluate the effect of rickshaw pulling on physiological and
(Polar, Finland). The average working heart rate (AWHR) was
biochemical parameters of cycle rickshaw pullers and compare the
calculated from the fourth minute of work7. The peak working heart
data with that of control subjects. rate of the subjects was recorded. Recovery heart rates were also
recorded every minute and sum of recovery heart rate was calculated.
Materials and Methods The energy expenditure for cycle rickshaw pulling was calculated
from the peak working heart rate (PWHR) using the equation of Datta
Subjects: Fifteen cycle rickshaw pullers in the age group of 20-39 and Ramanathan8. On the second day, the subject was allowed to do
years, were selected randomly. Eight control subjects (non rickshaw graded exercise in a bicycle ergometer (Biodex, USA) in the laboratory
pullers) of same age group were selected from the adjoining area of in order to determine maximal oxygen uptake (VO2max) and maximal
K olkata. They were engaged in jobs like gardening and farming. heart rate (HRmax).
59
Annual Report 2007-08
Control subject also participated in this study for two consecutive
days. On the first day, physical characteristics and resting heart were
noted. HRmax and VO2max were determined in the laboratory. Next
day, the subject was asked to pedal a bicycle ergometer (similar to
cycle rickshaw pullers) in three phases of 15 min of work and 15 min of
rest, each. The ergometer load was adjusted to produce heart rates in
the range of 80% to 90% of their maximal heart rate. PWHR was noted
and AWHR and recovery heart rates were also recorded every minute.
During experiments the dry bulb (DB) temperature was 30.6 ± 3.09 ° C
and wet bulb (WB) temperature was 27.2 ± 1.46 ° C. The VO2max study
was conducted in the laboratory in comfortable environment at DB
25.7 ± 1.54 ° C; WB 18.7 ± 2.16 ° C.
Biochemical analysis: In case of rickshaw pullers, on the first day,
2 ml of blood sample was collected from antecubital vein one hour
after completion of three phases of exercise. The state was
designated as post-exercise condition. Next day, blood was also
collected when the subject reported to the laboratory and was at rest
for one hour. This state was described as pre-exercise condition.The
control subjects, on the first day, were allowed to take rest for one hour
after reporting at the laboratory followed by collection of blood
samples (2 ml) from antecubital vein. This was pre-exercise condition.
Next day, it was collected after work (pedalling a bicycle ergometer).
This stage was defined as post exercise condition.
Each sample of blood was separated in two tubes: one containing
fluoride (1 ml) for glucose; and the other (1 ml) was allowed to clot for
serum separation. The following parameters were assayed by
Chemwell analyser (USA) using standard commercial kit following
methods of (i) Trinder9 for plasma glucose; (ii) Bakkar10 for Serum LDH;
(iii) Ellis11 for Serum CK ; (iv) Bowers12 for Alkaline Phosphatase and (v)
Bergmeyer13 for SGOT (vi) Fossati et al14 for Serum uric acid.
Results
The mean age and BMI of rickshaw pullers was 27.8 ± 5.95 years and
21.76 ± 3.12 kg m-2 and that of control subjects were 25.6 ± 5.53
years and 22.20 ± 3.56 kg m-2 respectively. There was no significant
difference in age as well as BMI. BMI values indicated that both the
groups belonged to normal category15,16.
The average HRmax of the rickshaw pullers was 187 ± 6.2 bpm
and it was significantly higher (p <0.05) than the value of control
subjects (179 ± 10.9). The VO2max of the rickshaw pullers (36.6 ±
-1 -1
4.32 ml.kg .min ) were significantly higher (p<0.001) compared to
control subjects (30.1 ± 3.14 ml.kg-1.min-1).
The heart rates during work varied from subject to subject in
different phases. The AWHR of rickshaw pullers in phase I, II and III
61
were 134 ± 12.4, 133 ± 14.9 and 134 ± 16.7 bpm respectively
suggesting that pulling cycle rickshaw is a job of heavy category17.
The mean PWHR of rickshaw pullers were found to be 145 ± 18.2
to 146 ± 14.9 bpm. This observation also substantiated that the job
18,19
was very heavy . As the sum of recovery heart rate (beats) did not
vary (Phase I-1250 ± 110.8, Phase II 1254 ± 117.4 and Phase III
1258 ± 130.8), it could be interpreted that the work stress imposed on
the rickshaw pullers were same in three phases. Repetition of work
has no effect on physiological functions. In case of the control subjects,
the values in three phases were 1339 ± 267.10, 1331 ± 248.50 and
1277 ± 251.98 beats respectively, which were not significantly
different from each other as also with those of rickshaw pullers.
The energy expenditure (EE) of rickshaw pullers in three phases
-1
were 21.7 ± 2.27.and 21.7 ± 2.82 and 21.3 ± 3.43 kJ.min
respectively and therefore the job of cycle rickshaw pulling can be
17,19
categorized as heavy . The results of the control subjects were
almost same with the rickshaw pullers.
Relative costs of work of the cycle rickshaw pullers and control
subjects as percentage of maximal energy expenditure have been
presented in Table 1. Result showed that there is an decreasing trend
of the values in rickshaw pullers compared to control subjects in all the
three phases of work. Significantly less value (p<0.05) was observed
in rickshaw pullers in phase I of work.
Table 1. Relative cost of work as percentage of maximal
energy expenditure
Work Control Subjects Rickshaw puller
Phase (n = 8) (n = 15)
Phase I 81.44 ± 9.58 74.18 ± 7.72*
Phase II  13.20
83.25 ± 74.26 ± 10.44
Phase III 79.88 ± 6.90 73.15 ± 12.35
The biochemical variables in pre-exercise condition were found
to be out of standard reference range, as manifested by elevated level
of LDH (53% cases in rickshaw pullers, 38% cases in control
subjects), plasma glucose (13% cases in rickshaw pullers, no cases in
control subjects), CK (33% cases in rickshaw pullers, 25% cases in
control subjects). Elevation of LDH was observed in 80% cases and
that of CK was noted in 60% cases as an effect of exercise in tested
population of rickshaw pullers. Among the control subjects, rise in LDH
was noted in 50% cases as an effect of work and elevated CK was
observed in 25% cases like pre-exercise condition.

Table 2 shows that effect of work on energy utilization among the cycle
rickshaw pullers and control subjects. The plasma glucose level was
found to be within normal range at both pre and post exercise session.
The level of serum LDH increased at post-exercise session in
comparison to that of pre-exercise condition among rickshaw pullers.
Similar trend was also noted in control subjects. Similarly, the level of
serum CK was also elevated after rickshaw pulling in comparison to
that at resting condition.
The effect of work on serum alkaline phosphatase (ALK P) and
serum aspartate transaminase (AST) is also given in Table 2. Pre and
post exercise values of ALK P were within normal range both in case of
rickshaw pullers and of control subjects. In case of AST, a significant
(p<0.001) difference was observed between control and rickshaw
pullers at both post and pre-exercise condition. In post exercise
condition, uric acid was noted above normal range in case of control
subject. On the other hand, uric acid was exhibited within the normal
range among rickshaw pullers at both pre- and post- exercise session.
Discussion
The study indicated that the present rickshaw pullers had similar work
capacity to that of those engaged in other occupations like steel mill
workers20, Indian soldiers21 and agricultural workers22. VO2max of the
rickshaw pullers were significantly higher than those of the control
subjects.
The job of cycle rickshaw pulling is considered as heavy to very
heavy based on peak working heart rate and energy expenditure17,19.
Data of different phases of work showed that there was no significant
Table 2. Effect of exercise on biochemical changes among control and cycle rickshaw pullers
Variable (Unit) Pre exercise
Control
(n=8)
Plasma Glucose (mg/dL) 69.3±12.16
Serum LDH (U/L) 373.1±76.02
Serum CK (U/L) 151.5±90.74
Serum ALK P (U/L) 243.63±68.17
Serum AST (U/L) 29.00±8.68
Serum uric acid (mg/dL) 5.69±1.78
Values are Mean ±SD for each group. Values in parenthesis indicate number of subjects
* = p<0.05, * * = p<0.01, * * * = p<0.001 (compared between control and rickshaw pullers
62
Annual Report 2007-08 Annual Report 2007-08
change in the mean values of different parameters in different References 14. Fosatti P , Precipe L, Berti G Use of 3,5-dichloro 2 hydroxy
phases. Energy requirement was same for both the groups while benzene sulpfonic acid/ aminophenazone choromogenic system
doing heavy work. Relative cost of work of the control subjects were 1. Pradhan CK , Thakur S, Mukherjee AK and Roy Chowdhury A: in direct enzymatic assay of uric acid in serum and urine. Clin.
significantly higher than those of the rickshaw pullers during work in Physiological assessment of cycle rickshaw pullers. Ind J Physiol Chem 1980; 26:227-231
phase I, suggesting the metabolic changes took place during course 15. World Health Organisation (WHO) Physical status: the use and
& Allied Sci.2004; 58 :113-118.
of work. interpretation of anthropometry. Technical Report Series No.
2. Mamus RT, Santos MGD, Campbell B and K reider R Biochemical
effects of carbohydrate supplementation in a simulated 854. Geneva : WHO 1995.
In both rickshaw pullers and control subjects, the glucose level
showed lower trend after post exercise session compared to that of competition of short terrestrial duathlon. J Int Soc Sports Nutrition 16. Weisell RC. 2002, Body mass index as an indicator of obesity. Asia
pre-exercise condition. The interesting observation noted was 2006; 3: 6-11. Pacific J Clin Nutr 2002; 11(Suppl) : S681 - S684.
significantly higher value (p<0.001) in post-exercise condition of cycle 3. Havas E, K omulainen J and Viko V Exercise induced increase in 17. Astrand PO and Rodahl K . Textbook of Work Physiology, 3rd edn.
rickshaw pullers compared to control subjects. Exercise induced rise serum creatine kinase is modified by subsequent bed rest. Int J New Y ork : Mc-Graw Hill Book Company; 1986.
in LDH was noted both in rickshaw pullers and control subjects. 18. Brouha L: Physiology in Industry. New Y ork : Pergamon Press.
Sports Medicine 1997; 18: 578-582.
Similarly the level of Serum CK was elevated after rickshaw pulling in
4. Costill DL, Fink WJ, Pollack ML Muscle fiber composition and 1960.
comparison to that of pre-exercise condition. Decreasing trend of CK
enzyme activities in elite distance runners. Med. Sci. Sport 1976; 19. Sen RN and Nag PK Work organization of heavy load handling in
at post exercise session was noted in control subjects. The findings
8 : 96 100. India. J Human Ergology 1975; 4, 103-113.
reveal that creatine phosphate, as coin of energy, during exercise was
recovered by CK in rickshaw pullers only. 5. Wu HJ, Chen K T, Shee BW, Chang HC, Y i-Jen Huang Y J, Rong-Sen 20. Saha PN. Aerobic capacity of steel workers in India. Ergonomics
Y ang RS Effects of 24 h ultra-marathon on biochemical and 1978; 21:1021-1025.
Liver function was assayed by estimating the ALK P and AST. In haematological parameters. World J Gastroenterol 2004; 10 : 21. Sengupta J, Joseph NT and Srinivasulu N Ergonomics studies of a
case of control subjects, AST and ALP level in pre and post exercise 2711-2714. digging tool. Ind J Physiol & Allied Sci. 1974; 28 : 22-28.
session maintain normal range. Higher than normal value of AST was
6. Banerjee S and Sen RN. Determination of the surface area of the 22. Nag PK and Pradhan CK : Ergonomics in the hoeing operation. Int J
observed among rickshaw pullers both at pre and post-exercise
condition. As the rickshaw pullers perform heavy work, the AST level, body of Indians. Journal of Applied Physiology 1955; 7: 585 - 588. Industrial Ergonomics 1992; 10: 341-350.
in pre and post-exercise session, might be maintained at normal level. 7. Nag PK , Sen RN and Ray US. Cardiorespiratory performance of
Analysis of serum uric acid revealed that heavy work induces oxidative porters carrying loads on a treadmill. Ergonomics 1979; 22 : 897-
stress in control subjects as manifested by increase in uric acid in post 907.
exercise session compared to pre exercise session whereas the 8. Datta SR and Ramanathan NL. Energy expenditure in work
rickshaw pullers habituated with this profession may not face such predicted from heart rate and pulmonary ventilation. J Appl
stress.
Physiol 1969; 26 : 297 - 302.
9. Trinder P Determination of Glucose in blood using glucose
oxidase with an alternative oxygen acceptor. Anal Clin. Biochem
Post exercise 1969; 6:24
Cycle Rickshaw Pullers Control Cycle Rickshaw 10. Bakker AJ, Bakker A, Bierma-Ram A, Dijkstra JT, Renting-Wiering
(n=15) (n=8) Pullers (n=15) H, Syperda H, Z ijlstra A. Improved reliability of measurement of
lactate dehydrogenase by IFCC method in heparin plasma. Clin
81.8* ±15.13* 61.1±10.32 75.4 ± 7.43* * *
Chem 2005; 51: 215-217.
** **
469.1 ±61.66 411.1±113.93 477.0 ± 58.20 11. Ellis G, Gold berg DM Automation of a kinetic spectrophotometric
182.9±69.96 133.9±69.57 202.9 ± 42.96
**
assay for ATP: creatine phosphotransferase activity suitable for
human serum and muscle extracts. Enzymologia 1972; 42:407-
227.00±150.58 236.25±82.93 222.37 ± 92.77 422.
*
44.50±6.28* * 29.88±14.28 44.10 ± 8.35 12. Bowers GN Jr., McComb RB. A continuous spectrophotometric
method for measuring the activity of serum alkaline phosphatase.
6.30±1.81 5.79±1.35 5.30 ± 1.23
Clin Chem 1966; 12:70-89.
13. Bergmeyer HU, Bowers GN, Horder M, Moss DW IFCC method for
aspartate aminotransferase. Clin Chim Acta 1976; 70:F31-40.
63
Annual Report 2007-08

Blood Methemoglobin Levels in the Dye-Stuff Workers

AB Patel, R Beniwal, BC Lakkad and VK Shivgotra

Abstract

A number of reports from the developed countries indicate that drugs, cosmetics or substances of abuse are
the commonest causes of acquired methemoglobinemia. There are very few reports of occupational
methemoglobinemia from these countries that may be due to better regulation of manufacturing processes. In
a developing country like India, the chemical industry is growing rapidly and is highly concentrated in some
parts of the country including Gujarat. The present study was done on 865 workers of various departments of
a chemical manufacturing unit from Gujarat, which is engaged mainly in the production of dye or dye
intermediates. A complete history was recorded with respect to duration and nature of occupation,
demographic data, clinical symptoms etc on predesigned proforma. Samples were collected for routine
analysis of urine and blood methemoglobin levels. The blood methemoglobin (MeHb) levels were not found to
change significantly with the increase in the duration of employment.

Keywords: Dye stuff-workers, dye and dye intermediates, blood methemoglobin

Introduction Materials and Methods

There are larger numbers of dye and dye intermediate units in India
and Gujarat accounts for about three fourths of them. These units are
engaged in the production or utilization of various chemicals
including aromatic amines like p-aminophenol, p-phenylenediamine,
chlorine derivatives of o- and p-toluidine, toluenediamine,
diaminodiphenylmethane and p-nitroaniline. Various pathological
effects by these chemicals have been documented most frequent
being methemoglobinemia.
Hemoglobin can accept and transport O2 only when the iron
atom is in its ferrous form. When hemoglobin becomes oxidized,
3+
it is converted to the ferric state (Fe ) or methemoglobin (MeHb)
resulting in to a condition called methemoglobinemia in which
there is inhibition of binding and delivery of oxygen by a red blood
cell1. MeHb lacks the electron that is needed to form a bond with
oxygen and thus is incapable of O2 transport. Exposure to
chemicals such as certain dyes and dyestuff intermediates with
nitrate and nitrite groups may cause rise in MeHb levels. This
condition is mainly caused by the intentional or non-intentional
exposure of oxidizing agents such as nitrates and nitrites
2
including occupational exposures . This study was conducted to
assess the blood MeHb levels in the dyestuff workers. The levels
were correlated with the duration of employment.
Subjects: A total of 865 subjects enrolled for the study from an
industry in Gujarat manufacturing and exporting over 500 chemical
products including dyes and dye intermediates bulk drugs and
intermediates, cresol and cresol derivatives, epoxy resins and epoxy
hardeners, formaldehyde etc. were included in the study. These
workers were employed in different departments and worked in
three shifts during a day. A written consent was obtained from each
subject. A complete history was recorded with respect to duration
and nature of occupation, demographic data, etc. on a predesigned
proforma.
Analysis of methemoglobin (MeHb) from blood: The blood
samples of the workers were collected from antecubital vein in the
vacutainer tubes containing EDTA. The MeHb levels from the blood
samples were analyzed immediately by the method of Evelyn and
Mallay3 as modified by Henry4. Hemoglobin estimation was carried out
5
by using the Drabkins method and the levels of MeHb were expressed
as % of hemoglobin. 54/837 subjects with non-detectable levels of
blood MeHb were excluded from statistical analysis.
Routine analysis of urine: Analysis of urine samples for routine
parameters including bilirubin, urobilinogen, glucose, ketone, urinary
blood and proteins were carried out by using the reagent strips
(Multistix SG, Bayer Ltd).

65
 Annual Report 2007-08 Annual Report 2007-08

Necessary ethical clearance was obtained from the Institutional Discussion
Ethical Committee for the study. Statistical Significance for difference
In the present study, certain cases showed microscopic hematuria but
among the groups was analyzed by using one-way ANOVA.
it did not show any significant difference with respect to the duration of
employment. When blood in urine is not visible by a naked eye but
visible only under the microscope is called microscopic hematuria.
The potential causes of this condition includes urinary tract infection,
bladder or kidney stone, enlarged prostrate, glomerulo-nephritis,
cancer of kidney, bladder or prostrate and strenuous exercise.
Exposure to exogenous oxidizing drugs (benzocaine,
trimethoprim, sulphonamides etc) and chemicals such as certain dyes
and dyestuff intermediates with nitrate and nitrite groups (like aniline
dyes, nitrobenzene, nitroaniline etc) may accelerate the rate of
formation of methemoglobin up to one-thousandfold, overwhelming
the protective enzyme systems and acutely increasing
methemoglobin levels. Though blood MeHb is one of the good
biomarkers to assess the exposures of the workers of the dyestuff
industry. The study did not show any significant change in the blood
MeHb levels of the workers indicating that the workers are not
significantly exposed to methemoglobinemia inducing compounds.
Table 1: Blood methemoglobin levels of the workers in relation to their
total period of employment Acknowledgment: Ms SK Patel, Mr BS Solanki and Ms H Ahir are
Duration of acknowledged for their assistance in the work.
No. of Blood MeHb Range
employment
(yrs) workers (% of Hb) * (%) of Hb References
# 0.09 -2.78
<10 72 0.88  0.082 1. Wright RO, Lewander WJ, Woolf AD: Methemoglobinemia:
Environmental and Biological Monitoring of Chromium Exposure
Near Dumping Sites of Non-Recyclable Waste by
Chromium Based Industry
LJ Bhagia, NG Sathawara, RA Rathod, JB Vyas, MI Shaikh, P Sekhar
Abstract
The aim of this study was to evaluate total chromium content in biological (blood, urine and hair) and
environmental (soil, air and water) samples collected from dumping and control sites using Flame Atomic
Absorption spectroscopy (AAS) technique. The chromium levels in air, water and hair samples of both control
and exposed sites/subjects were within normal limits. Chromium levels in soil samples were found to be much
higher in exposed (dumping) sites as compared to control sites but lower than the normal values reported for
residential localities. In exposed subjects the concentrations were higher (statistically not significant) than
those in control subjects. Nevertheless, higher values observed in blood and urine samples might be due to
combined exposure to chromium from soil and food.
Keywords: Chromium exposure, Biological and Environmental Samples, AAS
Introduction Collection of biological and environmental samples: About four
ml venous blood samples of 634 subjects (373 exposed and 261
Chromium is a naturally occurring element found in rocks, animals,
control), 349 hair samples (269 exposed and 80 control) from nape of
plants, soil, and in volcanic dust and gases1. Chromium exists in the the neck region and 707 urine samples (412 exposed and 295 control)
environment primarily in two valence states 1) trivalent (chromium 3+ ), were collected and analyzed for chromium using standard methods
11-

# 0.02 -3.15 etiology, pharmacology and clinical management Ann. Emerg. which is biologically active, occur naturally in environment and in food 13
. Similarly, 35 drinking water samples (8 control and 27 exposed) and
10 to <20 281 0.83  0.041
#
Med 1999; 34: 646-56. and 2) hexavalent (chromium 6+ ), is a toxic2 are generally produced 34 soil samples (11 control and 23 exposed) were also collected using
20 to <30 199 0.79  0.051 0.04 -3.44 from industrial pollution and anthropogenic activities3,4. Chromium (III) standard methods. A total of 122 air samples were collected using
# 0.02 -3.20 2. Dewan A, Patel AB and Saiyed HN: Acute methemoglobinemia- A in trace amount is an essential nutrient. In the chemical industry,
> 30 225 0.80  0.045 both high volume sampler (outdoor) and personal sampler (indoor)
common occupational hazard in an industrial city in western chromium is used primarily in pigments, metal finishing, leather
0.02 -3.44 from various locations. 25 air samples (8 control and 17 exposed)
Total 777 0.82  0.025 India. J. Occup. Hlth 2001; 43(3): 168-171. tanning and wood preservatives. Chromium pigments are used in were collected on glass fiber filter paper EPM 2000 (24 cm X 20 cm)
* The values are Mean  SEMs; paints, inks and plastic coloring. Workers employed in such industries 3
using PM10 high volume sampler at the rate of 1.1 m /min for 8 hrs. A
# F (3, 776)=0.38, p> 0.05
3. Evelyn K A and Malloy HT: Micro-determination of oxyhemoglobin, may have a higher chance of exposure to chromium than the general
methemoglobin and sulfhemoglobin in a single sample of blood. total of 97 samples (29 control and 68 exposed) were collected on
population. Breathing high levels of chromium (VI) can cause irritation membrane filter (37mm, 0.8mm pore size) and in 1M HNO3 (analytical
Results J. Biol. Chem 1938; 126: 655-662. to the nose, such as runny nose, nosebleeds, and ulcers and holes in
grade) using personal sampler (SK C) at flow rate of 2 LPM for 8hr/day.
the nasal septum. Ingesting large amounts of chromium (VI) can cause
The occupational groups with limited numbers and those with higher 4. Henry R, Cannon DC, Winkleman JW: Clinical Chemistry, stomach upsets and ulcers, convulsions, kidney and liver damage,
Principles and Practice. New Y ork, Harper and Row, 1974; 1 Pre-treatment / Digestion of samples:
mean age are excluded from further analysis. Urine examination and even death . Epidemiological studies have shown that workers
showed microscopic hematuria among 4.17 % (35/837) of the pp1149. occupationally exposed to chromium (VI) are considered to be at risk Biological samples: 2ml of whole blood was digested in wet digestion
workers with no significant difference among different departments of for developing lung cancer5-10. system (Ethios 1600, advanced Microwave lab station, Italy) using a
the industry. 5. Drabkin DL, Austin JH: Spectrophotometric studies: Preparation mixture of 2ml of conc. HNO3 (ultra pure) and 0.2ml H2O2. The digested
from washed blood cells; nitric oxide hemoglobin and Materials and Methods sample was diluted and make up the volume for 4ml with triple
Blood MeHb levels did not change significantly (F=0.38, p > 0.05) sulfhemoglobin. J Biol Chem 1935; 112: 51-65 distilled water and centrifuged14.
with the increase in the duration of employment (Table 1). No Selection of sites: The exposure sites were selected where dumping of
significant change was found in blood MeHb of workers working in non-recyclable waste was done. Control sites were selected 5 Hair samples were washed twice with acetone and triple distilled
different departments. kilometers away from the exposure sites. water and dried in dust free oven at 50 º C. Approx 0.2 g hair sample

66 67
 Annual Report 2007-08 Annual Report 2007-08

was digested with 2ml conc. HNO3 (ultra pure), 1 ml triple distilled water Table 1: Mean chromium level of environmental samples Biological Samples: The mean blood Cr level of the exposed 8. Mancuso TF and Hueper WC. Occupational cancer and other
and 0.2ml of H2O2 by wet digestion (Ethios 1600, advanced Microwave subjects (4.34 ± 0.21 mg/dL) was higher as compared to control health hazards in a chromate plant: A medical appraisal. I. Lung
Samples Exposed Control
lab station, Italy) and make up the volume for 4ml with triple distilled subjects (3.71 ± 0.18 mg/dL) (Table 2) and values reported by ATSDR1 cancer in chromate workers. Ind. Med. Surg. 1951; 20: 358-363.
water11,15,16. Indoor 
(i.e. 2.0 to 3.0 mg/100ml). The mean urine Cr concentration of exposed
(Filter Paper 0.26  0.03 0.2  0.05 9. Mancuso TF. International Conference on Heavy Metals in the
(34) (8) subjects (27.93 ± 0.82 mg/L) was slightly higher than that of control Environment. Toronto, Ontario, Canada. 1975; (Cited in U.S. EPA,
Air media)
Environmental Samples: Approx. 2g sieved and dried soil samples
(mg/m3) Indoor  0.09 subjects i.e. 21.12±0.51 mg/L (Table2). Assuming that urine excreted 1991; and in U.S. EPA, 1984a)
were digested using 4ml of conc. HNO3 (ultra pure) and 5ml of distilled 0.63   0.06 0.5 
(HNO3 in (21) per day to be one liter, the values for both control and exposed 10. Sano T. and Mitohara I. Occupational cancer among chromium
water on a hot plate at about 60-70 º C. The samples were evaporated to (34)
bubbler) subjects are higher than the value recommended by ATSDR workers. Jap. J. Chest Disorders 1978;37: 90-101 (Cited in U.S.
dryness and the final volume was made up to 10ml with 1M HNO3 Outdoor 0.07   0.01 0.018   0.002 (10mg/day)1. Cr from soil in exposed sites may be responsible for high EPA, 1984a)
solution (ultra pure). (High volume (17) (8) concentrations of Cr in blood and urine of exposed subjects. But high 11. Harrison, WW, Y urachek JP and Benson CA: The determination of
Water sampler)
Cr concentration in urine and blood samples of subjects from control trace elements in human hair by atomic absorption spectroscopy.
The collected air samples were wet digested using 4ml of conc. ( mg/L) Tube well 30.53  4.98 4.36  0.53
(8) sites cannot be explained from lower values of Cr observed in soil Clin. Chem. Acta, 1969; 23: 83-91
HNO3 (ultra pure) and 5 ml of distilled water on a hot plate at about (10)
samples from control sites. It seems that Cr from food also might
60-70 º C and the filtrate was made to 4ml volume using 1M HNO3 (ultra Municipality 6.43  0.55 12. Petering HG, Y eager DW and Wither SO: UP Trace metal content of
contribute to Cr concentration in blood and urine samples.
pure). (17) hair\ -II Cadmium and lead of human hair in relation to age and
Soil 234.77  54.22 33.37   3.68 sex. Arch. Environ Hlth., 1993; 27: 327- 330
The mean hair chromium level of exposed subjects (0.68 ± 0.06
Calibration/analysis: All the digested samples were analyzed by ( mg/g) (23) (11) 13. Jenkins, DW. Toxic trace metals in mammalian hair and nails.
mg/g) was slightly higher as compared to control subjects (0.53 ± 0.08
flame Atomic Absorption Spectrophotometer (Perkin Elmer, USA, Values expressed as Mean SE, 1979; EPA-600/4-79-049
mg/g). The populations with no known exposure to chromium
double beam with background corrector, model 3100) at the Values in parenthesis indicate number of samples
reportedly have hair levels ranging from 50-100 ppm1. 14. Sathawara NG, Patel K G, Vyas JB,et al. Chromium exposure study
wavelength 283.3 nm. The method was standardized by preparing
Table:2: Mean chromium level of biological samples in chemical based industry. J. Environ. Biol. 2007; 28(2): 405-
solutions of different standard chromium concentrations like 0.5,1 and
References 408
4 ppm. Samples Exposed Control
Blood 15. Buckey, RA, Chem. HCA and Dreosti, IE. Radio-isotopic studies
4.34  0.21 3.71  0.18
1. Agency for Toxic Substances and Disease Registry (ATSDR). concerning the efficacy of standard washing producers for the
(g/dL) (373) (261)
Chromium Toxicity. U.S. Department of Health and Human cleansing of hair before zinc analysis. Amer. J.Clin. Nutr., 1984;
Hair 0.68  0.06 0.53  0.08 40:840-846.
Services, Atlanta, G.A. 2000
(g/g) (269) (80) 2. US EPA (US Environmental Protection Agency). Health 16. Y ung-Y ung T, Chin- Thin W, Wei-Tun C, Chin-Wang H.
Urine 27.93  0.82 21.12  0.51 Assessment Document for Chromium. Environmental Criteria Determination of Trace Elements in Hair Specimens of Workers in
(g/L) (412) (295) and Assessment Office, US Environmental Protection Agency, Paint Factory. Analytical Science 1997; Vol. 13 Supplement
Research Triangle Park, NC. EPA 600/8-83-014F. NTIS PB 85- 17. Canadian Soil Quality Guidelines for the Protection of
Values expressed as Mean  SE,
Values in parenthesis indicate number of sample 115905; 1984b. Environmental and Human Health, 1997; Cepg-
3. World Health Organization. Chromium Environmental health rcqe.ccmc.ca/download/en/262
The mean soil Cr levels in the exposed samples 234.77±54.22 mg/g
Criteria 61. Geneva, Switzerland. 1988 18. ACGIH: Threshold Limit Values for chemical substances and
were much higher than control samples 33.37±3.68 mg/g. Canadian
Soil Quality Guidelines for the Protection of Environmental and Human 4. Hertel RF. Sources of exposure and biological effects of physical agents and biological exposure indices, Cincinnati,
Health17, reported that normal soil samples have Cr concentration of chromium. In: Environmental Carcinogens Selected Methods of 2008.
64 mg/g. Samples collected from control sites have concentrations Analysis, International Agency for Research on Cancer, Lyon, 19. Mertz, W. Chromium-Occurrence and function in biological
lower than 64 mg/g whereas exposed sites had much higher 1986; 8:79-92. IARC Scientific Publication No. 71. systems. Phys. 1969; Rev 49,163-239.
concentration. High levels of Cr in soil of exposed sites were expected 5. Leonard A. and Lauwerys RR. Carcinogenicity and mutagenicity
because of dumping of non-recyclable waste of the industry. of chromium. Mutat. Res. 1980; 76: 227-239
Results and Discussion 6. Langard S. The carcinogenicity of chromium compounds in man
Table 1 depicts the mean chromium levels of indoor air
and animals. In: Burrows, C., Ed. Chromium: Metabolism and
Environmental Samples: The mean chromium level was found to be samples taken by personal samplers in the exposed and control
higher in water from exposed sites in both tube well water Toxicity. CRC Press, Inc., Boca Raton, FL, 1983; 13-30.
sites. The Cr concentrations in indoor air samples are much lower
(30.53±4.98mg/L) and municipality water (6.43±0.55mg/L) as than TLV18 of 0.5 mg/m3. The mean chromium levels of outdoor air 7. Mackison FW, Stricoff RS and Partridge LJ. Occupational Health
compared to control samples (4.36 ±0.53mg/L) (Table 1). However 3
samples in the control site was 0.018±0.002 mg/m while that at Guidelines for Chemical Hazards. National Institute for
these values were lower than the prescribed standards i.e. 100 mg/L exposed site was 0.07±0.01 mg/m3. There is no specific standard Occupational Safety and Health, DHHS (NIOSH) Washington, DC.
in exposed as well as control samples1. for Cr in community environment .
1
1981; Pub. # 81-123.

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Annual Report 2007-08

On-line Simultaneous Detection of Tri- and Hexa-valent Chromium

in Urine by Ion Chromatography
SSA Zaidi, SJ Gandhi and KA Patel
Abstract

Online separation, post-column derivatization and spectrophotometric determination of chromium species

(chromium III and chromium VI) in human urine were carried out by ion chromatography. Study groups
included healthy volunteers control, and occupationally exposed workers (welders, spray painters and coach
manufacturers). Spot urine samples during the work-shift were collected and analysed within 24 hrs. Prior to
analysis pH of all the urine samples was adjusted to 6.7 to 6.8. Calibration graph for chromium III and
chromium VI revealed coefficient determination 0.998 and 0.994 with RSD 3.979 and 9.213%, respectively.
The average level of Cr III (mean + S.D.) in control group (N=20) was found to be 37.54 + 21.63 µg/l or ppb.
Chromium VI was also detected in 6 of the 20 control subjects and the values were 0.414 + 0.194 ppb.
Chromium III content in welders (N=17), spray painters (N=13) and coach manufacturers (N=11) were
registered to be 56.83 + 37.32, 34.06 + 19.55, and 92. 35 + 53.47 ppb, respectively. Chromium VI content in
welders (N=15) and coach manufacturers (N=8) was measured to be 0.58 ± 0.39, and 0.35 ± 0.22 ppb
respectively. Only 2 of the 13 spray painters exhibited chromium VI levels in their urine, and the values were
0.19 and 2.18 ppb. The level of Cr III was significantly higher (p<0.01) in coach manufacturers and welders
(p<0.05) as compared to control.
Keywords: Trivalent and hexavalent chromium, ion chromatography, welders, spray painters

Introduction
India is one of the third largest producers of chromium compounds
and contributes about 10% of the total world's production. People may
also be exposed to higher levels of chromium if living near to the
landfill sites with chromium-containing wastes, industrial facilities
that manufacture or use chromium and chromium-containing
compounds, cement-producing plants, industrial cooling towers that
previously used chromium as a rust inhibitor, waterways that receive
industrial discharges from electroplating, leather tanning and textile
industries and busy roadways. Breathing in higher levels of chromium
VI (greater than 2 µg/m3) can cause irritation to the nose, such as runny
nose, sneezing, itching, nosebleeds, ulcers, and holes in the nasal
septum. Long-term exposure to chromium has been associated with
lung cancer. Breathing in chromium III does not cause irritation to the
nose or mouth in most people. In the same way, small amounts of
chromium VI that swallowed will not hurt, however, accidental or
intentional swallowing of larger amounts has caused stomach upsets
and ulcers, convulsions, kidney and liver damage, and even death.
The concentration of total chromium in air (both Cr III and Cr VI)
generally ranges between 0.01 and 0.03 µg/m3. Chromium
concentrations in drinking water (mostly as Cr III) are generally very
low, less than 2 ppb. A wide variation in the urinary level of chromium
was reported, however for unexposed population it ranges > 1-2 µg/L
or ppb. NIOSH considers all chromium VI compounds (including chromic
acid) to be potential occupational carcinogens and recommends an
exposure limit of 1 µg chromium (VI)/m3 for a 10-hour workday, 40-hour
workweek. Worldwide reports are very scanty on the excretion of
chromium in unexposed population and occupationally exposed
workers. Even recent reports of ATSDR 2008 do not provide much
information on the excretion of chromium species in urine of unexposed
and chromium workers. In one of the recent studies1 conducted in 2004
by “ Eco Friends” in a village of Noraiakheda, K anpur, North India
poisoning of ground water was reported 100 to 1000 times higher than
the desirable limits for drinking water. The concentration of hexavalent
chromium has been reported up to 16.3 mg/l that is 1630 times more
than the WHO standards2 (0.01 mg/l) for drinking water at a depth of 140
ft in this village. With the aims of chromium toxicity background and
health effects and the scarcity of the data in context of present Indian
scenario it is urgently needed to measure chromium levels in our Indian
environment. Total chromium has been reported by few investigators,
however, chromium species i.e. Cr III and Cr VI level have hardly been
attempted by researchers earlier. The present study describes the
levels of dissolved chromium compounds (Cr III and Cr VI) in urine using
ionchromatography in healthy control and occupationally exposed
workers.

71
 Annual Report 2007-08 Annual Report 2007-08

Materials and Methods Statistical analysis: Instrument IC 2500 is equipped with

Chromeleon software (6.70 version programme) for automated
Sample preparation: Spot urine samples about 25 to 50 ml were data processing and quantifying the analytes. This facility was
collected in fresh 60 ml vial without using any preservatives. Fresh used during this study.
vials were washed with 0.1 M HNO3 followed by RO water and then + 3
1 - Cr - 3.517 2 - Cr
+ 6
- 5.592
finally with ultra pure water (Millipore). All urine samples were passed
Results
through 0.45 µm syringe filter (ACRODISC® ). After adjusting pH of the
samples to 6.7-6.8, 1 ml of the each filtered urine sample was applied US EPA method 218.6 as modified by Dionex in its Technical Note
on Dionex ion-chromatograph. Due to the relatively low levels of No. 24 was employed to determine chromium III and chromium VI
biological matrix in urine compared to blood, urine samples were
in urine samples. We, very first time employed this method for the
directly applied.
simultaneous detection of trivalent chromium and hexavalent
Chemicals: All the chemicals and solvents used in this study were chromium in urine. It provides a beautiful mean of separating tri-
of high purity grade. Milli Q water of 18 mega-ohms was used and hexavalent-chromium from the other species and from the
throughout the study. Reference standards of chromium III and other biological constituents present in the sample. This method
chromium VI, were purchased from Fluka (Germany). Other chemicals allows sufficient sensitivity to determine chromium (Cr III) as 3+ 6+
Figure 1: Chromatogram of standards (trivalent Cr ; 2 ppm) and hexavalent (Cr ; 10 ppb)
used in this study were of highly purified grade (more than 99.5 % chromic ion (Cr3+ ) and Cr VI as chromate (CrO42-) ion. After on-line
pure). Dionex Ion-chromatograph IC 2500 was used for the simultaneous separation of Cr III and Cr VI, these chromium
chromatographic studies. species are allowed to react with post column reagent,
diphenylcarbazide (DPC) to form derivatives, which are read
Assay Conditions spectrophotometrically at 520 nm employing PDA-100 detector.
Columns CG5A Guard column
On-line simultaneous separation and detection of Cr III and Cr
CS5A Analytical column VI is shown in Figure 1. Figure 2 shows absorption spectra. Figure
Eluent 2 mM PDCA 3 indicates five-point calibration graph. Coefficient determination
for Cr III and Cr VI were 0.998 and 0.994 with RSD 3.979 and
2 mM Na 2HPO4
9.213 %, respectively. Good reproducibility of the results with
10 mM NaI consistent retention time was obtained (Figure 4). The above
50 mM CH 3COONH4 calibration of chromium III and chromium VI was used for the
quantification of chromium species.
2.8 mM LiOH
Flow rate 1.0 ml/min Urinary Levels of Chromium III and Chromium VI in study
Post - column reagent 2 mM DPC, 10% CH 3 OH population: The levels of chromium III and chromium VI are
0.9 N H 2 SO4 summarized in Table 1. The average level of Cr III (Mean + S.D.) in
Reagent flow rate 0.5 ml/min control group (N=20) was found to be 37.54 + 21.63 µg/l or ppb.
Mixing device Reaction Coil Chromium VI was also detected in 6 of the 20 control subjects and
Detector wavelength 520 nm the values were 0.414 + 0.194 ppb. Chromium III content in
Sample loop volume 500 μL welders (N=17), spray painters (N=13) and coach manufacturers
(N= 11) were registered to be 56.83 + 37.32, 34.06 + 19.55 and
Calibration curve: A mixture of standards (Cr III and Cr VI) at varying 92. 35 + 53.47 ppb respectively. Chromium VI content in welders
concentrations as mentioned below was prepared to obtain a five points (N=15) and coach manufacturers (N=8) was measured to be 0.58
calibration curve. Solutions were prepared fresh each time of calibration. ± 0.39, and 0.35 ± 0.22 ppb respectively. Only 2 of the 13 spray
Concentrations were: A) 100 ppb Cr+ 3 + 0.5 ppb Cr+ 6, B) 200 ppb Cr+ 3 + painters exhibited chromium VI levels in their urine, and the
+ 6 + 3 + 6 + 3
1.0 ppb Cr , C) 400 ppb Cr + 2.0 ppb Cr , D) 1000 ppb Cr + 5.0 ppb values were 0.19 and 2.18 ppb. Some of the occupational workers
+ 6 + 3 + 6 + 3 + 6
Cr , E) 2000 ppb Cr + 10 ppb Cr , F) 5000 ppb Cr + 25 ppb Cr . One in each group did not show either Cr III or Cr VI content in their
ml of the above standard mixture of solution was injected and the urine. The level of Cr III was significantly higher (p<0.01) in coach Figure 2: Spectral representation of standards: Chromium III and Chromium VI
instrument received 500 mL as injecting volume of sample/ standard. manufacturers and welders (p<0.05) as compared to control.

72 73
 Annual Report 2007-08 Annual Report 2007-08

Table 1. Determination of Cr III and Cr* VI in urine by ion chromatography

4.00 Cr+3 External UV_VIS_1 4.00 Cr+6 External UV_VIS_1 Parameter Control Welders Spray painters Coach manufacturers
Area [mAU*min] Area [mAU*min]

Subject studied 20 17 21 13
3.00 3.00 Cr 3+ (ppb) 37.54 + 21.63 56.83 + 37.3 # 34.06 + 19.55 92.35 + 53.47 @
Detected (N=20) (N=17) (N=13) (N=11)

2.00 2.00 Range (8.5 - 46.70) (21.90 -152.24) (10.18 -77.75) (31.9 -225.8)
Cr 6+ (ppb) 0.414 + 0.196 0.58 + 0.397 0.194 & 2.17 0.35 + 0.22

1.00
Detected (N=15) (N=15) (N=2) (N=8)
1.00
Range (0.008 -1.43) -- (0.099-0.675)
* pH of all urine samples was adjusted to 6.7-6.8 ; Value represents Mean + SD; Figure in parenthesis indicate range
# p<0.05 when compared with control group; @ p<0.01 when compared with control group
ppb 0.00 ppb
0.00
0.0 5.0 10.0 15.0 20.0 25.0 30.0
0 1,000 2,000 3,000 4,000 5,000 6,000 chromium III and chromium VI were found quite high as compared to
2 - CALIBRATION OF CR+3 AND CR+6 AN 7 AUGUST 2008 SCALE #9
1 - Cr+3 2 - Cr+6
std Cr+3 1ppm + Cr+6 5 ppb EXT541NM
other studies reported from abroad. However, our results correlate
[A] Chromium III [B] Chromium VI 0.57
mAU WVL:520 nm
with the previous results reported earlier from this Institute from the
Figure 3: Calibration curve of standard chromium. [ A] Chromium III; [ B] Chromium VI 0.38
same place of this study. Higher levels may reflect indiscriminate
disposal of chromium waste and poor hygiene system and

1-2.417
0.25
occupational condition besides many other factors that may affect
exposure and measurement. Several methods, as summarised by

3-Cr+3-3.842
0.12

ATSDR3 are available for the analysis of chromium in different

. ,

2-3.033
1 - CALIBRATION OF CR+3 AND CR+6 AN 7 AUGUST 2008 SCALE #3 std Cr+3 100 ppb+Cr+6 0.5 ppb UV_VIS_1 3.17 CALIBRATION OF CR+3 AND CR+6 AN 7 AUGUST 2008 SCALE #36 Urine sample44 dil 1:1 UV_VIS_1 -0.00
2 - CALIBRATION OF CR+3 AND CR+6 AN 7 AUGUST 2008 SCALE #4 std Cr+3 200 pp+Cr+6 1 ppb UV_VIS_1 mAU WVL:520 nm
3 - CALIBRATION OF CR+3 AND CR+6 AN 7 AUGUST 2008 SCALE #8 std Cr+3 400 ppb+ Cr+6 2 ppb UV_VIS_1 biological media and many of them are approved by federal agencies

4-Cr+6-5.692
4 - CALIBRATION OF CR+3 AND CR+6 AN 7 AUGUST 2008 SCALE #9 std Cr+3 1ppm + Cr+6 5 ppb UV_VIS_1 -0.13

5 - CALIBRATION OF CR+3 AND CR+6

6 - CALIBRATION OF CR+3 AND CR+6
AN 7 AUGUST 2008
AN 7 AUGUST 2008
SCALE #5
SCALE #6
std Cr+3 2ppm+Cr+6 10 ppb
std Cr+3 5ppm+Cr+6 25 ppb
UV_VIS_1
UV_VIS_1 2.50
and organisation such as EPA and NIOSH. Earlier methods described
23.5 33 -0.25
2.3
mAU WVL:520 nm 1-
total concentration of chromium in environmental and biological
2.00 -0.38
samples, which seems not to be appropriate from the toxicological
1.50
-0.49
-0.8 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
min
10.3
implementation point of view, as Cr III is nutritionally required by the
3.4
92 - 5.5
67 body while Cr VI is highly toxic and carcinogenic. Therefore it is of
10.0
r+3
-
-C
r+6 00 Figure 6: Overlay of urine sample with standard trivalent
1 -C 2 1.00
-C
r+3
- 3.7
50
particular interest to explore toxicity associated with Cr VI. Earlier it
2
6 - 5.4 chromium (1 ppm) and hexavalent chromium (5 ppb)
3-
Cr+
was difficult to distinguish Cr III and Cr VI but now methods are
0.50
0.0 available to quantify both forms of chromium species simultaneously.
-0.00 Therefore, estimation of Cr III and Cr VI is now gaining popularity to
accuracy. Decline or rise in pH affects the estimation. Therefore
assess chromium toxicity and health effects associated with
chromium in this study was estimated at pH 6.8. All urine samples
particular forms of chromium.
-13.1 min
-0.82 min were adjusted to p H 6.7 to 6.8 prior to the analysis and no sample
-0.4 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.2 -0.4 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.2
treatment was given. Some urine samples were diluted wherever
Figure 4: Standard chromatogram of Cr III and Cr VI showing Figure 5: Chromatogram of urine sample of study subject required. Results of this study indicated that chromium III was References
reproducibility of retention time in overlay of standards indicating presence of trivalent and hexavalent chromium detected in all control and exposed subjects. Concomitant level of
1. Eco Friends; Environmental Education Protection and Security:
hexavalent chromium in control group was detected to excrete
http:/www.ecofriends.org/reports /043Groundwater.htm
Discussion level of analytes. Analytical column CS5A has unique ability to chromium VI in urine, however it presumed that chromium when
separate anionic and cationic species in single run of column and entered the body is detoxified to chromium III that is excreted from the 2. WHO, 1988 World Health Organisation. Environmental Health
Automated on-line separation, derivatization and detection of both species were found well separated by the standard procedure body. But we provide evidence that concomitant excretion of Criteria 61: Chromium. Geneva: WHO 197.
chromium species (chromium III and chromium VI) by Dionex ion- used in this study. Though chromium species are highly pH hexavalent chromium does also appear in the urine (Figures 5 & 6),
chromatograph IC 2500 provide excellent opportunity to separate dependent and are inter-convertible; at acidic pH, chromium III which has not been reported earlier. Available reports indicate that 3. ATSDR 2002. ATSDR Tox Profiles: U.S. Department of Health &
chromium III and chromium VI species which is not possible in many of dominates while alkaline pH favours chromium VI form. However at excretion of hexavalent chromium in urine is rare and has almost not Human Services, Agency for Toxic Substances and Disease
the sophisticated instruments used to quantify chromium at ultra low pH 6.8 both forms of chromium could be eluted with high degree of been described earlier. This needs further investigations. Levels of Registry, Division of Toxicology, U.S.A.

74 75
 Annual Report 2007-08 Annual Report 2007-08

Chemically Induced Methemoglobinemia from Acute Poison Information Centreof the Institute for identification and the electron that is needed to form a bond with oxygen and thus is
blood samples for MeHb analysis. From the absorption spectra incapable of O2 transport. This condition is mainly caused by the
Nitrobenzene Poisoning (Unknown liquid and Nitrobenzene, pure were diluted 1:14,000 and 1: intentional or non-intentional exposure of oxidizing agents such as
AB Patel, A Dewan, KJ Upadhyay,* SA Patel * and JK Patel* 30,000 respectively with ethanol 99.9% for scanning) which showed nitrates and nitrites including occupational exposures6.
wavelength peak of 258 nm matching with the pure nitrobenzene
Abstract (Figure 1) and from its typical bitter-almond smell; it was identified as Although not completely understood, reduced nitrobenzene
nitrobenzene. metabolites are believed to be responsible for nitrobenzene-induced
Methemoglobinemia is an unusual and potentially fatal condition in which hemoglobin is oxidized to methemoglobinemia. Studies with laboratory animals demonstrated
methemoglobin lowering its ability to bind and transport oxygen. The most common cause of Serum and RBC cholinesterase levels were within normal limits that orally administered nitrobenzene is reduced in the intestine and
methemoglobinemia is the ingestion or inhalation of oxidizing agents such as nitrates and nitrites. A rare case excluding the involvement of organophosphate pesticide. The MeHb that intestinal microfloral metabolism is essential for the production of
of nitrobenzene poisoning after oral ingestion which presented with altered sensorium and cyanosis is levels from the blood samples were analyzed on the same day and the methemoglobin7. We have observed extremely high levels of
reported. Timely identification of the compound and the estimation methemoglobin (MeHb) were helpful in levels were found to be 66.7 % of Hb (normal: 0-3 % of Hb). On methemoglobin (66.7%) and these levels reduced near to normal after
saving the patient. The importance of laboratory support of a poison information center in evading death due confirmation of chemically induced methemoglobinemia, he was a dose of methylene blue (100 mg, i.v.). The levels again rose after 18
administered 100 mg methylene blue i.v. over 10 minutes at 16:10 hrs of antidote administration. Delayed release of nitrobenzene from
to unknown poisoning has been highlighted.
hrs. His MeHb level reduced to 5.4 % after 1 hour of methylene blue stores in the adipose tissue and gastrointestinal tract is commonly
Keywords: Nitrobenzene poisoning, methemoglobinemia, poison information centre administration and gained consciousness at 22:00 hrs. Serial MeHb seen after severe poisoning8. This delayed rise in the MeHb levels may
levels at different time intervals are shown in Figure 2. He was be attributed to the release of nitrobenzene.
absolutely asymptomatic at the time of discharge.
Acknowledgment: We are thankful to K S Shah, GB Jingar, AA
Introduction RBC cholinesterase levels determined by spectrophotometry on a RA- Hemoglobin can accept and transport O2 only when the iron atom Pandya, SK Patel and BS Solanki for the technical assistance and help
50 chemistry analyzer by modified Ellman's were method using acetyl during the study.
is in its ferrous form. When hemoglobin becomes oxidized, it is
Suicide is a major cause of premature mortality globally and 113914 thiocholine as substrate3. The MeHb levels from the blood samples converted to the ferric state (Fe3+ ) or methemoglobin resulting in to a
suicides are recorded annually from India1 for which variety of were analyze by the method of Evelyn and Mallay as modified by References
condition called methemoglobinemia in which there is inhibition of
chemicals are used. The treatment of poisoning caused by an unknown Henry4. Analysis of the unknown liquid, cholinesterase and serial 5
binding and delivery of oxygen by a red blood cell . MeHb lacks the
compound is a challenge to a treating physician and the situation estimations of blood MeHb were performed at poison Information 1. Gunnell D, Eddleston M, Phillips MR, K onradsen F. The global
becomes graver when the laboratory support for certain specific poison center of the institute. distribution of fatal pesticide self-poisoning: Systematic review.
is unavailable. Nitrobenzene, an aromatic nitro-compound, occurs as a 4
BMC Public Health. 2007;7: 357.
pale yellow moderately water-soluble oily liquid with an odor Results and Discussion 2. ATSDR (Agency for Toxic Substances and Disease Registry).
resembling that of bitter almonds. It is used in the manufacture of 3 Toxicological profile for nitrobenzene. Prepared by Life System,
aniline, benzidine, quinoline, azobenzene, rubber chemicals, A worker (20 yrs) associated with screen printing work, ingested an 258nm Inc. under subcontract to Clement Associates, Inc., for ATSDR, US
pharmaceuticals and dyes2. It is also used as a solvent in shoe and unknown liquid used for screen printing in an intentional poisonin, had 2 Public Health Service under contract 205-88-0608. ATSDR/TP-
Nitobenzene,pure
metal polishes and in screen-printing. Its toxic effects are due to its vomiting and epigastric burning sensation after about 40 minutes of

Abs
90-19,1990.
ability to induce methemoglobinemia. A case study of acute ingestion was admitted to the Civil Hospital, Ahmedabad for 1 Unknown Liquid (Poison) 3. Ellman GL, Courtney K D, Andres V Jr, Featherstone RH. A new and
nitrobenzene poisoning in a suicidal attempt is discussed. treatment. Physical examination of fingers and tongue revealed a rapid colorimetric determination of acetylcholinesterase activity.
cyanosis with poor respiratory effort; pulse rate 120/min; blood 0
Ethanol,pure(base Line) Biochem Pharmacol. 1961;7:88-95.
Materials and Methods pressure 100/70 mm of mercury and pupils bilaterally dilated. On 200 250 300 350 350 400 4. Henry R, Cannon DC, Winkleman JW. Clinical Chemistry,
admission, the hemoglobin level was 13.7 g/dl (normal: 14-18 g/dl) Wavelength(nm) Principles and Practice. New Y ork, Harper and Row, 1974;
A worker (20 yrs) associated with screen printing work, ingested an and total leucocyte count (20,000/cu mm) showe polymorphonuclear Fig.1: Absorption spectra of the unknown liquid used by the patient for ingestion
pp1149.
unknown liquid used for screen printing in an intentional poisoning at leucocytosis. There was no jaundice and toxicity profile for cannabis, 5. Wright RO, Lewander WJ, Woolf AD. Methemoglobinemia:
MeHb levels (% of total Hb) 80
around 6:00 hours in the morning. He was admitted to the Civil Hospital, morphine, cocaine, amphatamine, benzodiazepine and 70 66.7 etiology, pharmacology and clinical management. Ann.
Ahmedabad at 12:00 hours. Physical examination; routine phenobarbitone gave negative results. Routine urine examination, 60
Emergency Med. 1999; 34: 646-56.
ECG, X-ray chest and biochemical profile which included SGPT, RBS, 50
examination; routine hematological and urine examination, blood gas 6. Dewan A, Patel AB, Saiyed HN. Acute methemoglobinemia- A
40
analysis,ECG X-ray chest were performed on admission. The blood blood urea, serum billirubin, sodium and potassium were within 30 common occupational hazard in an industrial city in western India.
samples were analyzed for routine biochemical investigations which normal limits. 20 J Occ Hlth. 2001;43,168-71
15.8
included blood Hb, SGPT, RBS, blood urea, serum-billirubin,-sodium 10
5.4 6 7. Goldstein RS, Chism JP, Sherill JM, Hamm Jr. TE. Influence of
Arterial blood gas analysis showed pH 7.34, PaO2 70.6 mm of 0 1.63
and-potassium and urine for drug screening. Unknown pale liquid used dietary pectin on intestinal microfloral metabolism and toxicity to
11h10m6h50m 28h 33h30m 53h30m
for ingestion by the patient was scanned through UV-Visible mercury, PaCO2 31.8 mm of mercury, HCO3 16.4 meq/l. The patient nitrobenzene. Toxicol Appl Pharmacol. 1984;75: 547-553.
Duration after ingestion (h:m)
wavelengths on a spectrophotometer (Cary-100, Varian). Serum and was given oxygen with venti-mask at the rate of 6-8 liters/min. The Fig.2: Methemoglobin levels during first 55 hours after nitrobenzene ingestion
8. Gupta G, Poddar B, Salaria M, Parmar V. Indian Pediatrics. 2000;
*
B J Medical College, Civil Hospital. Ahmedabad unknown pale yellow liquid consumed for suicide was sent to the 37: 1147-1148.

76 77
 Annual Report 2007-08 Annual Report 2007-08

Assessment of Human Exposure to PCBs Through Biological Monitoring

VK Bhatnagar, N Shukla, VK Shivgotra, SSA Zaidi, R Kashyap, S Kumar

Abstract

The blood samples from the general population of Ahmedabad (urban, n=70; and rural, n=80) and fatty food
samples (fish, n=50 and chicken, n=50) were analysed for the congeners of polychlorinated biphenyls. Sum
of CB congeners (∑CB) in Ahmedabad (urban) and Ahmedabad (rural) had a mean of 5.78 ng/mL and 2.69
ng/mL respectively. There was significant positive correlation between age and CB 118 (r=0.46; p<0.05) and
age and ∑CB (r=0.28; p <0.05). Average mean value of ∑CB in fish and chicken samples was 15.58 and
15.47 ng/gm-wet tissue respectively.

Key words: PCB, congeners, human blood, biological monitoring

Introduction Extracts are eluted with hexane through deactivated silica gel
in adsorption chromatographic colum. Fish and chicken samples
Polychlorinated phenyls (PCBs) are the complex mixtures of chlorinated
were extracted following conventional procedures. Analyte was
biphenyls that vary in degree of chlorination. These are used as
dissolved in appropriate volume of n-hexane and suitable volume
coolants and lubricants in electrical equipment such as capacitors and
injected into GC Agilent 6890N equipped with micro-ECD. GC
transformers due to their general inertness and heat stability. These are
conditions were: Capillary Column (J& W Scientific; 122-1262; DB-
also used in rubber, synthetic resin, ink and paint industries.
XLB, 60 m X 0.25 mm X 0.25 m nominal). Oven temp programming
However, upon searching the existing database on the residue level
of these contaminants in our country, we find paucity of information. was set to 150 0C for 1 min, then with 2 0C/min to 250 0C, kept for 20
Therefore, the present study has been designed with the following min. N2 was used as carrier gas (1.1 ml/min). The injector (mode: Figure 1: Standard Chromatogram of PCB's congeners on GC-ECD
0 0
objectives: split-less) and detector temp kept at 270 C and 300 C respectively.
 Evaluate the levels of PCBs congeners in blood samples of general Standard chromatogram of various CB congeners has been
population of Ahmedabad (urban and rural settings); and displayed in Figure 1. Correlation coefficient of various CB
 Determine the levels of PCBs congeners in fatty food samples congeners at level (0.5, 1, 5, 10, 25, 50, 100 pg/mL) was observed
(chicken and fish) collected from the local markets of Ahmedabad. 0.99. Recovery experiments carried out by spiking serum sample
with mixture of various congeners (# 16) at 0.1 ppb level and %
Materials and Methods recovery ranged from 54% to 163%. Statistical calculation was
performed using SPSS 16.0 for window.
The representative human blood samples from the general
population Ahmedabad (urban area; n=70 and rural area; n=80) have
been collected. It was ensured that the subjects have no
occupational exposure to pesticides and other persistent pollutants.
The objective of the study explained and informed consent received.
Subjects were requested to provide information on their
demographics, dietary habits and smoking status. These samples
were extracted and clean up for the analysis of PCBs congeners. The
representative fish samples (n=50), and chicken samples (n=50)
collected from the various markets of Ahmedabad. Serum samples
were extracted following the procedure as mentioned by Burse et al1.
Serum sample denatured with methanol and extracted with n- Figure 2: Lavels of PCB congeners in human blood of Ahmedabad (Urban) Figure 3: Lavels of PCB congeners in human blood of Ahmedabad (Rural)
hexane-diethyl ether (1:1).

78 79

Figure 4: Levels of PCB congeners in fish samples
Results and Discussion
Data in Figure 2 reflect the presence of various congeners in blood
samples (n=70) collected from Ahmedabad (urban). ΣCB (sum of
all congeners) ranged from 0.10 to 38.24 ng/mL with a
mean of 5.78 ng/mL in urban samples. CB 199 was not detected in
these serum samples. Serum Level of CB congeners in samples
(n=80) collected from Ahmedabad (rural) has been depicted in Figure
3. ΣCB in rural serum samples was 2.69 ng/mL, which ranged from
0.15 to 13.3 ng/mL. However, the congeners e.g. CB 46, CB 105, CB
167 and CB 199 were not detected in rural samples.
Fifty samples each of fish and chicken were analyzed for CB
congeners and levels have been depicted in Figure 3 and 4
respectively. Overall mean of Σ CB in fish and chicken was 15.58
ng/gm (Range: 6.42 - 31.57 ng/gm) and 15.47 ng/gm (Range: 2.51
30.15 ng/gm). CB 138, CB 180 and CB 170 were present in all fish
samples (100%), whereas CB 28 and CB 74 in 49 fish samples (98%)
and CB 153 in 48 fish samples (96%). CB 167 and CB 199 were not
present in fish samples. Similarly, the congeners e.g. CB 153 were
present in 49 chicken samples (98%) followed by CB 28, CB 138 and CB
170 in 48 chicken samples (96%). Three congeners e.g. CB 46, CB 99
and CB 199 were not detected in chicken samples.
Conclusion
Overall mean of Σ CB in fish and chicken was 15.58 ng/gm (Range:
6.42 - 31.57 ng/gm) and 15.47 ng/gm (Range: 2.51 30.15 ng/gm).
80
Annual Report 2007-08
Figure 5: Levels of PCB congeners in chicken samples
CB 138, CB 180 and CB 170 were present in all fish samples (100%),
whereas CB 28 and CB 74 in 49 fish samples (98%) and CB 153 in 48
fish samples (96%). Sum of CB congeners (∑CB) in Ahmedabad
(urban) and Ahmedabad (rural) had a mean of 5.78 ng/mL and 2.69
ng/mL respectively. There was significant positive correlation
between age and CB 118 (r=0.46; p<0.05) and age and ∑CB (r=0.28;
p <0.05). Average mean value of ∑ CB in fish and chicken samples was
15.58 and 15.47 ng/gm-wet tissue respectively.
Acknowledgment: We are thankful to MR Variya, Y ogesh Shah, SN
Y adav, Ashok Parmar, NA Suthar and CM Makwana for their
technical assistance.
Reference
1. Burse VW, K orver MP, Needham LL, et al. (1989) Gas
chromatographic determination of polychlorinated biphenyls (as
Aroclor 1254) in serum. Collaborative study. J Assoc Anal Chem
72, 649-659.
Annual Report 2007-08
Cancer in North East India - Understanding the Role of Pesticides
VK Bhatnagar, U Mishra, MR Variya, VK Shivgotra,
P Sivaperumal and R Kashyap
Abstract
The serum samples of breast cancer and NHL subjects and control subjects have been received from various
cancer registries of NE region. The blood serum samples have been analyzed for the residues of DDT, HCH and
Endosulfan and metabolites. These serum samples (n=201) have reflected the presence of pp'-DDE. Analysis
of serum samples of control subjects is in progress.
Keywords: Organochlorine pesticides, serum, brest and NHL Cancer
Introduction
North Eastern (NE) states of the country have reported high incidence
of cancer of all anatomical associated with use of tobacco and some
sites, which have been related to pesticide exposures. NE region has
diverse ethic groups with different customs, food habits and different
life style. Moreover, there is extensive use of pesticides in tea gardens
in NE region that can lead to widespread occupational and
environmental exposures. A multi-institutional project involving RMRC
- Dibrugarh; NIOH - Ahmedabad; IOP - New Delhi; ICPO - Noida; NCRP-
Bangalore; and six population based cancer registries of NE region
located at Aizal, Guwahati, Gangtok, Imphal, Dibrugarh and Silchar
have been attempted to envisage the link between the magnitude of
exposure, causative factor and genetic variants involved.
Results and Discussion
Materials and Methods
The concentration of different organochlorine pesticide residues
detected in the breast cancer and NHL subjects has been given in
A total of blood serum samples (n=201) of breast cancer and Non-
Tables 2 & 3 respectively. Preliminary analysis of serum samples of
Hodgkin's lymphoma subjects from various cancer registries of NE
breast cancer and NHL subjects have reflected the presence of pp-
region have been received. Serum samples have been processed for
1
DDE, pp-DDT and -HCH in 96%, 78% and 29% of the samples
extraction and clean up following the procedure of Dale et al for the
respectively. The analysis of control subjects (n=125) and remaining
analysis of persistent pesticide residues (-HCH, -HCH, -HCH, -
serum samples is in progress.
HCH, op'-DDE, pp'-DDE, op'-DDD, pp'-DDD, op'-DDT, pp'-DDT, -
Endosulfan, -Endosulfan, Endosulfan Sulfate and Dicofol). Final
Acknowledgment: We are thankful to Y ogesh Shah, SN Y adav, Tejal
analysis was done on Shimadzu GC- 2010 equipped with ECD. GC
Manvar, CM Makwana and Chander Parmar for the technical
conditions were: Capillary column 60 m x 0.2 m x 0.2 mm; Oven
0 0 0 0 0 assistance.
100 C, 20 C/min 200 C hold 25 min; 1 C/min to 240 C; Injector
Temp. 270 0C (spilt mode); Detector Temp-300 0C; Carrier gas (N2) Flow
rate = 1.5 ml/min. For the purpose of validation of method for analysis Reference
of pesticide residues, the data on linearity, line of regression,
1. Dale WE, Curley A, Cueto C Jr. 1966. Hexane extractable
correlation coefficient, and coefficient of determination at a
chlorinated pesticides in human blood. Life Sciences 5: 47-54.
concentration (Range: 1-100 pg) is given in Table 1.
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 Annual Report 2007-08 Annual Report 2007-08

Table 1: Regression equation, correlation coefficient and determination of coefficient of various analytes Table 3: Levels of organochlorine pesticides in NHL cancer subjects from NE Region (ng/mL)

Compounds Concentration (pg) Regression Equation Correlation Coefficient of n -HCH -HCH -HCH -HCH pp'-DDE pp'-DDT -Endo
Centre -DDT -Endo
Coefficient (r) determination (r 2 )
-HCH 1, 5, 10, 25, 50, 100 -1169.44+ 1119.85* x 0.99933 0.99865 7.97 (1)*
7.97 114.68 54.13 168.81
Aizwal 01 ND ND ND ND
(1)* (1)* (1)* (1)*
-HCH 1, 5, 10, 25, 50, 100 1220.50+ 1316.03* x 0.99986 0.99973
1.82
1.82 18.94  8 24.94 
-HCH 1, 5, 10, 25, 50, 100 2625.07+ 1218.55* x 0.99723 0.99447 Dibrugarh 04 ND ND (1)* 5.89 4.11 8.54 ND
(1)* ND
-HCH 1, 5, 10, 25, 50, 100 -16 08.18+ 1143.82* x 0.99972 0.99945 (4)* (3)* (4)*
2.81  2.85  19.81  7.92  26.59  1.1 1.1
- Endosulfan 1, 5, 10, 25, 50, 100 796.13+ 1609.84* x 0.99988 0.99977 3.65
Guwahati 57 0.52 ND 0.50 3.05 1.39 4.22
(1)* (1)* (1)*
- Endosulfan 1, 5, 10, 25, 50, 100 381.51+ 1616.74* x 0.99989 0.99979 (23)* (24)* (56)* (48)* (56)*
Endosulfan - 5230.98+ 936.72* x 1.19 1.19 29.24  12.71  36.87 
1, 5, 10, 25, 50, 100 0.99857 0.99714 ND (1)*
Sulphate Imphal 15 (1)* ND 5.90 2.00 7.57 ND ND
(15)* (9)* (15)*
op' -DDE 1, 5, 10, 25, 50, 100 -704.79+ 1834.58* x 0.99988 0.99975
21.95  3.48  25.43 
pp' -DDE 1, 5, 10, 25, 50, 100 -1254.13+ 2408.38* x 0.99957 0.99914 11.41
Silchar 04 ND ND ND ND 10.84 0.79 ND ND
op' -DDD 1, 5, 10, 25, 50, 100 953.76+ 1438.72* x 0.99977 0.99953 (4)* (4)* (4)*
pp' -DDD 1, 5, 10, 25, 50, 100 516.88+ 1520.97* x 0.99986 0.99972 ND=Not detected; * Number of positive samples

op' -D DT 1, 5, 10, 25, 50, 100 1074.49+ 1692.76* x 0.99988 0.99976

pp' -DDT 1, 5, 10, 25, 50, 100 -826.15+ 1335.59* x 0.99995 0.9999
Dicofol 1, 5, 10, 25, 50, 100 -420.28+ 769.00* x 0.99996 0.99991

Table 2. Levels of organochlorine pesticides in breast cancer subjects from NE Region (ng/mL)
Centre n -HCH -HCH  -HCH -HCH pp'-DDE pp'-DDT - DDT
16 2.83 ± 0.85 1.32 1.12 3.32 ± 0.90 15.57 ± 3.68 11.23 ± 2.60 26.05 ± 6.12
Dibrugarh
(5)* (1)* (1)* (5)* (15)* (14)* (15)*
15 2.69 ± 0.51 2.69 ± 0.51 8 ± 3.17 4.99 ± 2.373 9.92 ± 4.21
Gangtok ND ND
(8)* (8)* (13)* (5)* (13)*
65 1.64 ± 0.12 1.25 1.61 ± 0.11 15.88 ± 1.91 11.06 ± 2.05 24.87 ± 3.44
Guwahati ND
(12)* (1)* (13)* (64)* (52)* (64)*
1.59 ± 0.19 1.59 ± 0.19 26.49 ± 7.31 11.66 ± 4.83 32.62 ± 10.66
Imphal 09 ND ND
(3)* (3)* (8)* (7)* (9)*

15 2.05 ± 0.44 2.05 ± 0.44 18.01± 5.66 8.97 ± 1.94 26.98 ± 7.41
Silchar ND ND
(2)* (2)* (12)* (12)* (12)*
ND=not detected; * Number of positive samples

82 83
 Annual Report 2007-08 Annual Report 2007-08

Cancer in North East India: Understanding the Role of Tobacco ice to the NIOH for the estimation of cotinine - metabolite of nicotine, Chromatographic Conditions:
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its
PB Doctor, S Sedha, HS Trivedi, RR Teli and PR Mansuri Glucoronides (NNAL-Gluc) - metabolites of NNK , 1-hydroxypyrene - HPLC Perkin Elmer Series 200 with Quaternary
metabolite of pyrene, exposure marker of PAHs, t,t-MA - metabolite of pump, Auto sampler and Peltier Column Oven
benzene and 8-OHdG as a DNA damage marker by HPLC and LC- Column X Terra MS C 18, Length 100mm,
Abstract
MS/MS techniques. ID 2.1, 5 µm Particle size
Determination of cotinine provides a useful tool for estimating uptake of nicotine and tobacco related Aliquots of urine samples were prepared in sterile centrifuge Guard Column X Terra 20 x 2.1 mm
toxicants. We measured the level of cotinine in urine samples of tobacco related cancer cases as well as tubes for each analytes and sediments of each sample were sent to
controls received from the six cancer registries of North East region of India. The urinary cotinine levels were Institute of Cytology and Preventive Oncology (ICPO) for further Column Oven 40 ° C
measured by liquid chromatography coupled with triple quadrupole tandem mass spectrometer (LC/MS/MS- investigation.
API 3000). Urine samples to which deuterium labeled internal standard had been added were extracted with Flow Rate 300 µl
solid phase extraction (SPE) procedure using Drug test 1 SPE cartridge. Instrumental linearity/calibration A total of 953 urine samples of cancer cases and 432 urine
samples of controls were received. Out of 953 samples of cancer Injection Volume 10µl
curves were prepared by processing various concentrations i.e. 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 100.0,
cases, 695 urine samples of tobacco related and 258 samples of
150.0 and 200.0 ng/ml (ppb) of cotinine, each containing 25ng/ml of deuterated internal standard (Cotinine pesticide related cancer cases were received. Mobile Phase A: 5mM Ammonium Formate
d3). The linear correlation coefficient (r2) was 0.9998 for cotinine. The recovery percentage (Mean±SD) of (Gradient) B: Acetonitrile
cotinine was obtained in the range of 88.42 % ± 2.9 % to 91.36 % ± 1.33 % with less than 4% RSD. Cotinine Measurement of Creatinine: Creatinine of spot or random urine C: HPLC water
was measured in 406 urine samples (295 male and 111 female subjects) of cancer cases and 185 samples sample is a measure of concentration or dilution of the urine. Urinary
(87 male and 98 female subjects) of controls. creatinine provides a good adjustment for the variations in
concentration of spot urine samples due to differences in fluid intake. Instrumental linearity/calibration curves: Instrumental
Therefore, creatinine of all the spot urine samples was measured by linearity/calibration curves were prepared by processing various
Key words: LC-MS/MS, Tobacco exposure, cotinine, cancer cases concentrations i.e. 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 100.0, 150.0
the Alkaline Picrate (Jaffe's) Method7.
and 200.0 ng/ml (ppb) of cotinine, each containing 25 ng/ml of
Estimation of Cotinine from Urine Samples by LC-MS/MS: deuterated internal standard (Cotinine d3) prepared from reference
Introduction Materials and Methods Measurement of human urinary cotinine level is a practical approach stock standards in mobile phase. Calibration standards were
for obtaining important information about tobacco exposure. Cotinine prepared by spiking 2ml of blank urine with suitable amounts of
The consumption of tobacco products is responsible for a significant This study is a case control epidemiological design at selected six different concentrations. All the samples were cleaned up and
is the major metabolite of nicotine and most widely used and reliable
portion of human cancer at various sites, in particular lungs, oral cavity, cancer registries of the North East Region of India. All incidence cases extracted by SPE. The peak areas of analyte were plotted versus the
biomarker of tobacco exposure.
larynx, oesophagus, stomach etc.1 In India about 60 to 70% of cancers of tobacco related cancers of the esophagus, lung, stomach and oral original concentrations and evaluated by linear least square
in males and 30 to 40% cancers in females have been associated to cavity and pesticide related Breast and Non-Hodgkin's Lymphoma regression analysis using Analyst, Version 1.4.1. The acceptable value
Extraction of Urine Samples: Drug test 1 mixed mode solid phase
tobacco consumption. Tobacco products contain a diverse array of (NHL) cancers, diagnosed and confirmed by microscopy between 1st extractions columns from Anal Chem (I) Ltd, Allahabad were used for of the correlation coefficient (r2) should be equal to or more than 0.99.
chemical carcinogens which are responsible for these cancers. More February, 2006 to 19th June, 2008 from cancer registries located at the clean up and extraction of analyte from urine samples.
than 60 known carcinogens have been identified among constituents of Civil Hospital Aizwal, Regional Institute of Medical Sciences, Imphal, Instrumental limit of detection (LOD) and limit of quantification
tobacco smoke. These include polynuclear aromatic hydrocarbons BB Baruah Cancer Hospital, Guwahati, Silchar Medical College and Mass Spectrometry: Optimization of compound and source (LOQ): LOD & LOQ were defined as three and ten times the standard
(PAHs), aromatic amines, aldehydes, alkenes, metals and N-nitroso Hospital, Silchar, Sir TNM Hospital, Gangtok and Assam Medical dependent parameters: For analysis of urinary cotinine the MS deviation of the LC-MS/MS peak areas detected at the retention times
compounds. The most prevalent N-nitroso compounds in tobacco College, Dibrugarh have been included. A specially designed analysis was performed with API 3000 triple Quadrupole Mass of the analyte of interest in blank urine sample (S/N ratio ³3 and ³10
smoke and unburned tobacco products are the so called “ tobacco questionnaire has been used to record tobacco habits and spectrometer (Applied Biosystems/ MDS Sciex) equipped with respectively). Recovery was evaluated by adding five different
specific nitrosamines” (TSNAs)2-4. They are formed from nicotine and occupational history.
the other tobacco alkaloides during fermentation of tobacco. Two of the electrospray ionization interface. The electronization interface was concentrations- 10, 50, 100, 200, 500 ng/ml of cotinine in the urine of
TSNA, 4-(Methylnitrosamine)-1-(3-pyridyl) 1-butanone (NNK ) and 4- operated in positive ionization mode with an ion spray voltage of 5000 non-tobacco user. Five replicates of spiked urine samples were
Sample Collection: Collection of the biological samples is K V. Full scan mass spectra were recorded in order to select the most extracted by SPE and same number of concentrations of cotinine
(Methylnitrosamine)-1-(3-pyridyl) 1-butanol (NNN) are present hospital based and samples of controls matched for age, sex and
relatively in high concentrations and are the strongest carcinogens 5. abundant mass to charge ratio (m/z). Full scan product mass spectra prepared in mobile phase were analysed on LC-MS/MS. Ratio of the
place of residence were also collected from 5 centers. The spot urine was obtained with continuous infusion of cotinine standard (Sigma, results of unextracted and extracted samples were used to estimate
Urinary cotinine level is the most widely used and reliable samples around 50-60 ml. were collected in polyethylene cryo USA) (500 ng/ml) in product ion scan mode keeping Q1 constant on the recovery percentages.
biochemical marker to quantify exposure to tobacco products6. containers from selected patients and controls and preserved in m/z value. The most abundant product ion for cotinine was chosen for
0
Carcinogen biomarkers are important in establishing carcinogen dose (-20 C) deep-freezer at selected hospitals and were transported in LC-MS/MS analysis in the multiple reaction-monitoring (MRM) mode. Results and Discussion
in people who are exposed to tobacco products and in understanding frozen condition to IOP, New Delhi from Gangtok (Sikkim), Guwahati Optimization of compound and source dependent parameters have
mechanism of carcinogenesis and might ultimately be useful in (Assam) and Aizwal (Mizoram) and to ICPO, Noida from Silchar been carried out by continuous infusion mode and flow injection The extracted ion chromatogram (XIC) of cotinine is shown in
predicting cancer risk. (Assam) and Imphal (Manipur) and then were air-shipped under dry analysis (FIA). Figure-1. Under the conditions of LC-MS/MS selected, the linear range of

84 85
 Annual Report 2007-08 Annual Report 2007-08

Table 1 Percentage wise distribution of cancer cases and controls according to urinary cotinine level

Percentages of cotinine levels (ng/ml)

Types of cases
<5 5-19.9 20 - 49.9 50 - 999 1000-8000 > 8000
Lung 26.92 24.36 3.85 42.31 1.28 1.28
(78) (21) (19) (3) (33) (1) (1)
Stomach 24.00 20.00 41.33 14.67
- -
(75) (18) (15) (31) (11)
Oesophagus 15.48 29.76 3.57 40.48 10.71 -
(84) (13) (25) (3) (34) (9)
Oral 33.67 17.35 7.14 30.61 11.22 -
(98) (33) (17) (7) (30) (11)
Others 26.76 21.13 2.82 36.62 12.68 -
(71) (19) (15) (2) (26) (9)
Control 18.38 18.38 4.86 24.32 34.05 -
(185) (34) (34) (9) (45) (63)
8,9
Number in parentheses indicate the number of cases, <5.0 ng/ml – Non Tobacco user (Non Smoker)
5.0 -19.9 ng/ml – Passive exposure, 20.0 -49.9 ng/ml – Abstinent user for greater than 2 weeks
50.0 - 999 n g/ml – Light to Moderate Tobacco users (smoker), 1000 -8000 ng/ml – Active tobacco user or heavy smoker

Figure 1. Extractedion chromatograph of cotinine
cotinine was determined. As shown in linear correlation coefficient (r2)
was 0.9998 for cotinine. Instrumental Limit of Detection (LOD) and Limit
of Quantification (LOQ) for cotinine were 0.2 ppb & 0.5 ppb respectively.
The recovery percentage (Mean±SD) of cotinine was obtained in the
range of 88.42 % ± 2.9 % to 91.36 % ± 1.33 % with less than 4% RSD.
Quantitation of Urinary Cotinine: Out of 1127 urine samples of
cancer cases and controls, 1040 samples were processed for the
measurement of creatinine. The creatinine of 807 urine samples (488
Cancer cases and 319 Controls) was found in the normal range. Out of
488 samples of cancer cases (365 male and 123 female) and out of 319
samples of controls (139 male and 180 female), cotinine was measured
in 406 urine samples (295 male and 111 female subjects) of cancer
cases and 185 samples (87 male and 98 female) of controls (Table 1).
Acknowledgment: The investigators are grateful to PM K handvi and V
Patani for the help rendered during the study.
References:
1. IARC: Tobacco smoking, In IARC Monographs on the evaluation of
carcinogenic risk of chemicals to humans. Lyon, International
Agency for Research on cancer, 1986; pp. 199-298.
2. Hoffman, D, Adams, JD, Lisk D, Fisenne, Brunnermann and K D, I.
Toxic and carcinogenic agents in dry and moist snuff. J. Nat Cancer
Inst. 1987; 79: 1281-1286.
3. Hoffman D, Hecht, SS. Advances in tobacco carcinogenesis, In
Cooper CS, Grover PL (eds): Handbook of Experimental
Pharmacology, Heidelberg, Springer Verlag, 1990; pp. 63-102.
4. Hecht, SS. Tobacco carcinogens, their biomarkers and tobacco
induced cancer. Nature Rev. Cancer, 2003; 3: 733-744.
5. Hecht, SS. Human urinary carcinogen metabolites: Biomarkers for
investigating tobacco and cancer. Carcinogenesis, 2002; 23: 907-
922.
6. Lee PN. Uses and abuses of cotinine as a marker of tobacco smoke
exposure. In: Gorrod JW, Jacob P III (eds). Analytical determination
of nicotine and related compounds and their metabolites.
Amsterdam: Elsevier Science, 1999, pp. 669 719.
7. Harold Varley Determination of creatinine in urine. Practical Clinical
Biochemistry, Vol 1, Allan H Gowenlock and Maurice Bill (Eds.), 5th
Edition, 1980; 484-85.
8. Moyer TP, Charlson, JR, Enger, RJ, Dale, LC, Ebbert, JO, Schroeder,
DR and Hurt, RD. Simultaneous analysis of nicotine, nicotine
metabolites and tobacco alkaloids in serum or urine by Tandem
Mass spectrometry with clinically relevant metabolic profiles. Clin.
Chem. 2002, 48: 1460 - 1471.
9. Nicotine and Metabolites, Urine. “ Arup's Guide to Clinical Laboratory
Testing.” 2006, 0092356, CPT codes 83887.

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Annual Report 2007-08

Experimental Studies on Geno- and Feto-Toxicity of Panmasala

Sunil Kumar, Archana Kumari, BN Mojidra,
AK Gautam, Y Verma and BC Lakkad

Abstract

The study aimed to assess the possible effects of panmasala in somatic as well as reproductive cells and its
modification by natural compounds. For in vitro study lymphocyte culture was treated with 3 different
concentrations of aqueous extract of panmasala to find out panmasala induced chromosomal aberrations and
micronuclei induction (MN), if any. Adult Swiss albino male and female mice were exposed to three different
doses of panmasala plain (PMP) and panmasala with tobacco (PMT) (0.5, 1.5 and 3%) through feed for a
period of 3 and 6 months and mating was allowed in order to study reproductive performance. In another
experiment, 3 and 6% of both PMP and PMT were given through feed from gestation day (GD) 0, 6 and 14 till
weaning to assess the fetotoxic potential of panmasala. To assess the modifying role of curcumin against
panmasala induced mutagenicity, 2.5 g curcumin /kg feed for 8 weeks was given after 12-week panmasala
exposure. The preliminary data showed an increase in MN induction in binucleated cells of human
lymphocytes while observations on CA is in progress. Higher dose of PMT (1.5 and 3.0%) for 3 and 6 months
adversely affected the reproductive performance while 3% dose of both types of panmasala for 6 months also
negatively influenced the pregnancy outcome. In utero exposure from GD 0 with both doses of PMT resulted in
pre term birth. Lower birth weight was observed among the offspring of dams treated from GD 0, 6 and 14.
Curcumin has showed its efficacy against panmasala induced clastogenecity and spermatotoxicity.
Panmasala is having spermatotoxic and genotoxic potential and more damage is seen with gutkha as
compared to panmasala plain.

Keywords: Gutkha; panmasala; fetotoxicity, micronuclei; reproductive performance

Introduction Previously, significant increase in sperm head shape abnormality

Panmasala plain (PMP) is a dry mixture of areca nut, catechu, lime,
cardamom and unspecified flavouring agents. Gutkha (PMT) is another
popular variant of panmasala containing tobacco. Most of the ingredient
of panmasala are reported to possess cytotoxic, mutagenic and
genotoxic properties1. Even though data are available on mutagenicity
and carcinogenecity of its major ingredients i.e., areca nut and tobacco,
there is need to study the geno- and fetotoxic potential of their complex
mixture like panmasala and its modification by natural compounds.
Therefore, study is in progress with the following main objectives:
was observed after short term (12 week) and long term (6 months)
exposure of panmasala. Sperm and spermatid count as well as daily
sperm production was found to be significantly low after 3% PMT
treatment for 6 month2. In dominant lethal test, 4 months treatment with
3 and 6% of panmasala reduced the fertility potential of treated mice.
Data summarized for one spermatogenic cycle revealed significant
reduction in total and live implants / female in PMT treated mice.
Preimplantation loss was increased among all treated groups while post-
implantation loss was higher only in 6% dose level3.

Frequency of micronuclei (MN) induction in bone marrow cells of

 In vivo and in vitro evaluation of mutagenicity of panmasala.
 To study fetotoxic potential of panmasala.
 To study the role of -carotene & curcumin in panmasala induced
mutagenicity.
mice was significantly high at the dose of 3% PMP and PMT after 6 and 12
week exposure as compared to control. Further, chromosomal
aberrations (CA) was significantly elevated after both types of panmasala
treatment with higher doses (1.5 and 3%) given for 6 weeks and at all the
three doses given for 12 weeks with respect to control.

89

-carotene has been shown to have protective role against panmasala
induced toxicity as it lowered the sperm head shape abnormality caused
by 3% of both types panmasala and also reduced significantly the
abnormal morphology at 6% PMP as compared to panmasala exposed
animals maintained on standard diet. The present study conducted with
following objectives:
 In vitro genotoxic potential of aqueous extract of panmasala
 To note the effect of panmasala treatment on reproductive
performance
 To evaluate fetotoxic potential of panmasala
 To assess the modifying property of curcumin against the mutagenic
potential of panmasala
Materials and Methods
In vitro mutagenicity of panmasala: For in vitro study, aqueous extract of
panmasala was prepared4. The final yield of extracted material was 2.66
gm and 2.89 gm from PMP and PMT respectively. Stock solution (1
mg/ml) was prepared in triple distilled water and added to standard
blood lymphocyte culture at the concentration of 7.5, 15 and 30 µg/ml of
PMP and PMT extract. After 72 hours, cells were harvested for CA and
MN study.
In vivo mutagenicity of panmasala: Swiss-albino mice were
maintained in Institute's animal house under controlled conditions of
photoperiod, temperature and humidity in accordance with the
guidelines of the Committee for the Purpose of Control and Supervision
on Experiments on Animals (CPCSEA). Animals were provided with
standard mice feed and water ad libitum. Ethical clearance for the study
th
was obtained from the Institute's animal ethical committee held on 4
March 2005.
Reproductive performance /fertility study: In order to evaluate
reproductive fertility potential, adult Swiss albino male and female mice
were treated with 1.5 and 3% of both PMP and PMT through feed for 3
and 6 months. In addition, a separate group was treated with lower dose
i.e., 0.5% of both types of panmasala for six months. Thereafter they
were allowed to mate for 5 days. Mating was confirmed by the presence
of vaginal plug and considered as day 0 of pregnancy. Pregnant dams
were maintained on standard mice feed. All the relevant parameters
such as mating, fertility and pregnancy indices, gestation length, litter
size, sex ratio and weight, survival, mortality etc. of the offspring were
recorded.
Fetotoxicity: To note the fetotoxic potential of panmasala at
different stages of gestation, pregnant mice were treated with two
different doses of panmasala (3 and 6% PMP and PMT) through feed
from GD 0, 6 and 14 of pregnancy till weaning. Body weight of dam was
taken every 3rd day. Pregnancy outcome was documented.
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Annual Report 2007-08 Annual Report 2007-08
Modifying role of curcumin against panmasala induced mutagenicity: To
study the modifying property of curcumin, it was given at the dose of 2.5
g/kg feed for 8 weeks post panmasala treatment with 3 & 6% PMP and
PMT for 12 weeks. Another group was allowed to maintain on basal diet
(standard mice feed) post panmasala exposure. Control group was also
maintained for 20 weeks without any exposure. Body weight was
monitored weekly. After the scheduled treatment, animals were
sacrificed. Slides were prepared for MN and sperm morphology assay.
Statistical analysis One-way ANOVA and Tukey's post-hoc test,
student t- test and Fisher exact test were performed to compare
differences between control and treated groups.
Results
Figure 1: Body weight gain (%  SE) of panmasala treated male and female for 3 months
In vitro mutagenicity of panmasala: Preliminary observation on in vitro
study revealed an increase in MN induction in binucleated cells of human
lymphocytes culture after panmasala treatment. Further analysis and
observation on CA is in progress.
Reproductive performance/ fertility study: Data collected on weight
gain indicated that panmasala treatment for 3 and 6 months reduced the
body weight gain non-significantly in both the sexes as compared to
control (Figure 1; Table 1). Mating indices were considerably declined at
both the doses of PMT (1.5 and 3%) while it was comparable at PMP
treatment groups after 3 and 6 months exposure with respect to control
(Figure 2a). Fertility and pregnancy indices were non-significantly
reduced at all the doses of PMT at both the durations of panmasala
exposure while pregnancy index was lowered only at 3% of PMP (Figure
2b) but all these changes were statistically non significant. However
negative effects indicated the potential adverse effects of panmasala on
reproductive performance. Figure 2: Reproductive performance in panmasala treated male and female a) Mating index b) Fertility and Pregnancy index
All the panmasala treated pregnant dams (resulting from the mating Fetotoxicity: Results on fetotoxicity showed that body weight gain dams from gestation day 0, 6 and 14 as compared to control. Sex ratio
of panmasala exposed male and female) had almost similar body weight during pregnancy was more or less similar in all the treated group i.e., declined marginally in all the panmasala treated dams from GD 0 with
gain during pregnancy. The results on pregnancy outcome of 3 and 6
3 and 6% of both PMP and PMT from GD 0 and 6 with respect to control respect to control. However, exposure from other gestation days had
months panmasala treated group are presented in Table 2 and 3 (Fig. 3 a,b) except slightly lower weight in 6% PMT treated group on more or less similar sex ratio in treated and non treated groups.
respectively. Gestation length, litter size and live pups within a litter were
day 18 of pregnancy. Results of the pregnancy outcome in panmasala
more or less similar in all the panmasala treated group as well as non treated dams from GD 0, GD 6 and GD 14 upto weaning are presented Results on the modifying role of curcumin against panmasala
treated group. Birth weight of pups born to 3 and 6 months panmasala
in Table 4-6. The results indicated that PMT exposure at both the dose showed that treatment with curcumin for 8 week was able to reduce
treated parents were decreased as compared to control and it was
level i.e., 3 and 6% from GD 0 significantly decreased the gestational the sperm head shape abnormality in all the panmasala treated groups
significantly lowered at 3% PMT treatment for 3 months with respect to
length with respect to control (Table 4). Weight of the offspring born with respect to group maintained on basal diet indicating the efficacy
control (Table 2).
from 3 and 6% PMT treated dam from GD 0 and GD 6 was significantly of curcumin towards panmasala induced spermatotoxicity (Table 7).
Live birth and viability index were more or less similar among 3 lower as compared to control. It was also decreased significantly at Data on micronuclei induction among curcumin treated (for 8 weeks)
months panmasala treatment at both the dose level while it reduced after 3% PMT treated dam from GD 14. In comparison to control, live birth after panmasala treatment for 12 weeks and control animals is shown
six months exposure at 1.5% PMT and 3% of both PMP and PMT treated index was reduced in GD 0 and GD 14 groups treated with 6% of both in Table 8. Curcumin has reduced percentage of micronucleated
group with respect to control (Table 2 & 3). Weanling index was also types of panmasala i.e., PMP and PMT while viability index was polychromatic erythrocytes at lower dose (3%) of both types of
lowered at 3% dose of PMP and PMT in both 3 and 6 months exposure decreased at both the dose of PMT i.e., 3 and 6% and high dose of PMP panmasala treated groups as compared to group maintained on basal
and also decreased at 1.5% of PMP and PMT after 6 months exposure i.e., 6% among all the groups i.e., GD 0, 6 and 14. Weanling indices diet. However both the treatments viz. curcumin and basal diet
group. However, all these changes were statistically non significant. were considerable lowered at 6% of both types of panmasala treated showed more or less similar induction of micronuclei in PCE and NCE
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 Annual Report 2007-08 Annual Report 2007-08

Table 1: Body weight gain (%  SE) of panmasala treated male and female for 6 months
Male Female 90 80
Control 3 PMP 3 PMT
Control 3 PMP 3 PMT
Control 19.40  1.26 8.14  2.08 80
6 PMP 6 PMT 70
6 PMP 6 PMT
0.5% PMP 20.01  2.49 5.41  1.29 70
60

% weight gain

% weight gain
0.5% PMT 17.66  1.30 5.56  1.35
60
1.5% PMP 18.63  1.94 5.51  0.78 50
50
1.5% PMT 14.59  2.17 4.92  1.08 40
3% PMP 15.98  2.10 4.56  3.68 40
30
3% PMT 17.49  1.77 4.70  2.42 30
20
Table 2: Pregnancy outcome of 3 months panmasala treated animals 20

10 10
Control 1.5% PMP 1.5% PMT 3% PMP 3% PMT
Gestational 19.00  0.17 19.20  0.20 19.71  0.29 19.09  0.28 19.67  0.26 0 0
length (days) 3 6 9 12 15 18 9 12 15 18
(a) Gestation day (GD) (b) Gestation day (GD)
Litter size (n) 7.67  1.20 7.00  0.55 6.71  0.64 8.09  0.74 7.42  0.85
Live pups (n) Figure 3: Body weight gain during pregnancy in panmasala treated dams (a) GD 0 (b) GD 6.
7.33  1.25 6.60  0.75 5.57  0.84 7.45  0.92 7.00  0.86
Birth weight (g) 1.44  0.02 1.47  0.02 1.47  0.02 1.46  0.01 1.38  0.01*
Live birth index (%) 94.55  4.43 93.14  4.30 93.39  4.75 90.40  6.45 94.67  3.61
Viability index (%) 93.32  3.85 100.00 0.00 100.00  0.00 90.00  10.00 87.92  4.50 Table 4: Pregnancy outcome of dam treated with panmasala from GD 0
Weaning index (%) 93.32  3.85 97.14  2.86 96.88  3.13 88.18  9.87 82.41  8.61 Control 3% PMP 3% PMT 6% PMP 6% PMT
* p<0.05; Data are expressed as mean  SE Gestational Period
19.78 ± 0.28 19.00 ± 0.26 18.17 ±0.11* 18.83 ± 0.17 18.67 ±0.24*
Table 3: Pregnancy outcome of 6 months panmasala treated animals (days)
Litter Size (n) 9.89 ± 0.42 9.80 ± 0.70 9.67 ± 0.73 9.33 ± 1.05 9.11 ± 1.17
0.5% 0.5% 1.5% 1.5% 3% 3%
Control
PMP PMT PMP PMT PMP PMT Live pups (n) 9.78 ± 0.43 9.60 ± 0.75 9.33 ± 0.75 9.17 ± 1.17 8.33 ± 1.08
Gestation 19.40  19.40  20.40  19.22  19.20  19.89  19.27 
length (days) 0.24 0.24 0.75 0.36 0.25 0.20 0.14 Live birth index (%) 98.89 ± 1.11 97.42 ± 1.81 96.07 ± 2.25 97.22 ± 2.78 93.33 ± 4.57
6.20  6.00  5.60  6.44  6.60  7.33  6.22 
Litter Size (n)
0.49 0.71 1.08 0.94 0.82 0.58 0.70 Viability index (%) 97.84 ± 1.45 95.94 ± 2.38 93.88 ± 3.76 92.73 ± 3.4 95.48 ± 2.93
Live pups (n) 5.80  5.00  4.40 6.11  5.40  6.33  4.89 
0.66 1.10 1.33 1.06 1.02 0.87 0.84 Weaning index (%) 93.58 ± 2.33 89.87 ± 4.39 89.34 ± 6.62 89.09 ± 4.45 77.62 ± 16.2
Birth weight (g) 1.51  1.61  1.40  1.44  1.47  1.46  1.47 
0.02 0.03 0.02 0.02 0.04 0.02 0.03 1.78 ± 0.43 1.32 ± 0.21 1.28 ± 0.15 1.24 ± 0.36 1.19 ± 0.16
Sex ratio (M/F)
Live Birth 90.00  90.18  88.19  91.01  82.65  83.64  82.29 
Index (%) 10.00 6.08 6.84 7.38 3.00 7.47 5.55
Birth weight (g) 1.47 ± 0.01 1.43 ± 0.01 1.39 ± 0.01* 1.42 ± 0.02 1.39 ± 0.01*
Viability 100.00  95.83  100.00  94.87  80.83  84.13  78.13
Index (%) 0.00 4.17 0.00 2.48 12.37 10.48  7.54 * p<0.05; Data are expressed as mean  SE;
Weaning 100.00  95.83  100.00  91.05  76.67  80.00  77.08
Index (%) 0.00 4.17 0.00 4.79 13.69 14.58  12.07
Data are expressed as mean  SE

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 Annual Report 2007-08 Annual Report 2007-08

Table 5: Pregnancy outcome of dam treated with panmasala from GD 6 Table 7: Alterations in mice sperm morphology with curcumin (8 week) post 12 weeks panmasala treatment
3% PMP 3% PMT 6% PMP 6% PMT Pretreatment % Sperm head abnormality  SE
Gestational Period Control Basal diet Curcumin
19.50 ± 0.5 18.71 ± 0.18 18.86 ± 0.26 18.67 ± 0.33
(days)
1.88  0.10
Litter Size
(n)
8.00 ± 0.82 8.14 ± 1.1 9.14 ± 1.12 9.67 ± 0.56 3% PMP 3.95  0.17 3.81  0.39

Live pups 6% PMP 4.9  0.23 3.83  0.49

8.00 ± 0.82 8.00 ± 1.09 9.14 ± 1.12 9.67 ± 0.56
(n) 3% PMT 4.35  0.22 4.88  0.68
Live Birth Index 6% PMT 6.04  0.83 4.06  0.32
100.00 ± 0.0 98.41 ± 1.59 100.00 ± 0.00 100.00 ± 0.00
(%)
Table 8: Alteration in micronuclei in PCEs and NCEs after curcumin post treatment (8week) to panmasala (12week)
Viability Index
(%) 96.06 ± 2.5 93.37 ± 4.61 90.28 ± 5.01 94.63 ± 3.75 % MNPCE % MNNCE
Weaning Index Basal Diet Curcumin Basal Diet Curcumin
(%) 93.98 ± 4.21 91.70 ± 4.42 87.50 ± 4.69 91.57 ± 4.23
CONTROL 0.66  0.08 0.25  0.03
Sex ratio
2.37 ± 1.20 1.68 ± 0.37 1.96 ± 0.70 2.38 ± 0.66 3% PMP 0.83  0.07 0.64  0.22 0.35  0.03 0.27  0.04
(male/female)
Birth weight 3% PMT 0.84  0.08 0.74  0.10 0.37  0.04 0.23  0.04
(g) 1.45 ± 0.04 1.38 ± 0.07* 1.39 ± 0.06 1.38 ± 0.06*
6% PMP 0.81  0.16 0.89  0.14 0.33  0.05 0.37  0.03
* p<0.05; Data are expressed as mean  SE;
6% PMT 1.07  0.16 1.21  0.13 0.36  0.03 0.39  0.07
Table 6: Pregnancy outcome of dam treated with panmasala from GD 14
after 6% of PMP and PMT exposure. All these change were non- References
3% PMP 3% PMT 6% PMP 6% PMT significant.
1. K umar S (2008) Panmasala chewing induces deterioration in oral
Gestational Pd.
19.00 ± 0.26 18.75 ± 0.25 18.67 ± 0.33 18.80 ± 0.49 Discussion health and its implications in carcinogenesis. Toxicol Mech
(days) Methods 18: 665 - 677.
Litter Size The results obtained so far suggests that panmasala is having 2. K umar S, Archana K , Gautam AK , Mojidra BN, Verma Y , Lakkad
8.67 ± 0.71 10.60 ± 0.51 9.33 ± 0.67 8.60 ± 0.51 mutagenic and clastogenic potential as evident by the in vitro BC. Reproductive toxicity of panmasala in male Swiss albino
(n)
Live pups lymphocyte culture and in vivo increase in MN in swiss albino mice. mice. In XXVII Annual STOX; 2007, Oct 6-7; Bangalore, p 54.
8.67 ± 0.71 10.60 ± 0.51 8.67 ± 0.84 7.80 ± 0.86 Our study corroborates with earlier work done on in vitro CHO cell 3. Archana K , Gautam AK , Mojidra BN, K apurkar U, Joshi M, Lakkad
(n)
lines5 and among panmasala chewers6. In addition, exposure of BC, K umar S. Germ cell mutagenic potential of Panmasala in
Live Birth Index panmasala mainly PMT is shown to have fetotoxic potential as the in male Swiss albino mice. Natl Seminar on Teratology & Genetic
100.00 ± 0.00 100.00 ± 0.00 92.21 ± 5.11 91.11 ± 8.89
(%) utero PMT treated dams had small gestation period along with lower Disorders; 2008 Feb 8-9, BHU, Varanasi; p 18.
Viability Index birth weight. It is well documented that smoking adversely affects the 4. Jaju RJ, Patel RK , Bakshi SR, Trivedi AH, Dave SJ and Adhvaryu
97.92 ± 2.08 95.00 ± 5.00 88.48 ± 6.55 88.00 ± 12.00 pregnancy outcome. Thus, tobacco consumption resulting in pre-term SG. Chromosome damaging effects of panmasala. Cancer Lett.
(%)
birth along with lower birth weight is a consistent finding irrespective 1992; 65, 221 -226.
Weaning Index of the mode of intake. Recently arecoline, major alkaloid of areca nut, 5. Patel RK , Jaju RJ, Bakshi SR, Trivedi AH, Dave BJ and Adhvaryu SG.
97.92 ± 2.08 90.78 ± 4.60 83.05 ± 7.46 75.00 ± 25.00
(%) have been associated with adverse pregnancy outcomes7. Further, (1994). Panmasala a genotoxic menace. Mutat. Res. 320, 245-249.
Sex ratio both types of panmasala demonstrated reproductive toxic potential as 6. Dave BJ, Trivedi AH and Adhvaryu SG (1991) Cytogenetic studies
1.59 ± 0.59 2.57 ± 1.37 1.60 ± 0.36 1.80 ± 1.10
(male/female) revealed by the impairment in reproductive fertility performance and reveal increased genomic damage among 'panmasala'
Birth weight pregnancy outcome. Data so far obtained indicated the male and consumers. Mutagenesis 6, 159-163.
1.46 ± 0.02 1.38 ± 0.01* 1.44 ± 0.01 1.45 ± 0.01 female reproductive toxic potential of panmasala in Swiss albino mice. Garcı a-Algar O, Vall O, Alameda F, Puig C, Pellegrini M, Pacifici
(g)
The preliminary results on modifying role of curcumin suggest that the R and Pichini S (2005) Prenatal exposure to arecoline (areca
* p<0.05; Data are expressed as mean  SE;
adverse spermatotoxic effect of panmasala can be reduced by nut alkaloid) and birth outcomes. Arch. Dis. Child Fetal
curcumin treatment. Neonatal Ed 90, F276F277.

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 Annual Report 2007-08 Annual Report 2007-08

Effect of Arsenic on Brain and Testicular Tissue: Histopathological and species (ROS), nitric oxide (NO) and apoptosis was also prominent in
10
Histological Study: The testes were fixed in Bouin's fixative,
fetal brain tissue . Arsenic could reduce neurological viability; nuclear embedded in paraffin and 5m thick sections were stained with routine
Biochemical Study fragmentation and condensation as well as DNA degradation in primary hematoxylin-eosin. Histopathological changes in the testes were
11
A Roy Choudhury and Sanghamitra Sarkar culture of rat neuron . Significant changes in the steady state level of examined under optical microscope.
three major neurotransmitters, dopamine, norepinephrine and 5
hydroxytryptamine and monoamine-oxidase were observed following Morphometric Study: Quantitative analysis of spermatogenesis
Abstract 12
chronic arsenic exposure (III) in male albino rats . was carried out from 5 perfect transversely cut tubules at VII-stage of
spermatogenesis from each testis of respective groups. The relative
Arsenic exposure to Swiss mice, in the present study, gradually decreased the testicular weight compared to Arsenic causes reproductive effect on human and animal species. number of spermatogonia, resting spermatocyte, pachytene
control suggesting cellular regression of the testicular tissue. Testicular histology exhibited severe cellular Few studies have shown that arsenic exposure produces ovarian and spermatocyte and step-7 spermatids per tubular cross section were
damage in spermatogenic cell. Degeneration of interstitial (Leydig) cells was observed in the testis of arsenic- uterine disorders13, testicular steroidogenic dysfunction14, and quantified at 1000X magnification18. Correction for changes in nuclear
treated mice. Moreover Leydig cell population significantly decreases in both the doses over a period of 60 spermatogenic disorder in experimental animal15. The available data diameter was made using Abercrombie formula19. Seminiferous
days. The Leydig cell nuclear diameter increased significantly in both the doses in 30 days followed by gradual point to dose dependent effects on growth and viability of the tubular diameter was determined at 400X magnification by ocular
diminution of the Leydig cell diameter in 45 and 60 days. The exposure to experimental mice in two doses (30 conceptus and offspring but no effect on fertility16. In human study a micrometer. The morphometric measurement of Leydig cell nuclear
& 40 mg/L) for 30, 45 and 60 days showed a significant alteration in the activity of many antioxidants. The GSH correlation has been observed between arsenic exposure and diameter was done by using ocular micrometer at 600X magnification
content of brain and testicular tissue in both arsenic-treated mice revealed significant alteration with the incidence of abortion17. and Leydig cell population was analyzed per square cm area from
sagital plane area of the section at same magnification20.
increase in duration. In both the dose group for 30 days, there was marked increase in GSH content that Although various tissue toxicity caused by arsenic has been
afterward decreased with the duration of 60 days. GSH is known to protect cell against oxidative stress and intensively investigated, the arsenic induced testicular and brain Lipid Peroxidation: Extent of lipid peroxidation was estimated by
any alteration in GSH level (either increase or decrease) indicates a disturbed oxidative status. When cells are tissue toxicity and generation of oxidative stress in testicular and brain the method of Buege and Aust21. Brain and testes were homogenized
oxidatively challenged, GSH synthesis increases and this may a cause of increase in GSH content after initial tissue are still unclear. Therefore, the effect of arsenic (30 & 40 mg/L) in ice-cold 0.25 M phosphate buffer (pH 7.4). The homogenate and the
treatment of arsenic in mice. As oxidative stress continues, GSH synthesis cannot efficiently supply the in drinking water ad libitum in Swiss albino mice and the possible 5000g supernatant were used. To this homogenate, TCA-TBA-HCl
demand, therefore GSH depletion occur. This may be the possible cause of further diminution of GSH content generation of oxidative stress and testicular and brain tissue [Trichloroacetic acid (TCA) 15 % w/v, thiobarbituric acid (TBA)
in mice after arsenic exposure. GSH depletion can impair a cell's defense against the toxic actions of many histopathology were investigated. 0.375%, and hydrochloric acid (HCl) 0.25N] were added and mixed
compounds and may lead to cell injury and death. Arsenic exposure in both the dose for 60 days duration thoroughly. The solution was heated for 15 min in a boiling water bath.
enhanced significantly the lipid peroxidation of the testis, suggesting the generation of reactive oxygen After cooling, the flocculent precipitate was removed by centrifugation
Materials and Methods
radicals and testicular oxidative stress. at 1000g for 10 min. Levels of thiobarbituric acid reactive substances
Animals and Treatment: Adult Swiss male albino mice weighing (TBARS) were measured spectrophotometrically at 532nm.
Measurement of reduced glutathione: The reduced glutathione
Keywords: Arsenic toxicity, mice, oxidative stress, Leydig cell, lipid peroxidation approximately 25- 30 g were obtained from the animal facility of Central
content of the tissue was measured by the method of Ball RC et al22.
Research Institute (Ayurved), Ministry of Health and Family Welfare,
For calibration, a standard curve of reduced glutathione was prepared
Kolkata. They were housed in a room with ambient temperature of
0 using varied concentrations of glutathione treated with DTNB. Tissue
24±1 C in 12 h light/darkness cycle. Mice were allowed standardized 23
protein was estimated by the method of Lowry et al .
pellets supplied by NIN, Hyderabad and sterilised water ad libitum.
Introduction to drinking of arsenic contaminated water has been reported from Table 1: Effect of sodium arsenite on body weight (g) and organ
many countries. Recently large population in West Bengal in India and Sodiummetaarsenite was dissolved in drinking water and was weight (mg/g body wt)
Arsenic (As) is a common environmental contaminant widely Bangladesh has been reported to be affected with arsenic5,6. administered at 30mg/L and 40 mg/l (30ppm and 40ppm respectively) Body wt. 30 days 45 days 60 days
distributed around the world as a result of natural and man-made
origin. Groundwater with elevated concentrations of Arsenic has been Arsenic causes acute and chronic toxicity in human. The through drinking water ad libitum. Animals were mainly divided into Control 28.7 4.34 32.5 0.95 35.5 5
1-3
recognized as a problem of global concern . Arsenic contamination of neurological system is the major target for the toxic effect of arsenic. major 3 groups: 35.1 10.6
30 mg/l 38.5 2.3 36 4.6
groundwater is one of the principal pathways of human exposure to Various studies confirmed that arsenic could cross blood brain barrier 40 mg/l 35.6 4.28 34.1 4.91 35 5.7
Group I: Control, drinking water ad libitum only, Group II: Sodium-
inorganic Arsenic. Elevated concentrations of Arsenic have been and produces alteration in whole rat brain biogenic amines level in
meta-arsenite dissolved in drinking water at dose level of 30mg/L for
reported from several regions of the world4 resulting primarily from 7
animal chronically exposed to arsenic . Histopathological observation in Results
30, 45 and 60 days respectively. Group III: Sodium-meta-arsenite
natural sources, such as erosion and leaching from geological rat brain after exposure to multiple gallium arsenides revealed mild
8 dissolved in drinking water at dose level of 40 mg/L for 30, 45 and 60 Body and Organ Weight: The body weight gain was not significantly
formations, although sometimes from anthropogenic sources, such as effect on cerebral cortex region . Male Sprague-Dawley rat exposed to
uses of Arsenic for industrial purposes, mining activities and metal days respectively. After the experimental period, rats were weighed and changed after arsenic exposure in both the treated groups over a
arsenic showed decreased locomotor activity and behavioural
processing, and application of pesticides and fertilizers containing As. disorders9. Studies on human fetal brain tissue indicated regression in sacrificed by cervical dislocation. Testes and brain were removed, period of 60 days in comparison to the control (Group I) [Table 1].
The risk of Arsenic contamination is generally much higher in morphometric development and disturbance in the glutathione cycle cleaned of the adhering tissues and weighed. Tissues were immediately Significant reduction in testicular weight in Group II (Arsenic with
groundwater compared to surface water. Chronic arsenic toxicity due enzymes and neuronal development. Generation of reactive oxygen and subjected for biochemical and histopathologial study. 30mg/L) was observed from 45 days onward. But more conspicuous

96 97

decrease in testicular weight was noted in Group III (Arsenic with
40mg/L) after 30 days and significant diminution was found on day 60
(Table 2). In brain tissue, the weight was not significantly reduced after
arsenic treatment with 30mg/l over a period of 60 days. Similarly
insignificant change was noted in animals treated with 40mg/l arsenic
for 30 and 45 days, but significant reduction was observed after 60
days (p<0.005) [Table 2].
Histological Finding: Control testis (Group I) showed normal
histoarchitectural organization of germ cells and Leydig cell in
seminiferous tubules on day 30 through 60 (Figure 1a). Lower dose
group i.e. 30mg/L (Group II) for 30 days showed disintegration of
peritubular membrane, with disorganization of germ cells. After day 45
of Group II, vacuolization of seminiferous epithelium, loss of germ cells
population along with Leydig cell atrophy were noted (Figure 1b). In the
same group on day 60, histopathological changes included
degeneration and loss of germ cells, disarrangement of seminiferous
epithelium and atrophy of seminiferous tubules. Leydig cell showed
marked atrophy. Treatment with 40mg/L arsenic (Group III) produces
similar effect on testicular tissue like Group II, but of higher intensity. The
effect followed 30 day onward and on 45 (Figure 1c) and60 days. Arsenic
exposure over a period of 60 days also exhibited appearance of giant cell
in the seminiferous tubules (Figure 1d). In the cerebellar cortex,
morphological and structural variation occurs after arsenic exposure.
Histologically, the cerebellum of the control mice showed three layers:
outer molecular layer, internal granular layer and between the two a
single row of large flask- shaped cells, the cell of Purkinje (Figure 2a).
The low dose of arsenic, 30 ppm has effect on the cerebellar region of
brain. In group for 30 days, there was degeneration of the granular cell
layer. The purkinje cell layer was intact with a small area of granule cell
loss. In 45 days for the same group, moderate vacuolation in white
matter occur and there is purkinje cell degeneration was observed along
with granular cellular degeneration. In the group II for 60 days, numerous
vacuolation occur and are very apparent in the white matter along with
granular and purkinje cell loss (Figure 2b).
Similarly the higher doses, i.e. 40 ppm the effect are more severe
than the lower dose. In 30 days for 30mg/L arsenic, the granular cell
layer thickness decreases when compared with the control. There is
granular cellular and purkinje cell degeneration and moderate but
prominent vacuolation. In 45 and 60 days (Figure 2c), severe
degeneration in white matter along with extensive granular cell and
purkinje cell loss occur. Vacuolation in the molecular layer and cellular
degeneration in the molecular layer was more prominent in 45 and 60
days of 40mg/L arsenic treatment.
Morphometric analysis: In Group II, spermatogonial count was not
altered from that of control after 30 days through 60 days. But gradual
98
Annual Report 2007-08 Annual Report 2007-08
significant decrease in resting and pachytene spermatocyte was
observed in 30, 45 and 60 days respectively. The population of step-7- Figure 1:
spermatid was conspicuously decreased from 30 day onward
compared to the control in group II. Similarly in higher dose group a) Control testicular tissue in mice
(Group III), significant and gradual reduction in different stages of showing normal organization of germ
spermatogenesis i.e. resting and pachytene spermatocyte, 7- cells and Leydig cell in seminiferous
tubules. Õ (H& E, x 600).
spermatid was noted after 30, 45 & 60 days but spermatogonial cell
b) Treatment with 30mg/L arsenic on mice
count was unaltered (Table 3). for 45 days showing disintegration (D)
of seminiferous tubules, vacuolization
Seminiferous tubular diameter showed a dose and duration of seminiferous epithelium (V ) & loss of
dependent reduction in both the treated groups over a period of 60 days germ cells population. (H & E, x 600).
(Table 3). Gradual significant reduction in Leydig cell population c) Treatment with 40 mg/L arsenic on
compared to that of control in both the treated groups (Group II & III) over a mice for 45 days showing severe
degenerative (SDG) changes in the
period of 60 days was observed (Table 3). The Leydig cell nuclear
testicular tissue where loss of germ cell
diameter showed a marked variation in the treated groups i.e. Group II & is apparent in seminiferous tubule. (H &
III. The nuclear diameter significantly increased in both the treated group E, x 600).
initially in 30 days, but followed a slow and gradual decrease over 60 days. Treatment with 40mg/L for 60 days
showing complete degeneration of
Testicular Tissue Biochemistry: testicular glutathione (GSH): GSH spermatogenic (DGSP) cell with the
appearance of giant cell in seminiferous
content varied markedly in both the arsenic treated groups with
tubules. (H & E, x 600).
increase in duration. In both the groups, i.e. 30 and 40 mg/l arsenic,
GSH content increase after 30 days of treatment (p<0.001) followed by
gradual significant decrease in 45 and 60 days. A significant and
conspicuous decrease in GSH content was noted in higher arsenic
treated group i.e.40 mg/L (Figure 3).
Testicular Total Thiol: Similar effect was noted in total thiol
content of testicular tissue after arsenic exposure. The value varied
markedly in both the arsenic treated groups i.e. 30 & 40 mg/l arsenic
with increase in duration. In both the groups, total thiol content
increase after 30 days of treatment but gradual significant decrease
was observed in 45 & 60 days (Figure 4).
Testicular Lipid Peroxidation: The result shown in fig. 5 indicate
that the lipid peroxidation level increased significantly in testicular
tissue after arsenic exposure in both the arsenic treated groups i.e.
30 & 40 mg/l arsenic than control with increase in duration. The
increase was highly significant for 60 days (p<0.001) in both the Figure 2:
arsenic treated groups. a) The cerebellum showed normal structure with three layer- Molecular
layer,Inner granular layer and in the middle of it the Purkinje cells. (H& E, x
Brain Biochemistry; Figure 6 indicate that the GSH content in 10).
the brain after arsenic treatment revealed a significant increase in
both the treated groups i.e. 30 & 40 mg/l arsenic after 30 and 45 b) The cerebellum of mice exposed to 30mg/L arsenic for 60 days showed
days but a gradual decrease was noted with in duration of 60 days. numerous vacuolation occur and are very apparent in the white matter
along with granular and purkinje cell loss. (H& E, x 10).
Brain Lipid Peroxidation: The malondialdehyde content (a
measurement of lipid peroxidation) in brain was significantly The cerebellum of mice exposed to 40mg/L arsenic for 60 days showed
degeneration in white matter along with extensive granular cell and
increased in both the arsenic treated groups i.e. 30 & 40 mg/l arsenic
purkinje cell loss. (H& E, x 10).
on 30 days, but significantly declined after 45 and 60 days of treatment
in both the dose groups (Figure 7).
99
 Annual Report 2007-08 Annual Report 2007-08

Table 2: Effect of sodium arsenite on absolute organ weight (g) and relative weight (mg/g body weight) of brain and testicular tissue of
experimental mice
Exposure Testicular weight Brain weight
Absolute weight Relative weight Absolute weight Relative weight
30 days
Control 0.19  0.008 5.64  0.408 0.41  0.008 10.57 0.18
NA
30mg/l 0.18  0.017 NA 4.91 0.226 0.39  0.036 10.24 1.348
40mg/l 0.16  0.023* 5.251.36 0.39  0.012 NA 9.22 0.52
45 days
Control 0.22  0.008 5.3  0.66 0.42  0.007 10.95 1.23 Figure 6: Glutathione content in brain tissue after arsenic exposure
30mg/l 0.18  0.017 NA 4.95  0.775 0.40  0.026 NA 10.08 1.36
Figure 3: Glutathione content in testicular tissue after arsenic exposure
40mg/l 0.17  0.022** 5.311.8   0.36  0.045 * 12.45   1.6
60 days
Control 0.22  0.007 5.9 1.02 0.46  0.008 11.8  1.57
30mg/l 0.19  0.034** 5.2 0.62 0.44  0.041 NA 9.67  2.316
40mg/l 0.16  0.019** 4.711.138 0.38  0.033 ** 12.12 1.65

Table 3: Effect of arsenic at a dosage of 30 and 40mg/L arsenic for 30,45 & 60 days respectively on testicular tissue
Seminiferous Spermatodynamic count per 6.4 sertoli cell in each case no. of Leydig cell
Leydig cell
tubular observation was 10 at the 1000x magnification nuclear 2
count/cm
diameter ( m) diameter (n= 10) Figure 7: Lipid peroxidation values in brain tissue after arsenic exposure
[n= 50] Spg R-Spcyt Pachytene 7- sptd (  m) [n= 10]
30 days Discussion
Control 171.11  5.518 5.711.86 21.06 2.53 23.06 1.86 42.25 9.037 6.2 2.70 5.4 0.71 Arsenic is considered as a toxic metal, which reflects on human
NS
19.46 3.74 NA 20.37 2.57 NA 39.27 3.02 NA 8.73 2.84 ** Figure 4: Total thiol content in testicular tissue after arsenic exposure health. Various workers have observed systemic disorders 24-26, but
30mg/l 168.74  3.18* 5.95 1.76 4.75 .0.45 **
male reproductive study in relation to arsenic toxicity is scanty. Earlier
40mg/l 164.01 11.57 ** 5.76 1.09
NS
14.26 3.53 *** 17.96 2.52 *** 24.15 5.76 *** 8.3 1.828 ** 4.48 0.48 * study indicated that heavy metals like lead, mercury and chromium
27
causes cytotoxic effect in the male reproductive function . Arsenic
45 days
exposure to Swiss mice, in the present study, gradually decreased the
Control 182.6 3.75 5 1 1.86 45.6 7.45 6.5 2.43 5.3 0.52 testicular weight compared to control suggesting cellular regression
22.34 1.56 22.4 0.95
of the testicular tissue. This observation is in corroboration with the
30mg/l NS
11.51 2.96 ** 14.02 1.78 *** 19.15 8.08 *** 5.35 1.27 NA 4.43 0.57 ***
161.87  3.20 *** 5.884 1.39 earlier finding of Pant et al.28. Testicular histology in this study
exhibited severe cellular damage in spermatogenic cell. Moreover, the
40mg/l 159.18  4.13*** 5.12 1.41
NS
11.09 2.95 *** 13.82 2.05 *** 16.8 3.56 *** 7.7 1.946 NA 4.3 0.36 * appearance of eosinophilic multinucleated giant cell in the
60 days seminiferous tubule in higher treated group indicated cellular
degeneration. A significant gradual dose dependent regression was
Control 199.2 7.34 5.6  1.22 NS 23.42 2.6 23.3 2.16 44.2 5.25 6.5 2.43 5.3 0.52
observed in the number of resting spermatocyte, pachytene and round
NS
30mg/l 154.20  2.75 ***
5.82  1.23 10.74 3.46 *** 10.5 3.22 *** 16.15 5.76 *** 6.0 1.61 NA 4.59 0.93 ** spermatid in 30 and 40 mg/L over a period of 60 days, whereas there
NS
was no significant decrease in the number of spermatogonia. These
40mg/l 157.85  4.96*** 5.03  1.30 10.97 3.56 *** 9.79 3.89 *** 12.95 4.56 *** 5.57 1.27 NA 3.99 0.63 * finding acts as an indicator that the maturation of spermatogonia
Value are mean  SEM, NS Non significant; * p<0.05; ** p<0.01; *** p<0.001;., Spg: Spermatogonia, R-Spcyt: Resting spematocytes, Pachyt: Pachytene Spermatocytes, Figure 5: Lipid peroxidation values in testicular tissue after arsenic exposure through the process of meiosis has been severely distrupted following
7-Sptd: Spermatids. Number of observation is in parenthesis.

100 101

arsenic exposure. The above observation is in agreement with the
recent finding of Omura et al.29. Degeneration of interstitial (Leydig)
cells was observed in the testis of arsenic-treated mice. Moreover
Leydig cell population significantly decreases in both the doses over a
period of 60 days. The Leydig cell nuclear diameter increased
significantly in both the doses in 30 days followed by gradual
diminution of the Leydig cell diameter in 45 and 60 days. Inspite of a
testosterone assay in this study, it may be suggested that the
degeneration of Leydig cell with significant decrease in the Leydig cell
population probably would have resulted in decreased synthesis of
testosterone, which in turn disturb the process of spermatogenesis. It
has already been established that Leydig cell plays an important role in
the structural and functional integrity of seminiferous tubules and
synthesis of testosterone, which is one of the main component of
regulation the post meiotic stage of spermatogenesis 30-32. The
exogenous arsenic exposure may cause a chemical stress on the
cellular function. The initial increase in Leydig cell diameter may be a
better indication to adopt the metal induced stress but due to
continuous stress effect, cellular exhaust may be a result of Leydig cell
atrophy.
In the present investigation, arsenic exposure to experimental
mice in two doses (30 & 40 mg/L) for 30, 45 and 60 days showed a
significant alteration in the activity of many antioxidants. The GSH
content of brain and testicular tissue in both arsenic-treated mice
revealed significant alteration with the increase in duration. In both the
dose group for 30 days, there was marked increase in GSH content
that afterward decreased with the duration of 60 days. GSH is known
to protect cell against oxidative stress and any alteration in GSH level
(either increase or decrease) indicates a disturbed oxidative status.
When cells are oxidatively challenged, GSH synthesis increases and
this may a cause of increase in GSH content after initial treatment of
arsenic in mice. As oxidative stress continues, GSH synthesis cannot
efficiently supply the demand, therefore GSH depletion occur. This
may be the possible cause of further diminution of GSH content in mice
after arsenic exposure. GSH depletion can impair a cell's defense
against the toxic actions of many compounds and may lead to cell
injury and death33,34. Cellular toxicity of arsenic was found to be
inversely related to intracellular GSH levels and thus may be enhanced
by GSH depletion35. Acute administration of arsenic to rats produced a
significant reduction in hepatic GSH36. Chronic exposure of rats or mice
to arsenic via injections caused up to 35% depletion in hepatic GSH,
37,38
along with liver injury . Similar effect was observed in the total thiol
concentration. Arsenic exposure in both the dose for 60 days duration
enhanced significantly the lipid peroxidation of the testis, suggesting
the generation of reactive oxygen radicals and testicular oxidative
stress. Increased level of lipid peroxidation and consequent damage to
the cell membrane due to arsenic and its compounds was reported by
102
Annual Report 2007-08
39
a number of investigators . But in brain tissue, lipid peroxidation
showed a variable response after arsenic exposure for 30, 45 and 60
days. The LPO content increased significantly in both the arsenic-
treated groups after 30 days but a significant decrease in LPO content
in brain was found in arsenic treated mice after 45 and 60 days.
Arsenic induced elevation of oxidative stress reported previously by
40,41
other workers but at this point of time, the possible reason of
sudden decrease in LPO content of arsenic exposure is not
understood.
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2002. Arsenic induced changes in growth development and
apoptosis in neonatal and adult brain cells in vivo and in tissue
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41. Nag Chaudhuri,A., Basu, S., Chattopadhayay, S., Das Gupta, S.,
1999. Effect of high arsenic content in drinking water on rat brain.
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 Annual Report 2007-08

Load of Environmental Metals in Pregnant Women and

Reproductive Outcome
S Kumar, S Shaikh, V Mishra*, HU Doshi*, NG Sathwara, LJ Bhagia
Abstract
Developing fetus is vulnerable to the exposure to persistent chemicals, as developing organisms may not
have adequate defense mechanism. Certain environmental chemicals including metals might influence the
female reproductive/endocrine system. The study is in progress to find out the load of environmental metals in
pregnant women and reproductive outcome. One hundred and sixty four pregnant women were enrolled from
OPD of Obstetrics and Gynecology, of an hospital of Ahmedabad. Toxic heavy metals i.e. lead, cadmium and
essential metals like zinc, iron and calcium were analyzed in the blood of pregnant women. They were
followed in all trimesters and after delivery. Reproductive outcome was recorded in terms of neonatal
anthropometric indices. The mean blood lead level in the sample analysed so far, 8.15 ± 0.63 mg/dL and the
range was 1.21 to 28 mg/dL. The mean serum zinc, iron and ionic calcium level was 63.81 ± 2.53 mg/dL,
47.44 ± 2.72 mg/dL and 1.04 ± 0.01 mmol/L respectively. A slightly lower birth weight and birth length was
observed in the offspring of subjects having higher lead levels i.e. > 10 mg/dL as compared to <10 mg/dL.
There is considerable lead level in the women of this area and preliminary analysis revealed that there was
slightly lower birth weight and body length among the offsprings of higher maternal blood lead levels.
Key words: Pregnant women, metals, birth weight, body length, head circumference

50 Introduction Materials and Methods

45 44.26 A number of occupations are being reported to be associated with

1
reproductive dysfunction in males as well as in females . Women are
st
One hundred and sixty-four pregnant women, in their 1 trimester of
pregnancy have been enrolled randomly, from the OPD of Obstetric and
Blood Pb < 10 40.75 exposed to toxic environmental chemicals during their day-to-day
40 activities often without their knowledge. General population or even
Gynecology department, of a hospital at Ahmedabad. A written consent

35
33.3 33.5 pregnant women/fetuses or children are exposed to various toxic
metals and their oxides in a number of ways. Metals are present in
Blood Pb > 10 the environment because of their release from a wide variety of
30 sources. Metals enter into the food chain often by way of bio-
concentration, and some of them like mercury bio-accumulates in the
25 tissues of aquatic organisms and enter into the food chain.
Experimental studies indicated that both male and female
20 reproductive system is vulnerable to the effects of some of the
15 metals, which depend upon dose, duration, and timing of exposure2.
Metals like lead, cadmium, and mercury have toxic effects on
10 reproduction. However, data pertaining to reproductive outcome in
2.67 2.34 human are less documented. Certain environmental chemicals
5 including metals might influence the female reproductive/ endocrine
system leading to increasing infertility as well as adverse
0 reproductive outcome. The present study is in progress i) to find out
the load of environmental toxic heavy metals in pregnant women and
Birth weight Body length Head circumference in cord blood samples of newborn and ii) the role of heavy metals if
(kg) (cm) (cm) any in reproductive outcome.
Dept. of Obstetrics and Gynecology, IKD*, Civil Hospital*, Ahmedabad
has been taken from each subject after explaining the objectives and
possible benefit of the study to individual as well as to the society.
Among enrolled subjects, follow-up study has been undertaken in 79 in
2nd, 60 in 3rd trimester of pregnancy and 45 women after delivery. The
subjects were interviewed and information was recorded about
personal characteristics such as name, age, income, education, habits
such as tobacco chewing, smoking, drinking and occupational and
environmental exposure especially, possible exposure to metals,
pesticides and organic solvents, area of residence, medical and
reproductive history of the woman and her partner on predesigned
proforma. Blood samples were collected in each trimester of
pregnancy and also after delivery. Routine hematological parameters
like total erythrocyte, leucocyte and hemoglobin levels were
estimated. Blood samples were digested with HNO3 and H2O2 for the
estimation of metals like lead, cadmium and zinc using Atomic
Absorption Spectrophotometer (AAS, Perkin Elmer Model No. 3100,
U SA). The analysis of ionic cadmium was Carried out using electrolyte
analyzer (AVL, 9181, Germany). Few cord blood samples were also
collected for metal analysis. Information related to newborn i.e. birth

105
 Annual Report 2007-08 Annual Report 2007-08

Table 1: Subjects enrolled in first trimester and follow-up and obstetric history Table 5: Pregnancy outcome of the study subjects according to area of residence Table 6: Birth weight, body length and head circumference of newborn
Residential Area Birth weight (kg) Body length (cm) Head circumference (cm) according to maternal blood lead levels
Age group (yrs) Details of subjects Gravidity
Rural (5) Lead level (µ g/dL) Birth weight (kg) Body length (cm) Head circumference (cm)
1st trimester 2nd trimester 3rd trimester After Delivery 1 2 3 4 5 Total 2.60 ± 0.16 43.2 ± 0.86 34.0 ± 0.54
≤ 25 108 49 35 25 63 29 15 1 - 108 U rban (20) 2.60 ± 0.07 44.23 ± 0.31 33.35 ± 0.17 10 2.67 ± 0.10 44.26 ± 0.43 33.30 ± 0.28
26 -30 44 23 18 16 15 14 11 3 1 44 Industrial (20) 33.26 ± 0.40 10 2.34 ± 0.54 40.75 ± 1.79 33.50 ± 1.25
2.59 ± 0.12 42.57 ± 0.6
≥ 31 12 7 7 4 4 4 3 1 - 12 Numbers in parenthesis indicate the number of subjects; Values are expressed as Mean ± SE Values are expressed as mean ± SE

Total 164 79 60 45 82 47 29 5 1 164 weight (soon after birth), and body length and head circumference were ³10 µ g/dL and preliminary analysis revealed slightly lower birth weight
recorded within 24 hrs. after delivery. and body length among the offspring of higher maternal blood lead
groups (³10 µ g/dL) as compared to <10 µ g/dL (Table 6).
Table 2 : History of spontaneous and induced abortion of the study subjects Results
Discussion
The information about subjects enrolled, and their follow-up and obstetric
Age group (yrs) n Spontaneous abortion MTP Total Exposure to lead for women of childbearing age can have adverse effect
information indicated that eighty-two women were in first pregnancy,
rd on their offspring as it is known that lead deposited in the bone can be
< 25 forty-seven in second, twenty nine in 3 and five were in fourth pregnancy
108 18 7 25 mobilized during pregnancy and also transfer from mother to the fetus3. A
(Table 1). The mean age, weight and height of the study population was
considerable level of lead is observed in the study and preliminary
26 – 30 44 12 9 21 24.8 ± 0.25 yrs, 51.16 ± 0.68 kg, and 153.6 ± 4.8 cm. respectively. Data
analysis indicated slightly lower birth weight and body length of the
pertaining to distribution of subjects according to residential area
³ 31 12 2 2 4 newborn. There is a report, which indicated that prenatal lead exposure
indicated that, 89 subjects were residing in urban area, 21 in rural area
might reduce neonatal weight gain4. Earlier, Saxena et al., observed mean
Total 164 32 18 50 and 54 in industrial area. The socioeconomic status indicates that about
blood lead level of 22.53 among pregnant women with various adverse
55% subjects belong to the lower economic group (Rs <3000/month). outcomes such as pre-term delivery, stillbirth and spontaneous abortion
MTP- Medical termination of pregnancy
About half of the subjects were vegetarian. History of partner indicated 5
as compared to 19.4 in normal delivery cases . In the present study mean
that there were 7.3% smokers, 28.6% chewers and 5.5% occasional blood lead level of 8.5 ± 0.63 µ g/dL (1.21±28.0 µ g/dL) was observed in
alcohol users. Most of the women were housewives (93.2%). the women of Ahmedabad, indicating that some of the pregnant women
Table 3: Haematological parameters among study subjects
Complication during previous/present pregnancy showed that about 21 carried higher blood lead level and had the potential of adverse effects.
Age group (yrs) Different trimester (12.8%) of women, had complication in the present or previous The preliminary data indicated that birth weight and body length of the
Hematological
pregnancy and 32 subjects having history of spontaneous abortion in the newborn were decreased slightly when maternal blood lead levels
parameters study group (Table 2). The haematological profile of the study population
< 25 26 - 30 ? 31 I trimester II trimester III trimester increases. Presently, we are following the enrolled subjects till delivery
as per the age group and trimester is depicted in table 3. The mean and also enrolling more women in first trimester of pregnancy. The
Hb (g/dL) 10.39± 0.10 10.43± 0.16 10.52± 0.27 10.41± 0.08 10.43±0.16 10.28± 0.20 haemoglobin level was more or less similar in all the groups. However relationship, if any, between metal level and reproductive outcome will be
6 3 slightly lower haemoglobin level was noted in the third trimester with assessed in near future, after the analysis of metals in remaining and
RBC 10 /mm 3.93± 0.05 4.0± 0.08 4.14± 0.14 3.97± 0.04 4.11 ± 0.06 4.13 ± 0.07
respect to first and second trimesters. No significant changes were follow up subjects.
3
WBC /mm 7,350± 169 7,722± 296 7,283± 425 7,445±140 7,495 ± 173 7,283 ± 425 observed for RBC and WBC counts. The load of metals in the study
subjects is shown in Table 4. The mean blood lead level in the sample References
Values are expressed as mean ± SE /
analysed so far was 8.15 ± 0.63 mg/dL (range 1.21 to 28 mg/dL). The 1. Kumar S. Occupational exposure associated with reproductive
mean serum zinc and iron level in the study group was 63.81 ± 2.53 dysfunction. J Occup Health 2004; 46: 1-19.
Table 4: Metal level among study subjects mg/dL (range 37.80 to 108.07 mg/dL) and 47.44 ± 2.72 mg/dL (range- 2. Kumar S. Is environmental exposure associated with reproductive
18.2 to 120 mg/dL) respectively. The mean serum ionic calcium level health impairments? J . Turkish-German Gynecol Assoc. 2008; 9(1):
Metals n Mean ± SE Range was 1.04 ± 0.01 mmol/L (range-0.75 to 1.19 mmol/L). Cadmiumcould be 60-69.
analysed only in few samples i.e. 7 subjects and the mean level was 0.94 3. Gulson BL, Mizon KJ , Korsch MJ , Palmer J M and Donnelly J B.
Lead (µ g/dL) 77 8.15 ± 0.63 1.21 - 28.0 Mobilization of lead from human bone tissue during pregnancy and
± 0.20 mg/dL.
Cadmium (µ g/dL) 7 0.94 ± 0.20 0.3 - 1.8 lactation--a summary of long-term research. Sci Total Environ.
2003; 303: 79104.
The preliminary data on birth weight, body length and head
Z inc (µ g/dL) 42 63.81±2.53 37.8 - 108.07 4. MDH (Minnesota Department of Health). Blood lead screening
circumference of newborn according to area of residence indicated that guidelines for pregnant women in Minnesota.
Calcium (mmol/L) 164 1.04± 0.01 0.75 -1.19 no considerable difference in birth weight was found according to area of www.health.state.mn.us/divs/eh/lead, U pdated 12/2007].
residence. However, body length of newborn was slightly lower in the 5. Saxena DK, Singh C, Murthy RC, Mathur N and Chandra SV. Blood
Iron (µ g/dL) 62 47.44±2.72 18.2 -120.0 industrial area as compared to rural and urban area (Table 5). The data and placental lead levels in an Indian city: a preliminary report. Arch
further analysed as per the maternal blood lead level i.e. <10 µ g/dL and Environ Health 1994; 49: 106-110.

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