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

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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

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

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

15
Annual Report 2007-08 Annual Report 2007-08

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

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

19
Annual Report 2007-08 Annual Report 2007-08

Table1: Dust concentrations (mg/m3) in ball mills Environmental cum Epidemiological Survey in Asbestos Jointing
Mill Feeding Bagging Material Manufacturing Industry
Total dust Respirable dust Total dust Respirable dust A Saha, LJ Bhagia, YK Sharma, PK Majumdar and JB Vyas
A 10.74±6.42 (5) 2.45±0.71 (6) 12.90±3.32 (5) 3.70±1.26(6)
B 9.88±2.68 (4) 2.37±0.69 (6) 11.78±1.02 (4) 3.44±0.97 (6)
Abstract
C 10.03±2.97 (6) 2.31±1.76 (6) 17.65±2.04 (6) 3.51±0.40 (6)
D 12.88±2.88 (5) 2.19±1.62 (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
E 13.14±4.29 (4) 2.48±1.57 (6) 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.
Numbers in parenthesis indicate number of samples 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
Design for the dust control system: Dust control system is 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
designed by NIOH using ACGIH ventilation guidelines10. Dust control 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
system involves hood (one each at feeding and bagging points) units, Khambhat, India. Indian journal of Occupational Hygiene obstructive and 2.9% were having restrictive abnormality. Fiber level in all the workplaces were below
ducting, centrifugal fan and bag filters with reverse pulse jet system. and Safety. 1;2: 20-27, 2007. national and international standards. No worker was found to have radiographic features suggestive of
The reverse pulse jet systems are most commonly used. The pulse jet interstitial lung fibrosis.
cleaning mechanism uses a high pressure jet of air to remove dust 4. Sadhu HG, Parikh DJ, Sharma YK, et al. A follow up study of health
from the bag. Bags in the bag house compartment are supported status of Small-scale Agate Industry Workers. Indian J. of Ind.
Keywords: Asbestos, jointing material, pulmonary function
internally by rings or cages. Bags are held firmly in place at the top by Med. 41;3: 101-105,1995.
clasps and have an enclosed bottom. Dust laden-air is filtered through 5. Saiyed HN, Parikh, DJ, Ghodasara, NB, et al: Silicosis in slate
the bag, depositing dust on the outside surface of the bag. The dust pencil workers. An environmental/medical study. Am. J. Ind.
cake is removed from the bag by blast of compressed air injected into Med. 8: 127-133,1985. Introduction accumulate and cause scarring and inflammation, which can
the top of the bag tube. The blast of high pressure air stops the normal affect breathing and lead to serious health problems4. Asbestos has
flow of the air through the filter. The air blast develops into a standing 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
wave that causes the bag to expand as the bubble travels down the bag 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,
tube. As the bag flexes, the cake fractures and deposited particles are 135-142. mining, milling, processing, and transportation of asbestos and also the EPA, and the International Agency for Research on Cancer5-8. The
discharged from the bag. Total flow of the system is 1200 Cubic feet 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
per minute (CFM). Horse-Power of the motor is 5 hp (1440 RPM). 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
Speed of the fan is increased to 2300 RPM by using pulleys. Owners 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.
were encouraged to install dust control system for feeding and asbestosis. All types of asbestos fibres are responsible for human amphibole variety the target cells10. Murlidhar and Kanhere11 reported
bagging operations. One of the owners has agreed to install the system 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
on experimental basis. MoU has been signed with the mill at Beawar 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
for installation of dust control system. The work is in progress for 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
testing the efficacy of dust control system. 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%
References 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
1. Bhagia LJ, Tiwari RR, Sharma YK, Saiyed HN. Silicosis and its 25th edition, Signature Publications, 2004. Asbestos-cement industries manufacturing corrugated and flat desperately felt to assess the present day asbestos handling
control in small-scale silica mills in India. WHO newsletter Gohnet sheets, moulded goods, pipes etc. are the major consumers of workplaces and workers. The Company manufactures Compressed
(Global Occupational Health Network), 12; 12-15, 2007. 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
2. NIOH Report. Prevention, control and treatment of silicosis and
the Calendar Machine to form Compressed Asbestos Fibre Jointing
silico tuberculosis in agate industry- report submitted by NIOH to
Asbestos fibers when breathed in may get trapped in the lungs Sheets. These sheets are cut, as par required size, graphited, if
the Ministry of Health and family welfare, Govt. of India, 2004. and may remain there for a long time. Over time, these fibers can required printed, bundled and packed.

20 21
Annual Report 2007-08

Materials and Methods Fiber concentrations in the workplaces were evaluated using SKC
personal samplers with a flow rate of 1-2 ltr./min. The membrane filter
In India the chief use of asbestos is in roofing, pipeline and fireproofing. method using phase contrast microscopy was used. Samples were
Asbestos cement product manufacturing industries producing AC collected on cellulose acetate membrane filters (diameter 25mm,
sheets and pipes are the major user of asbestos in our country. Apart pore size 0.8mm) using personal samplers with flow rates of 1-2 LPM.
from this a portion of asbestos use in our country is committed by Samples were shifted to the laboratory and slides were prepared using
manufacturers of other asbestos containing materials (e.g. friction acetone-triacetin method. Fibres (length >5 m, width <3 m and
materials, jointing materials etc.). This study was carried out among aspect ratio ³3:1) were counted using Walton-Beckett graticule at
the workers of asbestos jointing material industry with the aim of magnification of 400X. Fibre levels were then compared with the
evaluating the working environment as well as the workers. Seventy permissible levels mentioned in the Indian Factories Act, 1948 (1
workers participated in this study. The aim and purpose of the study fibre/ml) and the recommended exposure levels of OSHA (0.1
was explained to the participants and consent was obtained fibre/ml), NIOSH (0.1 fibre/ml).
accordingly. Every individual subject was interviewed with a pre-
designed questionnaire to collect information in relation to personal, Results and Discussion
occupational and morbidity details of the workers. Lung function test
was carried out in all subjects. Forced vital capacity (FVC) was Mean age of the study subjects was 37.8±9.02 years (range 22-60
recorded by Spirovit-sp-10 (Schiller Health Care Ltd, Switzerland). years). Majority of the workers were more than 30 years of age.
Three successive recording were made in standing posture and the Twenty-four workers (39.3%) were of more than forty years age.
nose clip was used. The best of the three performances was Thirty workers (42.9%) were in 31-40 years age group. Sixteen
considered for calculation purpose. Other different parameters like workers (22.9%) were up to thirty years of age. Among the total
FEV1, FEF25-75 were also calculated from the same tracings. All volumes number of workers 63 (90%) were married and the rest were not. Only
obtained were expressed in body temperature on atmospheric 27.1% workers had more than secondary level education and about
pressure of air saturated with water vapour (BTPS). Body height and 14.3 % workers illiterate. Mean body mass index of the workers was
body weight were measured in bare feet on a standard scale. 20.6±4.4. Majority (57.1%) had normal body mass index values
Pulmonary function test values were predicted from the standard (18.5-24.99) whereas 30% and 12.9% workers had values lower and
prediction equation. The instrument was calibrated every day before higher than normal respectively. Among the total number of workers,
starting the experiment. FVC and FEV1 were expressed in litres, FEF25-75 10 (14.3%) were smokers and 28 (40%) workers were chewers.
in litres/sec and FEV1% was presented as the ratio of FEV1 and FVC. Majority of the workers (51.4%) were from Process Division followed
by Maintenance (24.3%) and Packing/Loading (8.6%). Other workers
All the study subjects were evaluated by radiological examination participated in the study were from Stores and other departments
as per ILO guidelines. Chest X rays (PA view) were obtained with the including persons employed for cleaning (15.7%). Mean job duration
subject in inspiration. These X rays were examined using ILO of the workers was 11.77 ± 7.71 years. Minimum experience was 1
Classification of Pneumoconioses Radiographs. Data were analyzed year and maximum experience was 30 years. Majority of workers
using Epi Info 5 and SPSS software. (45.7%) had experience of more than 10 years, whereas 25.7%

Table 1: Distribution of pulmonary function test parameters according to age and experience
Age Group (yrs.) FEV1 FVC FEF 25%- 75%
Up to 30 3.57 ± 0.62 4.07 ± 0.75 4.22 ± 1.36
31-40 3.29 ± 0.55 3.76 ± 0.65 3.94 ± 1.18
41-60 2.87±0.75 3.43 ± 0.97 3.15 ± 1.23
Significance (One way ANOVA) F=5.98; p<0.01 F=3.19; p<0.05 F=4.34; p<0.05
Experience Group (yrs.)
Upto 5 3.19 ± 0.61 3.65 ± 0.71 3.98 ± 1.67
6-10 3.33 ± 0.76 3.85 ± 0.84 3.67 ± 1.46
11 and above 3.16 ± 0.7 3.69 ± 0.89 3.61 ± 1.30
Significance (One way ANOVA) NS NS NS

23
Annual Report 2007-08

workers had duration of work of 6-10 years. Almost twenty nine 2. HSDB. 2003. Hazardous Substances Database. National Library of
percent of workers were having experience of up to 5 years of work. Medicine. http://toxnet.nlm.nih.gov/cgibin/sis/htmlgen/HSDB.

So far as morbidity is concerned, 5.7 % of the workers were 3. Pigg BJ (1994). The uses of chrysotile. Ann Occup Hyg 38: 453-
suffering from frequent cough during work, 2.9% from backache and 458.
2.9% from joint pain. Headache (5.7%), eye irritation (2.9%) and nasal 4. Agency for Toxic Substances and Disease Registry (June 2006).
irritation (2.9%) during or immediately after work were the other
Asbestos:Health Effects http://www.atsdr.cdc.gov/asbestos/
complaints of the workers. Pulmonary function test revealed that
11.4% workers were having obstructive disorder, 2.9% were having asbestos/health_effects/index.html.
restrictive abnormality and rests were normal. Significant effect of age
5. Agency for Toxic Substances and Disease Registry (September
(Table 1) on pulmonary function test parameters' values was observed
but no such significant effect was observed in relation to work 2001). Public Health Statement for Asbestos.
experience. Radiological findings suggestive of Koch's infection (Right http://www.atsdr.cdc.gov/toxprofiles/phs61.html.
Upper Zone) were observed in two workers. One of them had right-
sided pleural effusion also. 6. National Toxicology Program. Report on Carcinogens. Eleventh
Edition. U.S. Department of Health and Human Services, Public
Environmental monitoring was carried out at four sampling Health Service, National Toxicology Program, 2005.
locations. Fiber concentrations are shown in Table 2. Permissible 7. U.S. Environmental Protection Agency. Health Effects Assessment
Exposure level (PEL) in India is 1 fiber/ml whereas other agencies like for Asbestos. U.S. Environmental Protection Agency, 1984.
NIOSH, ACGIH and OSHA recommend 0.1 fiber/ml. It can be seen from
EPA/540/1-86/049 (NTIS PB86134608).
table 2 that fiber concentrations are lower than PELs.
8. International Agency for Research on Cancer. Asbestos. IARC
Monographs on the Evaluation of Carcinogenic Risks to Humans,
Table 2: Mean Fiber Concentration (fibers/ml) at different locations vol. 14. Lyon, France.
Location No. of Fiber levels
http://monographs.iarc.fr/ENG/Monographs/vol14/volume14.pdf.
Samples (Mean±SD)
Fiber Feeding 12 0.0382 ± 0.0224 9. Harvey G, Page M, Dumas L. Binding of environmental
Calendermachine 12 0.0135 ± 0.0128 carcinogens to asbestos and mineral fibres. Br J Ind Med 1984,
41: 396-400.
Cutting/finishing 6 0.0107 ± 0.0071
Near godown 6 0.0217 ± 0.0114 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
Conclusion
AP & Felton DV ed. Biological interaction of inhaled fibers and
Fiber level in all the workplaces were below the national and cigarette smoke. Richland, Washington, Battelle Pacific, pp 97-
international standards. Only three percent workers were found to have 120.
restrictive type of pulmonary function impairment. No worker was
11. Murlidhar V, Kanhere V. Asbestosis in a composite mill at Mumbai:
found to have radiographic features suggestive of interstitial lung
a prevalence study. Environmental Health: A Global Access
fibrosis.
Science Source 2005; 4:24.
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

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


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

25
Annual Report 2007-08

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

27
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


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

33
Annual Report 2007-08 Annual Report 2007-08

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

34 35
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
Annual Report 2007-08 Annual Report 2007-08

98.4%, 81.3 104.5%, 91.8 100.8%, 80.5 104.5%, 80.5 96.4%, 81.4 8. V. Muzyka, S. Bogovski, A. Viitak, T. Veidebaum, Alteration of Biomonitoring of Exposure to Urban Air pollutants in Traffic Policemen
92.8% respectively for tt-MA, SPMA, MA, HA and MHA. Urinary benzene heme metabolism in lymphocytes and metal content in blood
metabolites, tt-MA and SPMA are well-established biomarkers of plasma as markers of diesel fuels effects on human organism. HR Rajmohan and B Ravichandran
benzene exposure at level below 5 ppm. It is also reported that at The Science of the Total Environment, 2002; 286: 73-81.
exposure below 1 ppm (3.2 mg/m3) SPMA is very specific biomarker of 9. AM Mohon Rao, GG Pandit, P Sain, S Sharma, T M Krishnamoorthy Abstract
benzene than tt-MA20. Estimation of these metabolites, tt-MA and SPMA and KSV Nambi. Non-methane hydrocarbons in industrial
simultaneously in this study revealed a better picture of benzene locations of Bombay. Atmospheric Environ. 1997; 31: 7, 1077-1085.
exposure among the petrol pump workers in the range, <1ppm to 5 The urinary excretion of 8-hydroxy deoxyguanosine has been used as a biomarker of oxidative DNA damage in
10. Gautam Samanta, Gautam Chattopadhyay, Badal K Mondal, Tarit
ppm. The benzene levels both, personal exposure and static outdoor air, Roy chowdhuri, Partha P. Chowdhury, Chitta R. Chanda, Prabal both the clinical and occupational setting. The urinary 8-hydroxydeoxyguanosine in traffic policemen posted
at the petrol pumps showed values much below 1 ppm. The lower Banarjee, Dilip Lodh, Dipankar Das and dipankar Chakraborty, Air at busy traffic junctions were estimated along with the control population away from the busy traffic junctions
values of tt-MA compared to SPMA may be accounted due to the fact those doing administrative job. A total of 100 urinary samples (56 samples of traffic policemen and 44
pollution in Calcutta during winter A three year study; Current
that SPMA is comparatively more sensitive at levels < 1ppm (Table 3). It
science , 1998; vol. 75 no. 2. samples of control population were collected for estimation of 8-OHdG and analysed using enzyme linked
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 11. PK Padhy, C.K. Varshney, Total non-methane volatile organic immunosorbent assay (ELISA). The mean 8-OHdG significantly higher (14.24 ± 12.53 mg/g creatinine) than
SPMA respectively. Table 4 shows the urinary metabolites of aromatic compounds (TNMVOC) in the atmosphere of Delhi. Atmospheric level of those of control group (8.34 ± 6.24 mg/g creatinine) (p<0.01). The comparison made between the
hydrocarbons among the occupationally unexposed population. On Environment 34 (2000) 577-584.
study and control groups taking into consideration over all experience the results showed 13.88 ± 12.35 mg/g
comparing the levels of urinary metabolites in post-shift samples of 12. A K Mukherjee, SK Bhattacharya, S Ahmed, SK Roy,
Roychowdhury, A, S Sen “Occupational exposure of drivers and creatinine 8-OHdG in traffic policemen and 8.34 ± 6.24 mg/g creatinine 8-OHdG in control subjects which was
exposed worker, engaged in petrol pumps to that of the occupationally
unexposed population, it was observed that the levels of tt-MA (p< conductors to noise, heat dust and Volatile Organic Compounds in statistically significant (p<0.01). The 8-OHdG levels were significantly higher in non smokers of study group
0.001), SPMA (p< 0.05), MA (p< 0.001) and HA (p< 0.001), expressed the state transport special buses of Kolkata City”. Transportation compared to the control group (p<0.01). The study showed that urinary 8-OHdG is also associated with
in mg/L urine among exposed group were significantly higher. Research Part D Elsevier Science Ltd, 2003; 8: 11-19. occupational and other lifestyle factors.
However, in case of the values of urinary metabolites expressed in mg/g 13. P Kumar, BN Gupta, KP Pandya, SH Clerk. Behavioral studies in
creatinine, the levels of tt-MA (p< 0.001) and HA (p< 0.01) were found to petrol pump workers. Int. Archives of Occupational and Keywords: Urinary 8-hydroxy deoxyguanosine, traffic policemen, ELISA
be significantly higher. Environmental Health, 1988; 61: 1-2.
References 14. M Singhal, F Khaliq, S Singhal and O.P. Tandon. Pulmonary
functions in petrol pump workers: A preliminary study. Indian J. Introduction benzene concentration was in the range of 123.20 - 4048.00 mg/g
1. Boogaard PJ and Sittert NJV. Suitability of S-phenyl mercapturic Physiology Pharmacology, 2007; 51: 3, 244-248. creatinine in traffic policemen and 30.30 - 2462.90 m/g creatinine in
acid and trans trans-muconic acid as biomarkers for exposure to 15. C. Kesavachandra, S. K. Rastogi, M. Anand, N. Mathur, A. Urban air contains a diversity of chemical compounds, some of which petrol filling station workers. It is recognized that these population are
low concentrations of Benzene. Env. Health Perspectives, Dhawan. Lung function abnormalities among petrol pump are genotoxins. The urinary excretion of 8-hydroxy deoxyguanosine at high risk. Hence, biomonitoring of urinary 8-OHdG, an indicator of
1996;104: Suppl. 6. workers of Lucknow, North India. Scientific Correspondence, has been used as a biomarker in both the clinical and occupational oxidative DNA damage might indicate the likely extent of harmful
2. Duydu Y, Suzen S, Erdem N, Uysal H, Vural N. Validation of hippuric Current Sci, 2006;90: 9, 1177-1178 setting.1-6. Exposure to various environmental pollutants such as
effect that may occur on these groups. Urinary 8-OHdG is easy and
acid as a biomarker of toluene exposure. Bulletin of Environmental 16. Y Verma and SVS Rana. Biological monitoring of exposure to polyaromatic hydrocarbons, fly ash and fine particulate matter 2.5
non-invasive to collecting compared to collect tissue or leukocyte
Contamination and Toxicology 1999; 63: 1, 1- 8. benzene in petrol pump workers and dry cleaners. Industrial containing metal fumes of vanadium, chromium, manganese, nickel,
samples.
3. Angerer, J. “Occupational chronic exposure to organic solvents.” Health, 2001; 39: 4, 330-333. copper and lead increased the level of urinary 8-OHdG 7-9.
XII. o-cresol excretion after toluene exposure”. Int. Archives of 17. American Conference of Governmental Industrial Hygienists Occupational exposure to benzene is a known carcinogen. A dose
Occupational and Environmental Health 1985; 56 (4):, 323- 328.
Materials and Methods
(ACGIH). 1999 TLVs and BEIs. Threshold Limit Values for response relationship was found between personal exposure to
4. J. Angerer, A. Kramer, Occupational chronic exposure to organic Chemical substances and Physical Agents. Biological Exposure benzene and urinary 8-OHdG concentrations3. In order to expound the The urinary 8-hydroxydeoxyguanosine in traffic policemen posted at
solvents XVI. Ambient and biological monitoring of workers Indices. Cincinnati OH. 1999. health effects arising out of occupational exposure, monitoring of busy traffic junctions were estimated along with the control population
exposed to toluene. International Achieves of occupational and 18. J W S Longhurst, S J Lindley, A F R Watson, D E Conlan. The biomarkers of early genetic effect will be a useful tool. away from the busy traffic junctions those doing administrative job. A
environmental health, 1997; 69: 91-96. introduction of local air quality management in the United total of 100 urinary samples (56 samples of traffic policemen and 44
5. Jacobson GA, and McLean S. Biological monitoring of low level Kingdom: A review and theoretical framework. Atmospheric Studies were carried out on air pollution revealed that urban samples of control population) were collected for estimation of 8
occupational xylene exposure and the role of recent exposure. Ann. Environment, 1996; 30: 23, 3975-3985. population is exposed to various inorganic and organic pollutants. An OHdG and analysed using enzyme linked immunosorbent assay
Of Occupational Hyg, 2003; 47: 4, 331- 336. 19. Parivesh, a news letter from ENVIS Centre Central Pollution increased risk of cancer has been reported in occupations with heavy (ELISA). The collected urine samples were centrifuged at 2000 rpm for
6. S J Shahtaheri, M Abdollahi, F Golbabaei, A Rahimi-Froushani, F Control Board, (2002). exposure to traffic related pollution10. Recently study on health risk 10 minutes and the supernatants were used for the determination of 8
Ghamari. Optimization of SPE for Analysis of Mandelic Acid as a 20. Barbieri A, Sabatini L, Accorsi A, Roda A and Violante FS. assessment of rural and urban population due to indoor / ambient air OHdG. Urinary 8 - OHdG levels and subsequently adjusted by urinary
Biomarker of Exposure to Ethyl Benzene. Iranian J Env Health Sci Simultaneous determination of t,tmuconic acid, S- pollution of Bangalore city showed that the concentration of benzene, creatinine levels.
Eng, 2004, Vol.1, No.2, Vppo.l7.10,-N80. phenylmercapturic and S-benzylmercapturic acids in urine by a toluene and m- xylene varied from 4.35-55.10, 8.73-103.61 and
7. J. Y. Jang, P.O. Droz, S. Kim, Biological monitoring of workers rapid sensitive liquid chromatography / eletrospray tandem mass 1.53-135.67 mg/m3 respectively in traffic policemen and 1.200- The data pertaining to personal habits like smoking, alcohol
exposed to ethylbenzene and co-exposed to xylene. International spectrometry method. Rapid Communications in Mass 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
Achieves Occup. Environ. Health, 2001, 74:31-37. Spectroscopy, 2004; 18, 1983-1988. workers. The trans, trans-muconic acid a known biomarker of were collected using a questionnaire survey.

38 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
Annual Report 2007-08 Annual Report 2007-08

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

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

46 47
Annual Report 2007-08 Annual Report 2007-08

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

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

52
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 many as 25000 children between the age of 10 and 15 yrs are mainly
employed in shoe making both for the domestic and international
There is strong evidence that benzene is hematotoxic and causes markets. Children work on soling (fixing upper portion of shoe to
various types of leukemia. It has been classified as a group - I leather or rubber soles) with glue. The children working are exposed to
1
carcinogen by IARC . Two minor urinary metabolites, trans, trans benzene that is used as a solvent in glues. Most children suffer from
muconic acid (t,t-MA) and s-phenylmercapturic acid (SPMA) are respiratory problems, lung diseases and skin infections through
specific biomarkers of benzene exposure and can be used to monitor constant exposure to glue and fumes.
the benzene exposure in working environment2-7.
Materials and Methods
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 Chemicals: t,t-MA and SPMA standards were procured from Sigma-
be influenced by the uptake of sorbic acid (a widely used food Aldrich, Methanol (MeOH), Acetonitrile (ACN) and HPLC water of LC
preservative). While SPMA is formed after conjugation of the highly grade were obtained from JT Baker ltd, USA, Iso propanol and
reactive benzene oxide with glutathione. Dietary or no other sources for disodium hydrogen phosphate (Merck (I) Ltd), Ethyl acetate,
excretion of SPMA than exposure to benzene have been reported so far. Creatinine, Formic acid from Fluka, Acetic acid and Hydrochloric acid
were obtained from Across Organic, USA. Durapore Membrane filter
In this study, we have used a liquid chromatography / from Milipore were used for the fitering of mobile phase. QSAX, 100
electrospray tandem mass spectrometry method for the mg/3cc cartridge for sample clean up and extraction of analytes was
determination of t,t-MA and SPMA in the same analytical run from the used from Analchem Ltd, India.
urine samples. This method was applied to monitor benzene exposure
among child laborers of footwear industry of Agra and diamond Sample collection: Spot urine samples from randomly selected
industry workers of Surat. Thefootwear industry is a significant 30 exposed child labourers working in footwear manufacturing at
segment of the leather industry in India and ranks second among Agra, 15 matched control groups from school children studying in
footwear producing countries next to china. According to estimate, as government school located in the neighboring area, 60 workers

53
Annual Report 2007-08

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

55
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

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

56 57
Annual Report 2007-08 Annual Report 2007-08

Discussion 3 Ghittori S, Maestri L, Rolandi, L. Lodola L, Fiorentino ML, Imbriani Occupational Work Stress Among Cycle Rickshaw Pullers
M. The determination of t,t-MA in urine as an indicator of
A sensitive and specific LC-MS/MS method has been standardized for exposure to benzene. Appl. Occ. Env. Hyg., 1996; 11:187-191 CK Pradhan, B Dalal, K Nayak and S Thakur
the simultaneous measurements of two urinary metabolites, t,t-MA 4 Ruppert T, Scherer G, Tricker AR, Adlkofer T. trans, trans-
and SPMA, which are known to be good biomarkers of low levels of muconic acid as a biomarker of non-occupational environmental Abstract
benzene exposure in humans. Previous studies have indicated that the exposure to benzene. Int. Arch. Occup. Environ Health, 1997;
exposure level could influence the ratio of benzene metabolites. At low 69:247-251. The study was undertaken to evaluate the effect of rickshaw pulling on physiological and biochemical
levels of exposure to benzene, excretion of t,t-MA can be relatively high 5 Van Sittert NJ, Boogard PJ, Beulink GD. Application of the urinary parameters of cycle rickshaw pullers. Fifteen cycle rickshaw pullers and eight control subjects, in the age
even when SPMA excretion is very low9,10. Urinary SPMA in workers S- phenylmercapturic acid test as a biomarker of low level of
exposed to 0.5-ppm benzene has been reported in the range from 7.2to
group of 20-39 years were selected randomly from K olkata. Each subject participated for two consecutive
exposure to benzene in industry. Br. J. Ind. Med. 1993; 50:460-469.
25 µg/g creatinine. Similarly, the predicted excretion of t,t-MA ranged 6 Ghittori S, Imbriani, M Mestri L, Capodaglio E, cavallen A. days for determination of maximal oxygen uptake (VO2max) and energy expenditure study for doing work in
from 0.39 to 1.1 mg/g creatinine for 0.5 ppm benzene exposure11. As Determination of s-Phenaymercapturic acid in urine as an three phases. Pre and post exercise blood samples were collected and analysed for biochemical parameters.
t,t-MA is a metabolite of sorbic acid12 the values of t,t-MA in urine are indicator of exposure to benzene. Tox. Lett. 1999; 108: 329-34. VO2max of the rickshaw pullers was significantly higher than those of the control subjects. Average working
strongly influenced by the consumption of food or drinks containing 7 Melikian AA, Qu Q, Shore R. et al. Personal Exposure to different heart rate, peak heart rate and energy expenditure of the cycle rickshaw pullers and the control subjects
sorbic acid which is used as a preservative, while SPMA is a specific levels of benzene and its relationships to the urinary metabolites showed that the workloads of both the groups are heavy to very heavy. The energy requirement was same for
biomarker of benzene exposure at low level. s-Phenylmercapturic acid and trans, trans- muconic acid. J both the groups while doing heavy work. Significant difference in glucose level was observed between
chromatogr. B Analyst Technol. Biomed. Life Sci. 2002; 778:
It is observed by Wiesel et al13 that at relatively low benzene
rickshaw pullers and control subjects at pre-exercise condition. The Lactate Dehydrogenase (LDH) in pre-
211-221.
exposure levels, the complexity of the urine matrix suppressed the 8 Harold Varley Determination of creatinine in urine. Practical exercise condition of cycle rickshaw pullers was higher than normal range. Increasing trend of Creatine
ionization. The suppression of monitored analytes by co-eluting matrix Clinical Biochemistry, Vol 1, Allan H Gowenlock and Maurice Bill K inase (CK ) was noted after exercise in pullers. Cycle rickshaw pulling is categorised as heavy to very heavy job.
th
compound was also observed in our method. Therefore, in HPLC (Eds.), 5 Edition, 1980; 484-485.
separation dual columns of different dimensions were used to reduce 9 Subourin PJ, Bechtold WE, Griffit WC Bimbaum LS, Lucier and Keywords: Rickshaw pullers, maximal heart rate, energy expenditure, creatine kinase
the matrix suppression on analytes at their elution times. Henderson RF. Effect of exposure concemntration, exposure rate
and route of administration on metabolism of benzene by F344
In a study, among Chinese footwear workers, it was reported that rats and B6C3F1 mice. Tox. Appl. Pharm.1989; 99: 421.
these workers were exposed to high levels of benzene, toluene and 10 Henderson RF, Subourin PJ, Bechtold WE, Griffith WC, Medinsky Introduction Approval of the ethical committee of the institution was obtained.
other toxic solvents contained in the adhesive used in the shoe making MA, Bimbaum LS and Lucier GW. The effect of dose, dose rate, Written consent was obtained from all subjects prior to study.
process and many workers have been affected with aplastic anemia, route of administration and species on tissue and blood levels of 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
leukemia and other health problems14. benzene metabolites. Environ. Health Perspect. 1989; 82:9.
11 Melikian A, O'Concor, R, Prahalad AK , Hu P, LI H, K agan M influence their physiological and metabolic responses. The workload consecutive days. On the first day, body height, weight and resting
The observed values of t,t MA and SPMA in our study are below the Thompson S. Determination of the urinary benzene metabolites
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
values of Biological Exposure Indices (BEI) i.e. 500 µg/g creatinine for S-phenylmercapturic acid and trans,trans-muconic acid by reported that significant fall in blood glucose takes place after heavy index (BMI) were calculated from the height and weight of the
t,t-MA and 25 µg/g creatinine for SPMA, described by ACGIH15 of
2
exercise . Earlier study indicated that serum creatine kinase (CK ) subjects. Subsequently, the subjects were asked to pull rickshaw with
liquid chromatography/electrospray tandem mass
exposure to benzene. Except in one exposed subject in the footwear increased during first hours after exercise3. Heavy exercise increases two passengers for a period of 15 minutes according to their own habit
spectrometry. Carcinogenesis, 1999; 20 (4): 719-726.
lactate production. Lactate dehydrogenase (LDH) activities enhances of pedalling frequency on the same route in order to follow a standard
industry the values of t,t-MA and SPMA, and in two subjects the values 12 Barbieri A, Sabatini L, Accorsi A, Roda A & saverio Violante F.
after heavy physical exercise or submaximal exercise for a long road condition. After completion of this work, they are allowed to take
of SPMA were found higher than BEI described by ACGIH. These results Simultaneous determination of t,t-muconic,Sphenylmercapturic
duration4,5. However, data on difference in physiological responses rest for 15 minutes. This phase of 30 minutes (work plus rest) was
also support that the diamond manufacturers have stopped the use of and S-benzylmercapturic acids in urine by a rapid and sensitive
considered as Phase I. Again the cycling was continued and
benzene in diamond processing, as exposure is very low. Observed liquid chromatography /electrospray tandem mass spectrometry during various work phases and biochemical characteristics induced
designated as Phase II and Phase III. During pulling cycle rickshaw,
values might be due to exposure through other sources of method. Rapid Communication in Mass spectrometry. 2004; 18: by cycle rickshaw pulling were not available. The objective of the
heart rates were monitored every minute by heart rate monitored
environmental pollution. 1983-1988. study is to evaluate the effect of rickshaw pulling on physiological and
(Polar, Finland). The average working heart rate (AWHR) was
13 Weisel c, Y u R, Roy A, Georgopoulos P. Env. Hlth Persp. 1996; biochemical parameters of cycle rickshaw pullers and compare the
calculated from the fourth minute of work7. The peak working heart
104:1141. data with that of control subjects. rate of the subjects was recorded. Recovery heart rates were also
References 14 Chen MS, Chan A China's “ Market economics in Command” recorded every minute and sum of recovery heart rate was calculated.
1 IARC Monograph on the evaluation of carcinogenic risk to humans: Footwear workers Health in Jeopardy. Int. J. Health Serv., 1999; Materials and Methods The energy expenditure for cycle rickshaw pulling was calculated
Occupational exposure in petroleum refining, crude oil and major 29 (4) : 793-811. from the peak working heart rate (PWHR) using the equation of Datta
petroleum fuels, IARC; Lyons, France 1989:45. 15 American conference of Governmental Industrial Hygienists. Subjects: Fifteen cycle rickshaw pullers in the age group of 20-39 and Ramanathan8. On the second day, the subject was allowed to do
2 Inoue O, Seiji K , Nakatsuka H, et al. Urinary trans, trans muconic Threshold Limit Values and Biological Exposure Indices for 2001, years, were selected randomly. Eight control subjects (non rickshaw graded exercise in a bicycle ergometer (Biodex, USA) in the laboratory
acid as an indicator of exposure to benzene. Br. J. Ind. Med. 1989; American Conference of Govt Industrial Hygienists, Cincinnati, pullers) of same age group were selected from the adjoining area of in order to determine maximal oxygen uptake (VO2max) and maximal
46:122-127. OH, 2001. K olkata. They were engaged in jobs like gardening and farming. heart rate (HRmax).

58 59
Annual Report 2007-08

Control subject also participated in this study for two consecutive were 134 ± 12.4, 133 ± 14.9 and 134 ± 16.7 bpm respectively
days. On the first day, physical characteristics and resting heart were suggesting that pulling cycle rickshaw is a job of heavy category17.
noted. HRmax and VO2max were determined in the laboratory. Next
day, the subject was asked to pedal a bicycle ergometer (similar to The mean PWHR of rickshaw pullers were found to be 145 ± 18.2
cycle rickshaw pullers) in three phases of 15 min of work and 15 min of to 146 ± 14.9 bpm. This observation also substantiated that the job
18,19
rest, each. The ergometer load was adjusted to produce heart rates in was very heavy . As the sum of recovery heart rate (beats) did not
the range of 80% to 90% of their maximal heart rate. PWHR was noted vary (Phase I-1250 ± 110.8, Phase II 1254 ± 117.4 and Phase III
and AWHR and recovery heart rates were also recorded every minute. 1258 ± 130.8), it could be interpreted that the work stress imposed on
During experiments the dry bulb (DB) temperature was 30.6 ± 3.09 ° C the rickshaw pullers were same in three phases. Repetition of work
and wet bulb (WB) temperature was 27.2 ± 1.46 ° C. The VO2max study has no effect on physiological functions. In case of the control subjects,
was conducted in the laboratory in comfortable environment at DB the values in three phases were 1339 ± 267.10, 1331 ± 248.50 and
25.7 ± 1.54 ° C; WB 18.7 ± 2.16 ° C. 1277 ± 251.98 beats respectively, which were not significantly
different from each other as also with those of rickshaw pullers.
Biochemical analysis: In case of rickshaw pullers, on the first day,
The energy expenditure (EE) of rickshaw pullers in three phases
2 ml of blood sample was collected from antecubital vein one hour -1
were 21.7 ± 2.27.and 21.7 ± 2.82 and 21.3 ± 3.43 kJ.min
after completion of three phases of exercise. The state was
respectively and therefore the job of cycle rickshaw pulling can be
designated as post-exercise condition. Next day, blood was also 17,19
categorized as heavy . The results of the control subjects were
collected when the subject reported to the laboratory and was at rest
almost same with the rickshaw pullers.
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
Relative costs of work of the cycle rickshaw pullers and control
after reporting at the laboratory followed by collection of blood
subjects as percentage of maximal energy expenditure have been
samples (2 ml) from antecubital vein. This was pre-exercise condition.
presented in Table 1. Result showed that there is an decreasing trend
Next day, it was collected after work (pedalling a bicycle ergometer).
of the values in rickshaw pullers compared to control subjects in all the
This stage was defined as post exercise condition.
three phases of work. Significantly less value (p<0.05) was observed
Each sample of blood was separated in two tubes: one containing in rickshaw pullers in phase I of work.
fluoride (1 ml) for glucose; and the other (1 ml) was allowed to clot for
serum separation. The following parameters were assayed by Table 1. Relative cost of work as percentage of maximal
Chemwell analyser (USA) using standard commercial kit following energy expenditure
methods of (i) Trinder9 for plasma glucose; (ii) Bakkar10 for Serum LDH; Work Control Subjects Rickshaw puller
(iii) Ellis11 for Serum CK ; (iv) Bowers12 for Alkaline Phosphatase and (v) Phase (n = 8) (n = 15)
Bergmeyer13 for SGOT (vi) Fossati et al14 for Serum uric acid.
Phase I 81.44 ± 9.58 74.18 ± 7.72*
Results
Phase II  13.20
83.25 ± 74.26 ± 10.44
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 Phase III 79.88 ± 6.90 73.15 ± 12.35
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 biochemical variables in pre-exercise condition were found
to be out of standard reference range, as manifested by elevated level
The average HRmax of the rickshaw pullers was 187 ± 6.2 bpm of LDH (53% cases in rickshaw pullers, 38% cases in control
and it was significantly higher (p <0.05) than the value of control subjects), plasma glucose (13% cases in rickshaw pullers, no cases in
subjects (179 ± 10.9). The VO2max of the rickshaw pullers (36.6 ± control subjects), CK (33% cases in rickshaw pullers, 25% cases in
-1 -1
4.32 ml.kg .min ) were significantly higher (p<0.001) compared to control subjects). Elevation of LDH was observed in 80% cases and
control subjects (30.1 ± 3.14 ml.kg-1.min-1). 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
The heart rates during work varied from subject to subject in was noted in 50% cases as an effect of work and elevated CK was
different phases. The AWHR of rickshaw pullers in phase I, II and III observed in 25% cases like pre-exercise condition.

61
Annual Report 2007-08 Annual Report 2007-08

Table 2 shows that effect of work on energy utilization among the cycle 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
rickshaw pullers and control subjects. The plasma glucose level was phases. Energy requirement was same for both the groups while benzene sulpfonic acid/ aminophenazone choromogenic system
found to be within normal range at both pre and post exercise session. 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.
The level of serum LDH increased at post-exercise session in 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
comparison to that of pre-exercise condition among rickshaw pullers. 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.
Similar trend was also noted in control subjects. Similarly, the level of of work. interpretation of anthropometry. Technical Report Series No.
serum CK was also elevated after rickshaw pulling in comparison to 2. Mamus RT, Santos MGD, Campbell B and K reider R Biochemical
effects of carbohydrate supplementation in a simulated 854. Geneva : WHO 1995.
that at resting condition. 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
The effect of work on serum alkaline phosphatase (ALK P) and pre-exercise condition. The interesting observation noted was 2006; 3: 6-11. Pacific J Clin Nutr 2002; 11(Suppl) : S681 - S684.
serum aspartate transaminase (AST) is also given in Table 2. Pre and 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.
post exercise values of ALK P were within normal range both in case of 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.
rickshaw pullers and of control subjects. In case of AST, a significant 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.
(p<0.001) difference was observed between control and rickshaw Similarly the level of Serum CK was elevated after rickshaw pulling in
4. Costill DL, Fink WJ, Pollack ML Muscle fiber composition and 1960.
pullers at both post and pre-exercise condition. In post exercise comparison to that of pre-exercise condition. Decreasing trend of CK
condition, uric acid was noted above normal range in case of control 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
subject. On the other hand, uric acid was exhibited within the normal 8 : 96 100. India. J Human Ergology 1975; 4, 103-113.
reveal that creatine phosphate, as coin of energy, during exercise was
range among rickshaw pullers at both pre- and post- exercise session. 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.
Discussion 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.
The study indicated that the present rickshaw pullers had similar work session maintain normal range. Higher than normal value of AST was
capacity to that of those engaged in other occupations like steel mill 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
workers20, Indian soldiers21 and agricultural workers22. VO2max of the 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.
rickshaw pullers were significantly higher than those of the control in pre and post-exercise session, might be maintained at normal level. 7. Nag PK , Sen RN and Ray US. Cardiorespiratory performance of
subjects. 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.
The job of cycle rickshaw pulling is considered as heavy to very exercise session compared to pre exercise session whereas the 8. Datta SR and Ramanathan NL. Energy expenditure in work
heavy based on peak working heart rate and energy expenditure17,19. rickshaw pullers habituated with this profession may not face such predicted from heart rate and pulmonary ventilation. J Appl
Data of different phases of work showed that there was no significant stress.
Physiol 1969; 26 : 297 - 302.
9. Trinder P Determination of Glucose in blood using glucose
Table 2. Effect of exercise on biochemical changes among control and cycle rickshaw pullers oxidase with an alternative oxygen acceptor. Anal Clin. Biochem
Variable (Unit) Pre exercise Post exercise 1969; 6:24
Control Cycle Rickshaw Pullers Control Cycle Rickshaw 10. Bakker AJ, Bakker A, Bierma-Ram A, Dijkstra JT, Renting-Wiering
(n=8) (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
Plasma Glucose (mg/dL) 69.3±12.16 81.8* ±15.13* 61.1±10.32 75.4 ± 7.43* * *
Chem 2005; 51: 215-217.
** **
Serum LDH (U/L) 373.1±76.02 469.1 ±61.66 411.1±113.93 477.0 ± 58.20 11. Ellis G, Gold berg DM Automation of a kinetic spectrophotometric
Serum CK (U/L) 151.5±90.74 182.9±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-
Serum ALK P (U/L) 243.63±68.17 227.00±150.58 236.25±82.93 222.37 ± 92.77 422.
*
Serum AST (U/L) 29.00±8.68 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.
Serum uric acid (mg/dL) 5.69±1.78 6.30±1.81 5.79±1.35 5.30 ± 1.23
Clin Chem 1966; 12:70-89.
Values are Mean ±SD for each group. Values in parenthesis indicate number of subjects 13. Bergmeyer HU, Bowers GN, Horder M, Moss DW IFCC method for
* = p<0.05, * * = p<0.01, * * * = p<0.001 (compared between control and rickshaw pullers aspartate aminotransferase. Clin Chim Acta 1976; 70:F31-40.

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

65
Annual Report 2007-08 Annual Report 2007-08

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

68 69
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 was reported, however for unexposed population it ranges > 1-2 µg/L
or ppb. NIOSH considers all chromium VI compounds (including chromic
India is one of the third largest producers of chromium compounds acid) to be potential occupational carcinogens and recommends an
and contributes about 10% of the total world's production. People may exposure limit of 1 µg chromium (VI)/m3 for a 10-hour workday, 40-hour
also be exposed to higher levels of chromium if living near to the workweek. Worldwide reports are very scanty on the excretion of
landfill sites with chromium-containing wastes, industrial facilities chromium in unexposed population and occupationally exposed
that manufacture or use chromium and chromium-containing workers. Even recent reports of ATSDR 2008 do not provide much
compounds, cement-producing plants, industrial cooling towers that information on the excretion of chromium species in urine of unexposed
previously used chromium as a rust inhibitor, waterways that receive and chromium workers. In one of the recent studies1 conducted in 2004
industrial discharges from electroplating, leather tanning and textile by “ Eco Friends” in a village of Noraiakheda, K anpur, North India
industries and busy roadways. Breathing in higher levels of chromium poisoning of ground water was reported 100 to 1000 times higher than
VI (greater than 2 µg/m3) can cause irritation to the nose, such as runny the desirable limits for drinking water. The concentration of hexavalent
nose, sneezing, itching, nosebleeds, ulcers, and holes in the nasal chromium has been reported up to 16.3 mg/l that is 1630 times more
septum. Long-term exposure to chromium has been associated with than the WHO standards2 (0.01 mg/l) for drinking water at a depth of 140
lung cancer. Breathing in chromium III does not cause irritation to the ft in this village. With the aims of chromium toxicity background and
nose or mouth in most people. In the same way, small amounts of health effects and the scarcity of the data in context of present Indian
chromium VI that swallowed will not hurt, however, accidental or scenario it is urgently needed to measure chromium levels in our Indian
intentional swallowing of larger amounts has caused stomach upsets environment. Total chromium has been reported by few investigators,
and ulcers, convulsions, kidney and liver damage, and even death. however, chromium species i.e. Cr III and Cr VI level have hardly been
The concentration of total chromium in air (both Cr III and Cr VI) attempted by researchers earlier. The present study describes the
generally ranges between 0.01 and 0.03 µg/m3. Chromium levels of dissolved chromium compounds (Cr III and Cr VI) in urine using
concentrations in drinking water (mostly as Cr III) are generally very ionchromatography in healthy control and occupationally exposed
low, less than 2 ppb. A wide variation in the urinary level of chromium 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).

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

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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

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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

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

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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


was observed after short term (12 week) and long term (6 months)
Panmasala plain (PMP) is a dry mixture of areca nut, catechu, lime, exposure of panmasala. Sperm and spermatid count as well as daily
cardamom and unspecified flavouring agents. Gutkha (PMT) is another sperm production was found to be significantly low after 3% PMT
popular variant of panmasala containing tobacco. Most of the ingredient treatment for 6 month2. In dominant lethal test, 4 months treatment with
of panmasala are reported to possess cytotoxic, mutagenic and
3 and 6% of panmasala reduced the fertility potential of treated mice.
genotoxic properties1. Even though data are available on mutagenicity
Data summarized for one spermatogenic cycle revealed significant
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 reduction in total and live implants / female in PMT treated mice.
mixture like panmasala and its modification by natural compounds. Preimplantation loss was increased among all treated groups while post-
Therefore, study is in progress with the following main objectives: 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.
mice was significantly high at the dose of 3% PMP and PMT after 6 and 12
 To study fetotoxic potential of panmasala. week exposure as compared to control. Further, chromosomal
 To study the role of -carotene & curcumin in panmasala induced 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
mutagenicity. three doses given for 12 weeks with respect to control.

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

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

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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
Annual Report 2007-08 Annual Report 2007-08

39
arsenic exposure. The above observation is in agreement with the a number of investigators . But in brain tissue, lipid peroxidation apoptosis in neonatal and adult brain cells in vivo and in tissue 27. Roy Chowdhury A. Male Reproductive Toxicology New
recent finding of Omura et al.29. Degeneration of interstitial (Leydig) showed a variable response after arsenic exposure for 30, 45 and 60 culture. Tox. Lett. (Special issue). 128. Perspective in Life Science. In: Life Science in Modern Perspective,
cells was observed in the testis of arsenic-treated mice. Moreover days. The LPO content increased significantly in both the arsenic- 11. Namgung, U ., Xia, Z ., 2001. Primary cultures of rat cerebellar U GC Academic Staff College, U niversity of Calcutta. 2004; 97-105.
Leydig cell population significantly decreases in both the doses over a treated groups after 30 days but a significant decrease in LPO content neurons were used to study mechanism of arsenic. Toxicol. Appl. 28. Pant N, Murthy RC, Srivastava SP. Male reproductive toxicity of
period of 60 days. The Leydig cell nuclear diameter increased in brain was found in arsenic treated mice after 45 and 60 days. Pharmacol. 174, 130-138. sodium arsenite in mice. Hum Exp Toxico 2004; 23 (8): 399-403.
significantly in both the doses in 30 days followed by gradual Arsenic induced elevation of oxidative stress reported previously by 12. Kannan, G.M., Tripathi, N., Dube, S.N., Gupta M., Flora, S.J .S., 29. Omura M, Y amazaki K, Tanaka A, Hirata M, Makita Y and Inoue N.
40,41
diminution of the Leydig cell diameter in 45 and 60 days. Inspite of a other workers but at this point of time, the possible reason of 2001. Toxic effect of arsenic (III) on some hematopoietic and Changes in the Testicular Damage Caused by Indium Arsenide and
testosterone assay in this study, it may be suggested that the sudden decrease in LPO content of arsenic exposure is not central nervous system variables in rats and guinea pigs. Clin. Indium Phosphide in Hamsters during Two Y ears after
degeneration of Leydig cell with significant decrease in the Leydig cell understood. Toxicol. 39:675-682. Intratracheal Instillations. J Occup Health 2000; 42: 196-204.
population probably would have resulted in decreased synthesis of 13. Chattopadhyay S, Pal S, Ghosh D and Debnath J . Effect of dietary 30. Bartke A. Pituitary-Testis relationship. Reprod. Biol 1991; 1: 136-
testosterone, which in turn disturb the process of spermatogenesis. It co-administration of sodium selenite on sodium arsenite induced 152.
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1. Bhattacharya, P.; Chatterjee, D.; J acks, G.Occurrence of As-
synthesis of testosterone, which is one of the main component of 14. Sarkar M, Chaudhuri G., Chattopadhayay A., Biswas NM. Effect of Endocrinology 1965; 76: 1184-1189.
contaminated groundwater in alluvial aquifers from the Delta
regulation the post meiotic stage of spermatogenesis 30-32. The sodium arsenite on spermatogenesis, plasma gonadotrophins and 32. Sharpe RM, Maddocks S & Kerr J B. Cell-cell interactions in the
Plains, eastern India: Option for safe drinking water supply. Int. J .
exogenous arsenic exposure may cause a chemical stress on the testosterone in rats. Asian. J . Androl 2003; 5: 27-31. control of spermatogenesis as studied using Leydig cell
Water Res. Dev. 1997, 13, 79-92.
cellular function. The initial increase in Leydig cell diameter may be a 15. Shukla J P, Pandey K. Impaired spermatogenesis in arsenic treated destruction and testosterone replacement. American J ournal of
2. Bhattacharya, P.; Frisbie, S.H.; Smith, E.; Naidu, R.; J acks, G.;
better indication to adopt the metal induced stress but due to freshwater fish, Colisa fasciatus (Bl. and Sch.).Toxicology Letters Anatomy 1990; 188: 3-20.
Sarkar, B. Arsenic in the Environment: A Global Perspective. In
continuous stress effect, cellular exhaust may be a result of Leydig cell 1984; 21(2): 191-195. 33. Deneke SM, Fanburg BL. Regulation of cellular glutathione. Am J
Handbook of Heavy Metals in the Environment; Sarkar, B., Ed.;
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elements on the reproduction of mice and rats. Arch. Env. Health. 34. Anderson ME. Glutathione and glutathione delivery compounds.
3. Bhattacharya P.; J acks, G.M.; Khan, A.A. Arsenic in groundwater of
In the present investigation, arsenic exposure to experimental 23: 102-106. Adv Pharmacol 38:65-79 (1996).
the Bengal delta plain aquifers in Bangladesh. Bull. Environ. Cont. 17. Tabacova, S., Baird, D.D., Balabaeva, I., Lolova, D., Petrove, I.,
mice in two doses (30 & 40 mg/L) for 30, 45 and 60 days showed a Toxicol. 2002, 69, 538-545. 35. Ochi T, Kaise T, Oya-Ohta Y . Gluthathione plays different roles in
significant alteration in the activity of many antioxidants. The GSH 1994. Placental arsenic and cadmiun in relation to lipid peroxides the induction of the cytotoxic effects of inorganic and organic
4. Naidu, R.; Bhattacharya, P. Management and remediation of ans glutathione levels in maternal-infant pairs from cooper smelter
content of brain and testicular tissue in both arsenic-treated mice arsenic from contaminated water. In. Managing Arsenic in the arsenic compounds in cultured BALC/c 3T3 cells. Experientia
revealed significant alteration with the increase in duration. In both the areas. 15: 873-881. 50:115-120 (1994).
Environment: From Soil to Human Health, Naidu, R.; Smith, E.; 18. Leblond CP and Clarmont Y . Differentiation of stages of the cycle of
dose group for 30 days, there was marked increase in GSH content Owens, G.; Bhattacharya, P.; Nadebaum, P. Eds.; CSIRO Publishing 36. Maiti S, Chatterjee AK. Effects on levels of glutathione and some
seminiferous epithelium. Ann.N.Y .Acad.Sci 1952; 55: 548-573.
that afterward decreased with the duration of 60 days. GSH is known Melbourne, Australia, 2006; 331-354. related enzymes in tissues after an acute arsenic exposure in rats
19. Ambercrombie M. Estimation of nuclear population from
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microtome section. Anat. Rec 1946; 94: 239-249.
(either increase or decrease) indicates a disturbed oxidative status. in West Bengal- worst calamity in the world. J Ind .Med Assoc 75:531-537 (2001)
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When cells are oxidatively challenged, GSH synthesis increases and 1998; 96: 4-7. 37. Liu J , Liu Y , Goyer RA, Achanzar W, Waalkes MP. Metallothionein-
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22. Ball CR (1966): Estimation and identification of thiols in rat spleen
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injury and death33,34. Cellular toxicity of arsenic was found to be Flora, S.J .S., 1997. Arsenic induce changes in certain measurement from phenol reagent. J . Biol.Chem. 193: 265-275, 26:865-869 (1999).
inversely related to intracellular GSH levels and thus may be enhanced neurotransmitters level and their recoveries following chelation in 1951. 39. Modi M, Flora SJ . Combined administration of iron and
by GSH depletion35. Acute administration of arsenic to rats produced a rat whole brain. Toxicol. Lett. 92: 201-208. 24. Tchounwou, PB, Patlolla AK, Centeno J A. Carcinogenic and monoisoamyl-DMSA in the treatment of chronic arsenic
significant reduction in hepatic GSH36. Chronic exposure of rats or mice 8. Flora, S.J .S., Dube, S.N., Sachan, A.S., Pant, S.C., 1994. Effect of systemic health effects associated with arsenic exposure- A critical intoxication in mice. Cell Biol Toxicol. 2007
to arsenic via injections caused up to 35% depletion in hepatic GSH, multiple gallium arsenide exposure on some biochemical indices in Review. Toxicol Pathol 2003; 31(6): 575-588. 40. Chattopadhayay, S., Bhaumik, S., Nagchaudhuri, A., Das Gupta, S.,
37,38
along with liver injury . Similar effect was observed in the total thiol rat brain. Indust. Health. 32:247-252. 25. Guha Majumdar DN, Das Gupta J , Santra A. Chronic arsenic toxicity 2002. Arsenic induced changes in growth development and
concentration. Arsenic exposure in both the dose for 60 days duration 9. Rodriguez, V.M., Carrizales, L.J , J imenz-capdeville, M.E., Dufour, in West Bengal- worst calamity in the world. J Ind .Med Assoc apoptosis in neonatal and adult brain cells in vivo and in tissue
enhanced significantly the lipid peroxidation of the testis, suggesting L., Giordano, M., 2001. Effect of sodium arsenite exposure on 1998; 96: 4-7. culture. Tox. Lett. (Special issue). 128.
the generation of reactive oxygen radicals and testicular oxidative behavioural parameters in the rat. Brain. Res. Bull. 15: 301-308. 26. Rossman TG. Arsenic. In: Environmental and occupational 41. Nag Chaudhuri,A., Basu, S., Chattopadhayay, S., Das Gupta, S.,
stress. Increased level of lipid peroxidation and consequent damage to 10. Chattoadhayay, S., Bhaumik, S., Nagchaudhuri, A., Das Gupta, S., Medicine, Rom. W.N. Ed, Lippencott-Raven, Philadelphia. 1998; 1999. Effect of high arsenic content in drinking water on rat brain.
the cell membrane due to arsenic and its compounds was reported by 2002. Arsenic induced changes in growth development and 1011-1019. Ind. J . biochem. Biophys. 36, 51-54.

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