Environmental Disease

: 2021  |  Volume : 6  |  Issue : 3  |  Page : 91--97

Urinary metabolites as exposure biomarkers of benzene, toluene, ethylbenzene, and xylene in footwear workers and assessment of pulmonary function

Anupa Yadav1, Asim Saha2, Amit Chakrabarti1, Geoffrey Nengzapum1, Anirban Das2, Surajit Das1,  
1 Department of Industrial Hygiene, ICMR-Center on Non-Communicable Diseases, Kolkata, West Bengal, India
2 Department of Occupational Medicine, ICMR-Center on Non-Communicable Diseases, Kolkata, West Bengal, India

Correspondence Address:
Anupa Yadav
Department of Industrial Hygiene, ICMR-Center on Non-Communicable Diseases, Block – DP, Sector – V, Salt Lake, Kolkata - 700 091, West Bengal


Aim: This pilot cross-sectional study focused on biological monitoring of the benzene, toluene, ethylbenzene, and xylene (BTEX) urinary metabolites trans, trans-muconic acid (tt-MA), s-phenyl mercapturic acid (SPMA), hippuric acid (HA), mandelic acid (MA), and methylhippuric acid (MHA) and measured the effects of workplace BTEX exposure on pulmonary function of workers engaged in footwear manufacturing. Materials and Methods: Urinary metabolites tt-MA, SPMA, HA, MA, and MHA concentration in urine samples of study participants (N = 35) were analyzed by reverse-phase high-pressure liquid chromatography. Pulmonary function parameters were measured by spirometer and peak flow meter. Demographic information and work exposure information of study participants were collected by questionnaire interview. Results: In exposed workers, concentration of urinary SPMA, tt-MA, and HA was significantly higher (P < 0.01, in all) than the control group. Reduction in both force expiratory volume in 1 s (Forced expiratory volume in one second) and peak expiratory flow rate (PEFR) were inversely associated (P < 0.01) with growing years of age among all workers. Inverse association was measured between urinary tt-MA and decline in forced vital capacity and PEFR (P < 0.05, for each) in workers. Based on the questionnaire interview, workers were not found to be aware of workplace exposure hazards. Conclusions: Exposure biomarkers of benzene (tt-MA and SPMA) and toluene (HA) were significantly higher in workers than the control group. Study results evident the presence of occupational exposure to benzene and toluene in footwear workers. Deterioration in FEV1 and PEFR were also measured among all workers with growing years of age. The sample size was small in the present study, so further research required to confirm our results.

How to cite this article:
Yadav A, Saha A, Chakrabarti A, Nengzapum G, Das A, Das S. Urinary metabolites as exposure biomarkers of benzene, toluene, ethylbenzene, and xylene in footwear workers and assessment of pulmonary function.Environ Dis 2021;6:91-97

How to cite this URL:
Yadav A, Saha A, Chakrabarti A, Nengzapum G, Das A, Das S. Urinary metabolites as exposure biomarkers of benzene, toluene, ethylbenzene, and xylene in footwear workers and assessment of pulmonary function. Environ Dis [serial online] 2021 [cited 2021 Dec 7 ];6:91-97
Available from: http://www.environmentmed.org/text.asp?2021/6/3/91/329041

Full Text


Footwear making is a labor-intensive job, most of the time working hours are very lengthy and not supported with required working condition. Workers not provided with adequate health protection against occupational hazardous from harmful chemicals and other materials. The footwear manufacturing industry consumes widely and freely raw materials such as primer, hardener, adhesives, glues, and thinners mainly to unite, assemble, and for finishing of footwear. Almost all these raw materials contain organic solvents such as benzene, toluene, ethylbenzene, and xylene (BTEX), and resulting in workers exposure to these solvents.[1],[2],[3] These organic solvents are highly volatile in nature, their main routes of exposure are through inhalation, skin absorption, and oral.[4],[5] As per reported toxicological studies, all BTEX compounds are neurotoxins and irritants.[6] However, the International Agency for Research on Cancer considers benzene and ethylbenzene as Class 1A and probable human carcinogen, respectively.[7],[8],[9] While toluene and xylene are classified as noncarcinogens, they can cause significant cell growth inhibition,[10] depression of central nervous system, and irritation of eyes and throat.[6] Occupational exposure of footwear industry workers to volatile organic compounds is likely a contributing factor to adverse health outcomes in this population.[11] Acute and chronic respiratory impairments are observed in shoe workers exposed to organic solvents from glues or other adhesives.[12],[13] Absorbed BTEX mostly (80% or more) metabolized and excreted through urine and small fraction (about 20%) through exhaled air.[14] Biomonitoring is an important tool to measure accurate exposure, to detect early sign of onset of the disease, and prevention of further progress of the disease among exposed workers.[15] For benzene trans, trans-muconic acid (tt-MA) and s-phenyl mercapturic acid (SPMA) have been established as the major exposure biomarkers at exposure level <5 ppm.[16] However, if exposure to benzene <1 ppm; SPMA is a more sensitive and reliable biomarker than tt-MA due to its longer elimination half-life.[17] A metal factory workers were exposed to ethylbenzene and xylene during painting and solvent mixing process, when their urine samples were analyzed a significant amount of mandelic acid (MA), and methylhippuric acid (MHA) was found.[18] A considerable amount of tt-MA, MHA, and hippuric acid (HA) was measured in urine samples of the petroleum refinery workers exposed to benzene, xylene, and toluene.[19] Therefore, biomonitoring of BTEX exposure biomarkers (tt-MA, SPMA, HA, MA, and MHA) in footwear workers is an important tool to determine the extent of exposure to prevent future adverse health effects among these workers. As per ACGIH (2017), biological exposure indices (BEI) recommended values are tt-MA (0.5 mg/g creatinine), SPMA (0.025 mg/g creatinine), HA (1.5 g/g creatinine), MA (0.15 g/g creatinine), and MHA (1.5 g/g creatinine).[20]

Available literature presents little information on the association between urinary exposure biomarkers of BTEX and respiratory health-related problems among occupational workers, especially in India. Therefore, the objective of the present study was to monitoring level of urinary metabolites tt-MA, SPMA, HA, MA, and MHA, and to find out the effects of occupational BTEX exposure on respiratory health of workers engaged in footwear manufacturing, in Kolkata city, India.

 Materials and Methods

In this cross-sectional study (it is a preliminary work), male (N = 22) footwear workers were randomly selected from the unorganized sector of the footwear manufacturing industry in Kolkata City, West Bengal, India. In Indian context as per the National Commission for Enterprises in the Unorganised Sector; definition of unorganized sector is “consisting of all unincorporated private enterprises owned by individuals or households engaged in the sale or production of goods and services operated on a proprietary or partisanship basis and with less than ten total workers.” This study has a limitation of small sample size. As far as unorganized footwear-making activity of Kolkata city is concerned, sporadically scattered units are existent in central part of the city. A walk-through survey was undertaken prior to the study to identify the units. Twenty-seven such units could be identified, and they had 86 workers in total. We intended to include half of the total workers (i.e., 43) in the study. Hence, 43 workers were selected randomly using a random number table (originating from Microsoft Excel software). We approached 43 works for study and 40 of them agreed to participate. Healthy males (N = 13) of similar age and socioeconomic status were selected (not exposed to BTEX) as control group. The study was approved by ethical committee of Regional Occupational Health Center (Eastern), Kolkata (IEC/No. 13th IEC/ROHC (E)/5.2 dated December 24, 2018). Demographic, socioeconomic, and occupational history was collected by interviewing all study participants using a questionnaire before collection of urine samples. Subjects with a medical history and taking any medicine were excluded; while who have minimum 4 years of working experience in footwear manufacturing were included in this study. Five urinary exposure biomarkers tt-MA, SPMA, HA, MA, and MHA (the former two being metabolites of benzene and later three being metabolites of toluene, ethylbenzene, and xylene) of BTEX were measured. Pulmonary function test (PFT) was performed by Spirovit SP-10 and Wright's peak flow meter. Written informed consent form was obtained from all study participant.


Urine sample collection

Postshift urine from workers was collected, similarly at the end of daily activities urine samples from the control group were also collected. Urine samples were collected in polyethylene containers; then brought to the laboratory under refrigerated conditions and stored at −20°C until analysis.

Analysis of urinary biomarkers

Sample cleanup and extraction procedure

Urine samples were thawed and cleaned by solid-phase extraction (SPE) using a strong anion exchange (SAX) cartridge (500 mg/3 ml, Agilent), attached with a vacuum elution system (VacElute). SAX cartridge was conditioned with methanol and water (3 ml each), followed by washing with phosphate buffer. Urine-phosphate buffer mixed (1:1) and passed through SAX cartridge at flow rate of 2–3 ml. Followed by washing with water and 1% acetic acid. Then the cartridge was dried, and finally, the analytes of interest were eluted with 10% acetic acid.[21]

Sample quantification

The analysis of extracted urine was performed with high-pressure liquid chromatography (HPLC) (Shimadzu, Japan), consisted of a liquid sampler (Model: LC-10AT), detector (Model: SPD-M 10A), column oven, and monitor. Reverse phase ODS-2 Hypersil Column (250 mm × 4.6 mm and 5 μm) with oven temperature 30°C was used. Trifluoroacetic acid Trifluoroacetic acid (TFA) 0.1% acetonitrile-water was used as mobile phase. Pump was operated at a programmable time range for 50 min, 1 ml/min flow rate. The sample injection volume was 10 μL. Chromatogram of each metabolite was detected at a specific wavelength of 265 nm (tt-MA), 253 nm (SPMA), and 205 nm (HA, MA, and MHA).[21]

Calibration curve and recovery%

Pooled urine sample (control) mixed with phosphate buffer (1:1) and spiked with 0.5–10 μg/ml of standard mixture of tt-MA, SPMA, MA, HA, and MHA, respectively. After SPE cleanup and extraction process 10 μl of each injected in HPLC. For recovery study, urine was spiked with standard mixture of 6 μg/ml.

Pulmonary function test

Study participants were explained the purpose of the study and procedure of PFT before measuring the PFT parameters. Height and body weight of participants were measured in bare feet on a standard scale. Spirometry was performed in standing position with nose closed by nose clips using Spirovit SP-10 (Schiller Health Care, Switzerland), and peak expiratory flow rate (PEFR, L/min) was measured by Wright's peak flow meter (Clement and Claeke, UK). The procedure was repeated to achieve three acceptable attempts. Pulmonary function was evaluated by measuring the following parameters, the slow vital capacity (SVC, L), i.e., the volume of air in liter that can be normally exhaled by unforced maneuver and forced vital capacity (FVC, L), i.e., the maximum volume of air in liter that can be forcefully exhaled. Forced expiratory volume in one second (FEV1, L), i.e., volume of air in liter forcefully exhaled in 1 s and forced exploratory volume in the first second as the percentage of FVC (FEV1%) were calculated from the tracing. All volumes obtained were expressed in body temperature on atmospheric pressure of air saturated with water vapor (BTPS). PFT values were predicted from the standard prediction equation. The instrument was calibrated every day before starting the experiment.[22],[23]

Statistical analysis

Statistical software SPSS (Manufacturer IBM, New York City, Country US.) was used for study data analysis. Levels of urinary tt-MA, SPMA, HA, MA, and MHA in both study groups (footwear workers and controls) were compared using Student's t-test to determine significant differences. The significance criterion was set at a P < 0.05. ANOVA was used to analyze relation between PFT parameters and variables such as urinary biomarkers, job duration, and age group. Logistic regression is applied to minimize the impact of baseline characteristics between two populations.


Socio-demographic and anthropometric data

The mean age of footwear workers and control subjects was 43.27 ± 11.38 (range 18–60 years) and 43.46 ± 12.20 (range 25–60 years) years, respectively. The mean body mass index (BMI) was 58.91 ± 10.20 and 63.23 ± 14.49 kg/m2 for workers and control subjects, respectively. No significant differences were observed in demographic characteristics of workers and the control group [Table 1]. Mean job experience of workers was 23.23 ± 12.24 years (range 4–45 years).{Table 1}

Workplace exposure-related complaint

Workers were used to apply glues or adhesives without wearing gloves in hands during footwear manufacturing and not using any other personal protective equipment (PPE). When they were asked about their health complaints, it was found that 45% of workers had burning eyes during/after work, 36% had nose or throat irritation, 18% had dry cough, 22% had occasional chest pain, 5% had skin irritation, 6% had breathing difficulty, and 68% had backache. When they were asked about awareness of health hazards related to chemicals exposure in the workplace, 72% of them replied that they are not so aware.

Exposure biomarkers

Trans, trans-muconic acid, MA, HA, MHA, and SPMA were detected at 14.2, 17.7, 19.04, 21.55, and 31.14 min relative retention time. The range of calibration was 0.5–10 μg/ml, and good linearity was observed (R2 ≥ 0.99) for all the five biomarkers. The limit of detection was 0.42, 0.79, 0.46, 0.44, and 0.34 μg/ml and the limit of quantification was 1.41, 2.65, 1.53, 1.47, and 1.13 μg/ml for tt-MA, MA, HA, MHA, and SPMA, respectively. Recovery % obtained were 80%–98% (tt-MA), 85%–100% (SPMA), 83%–101% (HA), 80%–91% (MA), and 81%–95% (MHA).

Measured mean value of tt-MA, SPMA, and HA is presented in [Table 2], values of these three metabolites were markedly high in workers group than the control group. However, there was not much variation in mean values of MA and MHA between the two groups. The urinary HA, MA, and MHA concentrations in workers and control subjects were found less than BEI ACGIH (2017).[17] Logistic regression analysis was undertaken to understand the effects of age, BMI, work status, cooking activity participation, use of cooking fuel, and exposure duration (i.e., work experience) on the level of tt-MA and SPMA. After adjustment of confounders, exposure duration (i.e., work experience) found to have significant effect RR= 1.1, 95% confidence interval [CI] = 1.009–1.198, B = 0.095, SE = 0.044) on tt-MA level. Similar results was found (although nonsignificant) for SPMA (RR = 1.0, 95% CI = 0. 935–1.160).{Table 2}

Spirometry results

The results of spirometry (SVC, FVC, FEV1, FEV1%, and PEFR) measurements for workers and control subjects are presented in [Table 1]. No significant differences in lung functions were observed between the exposed and control groups for any of the spirometry parameters. [Table 3] presents significant reduction in FEV1 and PEFR (P < 0.01, for each) with increase of age among all exposed workers. While analyzing PFT parameters values across the duration of employment for workers, no significant deterioration was observed in lung function. In control subjects, no significant difference is measured in pulmonary indices (SVC, FVC, FEV1, and PEFR) with age (<30; 30–45; 45 > years) categories. All the study participants were nonsmoker. Two cases of restrictive abnormality and three cases of obstructive abnormality were identified in workers; these results are supported by Mukherjee et al.'s study done in petrol pump workers.[21]{Table 3}

Among control subjects, the number was two for each abnormality (restrictive and obstructive pulmonary diseases). When PFT parameters of the workers compared with exposure biomarkers (tt-MA, SPMA, HA, MA, and MHA) of BTEX solvent, significant (P < 0.05) deterioration in FVC and PEFR was observed in respect of tt-MA values (<0.5 mg/g creatinine vs. >0.5 mg/g creatinine).


Based on the investigators' observations during sample collection in footwear industry, generally 2–3 workers were working in small workshops involved in different processes such as cutting, stitching, pasting or applying adhesives/glues, fitting, fixing, and finishing. They were not using any brush or piece of cloth to apply the glue or adhesive, doing shoe pasting with naked hand without wearing any gloves. They were not using any respiratory protection or mask while working. There were no partitions used to separate different working groups from each other. Workers were working in close proximity of each other. Working area had no proper ventilation. At some of the workshops, there was window and exhaust fan for ventilation, while in others, there was no provision for ventilation. They were not using PPE. When the reason was asked for not using PPE, the average explanation was they feel uncomfortable with mask while working. In addition, when working room temperature is hot, workers prefer to work freely without PPE and even sometimes use to take off their shirts because of sweating during work. Therefore they were exposed to a mixture of organic solvent directly by inhalation or dermal exposure.

According to data collected from the questionnaire interview, workers had common complaints for health-related symptoms such as burning eyes during/after work, nose or throat irritation, skin irritation, dry cough, occasional chest pain, and breathing difficulty. Reason for these symptoms could be exposure to volatile solvents (BTEX) at workplace. Ergonomic problem (backache) may be due to a prolonged seating posture during working hours.

It was evident from the literature that if workers were exposed to 1 ppm benzene during 8-h working shift, then reported level of tt-MA excreted in urine was 500–1,500 μg/g creatinine and maximum reached up to 1,900 μg/g creatinine.[24] In our study, the level of tt-MA among exposed workers increased up to 2,797 μg/g creatinine. These results were suggesting that footwear workers may had benzene exposure at workplace ≥1 ppm, and that concentration was greater than the limit (0.5 ppm) recommended by ACGIH (2017).[20] Exposure biomarkers monitoring of footwear works showed that measured mean values of tt-MA (1019 μg/g creatinine) and SPMA (164 μg/g creatinine) in urine, were higher than the recommended BEI, however the values for HA, MA, and MHA, were less than BEI ACGIH (2017), similar results were observed by Mukherjee et al. in petrol pump workers study.[21] In the present work, measured mean value of urinary tt-MA among footwear workers was less than the values reported in other occupational groups such as gasoline workers (tt-MA 1,450 μg/g creatinine), service station workers (tt-MA 1,087.63 μg/g creatinine), and chemical company workers (tt-MA 3,443 μg/g creatinine).[24],[25],[26] While the measured mean value of urinary SPMA in footwear workers was higher than the values found in other occupational settings such as service station workers (SPMA 19.44 μg/g creatinine), traffic police (SPMA 2.6 μg/g creatinine), footwear (SPMA 0.19 μg/g creatinine), and shoe workers (SPMA 26.07 μg/g creatinine).[26],[27],[28],[29] Significant differences (P < 0.01) in urinary tt-MA, SPMA, and HA level between footwear workers and control group were observed, but measured values of urinary MA and MHA were not much different for both the study groups. When logistic regression was applied to adjust the confounders (such as age, BMI, work status, cooking activity participation, use of cooking fuel, and exposure duration), it was found that duration of exposure had significant effect on the level of urinary tt-MA in workers. There was no significant difference in pulmonary function status between the two groups. The duration of employment had no effect on pulmonary function parameters in workers. Significant deterioration in PFT parameters among workers was found with increasing age. Urinary tt-MA was significantly and inversely associated with FVC and PEFR in footwear workers; finding supported by work of Mukherjee et al.[21] Mainly study found workers had the level of urinary metabolites of benzene (tt-MA and SPMA) much higher than the reference value of BEI ACGIH (2017). Study results suggested that benzene exposure could cause deleterious effects in lung function among occupationally exposed footwear workers. Our study also suggested that urinary metabolites of BTEX could be used as surrogate tool of environmental monitoring of these solvent, as supported by other researchers.[24]

Our work has several potential limitations. First of all, our study has a small sample size, and this may be reason that we could not find potential difference in PFT between workers and control. Information related to workplace exposure to hazardous solvents and health-related issues assessed by questionnaire interview only. In spite of small sample size, we could not do personal monitoring of BTEX at occupational setting because of logistic difficulties. Workers were not ready to wear the personal sampler. They said they were not comfortable to work while wearing this sampler. Therefore we failed to link correlation between exposure biomarkers and specific solvents BTEX. We could not found any deterioration in PFT parameters when compared with exposure biomarkers (SPMA, HA, MA, and MHA), except for tt-MA, and unfortunately, urinary excretion of tt-MA is influenced by sorbic acid originating from food.[30] In addition, our study has strength also, according to our knowledge, only a few study especially in India showing the association between urinary exposure biomarkers (tt-MA SPMA, HA, MA, and MHA) of BTEX solvents and PFT in occupational setting.[21] At the national level, this kind of cross-sectional study among footwear workers to find an association between urinary exposure biomarkers and lung function parameters has been explored very less so far in literature, in spite of the facts that biomonitoring of BTEX urinary metabolites could allow overall exposure more accurately.


We found that workers engaged in footwear manufacturing industry worked for 12 h/day, 6 days/week, and had occupational exposure to benzene and toluene. This result was confirmed when exposure biomarkers of benzene (tt-MA and SPMA) and toluene (HA) were significantly higher in workers than in control subjects. In workers, significant decline in FEV1 and PEFR was measured with growing years of age. However, no significant difference was found in PFT between workers and the control group. Therefore, further studies are needed to confirm our results.

Financial support and sponsorship

This work was carried out with intramural support of the institute.

Conflicts of interest

There are no conflicts of interest.


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