JRHS 2008; 8(2): 61-68

Copyright © Journal of Research in Health Sciences

Environmental Exposure to Xylenes in Drivers and Petrol Station Workers by Urinary Methylhippuric Acid

Bahrami A (PhD)a, Jonidi-Jafari A (PhD)a, Mahjub H (PhD)b

a Department of Occupational Health, Faculty of Health, Hamadan University of Medical Science, Iran

b Department of Biostatistics & Epidemiology, Faculty of Health, Hamadan University of Medical Science, Iran

*Corresponding author: Dr AR Bahrami, E-mail: A167R@yahoo.com

Received: 1 April 2008; Accepted: 30 September 2008


Background: The aims of this study were evaluation of exposed to xylenes in low concentration and com­pare urinary level of methyl hippuric acid in taxi drivers and petrol stations workers in West of Iran.

Methods: This observation study was carried out on samples of the exposed men to xylenes in two oc­cupational groups in Hamadan City (west of Iran) from March 2003 to March 2004. Subjects included 45 taxi drivers and 25 petrol station workers. The study group was selected from 54 workers at petrol sta­tions and 300 drivers by simple random sampling. Xylenes was analyzed using gas chromatography equipped with a Flame Ionization Detector (FID). The urinary methyl hippuric acid (MHA) was ana­lyzed with High performance Liquid Chromatography (HPLC) equipped with an ultraviolet (UV) de­tector.

Results: Total xylene exposure was 1.05±0.55 ppm (mean±SD) with a range of 0.20-2.55 ppm that was about 4 times more than taxi drivers' exposure. The poor correlation coefficient was seen between xy­lenes concentration and urinary MHA for drivers (r2= 0.09 to 0.42) but significant associations were noted between urinary MHA and xylene in the breathing zone of petrol station workers (r2= 0.69 to 0.77; P< 0.05).

Conclusion: High xylenes levels are emitted in petrol stations at Iran. Urinary MHA level has a poor correla­tion with exposure to xylenes in drivers but has good correlation in petrol station workers.

Keywords: Xylene, Methylhippuric acid, Drivers, Petrol station workers, Urine, Air, Iran


Xylene can exist in three isomeric forms. These three isomers possess similar proper­ties. Com­mercial xylene is a mixture of these three iso­mers: ortho, meta and para, with the meta form usually the principal component (45-70%). As no explosive aromatic hydro­car­bons, mix­tures of the three (techni­cal xy­lene) isomers are heavily used in the chemi­cal industry and in the petroleum indus­try as a solvent and ga­soline “anti­knock” additives. Xylene will cause depression of the central ne­-

rvous system. This narcotic effect will impair performance and af­fect cerebral function (1-3). Xylenes are ubiq­uitous in the environment, xy­lenes released to the atmosphere as fugative emissions from in­dustrial sources, in automo­bile exhaust through volatilization of xylenes used as solvents. Most of the released to the environment partition into air (4). The Ameri­can Conference of Govern­mental Industrial Hy­gienists (ACGIH) rec­ommends a threshold limit value (TLV) of 100 ppm for xylenes and also recommended a biological exposur index for MHA 1.5 g/crea­tinine measured in urine from end of the shift (5).

Xylene is metabolized at a half-life rate of 20 to 30 h. Only about 5% of xylene is ex­haled unchanged. The balance of xylene is me­tabolized by the oxidation of a methyl group to toluic acid. The toluic acid is converted to MHA and excreted in the urine (6-7). There are some investigations about the urinary level of MHA in subjects who were exposed to xylenes in manufactures of industrialized coun­try (8-10). There are not any researches about compare exposure to xylene among different occupations in developing countries.

The major environmental problem in Iran is air pollution, there are about of 6 million vehicles in Iran and 40% of them are at least two dec­ades old (11).  The government of Iran phases out leaded gasoline and replace it with un­leaded gasoline by January 2000 it cause ad­ded more aromatic hydrocarbons to gasoline.  The petrol pump stations in Iran are besides of street and workers in this location did the pumping and services of fuel. During their daily work, workers have direct contact with petroleum products, so occupational expo­sure to xylene cannot be avoided.

This research was carried out in Hamadan city, west of Iran. Hamadan is the centre of Hama­dan Province with high vehicular density in­cluding 1000-2500 per hour in the centre of city and 20-250 per hour in the gasoline stations.

The aims of this study were evaluation of ex­posed to xylenes in low concentration in taxi drivers and petrol stations workers and com­pare level of methyl hippuric acid in West of Iran.


This observational study was carried out on samples of the exposed men to xylenes in two occupational groups in Hamadan City (west of Iran) from March 2003 to March 2004. Subjects included 45 taxi drivers and 25 pet­rol station workers. The study group was se­lected from 54 workers at petrol stations and 300 drivers by simple random sampling. A de­tailed questionnaire was completed for sub­jects, providing information about per­sonal characteristics and smoking history. We re­move the subjects that used food interference but affect of smoking and age were reported in paper.

Personal monitoring of exposure

A charcoal adsorption tube from (SKC, USA) connected to a small pump (Negretti Auto­mation, Model NR645, England) was used to obtain personal samples (11). The charcoal tube was attached to the workers overalls as closely as possible to the face in order to de­termine the xylenes concentrations in the brea­thing zone. The pump was operated at 200 ml/ min and the duration of sampling was 2-4 h. Xylenes were extracted with carbon disul­phide (CS2) from the charcoal. A gas chro­matogra­phy (Model 4600-Unicam Company, England) equipped with a Flame Ionization De­tector (FID) was used for quantitative meas­ure­ment. Separation of the compounds was achiev­ed with glass packing column 1.5m×4 mm i.d packed with 10% SE 30 on Chromo­sorb W AW-DMCS 100-120. This column had a pro­gra­mmed temperature of 50° C for 2 min fol­lo­wed by an  increase to 180° C  at a rate of 4° C min-1 and finally during analysis a constant temperature of 180°C for 2 min.

Analysis of urine

Exposed subjects and non-exposed control group were asked to pass urine in the end of the shift. Samples were refrigerated immedi­ately, transferred to the analytical laboratory, and kept frozen until analyzed.

MHA in Urine

The determination of MHA was carried out ac­cording to National Institute for Occupa­tional Safety and Health (NIOSH) method (12). Initially, 40 µl of HCl and 0.3 gr so­dium chlo­ride were added to 1 mL of urine into a gra­duated centrifuge tube. Four mL of ethyl ace­tate added to tube and the samples were mixed centrifuged at 1200 r.p.m for 5 min, then the ethyl acetate layered transferred to tapered test tube by pasture pipette. Samples were evaporated to dryness using a gentle steam of nitrogen in a heating block at 45 ºC before reconstitution. The residue of samples redis­solved in 200 µl of distilled water and 20 µl was injected to High Performance Liquid Chro­matography (HPLC) system. A HPLC chro­mato­graph (Knauer) equipped with an ul­travio­let (UV) detector (Model K-2600 Knauer) was used for analysis. The UV detector was set at 254 nm. The HPLC column was an APEX ODS II 3µm, 25×4.6 mm. Chromatog­raphy was isocratic in a mobile phase consist­ing of water-acetonnitrile-acetic acid (89:10:1) at a flow rate of 1 mL min-1.

The urinary creatinine was measured by Jaffe kinetic method using a Boehringer Mann­heim Hitachi 917 automatic analyzer and was re­ported following adjustment for creatinine con­centration.

Data analysis was performed using SPSS sta­tistical software for windows (version 13.0). Kolomogorov-Smirnov test was used for nor­mality distribution of variables. If any vari­able was not normal, Log10 trasformation was used to get a normal distribution. For association between two non-normal variables, Spear­man's correlation coefficient was used. For fitting first-order or second-order factors, log trans­formation of variables was used. In addition, for non-normal data, median and geometric mean was calculated. For comparing be­tween smokers and non-smokers Mann-Whitney U test was used.


The levels of MHA in urine and xylenes con­centration in the breathing zone of drivers and petrol station workers are shown in Table 1. There was a significant difference between the levels of MHA in urine of drivers and petrol station workers (P< 0.05). The mean concen­trations of xylenes in the ambient air of driv­ers, and petrol station workers were 0.24 and 1.05 ppm respectively. The urinary level of m-MHA is more than o- and p-MHA in both occupations. The urinary MHA in three sam­ples of drivers were not observed in subjects exposed to xylene less than 0.02 ppm.

Table 1: Results of xylenes in ambient air and biological monitoring of MHA at different occupations


Taxi Drivers  n=45

Petrol station workers  n=25



x ± SD1









































(mg/g creatinine)














(mg/g creatinine)














(mg/g creatinine)














(mg/g creatinine)













1 SD; Standard division

2 GM; Geometric means

Figure 1 and 2 show the scatter diagrams be­tween xylene concentration in the breathing zone, and urinary MHA of drivers and petrol station workers, respectively.

Figure 1: Correlation between MHA in urine and xylenes in the breathing zone of drivers

Figure 2: Correlation between MHA in urine and xylenes in the breathing zone of petrol station workers

There was not any significant difference for the levels of urinary o-MHA with o-xylenes and poor correlations were observed be­tween m & p-xylenes with urinary m & p-MHA in driv­ers (Table 2 and Fig. 1). Significant asso­ciations were noted between urinary MHA and xylene in the breathing zone for petrol station workers (Table 2 and Fig. 2). No dif­ferences were detected between smokers and non-smokers and also age groups either in driv­ers or in petrol station workers. None of sub­jects in both groups used drinks because using of alcohol banned in Iran.

Table 2: The regression line between xylenes in ambient air exposure and urinary methyl hippuric acid at different occupations


Regression line Formula


Coefficient (R2)

Petrol station


o-xylene and o-MHA

Log o-MHA=1.75 +2.34 Log o-xylene + 1.50 Log (o-xylene)2


m & p-xylene and m&p-MHA

Log m&p-MHA=1.77 +1.08 Log m&p-xylene + 1.93 (Log m&p-xylene)2



o-xylene and o-MHA

Log o-MHA=0.78 +0.43 Log o-xylene


m & p-xylene and m&p-MHA

Log m&p-MHA=1.62 +0.71 Log m&p-xylene



The present study was undertaken to evalu­ate the relationship between MHA and at­mospheric xylenes among taxi drivers and pet­rol station workers. Data from this study showed that MHA had a poor correlation co­ef­ficient with low concentration of xy­lenes in drivers. The exposure to xylenes in some other environmental sources such as ex­posure to aromatic hydrocarbons at home, cigarette or sources from street other than in­side of vehicle, effected on urinary level of MHA.  The other factors that made variation in uri­nary of MHA included anatomical and phy­siological difference between people, indi­vidual work practice, difference between in­haled xylenes concentration. The mean con­centration of xylenes in ambient air for driv­ers in current study was 0.24 differences to other reports. Jacobson and Mclean found that bio­logical monitoring of occupational xy­lene ex­posure at level <15 ppm using uri­nary MHA had a good correlation with atmos­pheric levels and was a valid comple­ment to ambient moni­toring (8). Also Jang et al showed significant correlation between xy­lene and MHA wher­ever the mean concen­tration for xylenes was 12.77 ppm (14). The American Conference of Govern­mental Industrial Hygienists (ACGIH, 2006) has recommended a biological expo­sure in­dex for MHA of 1.5 g MHA/g crea­tinine meas­ured from an end of shift urine sam­ple af­ter a TWA exposure to 100 ppm (15). There are several studies in which correla­tion be­tween occupational xylene exposure and uri­nary MHA exist. The results of this study showed that urinary m-MHA was greater than o- and p- MHA, it may concern to metabolism of m-xylene. In a study, Miller and Edwards was found that m-xylene isomers undergo pref­erential metabolism com­pared with o-and p-xylene (6), but study by Kawai et al. on in­dividual xylene isomers showed that the slo­pes of the regression lines for o- and m-iso­mers were similar, whereas that for p-xylene was larger (10). Studies of rats indicate that m-xylene in some effi­ciently absorbed through the skin than tolu­ene, benzene or hexane (17). Takeuchi et al. found weakly correlation be­tween the concen­tration exposure to the xy­lenes and un­metabolized xylene concentra­tion in the end-of-the shift urine samples (17).

We have not find any significant difference be­tween urinary MHA smokers and nonsmok­ers but Huang et al. reported that me­tabolism of xylenes was significantly re­duced among smok­ers or drinkers compared with non-smok­ers and non-drinkers (18). The mean concen­tration of xylenes in breath­ing zones of pet­rol station workers was 4-5 times higher than that of the drivers. The mean concentration of xylenes in the breathing zone of drivers was greater than re­ported studies from Asia, Aus­tralia, and America (19-20). The Refueling at petrol sta­tions and staying inside the vehi­cles have been shown to contribute to in­creased expo­sure to aromatic hydrocarbons. Dur­ing refuel­ing, the exposure level of petrol station workers varies according to the xy­lenes con­tent of fuel and the amount of time spent at the station. The proportion of petrol stations to number of vehicles in Iran is low, and for cities with populations of between 0.5 to 1 mil­lion there are usually 7 to 10 stations. This cause vehicles stay for a long time (be­tween 5-30 min) waiting for refueling and there­fore hydrocarbons such as aromatic com­pounds may be emitted from car ex­hausts to these locations.

In conclusion our results suggest that high xy­lene levels are emitted in petrol stations at Iran. Urinary MHA could not be applying as good biomarkers in drivers wherever sub­jects exposed to low level of xylenes in ambi­ent air but it is good biomarkers for expo­sure of xylene in petrol station workers.


This research was supported by Hamadan Uni­versity of Medical Science. The authors de­clare that they have no conflict of inter­ests.


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