Archives of

1. Introduction
Benzene is one of the pollutants that is dispersed in the air due to the activity of the petrochemical industry. According to Occupational Safety and Health Organization (OSHA), National Institute for Occupational Safety and Health (NIOSH), International Labor Organization (ILO), the World Health Organization, American Conference of Governmental Industrial Hygienists (ACGIH), International Agency for Research on Cancer (IARC), Environmental Protection Agency (EPA), and American Industrial Health Association, it is classified as definite human carcinogens which can lead to many complications such as aplastic anemia, leukemia, acute myeloid, lymphoma, and leukemia in exposed workers [1,2]. Breathing is the main way of contact with benzene in the industry. As a chemical mediator, Benzene is used
Relationship Between the Concentration of Airborne Benzene Pollutant and the Amount of Urinary Metabolites of Trans, Trans-muconic Acid, and Hippuric Acid in Employees Working in Different Plants of Bou Ali Sina Petrochemical Company
Sima Sabzalipour1*ID, Siavash Cheraghi1ID, Elahe Zallaghi2ID, Mohamad Erbian Gharmsir3
1Department of Environmental Sciences, Ahvaz Branch, Islamic Azad University, Ahvaz, Iran
2Department of Environmental Sciences, Municipal University of Applied Sciences, Ahvaz, Iran
3Master of Islamic Azad University, Yazd Branch, Iran
*Corresponding Author: Sima Sabzalipour, Email: shadi582@yahoo.com
Abstract
Background & Aims: The petrochemical industry as a modern industry, despite the positive outcomes it has brought to mankind, is a source of gaseous and aerosol pollution and industrial effluents on a large scale, which can have direct and indirect destructive effects on the environment and human life. This study investigated the relationship between the amount of airborne benzene with the amount of trans,trans-muconic acid (ttMA) and hippuric acid metabolites in the urine of workers working in petrochemical complexes with different exposure times and methods.
Materials and Methods: For this purpose, breathing the air of different petrochemical plants of Bou Ali Sina was sampled by the National Institute for Occupational Safety and Health 1501 method, and the urine of workers (n = 24) was also sampled in these units. In addition, the amount of benzene in the air samples and the amount of urinary metabolites of ttMA and hippuric acid were analyzed in urine samples sent to the laboratory using a high-performance gas chromatography-mass spectrometry device and gas-liquid chromatography. Finally, urinary creatinine was measured by an ultraviolet-visible spectrophotometer.
Results: The results showed that the concentration of benzene in the aromatic unit had the highest value, which had a higher level of pollution than both standards. The xylene mixing unit with a concentration of 3.6 μg/m3, the loading unit with a benzene content of 3.4 μg/m3, and a tank unit with 2.8 μg/m3 had a lower amount of benzene pollution compared to the Occupational Safety and Health Administration permissible exposure limit-short-term exposure limit (OSHA PEL-STEL) standard but had higher pollution levels in comparison to the OSHA PEL-TWA (time-weighted average) standard. In the sampling unit of the laboratory with a benzene amount of 0.94 μg/m3 and in the technician unit of the laboratory, the amount of pollution was lower than both OSHA PEL-TWA and OSHA PEL-STEL standards. The aromatic unit demonstrated the highest amount of benzene, while the lowest amount was related to the laboratory section.
Conclusion: The results of the measurement of urinary benzene metabolites revealed that the concentration of urinary phenol and inhaled benzene in evening shift workers was higher than the corresponding amount in the morning shift workers, which may be due to the high level of pollution evenings compared to the morning. On the other hand, the results represented that the average hippuric acid in the exposed people (n = 24) was higher than the control (n = 20) so that it was 0.35 in the exposed and 0.26 in the control subjects. In addition, the average muconic acid in the exposed and control subjects decreased to 1.57 and 0.89, respectively. The minimum and maximum amounts of muconic and hippuric acids in the exposed subjects were 0.97 and 2.62, as well as 0.14 and 0.83, respectively. The maximum and minimum concentrations of muconic and hippuric acids were 2.62 and 0.97, as well as 0.83 and 0.14 in exposed subjects, respectively, which was less than muconic acid.
Keywords: Benzene, Muconic acid, Hippuric acid, Respiration Air F-main urinary metabolite
Received: May, 9, 2021, Accepted: February, 5, 2022, ePublished: December 29, 2022
https://jhygiene.muq.ac.ir/
10.34172/AHS.11.4.349.1
Vol. 11, No. 4, 2022, 291-296
Original Article
Sabzalipour et al
292 Arch Hyg Sci. Volume 11, Number 4, 2022
in the production of chemicals such as styrene, cumene,
cyclohexane, synthetic rubber and adhesives and oils,
varnishes, polishing oil, paints, pharmaceuticals, and
agricultural chemicals. It is also employed as a solvent
in paints, thinners, inks, and adhesives. Occupational
exposure to benzene occurs in rubber industries, oil
refining, chemical factories, shoe manufacturing, gasoline
storage equipment, and gasoline stations. Exposure to
benzene can also occur by eating contaminated food
products, but the absorption of food is less than 1% of the
average daily benzene absorbed in the general population
[1]. The measurement of phenol in the urine is the usual
biological sign of exposure to benzene [3]. However,
studies have shown that when exposed to low levels of
benzene, which is less than 5 ppm, urine phenol may
not be a reliable biological sign. Therefore, trans,transmuconic
acid (ttMA) and hippuric acid seem to be
the most reliable benzene metabolites for monitoring
benzene exposure with low levels between 0.25 and 3.5
ppm. These acids are used as useful biological parameters
for exposure to benzene between 1 and 68 ppm in many
studies. Studies conducted in the petrochemical industry
of Iran indicate that workers in the petrochemical industry
are exposed to volatile organic compounds, including
benzene, through breathing [4]. In several studies, the
concentration of the metabolites of these compounds in
workers’ urine samples was investigated to evaluate the
level of workers’ exposure to benzene compounds and
the like [5,6]. In the petrochemical industry, workers
of various plants are exposed to benzene substances,
and there are many concerns about the adverse effects
of constant exposure to this substance; unfortunately,
a limited number of studies have so far focused on the
cancer risk of exposure to benzene in workers working in
petrochemical plants, refineries, and industrial factories
such as cement, steel, and iron smelting factories. Thus,
this study aimed to investigate the amount of airborne
benzene and its relationship with the amount of urinary
benzene metabolites, including phenol, ttMA, and
hippuric acid, in the urine sample of workers working in
petrochemical complexes with different exposure times
and methods.
2. Materials and Methods
This cross-sectional descriptive study was conducted to
evaluate the relationship between the amount of benzene
in breathing air and the amount of urinary benzene
metabolites in workers working in six petrochemical
plants of Bou Ali, Bandar Imam in the spring of 2017.
Data were collected using a questionnaire, as well as
analysis of the urine samples obtained from 24 people
working in the industry and 20 people as the control
group. To eliminate possible errors and contaminations
during sampling or transfer, a number of samples were
selected as witnesses from the employees of the study
department and prepared similarly.
2.1. Sampling and analysis of benzene in air samples
The sampling of air and workers was performed in
aromatic, mixing xylene, loading, tanks, sampler, and
laboratory units. After analyzing the relevant samples,
each worker completed a questionnaire in which his
demographic data were provided, including work
experience, age, current job details, drug use, smoking,
and the like.
The sampling and measurement of benzene compounds
were conducted using the American NIOSH method No.
1501. A gas chromatography method equipped with a
mass detector and a 20-microliter loop at a wavelength of
254 nm was employed to qualitatively and quantitatively
identify benzene. Isolation and identification of benzene
under the study was done using a siloxane column
with performance specification RR-c18 e 100*4.6 mm,
introduced by the US EPA [7]. Benzene, obtained from
Sigma-Aldrich Company at concentrations of 10, 30, 50,
100, 200, 500, and 1000 ppm, was applied to draw standard
curves to determine the type (using retention time) and
amount (using the area under the curve). After drawing
the standard curve of benzene, the original samples were
injected into the gas chromatography-mass spectrometry
device to determine the concentration. After determining
the concentration by Eq. (1), the concentration of benzene
in each of the air samples was determined.
( ) f b f b W W W B B B
C
V
+ + − − −
= (1)
where W and Wf represent the mass of the analyte found
on the filter and the amount of analyte in the front part
of the absorber, respectively. Moreover, Wf and B denote
the amount of analyte in the rear part of the absorber
and the mean value of the analyte on the blank filter bed,
respectively. Further, Bf, Bb, V, and C are the amount of
analyte in the front part of the blank absorber, the amount
of analyte in the back part of the blank absorber, the
sampled air volume in liters, and the concentration of the
analyte in mg/m3, respectively [8,9].
2.2. Measurement and determination of benzene
metabolites in employees’ urine samples
After informing the workers and obtaining consent
to receive urine samples from them, the information
related to the investigation of the influencing factors in
the concentration of urinary volatile organic compound
metabolites, including age, work experience, weekly
working hours, and smoking, was collected through a
questionnaire. According to ACGIH instructions, urine
samples were collected in polyethylene containers at the
end of the work shift on the same day. To determine the
concentration of ttMA in the urine sample, the solid
phase extraction method was employed by a strong
exchange cartridge. After calculating the area under the
peak of the unknown sample, the concentration of ttMA
and hippuric acid compounds in the original sample were
calculated using the calibration curve and considering the
Arch Hyg Sci. Volume 11, Number 4, 2022 293
Relationship Between the Concentration of Airborne Pollutant and the Amont of Urinary Metabolites
correction factor related to the ratio of the volume of the
urine sample to the volume of the extractant solution [10].
2.3. Data analysis
SPSS 16, Student’s t-test, and linear regression were used
to statistically analyze the collected data and to examine
the correlation of quantitative variables with each other
and the status of each of the studied variables based on
qualitative variables.
3. Results
The results of the amount of environmental contact
of employees in different workplaces are displayed in
Figure 1. As shown, the amount of benzene in the ambient
air of the aromatic and loading units was higher than that
of the other units.
According to Figure 1, the concentration of benzene
in the aromatic unit had the highest value, which has
a higher level of pollution than both standards. The
mixed xylene unit, loading unit, and tank unit with a
concentration of 3.6, 3.4, and 2.8, respectively, had lower
benzene pollution compared to the Occupational Safety
and Health Administration permissible exposure limitshort-
term exposure limit (OSHA PEL-STEL) standard,
but the amount of pollution was higher in comparison to
the OSHA PEL-time-weighted average (TWA) standard.
The laboratory sampling unit with 0.94 benzene and the
laboratory technician unit had less pollution than both
OSHA PEL-TWA and OSHA PEL-STEL standards.
4. Discussion
4.1. Complications and Effects of Benzene
The evaluation of the symptoms and side effects of
benzene demonstrated that among the acute side effects,
headache and burning eyes are more common among
24 exposed people than 20 controls, but the prevalence
of other acute and chronic side effects and symptoms
such as bad smell in the nose, abnormal taste in the
mouth, imbalance, and dizziness were more frequent
in the exposed subjects than the control ones. There
was no significant relationship between the two groups
at the 0.05 level. According to the research, benzene
vapors at medium and high concentrations (acute
poisoning) affect the central nervous system and cause
symptoms such as dizziness, weakness, headache, nausea,
vomiting, incoordination of body parts, blurred vision,
loss of consciousness, and confusion. Additionally, high
concentrations of benzene vapors have an irritating effect
on the mucous membranes of the eyes, nose, throat, and
respiratory system [8,9]. However, due to the correct use
of appropriate personal protective equipment such as
gloves, masks, and filters suitable for absorbing organic
vapors and working against the wind direction, most of
the symptoms and complications mentioned in the two
groups represented no significant differences.
The effect of benzene and its side effects on the
employees of a petrochemical complex were measured,
and it was revealed that the highest number and percentage
of the exposed people had headache symptoms, and then
the number of people exposed to eye-burning was the
highest. According to Table 1, the number of exposed
people was higher than that of the control, and the effects
of benzene were mostly related to headache and burning
eyes. The least number of burning in the nose and the
imbalance were observed in the exposed subjects and the
controls, respectively.
Based on the obtained results, it was observed that
short-term concentration (19.5 ppm), mixed xylene
occupational group (18.76), short-term concentration
loading (17.43 ppm), and the laboratory sampler (14.7
ppm) were more than the maximum standard in the
aromatic occupational group, and they were less than
the maximum standard in the breathing zone of the
laboratory technicians and tanks. Among the reasons for
the excessive concentration of the loading unit standard
is the mismatch of the tanker valve with the arm funnel;
due to the carelessness of the loading staff in discharging
the remaining liquid benzene in the arm and its trap or
the technical defect of the loading system after filling the
tanker and removing the arm from it, liquid benzene is
occasionally poured on the ground in a small amount
and sometimes drop by drop, causing environmental
pollution.
In the tank unit, to comply with safety and
environmental principles, the tanks in this unit are
covered with nitrone. In addition, the benzene tank,
which has a high vapor pressure, is built with an internal
floating roof. Therefore, due to the closed system, there is
no pollution of benzene vapors in this unit. There is no
air pollution in the laboratory area due to the presence
of a local (range hood) and general ventilation system
and the performance of laboratory work under the hood.
However, the users of laboratory samplers are exposed to
benzene from different parts of the unit during sampling
and are exposed to the same concentration (14.7 ppm) in
the short term.
The results of the t-test showed that the average amount
of phenol in the urine after the working shift of the exposed
Figure 1. Concentration of benzene in the air of the breathing zone of
different petrochemical units in Bou Ali Sina Petrochemical Company.
Note. TWA: Time-weighted average.
Aromatic Mixed xylene Loading Tanks Lab Sampler Lab Technician
Sabzalipour et al
294 Arch Hyg Sci. Volume 11, Number 4, 2022
subjects was higher than that of the control subjects, but it
was still lower than the allowed amount (10 mg of phenol
per gram of creatine). Based on the test results, the average
amount of urine phenol after the work shift of the exposed
group was more than its value before the working shift of
the same group, but the average amount of urine phenol
before and after the work shift of the control group had no
difference, indicating that although there is contact with
benzene in the exposed group, due to the use of suitable
personal respiratory protective equipment and gloves,
as well as the presence of an engineering control system
(local ventilation) in the required places and compliance
with the principles of professional hygiene, its metabolite
in the urine is less than the allowed amount (Figure 2).
The average concentration of phenol, which is
determined as a biological indicator of contact with
benzene, is lower in the morning shift, while it is higher
in the evening shift than the standard recommended by
Iran’s Technical Committee of Occupational Health. The
standard limit of phenol in the urine is 25 mg/g of creatine.
Moreover, the concentration of benzene is lower than the
8-hour standard (0.5 ppm), which is recommended by
Iran’s Technical Committee of Occupational Health and
the American Industrial Hygiene Association [10]. In
this research, the concentration of phenol in the workers’
urine and the inhaled benzene in the evening shift
workers is more than in the morning shift workers, which
may be related to the higher pollution in the evenings.
The pollutant concentration in the air in the evening is
higher than in the morning air due to the concentration
of the pollutant during the daytime hours. Therefore,
evening shift workers inhale pollutants more than their
morning shift counterparts, and the concentration of
phenol excreted in this group is usually higher. Phenol is
expected to further increase when the amount of benzene
pollutants is higher in the work environment. Research
has confirmed the relationship between phenol excreted
from workers’ urine and benzene in the air (Table 2).
The low amount of urine phenol in the morning shift
workers can be related to the concentration of benzene in
the air of different units because, in the morning, the air
of benzene production units has a lower concentration of
pollutants such as benzene. This concentration increased
with the increase of the production time. The absence of
significant changes in the amount of phenol in the urine
during the work shift may be associated with the half-life
of phenol, which is one of the indicators of benzene and is
28 hours. Considering that the length of each work shift
is 8 hours, which is far less than the half-life of phenol,
phenol does not change much during the work shift. In
their study, Rastkari et al found that if benzene in the
work environment is less than 1 ppm, the excretion of
phenol from the urine is the same, while ttMA, which
is also produced from the metabolism of benzene,
represents an increase [1]. Fang et al reported that if the
concentration of benzene is lower than 1 ppm in the air,
the concentration of phenol after starting work and phenol
before starting work does not differ significantly [3]. The
Table 1. Mean values measured in the studied groups
Acute symptoms
Study group
Exposure Control P value
No. % No. %
Burning eyes 12 50 10 41.66 0.604
Burning nose 3 12.5 2 8.33 0.397
Abnormal sense of smell 9 37.5 3 12.5 0.013
Sore throat 10 41.66 2 8.33 0.0362
Imbalance 5 20.83 1 4.16 0.091
Burning 6 25 2 8.33 0.282
Dizziness 8 33.33 5 20.83 0.397
Abnormal taste in the mouth 5 20.83 3 12.5 0.469
Blurred vision 10 41.66 9 37.5 0.822
heavy-headedness 4 16.66 10 41.66 0.159
Headache 19 79.16 18 75 0.715
Figure 2. Comparison of the average amount of phenol before and after the working shift in the exposed and control subjects using Duncan’s mean comparison
test (α = 1 %).
Arch Hyg Sci. Volume 11, Number 4, 2022 295
Relationship Between the Concentration of Airborne Pollutant and the Amont of Urinary Metabolites
findings of another study by Maghsoodi Moghadam et al
demonstrated that at the concentrations of 0.7-13.6 μg/m3
of benzene in the air, phenol in the urine of workers was
constant during work [4], but the concentration of ttMA
and phenylmercapturic acid increased during the work
shift in this study (Table 3).
Based on the results (Figure 3), the maximum and
minimum concentrations of muconic and hippuric
acids were 2.62 and 0.97, as well as 0.83 and 0.14 in
the exposed people, which is less than muconic acid.
Urine phenol levels in workers with high experience
(15-23 years) were lower than phenol in workers with
low experience (less than 15 years). This can be related
to the kinetics of benzene in the body. Long-term contact
with organic solvents increases the possibility of creating
other metabolic forms in the body so that in addition
to phenol, catechol, hydroquinone, muconic acid, and
phenylmercapturic acid are excreted from the urine.
Therefore, by increasing the kinetic pathways in the body
of workers with high experience, the phenol excreted
in their urine will be significantly reduced compared to
workers with low experience. Considering the constancy
of phenol in the urine during shift work and the studies
conducted on ttMA and phenylmercapturic acid at
concentrations less than 1 ppm, the measurement of the
above metabolites in the urine in conditions where the
concentration of benzene in the air is less than 1 ppm,
benzene is preferable in measuring urine phenol.
It was expected that smokers would be more exposed
to benzene due to smoking and being in the area of the
site, thus an independent t test was used to compare
the variable of exposure to benzene and the variable of
smoking, and the result of the test showed no statistically
significant relationship between smoking and nonsmoking
and exposure to benzene in the air of the
workplace. Moreover, the statistical t test/limit value of 5
one-sample tests was employed to compare the amount of
exposure to benzene with 1 ppm, and considering the 1
ppm exposure to benzene in the air/mean of 52 working
environments, it was found that there was a significant
difference with the permissible benzene threshold, and
it was equal to this permissible threshold. Additionally,
the results of personal exposure evaluations conducted
among different units, shifts, and work groups to
determine the concentration of benzene in the air of the
work environment demonstrated that the concentration
of benzene in the evening shift, compared to the morning
shift, had a higher mean concentration in different work
groups. This can be considered due to the heat of the air
and environmental conditions in the morning shift when
the workers and employees spend less time on the site, as
well as in the night shift considering that the operational
power of the unit is lower than its normal level and the
mean concentration of the exposure of people in this shift
will be lower than the other two shifts.
5. Conclusion
The results revealed that the concentration of benzene
in the aromatic unit had the highest value, which had a
higher level of pollution than the other two standards.
The mixed xylene unit with a concentration of 3.6,
loading unit with a benzene amount of 3.4, and tank
Table 2. Average results of biological monitoring of benzene (urine phenol)
before and after the shift in the exposed and control subjects
Measured
parameters
Study
group
Status
Before work shift After work shift P-value
Mean ± SD Mean ± SD
Phenol
Exposure 4.76 ± 1.97 5.94 ± 3.46 0.016
Control 3.24 ± 2.18 3.57 ± 2.54 0.892
Note. SD: Standard deviation.
Table 3. Mean urinary metabolites of hippuric and muconic acids in the
exposed and control subjects
Variable Status No. Min. Max. Mean SD
Hippuric acid
Exposure 24 0.14 0.83 0.35 0.16
Control 20 0.1 0.61 0.26 0.12
Muconic acid
Exposure 24 0.97 2.62 1.57 0.071
Control 20 0.56 1.53 0.89 0.21
Note. Min. Minimum; Max.: Maximum; SD: Standard deviation.
Figure 3. Mean concentration of hippuric and muconic acids in the exposed and control subjects. Note. Min.: Minimum; Max.: Maximum.
Sabzalipour et al
296 Arch Hyg Sci. Volume 11, Number 4, 2022
unit with an amount of 2.8, compared to the OSHA
PEL-STEL standard, had a lower amount of benzene
pollution; however, in comparison to the OSHA PELTWA
standard, the amount of pollution was higher.
The laboratory sampling unit with 0.94 benzene and
the laboratory technician unit had less pollution than
both OSHA PEL-TWA and OSHA PEL-STEL standards.
On the other hand, the evaluation of the symptoms
and side effects of benzene represented that among
the acute side effects, headache and burning eyes were
more common among the exposed subjects than the
control subjects, but the prevalence of other acute and
chronic side effects such as the sense of abnormal smell
in the nose, abnormal taste in the mouth, imbalance,
dizziness, despite their frequency, was higher in the
exposed subjects than in the control subjects. The mean
concentration of phenol, determined as a biological
indicator of contact with benzene, was lower in the
morning shift, while it was higher in the evening shift
than the standard recommended by Iran’s occupational
health technical committee. On the other hand, the
results showed that the concentration of benzene in
the evening shift, compared to the morning shift, had
a higher mean concentration in different work groups.
The concentration of phenol in the workers’ urine
and benzene in inhaled air was higher in evening shift
workers than in morning shift workers, which might
be related to the higher pollution in the evenings than
in the mornings. The pollutant concentration in the
air in the evening was higher than the morning air
due to the concentration of the pollutant during the
daytime hours. Therefore, evening shift workers inhaled
pollutants more than morning shift workers, and the
concentration of phenol excreted in this group of
workers was usually higher. According to the results of
measuring the concentration of benzene in the aromatic
working group, mixed xylene and the average loading of
exposure to this chemical substance were several times
higher than the standard, indicating the faster start of
control measures. Benzene is the final product of these
two units and it is impossible to remove it from the
production source, thus management control measures
such as reducing the hours of exposure to this substance
by increasing the number of workers can be suggested
as an effective management control solution. Another
control measure regarding this hazardous substance
is to check the gaskets of gasoline pumps at specified
intervals and replace them if there is any defect. It is
also recommended that direct reading devices should be
used to monitor the possibility of leakage.
Acknowledgments
The authors would like to express their gratitude to Islamic Azad
University, Ahvaz branch and Bou Ali Sina Petrochemical Company
for their cooperation in obtaining the required data.
Author Contributions
All authors contributed to conceptualization: data management:
formal analysis: funding acquisition: review: methodology:
project management: resources: software: monitoring: validation:
visualization: writing - original draft: writing - review and editing .
Conflict of Interests
The authors declare that there is no conflict of interests regarding the
publication of this manuscript. Furthermore, the ethical issues have
been completely observed by the authors including plagiarism,
informed consent, misconduct, data fabrication and/or falsification,
double publication and/or submission, and redundancy.
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