Introduction

Workers are frequently exposed to multiple occupational hazards, including chemical, physical, and biological agents. Excessive exposure to chemicals may lead to a wide spectrum of adverse health outcomes, ranging from reversible disorders to life-threatening or disabling illnesses ^{1 - 4} .

Benzene, a colorless liquid with a characteristic sweet odor, is one of the most widely used industrial solvents. It serves as a precursor in the production of chemicals such as styrene, cumene, and cyclohexane. It is also used in the manufacture of lubricants, dyes, detergents, pesticides, and certain types of rubber ⁵ . Due to its high volatility, inhalation is the primary route of occupational exposure; however, absorption through the gastrointestinal tract and, to a lesser extent, through the skin, has also been documented.

Benzene is classified as a Group I human carcinogen by both the International Agency for Research on Cancer (IARC) and the U.S. Environmental Protection Agency (EPA) ⁸ . The hematopoietic system is considered the critical target of benzene toxicity. Hematological effects have been observed following both acute and chronic exposures with decreased lymphocyte counts recognized as an early biomarker and a consistent finding among workers with evident benzene toxicity.

To limit occupational risks, the Occupational Safety and Health Administration (OSHA) has established a permissible exposure limit-time weighted average (PEL-TWA) of 1 ppm for an 8-hour work shift, while the American Conference of Governmental Industrial Hygienists (ACGIH) recommends a threshold limit value-time weighted average (TLV-TWA) of 0.5 ppm ¹¹ . Previous studies have reported hematological effects at benzene concentrations well above these limits ^{12 , 13} . The severity of such effects generally increases with both exposure concentration and duration.

However, increasing concern has been raised regarding the potential hematological effects of benzene even at concentrations below the current OSHA PEL-TWA.

There are three primary approaches to assessing occupational exposures: air monitoring, biological monitoring, and medical examinations ¹⁴ . Among these, medical examinations are essential for evaluating adverse health effects resulting from occupational hazards. The complete blood count (CBC) has long been used as a simple, inexpensive, and accessible method for assessing hematotoxicity ¹⁵ .

However, studies investigating the relationship between benzene exposure and hematological alterations have produced inconclusive results, particularly at low-level exposures. While reductions in white blood cell (WBC) counts and their subsets are well-documented in clinical cases of benzene-induced hematotoxicity, ¹⁶ emerging studies suggest that red blood cells (RBC) and their indices may be more sensitive indicators at low levels of benzene exposure ^{17 , 18} . For example, reduced RBC counts have been reported as the only hematological alteration among workers exposed to concentrations below 1 ppm ¹⁷ . Similar findings have also been observed in animal studies ^{19 , 20} .

In contrast, some studies have reported no significant associations between reduced blood parameters and benzene exposure ^{21 , 22} . Given the above, the present study was conducted to investigate the potential association between exposure to low concentrations of benzene and the levels of blood parameters in a group of workers exposed to this hematotoxic solvent.

Methods

Subjects and Study Design

This cross-sectional study was conducted at a petrochemical plant in Iran. A total of 512 male workers occupationally exposed to benzene were enrolled, along with 217 age- and sex-matched non-exposed subjects from a nearby gas power plant.

Data were collected using a structured questionnaire that gathered information on demographic characteristics, smoking habits, alcohol and medication use, history of exposure to chemicals, and prior occupational exposures, particularly those known to pose a risk of hematotoxicity.

Eligibility criteria included: (i) at least one year of employment in the current job (exposure for the benzene group and duration of employment for the non-exposed group), (ii) no past or present exposure to other hematotoxic agents, and (iii) absence of underlying hematological or systemic diseases such as thalassemia, hepatitis, favism, or hemophilia.

Ethical ConsiderationAll participants provided written informed consent before enrollment. The study protocol was approved by the University Ethics Committee (IR.SUMS.SCHEANUT.REC.1402.028) and conducted in accordance with the principles outlined in the Declaration of Helsinki (1964, revised 2000) ²³ .

Hematology Studies

Venous blood samples were collected from the brachial veins of participants at their workplaces using standard venipuncture procedures. Samples were processed for complete blood count (CBC) analysis, including: 1) total WBC and differentials (monocytes, lymphocytes, and neutrophils), 2) total RBCs and hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), and 3) platelets, mean platelet volume (MPV), and platelet distribution width (PDW).

Exposure Assessment

The study population was divided into 34 similar exposure groups (SEGs), each consisting of 2 to 36 workers. From these groups, 175 workers were randomly selected for personal air sampling. Full-shift personal air samples were collected using constant-flow personal sampling pumps and charcoal tubes (SKC 226-01), following the NIOSH Method 1501 protocol to determine time-weighted average (TWA) exposures.

Air samples were transported to the laboratory on ice packs. Benzene was desorbed with 1 mL of carbon disulfide for 30 minutes of agitation, and one μL of the extract was injected into a gas chromatograph (Varian CP-3800). Analysis was performed using a flame ionization detector (GC-FID) equipped with a CP-Sil 5 CB capillary column (30 m × 0.25 mm). The chromatographic conditions were as follows: injector temperature, 240 °C; detector temperature, 300 °C; oven temperature, initially held at 50 °C for 10 min, then increased at 20 °C/min to 250 °C and maintained for 1 min. Nitrogen was used as the carrier gas at a flow rate of 1 mL/min.

Statistical Analysis

Data analysis was performed using SPSS software (version 22.0). The student’s t-test and multiple linear regression analyses were used. A p-value < 0.05 was considered statistically significant.

Results

Table 1 presents the demographic characteristics and benzene exposure levels of the study groups. Seventeen workers were excluded from the study because they did not meet the inclusion criteria.

Significant differences were observed between exposed and non-exposed participants with respect to age, height, and job tenure. The mean TWA exposure to benzene among exposed workers was 0.07 ± 0.37 ppm, which is well below the current permissible exposure limit (PEL-TWA) of 1 ppm.

Table 2 summarizes the comparison of hematological parameters between exposed and non-exposed workers. Statistically significant differences were observed in platelet count, MPV, PDW, RBC, hemoglobin, MCV, and RDW levels between the two groups. In contrast, no significant differences were found in total WBC count or its subsets.

Table 3 presents the adjusted associations between benzene exposure and blood parameters among the studied workers. After controlling for potential confounders, including age, BMI, and job tenure, statistically significant associations were observed only for RBC and MCV. Specifically, benzene exposure was associated with a 0.0004-unit increase in RBC and a 0.01-unit decrease in MCV. A borderline association was observed for lymphocytes (B = 0.001, p = 0.057). No significant associations were detected for other hematological parameters.

Discussion

In the present study, we investigated a group of workers exposed to low levels of benzene to assess the potential association between benzene exposure and hematological parameters. A relatively large sample of benzene-exposed workers (n = 495) and a reference group were included. The exposed workers, employed in a petrochemical plant, experienced low-level benzene exposure (0.07 ± 0.37 ppm). Accurate exposure assessment is critical when studying the toxic effects of chemicals on biological systems. A major limitation of some previous studies is the lack of detailed exposure data, which complicates drawing clear conclusions regarding exposure-outcome relationships. In contrast, our study employed a rigorous exposure assessment program, providing a reliable estimate of benzene levels among the workers.

Benzene is known to cause bone marrow suppression, potentially leading to progressive reductions in RBCs, platelets, and WBCs. Leukopenia, or a reduction in WBCs, is commonly reported in benzene-exposed workers ^{12 , 24} . However, in the present study, no significant differences were observed in WBC counts or their subsets between the exposed and non-exposed groups (Table 2). This discrepancy may be explained by differences in benzene exposure levels across studies. The threshold dose of benzene required to induce leukopenia remains unclear. Previous research has reported leukopenia at a wide range of exposures, from 3.8 to 34 ppm ^{12 , 25 , 26} . For example, Kipen et al. observed decreased WBC counts in workers exposed to 75 ppm of benzene but not in those with 15-20 ppm exposure ²⁵ . These findings suggest that leukopenia is primarily associated with high-dose benzene exposure, whereas low-level exposures, such as those in the present study, may not significantly affect WBC counts.

Recently, several studies have investigated the effects of low-level (<1 ppm) benzene exposure on hematological parameters. Growing evidence suggests that RBCs and their indices are particularly sensitive to benzene exposure, even at low doses ^{17 , 19} . In the present study, all measured blood parameters remained within the normal range. However, compared with non-exposed subjects, significant differences were observed in platelets, MPV, PDW, RBC, hemoglobin (HB), MCV, and RDW (Table 2). To account for potential confounders, including age, BMI, and job tenure, multiple regression analyses were performed. After adjustment, statistically significant associations were identified between benzene exposure and RBC (B = 0.0004, p = 0.041) as well as MCV (B = -0.01, p = 0.044) (Table 3).

These findings are consistent with previous research ^{17 , 19 , 20 , 27} . For example, Koh et al. (2015) reported a reduction in RBC count as the sole hematological change among Korean male workers exposed to less than one ppm benzene. Similarly, Collins et al. observed no significant changes in other blood parameters in a study of 387 workers exposed to low levels of benzene. ²⁸ Tsai et al. also reported minimal effects on hematological indices in a study of 1,200 workers with low exposure. ²⁹

The present study has several strengths. First, a large number of workers were included. Second, a robust exposure assessment program was conducted to determine the workers’ exposure to benzene accurately. Third, none of the exposed workers had pre-existing conditions that could affect blood parameters, such as genetic disorders, infections, or inflammatory diseases.

However, the study also has important limitations. Non-occupational sources of exposure, such as smoking or environmental pollution, can be significant in cases of low-level benzene exposure. The potential effect of smoking was not considered. Additionally, workers with abnormal blood parameters may have been transferred to less contaminated areas or departments where no exposure was expected and, therefore, were not included in the study. Finally, the cross-sectional design limits the ability to establish a causal relationship.

Nevertheless, the following points support that the observed changes in blood parameters are likely attributable to benzene exposure:

1. The study included workers without pre-existing blood disorders,

2. The exposed subjects were not concurrently exposed to other hematotoxic chemicals, and

3. Regression analyses (adjusted for key confounders) demonstrated a significant relationship between benzene exposure and certain blood parameters.

Conclusion

The findings indicate that RBC and its indices (e.g., MCV) are more sensitive to low-level benzene exposure than other blood parameters. Further studies with extended follow-up periods and detailed data on long-term occupational exposure are recommended to confirm and expand upon these preliminary findings.

Acknowledgment

We are grateful to the petrochemical managers and employees for their helpful cooperation.

Author’s Contribution

Fereshteh Aliasghari: Methodology, data curation, writing original draft.

Seyedeh Mahsa Taghavipour, Fatemeh Rahimian, Anahita Fakherpour: Data collection, writing original draft.

Nadia Mohammadi and Mohammad Fararouei: Analyze and interpretation of the data; writing-reviewing and editing.

Esmaeel Soleimani: Funding acquisition, supervision, methodology, writing-reviewing and editing.

Funding

This work was supported by the Shiraz University of Medical Sciences (SUMS) under grant number .

Conflict of Interest

The authors declare that they have no competing interests.

References

1. Behnami F, Yousefinejad S, Jafari S, Neghab M, Soleimani E. Assessment of respiratory exposure to cypermethrin among farmers and farm workers of Shiraz, Iran. Environ Monit Assess. 2021; 193(4):187. DOI | PubMed
2. Neghab M, Amiri F, Soleimani E, Hosseini SY. The effect of exposure to low levels of chlorine gas on the pulmonary function and symptoms in a Chloralkali unit. J Res Health Sci. 2016; 16(1):41-5. Publisher Full Text | PubMed [ PMC Free Article ]
3. Neghab M, Soleimani E, Rajaeefard A. Assessment of occupational exposure to n-hexane: a study in shoe making workshops. Res J Environ Toxicol. 2011; 5(5):293-300. DOI
4. Sohrabi Y, Sabet S, Yousefinejad S, Rahimian F, Aryaie M, Soleimani E, et al. Pulmonary function and respiratory symptoms in workers exposed to respirable silica dust: a historical cohort study. Heliyon. 2022; 8(11):e11642. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
5. Wilbur SB, Keith LS, Faroon O, Wohlers D. Toxicological profile for benzene. U.S. Department of Health and Human Services Public Health Service, Agency for Toxic Substances and Disease Registry. 2007.
6. Snyder R. Overview of the toxicology of benzene. J Toxicol Environ Health A. 2000; 61(5-6):339-46. DOI | PubMed
7. Sohrabi Y, Rahimian F, Yousefinejad S, Aliasghari F, Soleimani E. Microextraction techniques for occupational biological monitoring: Basic principles, current applications and future perspectives. Biomed Chromatogr. 2024;e5883. DOI
8. Cancer IAfRo. IARC monographs on the evaluation of carcinogenic risks to humans. PCDFs. IARC Monogr Eval Carcinog Risks Hum. 1997.
9. Midzenski MA, McDiarmid MA, Rothman N, Kolodner K. Acute high dose exposure to benzene in shipyard workers. Am J Ind Med. 1992; 22(4):553-65. DOI | PubMed
10. Wiwanitkit V, Suwansaksri J, Soogarun S. The urine trans, trans muconic acid biomarker and platelet count in a sample of subjects with benzene exposure. Clin Appl Thromb Hemost. 2004; 10(1):73-6. DOI | PubMed
11. American Conference of Governmental Industrial Hygienists. Threshold limit values and biological exposure indices. Cincinnati (OH): ACGIH; 2023.
12. Qu Q, Shore R, Li G, Jin X, Chen LC, Cohen B, et al. Hematological changes among Chinese workers with a broad range of benzene exposures. Am J Ind Med. 2002; 42(4):275-85. DOI | PubMed
13. Qu Q, Shore R, Li G, Jin X, Chen LC, Cohen B, et al. Validation and evaluation of biomarkers in workers exposed to benzene in China. Res Rep Health Eff Inst. 2003; 115:1-72. PubMed
14. Soleimani E. Benzene, toluene, ethylbenzene, and xylene: current analytical techniques and approaches for biological monitoring. Crit Rev Anal Chem. 2020; 39(1):168-87. DOI
15. Kooshki F, Neghab M, Soleimani E, Hasanzadeh J. Low-level exposure to lead dust in unusual work schedules and hematologic, renal, and hepatic parameters. Toxicol Appl Pharmacol. 2021; 415:115448. DOI
16. McHale CM, Zhang L, Smith MT. Current understanding of the mechanism of benzene-induced leukemia in humans: implications for risk assessment. Carcinogenesis. 2012; 33(2):240-52. DOI
17. Koh DH, Jeon HK, Lee SG, Ryu HW. The relationship between low-level benzene exposure and blood cell counts in Korean workers. Occup Environ Med. 2015; 72(6):421-7. DOI | PubMed
18. Zhang X, Deng Q, He Z, Li J, Ma X, Zhang Z, et al. Influence of benzene exposure, fat content, and their interactions on erythroid-related hematologic parameters in petrochemical workers: a cross-sectional study. BMC Public Health. 2020; 20:1-13. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
19. Liang B, Chen Y, Yuan W, Qin F, Zhang Q, Deng N, et al. Down-regulation of miRNA-451a and miRNA-486-5p involved in benzene-induced inhibition on erythroid cell differentiation in vitro and in vivo. Arch Toxicol. 2018; 92:259-72. DOI | PubMed
20. Yuan W, Sun Q, Jiang Y, Zhang X, Chen L, Xie C, et al. MiR-146a affects the alteration in myeloid differentiation induced by hydroquinone in human CD34+ hematopoietic progenitor cells and HL-60 cells. Toxicol Res (Camb). 2016; 5(3):848-58. DOI
21. Bogadi-Šare A, Zavalić M, Turk R. Utility of a routine medical surveillance program with benzene exposed workers. Am J Ind Med. 2003; 44(5):467-73. DOI | PubMed
22. Collins JJ, Conner P, Friedlander BR, Easterday PA, Nair RS, Braun J. A study of the hematologic effects of chronic low-level exposure to benzene. J Occup Med. 1991; 33(6):619-26. PubMed
23. World Medical Association. Declaration of Helsinki: Ethical principles for medical research involving human subjects. Bull World Health Organ. 2001; 79(4):373-4.
24. Cody RP, Strawderman WW, Kipen HM. Hematologic effects of benzene: job-specific trends during the first year of employment among a cohort of benzene-exposed rubber workers. J Occup Environ Med. 1993; 35(8):776-82. PubMed
25. Kipen HM, Cody RP, Crump KS, Allen BC, Goldstein BD. Hematologic effects of benzene: a thirty-five year longitudinal study of rubber workers. Toxicol Ind Health. 1988; 4(4):411-30. DOI | PubMed
26. Ward E, Hornung R, Morris J, Rinsky R, Wild D, Halperin W, et al. Risk of low red or white blood cell count related to estimated benzene exposure in a rubberworker cohort (1940-1975). Am J Ind Med. 1996; 29(3):247-57. DOI | PubMed
27. Moro AM, Brucker N, Charão MF, Sauer E, Freitas F, Durgante J, et al. Early hematological and immunological alterations in gasoline station attendants exposed to benzene. Environ Res. 2015; 137:349-56. DOI | PubMed
28. Collins JJ, Ireland BK, Easterday PA, Nair RS, Braun J. Evaluation of lymphopenia among workers with low-level benzene exposure and the utility of routine data collection. J Occup Environ Med. 1997; 39(3):232-7. DOI | PubMed
29. Tsai SP, Fox EE, Ransdell JD, Wendt JK, Waddell LC, Donnelly RP. A hematology surveillance study of petrochemical workers exposed to benzene. Regul Toxicol Pharmacol. 2004; 40(1):67-73. DOI | PubMed