# Low-Dose Benzene Exposure Monitoring of Oil Refinery Workers: Inhalation and Biomarkers
## 2 Materials and Methods  
The occupational monitoring campaign was conducted from March to April 2019. The study population was a non-smoking cohort of 23 workers at an oil refinery: 15 field operators employed in routine operations inside the plant, five laboratory technicians, and three supervisors of fuel-tank loading. All the subjects gave their informed consent in compliance with the Helsinki Declaration [22; 23]. Personal sampling was performed in the breathing zone, in the space between the chin and shoulder, throughout the work shift. At the shift's end, the operators proceeded with urine collection; these were immediately sealed to avoid leakage and placed at \(-20\)\({}^{\circ}\)C. All participating workers filled out a questionnaire (see Supplementary Material Figure S1) to collect data on their professional and lifestyle habits, especially passive smoking exposure and diet. To better understand and exclude the possible contribution of dermal exposure, specific questions were asked in the questionnaire, and the conditions of the workers' gloves were checked at the end of the shift to verify internal contamination. In addition to U-B, _t,t_-MA, and SPMA, SA and U-C were analyzed to verify dietary intake and smoking habits, respectively. Furthermore, airborne n-hexane from personal air sampling and its specific metabolite in urine, the free fraction of 2,5-hexanedione (2,5 HD), were also determined. Since n-hexane is not affected by confounding factors, it helps to discriminate between exposure due solely to gasoline and other benzene sources of exposure. Airborne toluene was monitored as well, since it is a constituent of gasoline. All solvents and analytical standards were purchased by Merck KGaA. The standard method adopted for evaluating airborne benzene and n-hexane was EN 14662-2 (EN 14662-2, 2005), using active sampling with standard 150/75 mg charcoal sorbent tubes (Di.Elle, Padova, Italia) and GilAir Pumps (Gilian-Sensidyne, St. Petersburg, US) at a flow rate of 200 mL/min, collecting for 480 min during the work-shift. The tubes were then closed with PTFE caps, stored at \(-20\)\({}^{\circ}\)C, and analyzed no later than one week after collection. Prior to the gas-chromatography-mass spectrometry (GC-MS) analysis, the extraction step was carried out; 1 mL of carbon disulfide low-benzene (Merck KGaA) with a diluted internal standard (5 ppm of fluorotoluene, Merck KGaA) was added to each sample, and then they were shaken for 30 min. A Shimadzu QP2010 GC-MS, equipped with a three-axis autosampler AOC-5000 Plus with Multi Tools Exchange (Shimadzu Europa GmbH, Duisburg, Germany), was used with a Varian Factor FOUR VF-5 MS analytical column, 60 m, 0.25 mm internal diameter, and 1.0 \(\upmu\)m film thickness (J&W Agilent Technologies, Santa Clara, CA, USA). The MS acquired data in scan mode with intervals of 4.40-6.90 min (29-100 \(m/z\)) and 7.30-38.00 (40-250 \(m/z\)). Benzene, n-hexane, fluorotoluene, and toluene were quantified at \(m/z\) 78, 57, 109, and 91, respectively. The GC-MS quantification was performed using an internal standard calibration curve. Field blank tubes and spiked samples were used to build the calibration curve. Concerning the biological monitoring, only one urine sample for each enrolled worker was collected at the end of the work-shift by a 40 mL amber glass vial with PTFE/silicone septum (Merck KGaA), and it was stored immediately at \(-20\)\({}^{\circ}\)C. After thawing, the urine samples were portioned out and kept at \(-20\)\({}^{\circ}\)C for SPMA, U-C, _t,t_-SA, _t,t_-MA, 2,5-HD, and creatinine determinations, scheduled within 30 days, while urine aliquots for U-B and U-MTBE were stored at 4\({}^{\circ}\)C and analyzed within the following seven days. Creatinine was measured to adjust _t,t_-MA, _t,t_-SA, and SPMA concentrations, as \(\upmu\)g/g creatinine, for comparison with ACGIH limit values. The analytical methods applied, using GC/MS or LC/MS detection, are summarized in Tables 1 and 2.