Chakroun, Radhouane MSC; Kaabachi, Néziha MD; Hedhili, Abderrazek MD; Feki, Moncef MD; Nouaigui, Habib MD; Laiba, Mohamed Ben MD; Mebazaa, Abderraouf MD
Benzene is known to produce toxicological effects. The most frequently reported effect in both animal experiments and human epidemiological studies is bone marrow depression leading to aplastic anemia. 1 These studies have also clearly linked benzene exposure with leukemia. 2
In the European Union, a threshold limit value (TLV) for the time-weighted average (TWA) of 1 ppm was recommended for benzene. 3 The American Conference of Governmental Industrial Hygienists (AGCIH) suggested a TLV-TWA of 0.3 ppm. 4 Moreover, the National Institute for Occupational Safety and Health (NIOSH) proposed a lower value that did not exceed 0.1 ppm. 5
In Tunisia, health hazards because of occupational benzene exposure are clearly recognized. 6 Exposed workers have to be given special medical care by occupational doctor. 7 However, no published data relating both environmental and bio-monitoring studies to occupational exposure is available. The primary purpose of this study was therefore to monitor benzene exposure among a sample of Tunisian workers by the evaluation of the concentration of benzene inhaled by each worker during the work shift and by the analysis of urinary trans,trans-Muconic acid (t,t-MA). This metabolite was shown to be a more suitable bio-marker of low-levels exposure to benzene than phenolic metabolites. 8 Moreover, Hotz et al found t,t-MA followed by S-Phenylmercapturic acid to be better indicator of benzene exposure in the 0.1 to 1 ppm range than urinary Phenol, Hydroquinone, Catechol, and both blood and breath benzene. 9
The second purpose of the present study was to check the reliability of t,t-MA as a bio-marker for benzene exposure monitoring taking into account local culture, work conditions and environment.
Materials and Methods
The study included 30 workers, aged 25 to 55 years. 20 male tanker fillers, 9 male and 1 female filling station attendants.
During a work shift, a tanker filler makes 2 to 4 fillings of petrol. Each loading lasts 15 to 30 minutes, depending on the volume loaded. During this operation, the worker has to maintain the filling tube in the opened aperture on the top of the tank. Later, he delivers the petrol in the service station by means of a flexible he links to the station’s tank. All workers wear gloves, but no respiratory protection is provided.
The filling station attendants have to supply vehicles with petrol. They don’t wear any protection devices.
A nonoccupationally exposed control group of 18 males and 2 females, aged 27 to 44 years was also investigated.
For all subjects of both groups, information about work conditions, smoking habits, alcohol consumption, sport practice, odd jobs, personal medical antecedents, and medicine intake had been collected by questionnaire. Mensuration of height and weight had also been made for all the subjects studied.
Measurement of Benzene in Ambient Air
Individual exposure to benzene at the workplace was monitored with a 3M-3500 organic vapor monitor through the whole work shift. Early in the morning, before they entered the filling zone, the diffusive samplers were fixed at chest level of the workers. The samplers were detached at the end of the shift and stored at −4°C until analysis. 3M-3500 badges have diffusion and recovery rates for benzene 35.5 mL/minute and 102%, respectively.
Measurement of benzene adsorbed in the dosimeter was carried out within one week, according to the general schema of the NIOSH standard method 1501, 10 using a gas chromatograph Hewlett Packard (HP-5890 II) equipped with a flame ionization detector and an integrator (HP-3396 II). The separation was accomplished on a CP-SIL 8 CB capillary column (50 m × 0.32 mm, 0.4-μm film thickness). The initial column temperature was 30°C for 4 minutes, followed by a temperature gradient of 10°C/minute to 150°C held for 1 minute, followed by a gradient of 20°C/minute to 220°C held for 9 minutes. A detection limit of 0.002 ppm could be achieved.
Measurement of Urinary trans,trans-Muconic Acid (t,t-MA)
Urine samples were collected at the beginning (t,t-MAA) and the end of work shift (t,t-MAB) and stored at −20°C until analysis. For the control group, only pre-shift urine samples were collected (t,t-MAC).
Measurement of t,t-MA was performed according to the method of Ducos et al. 8 1 mL of urine sample was submitted to a cleanup procedure with a Bond-Elut solid phase extraction column filled with 500 mg Sax previously conditioned by 3 mL methanol and 3 mL distilled water. The column was later washed with 1% acetic acid solution. Finally t,t-MA was eluted with 10% acetic acid solution. The collected volume was adjusted to 5 mL with distilled water. 10 μl of this solution was injected into an HPLC (Shimadzu LC-7A), with an UV/VIS spectrophotometer detector (Shimadzu CR-6A). Chromatographic separation was performed on a 250x4 mm column packed with 5 μm Lichrospher 100RP-18, used with Lichrocart-RP18-5 μm pre-column. The mobile phase was a 1% acetic acid/methanol solution (90:10 V/V) with a flow rate of 1 mL/minute. The analytical wavelength for peak detection was set at 259 nm. Detection limit and linearity allowed to measure a t,t-MA concentration of 0.05 mg/l (R2 = 0.9998 for the range of 0.05 to 5 mg/L).
Statistical analysis was performed using Epi Info software. Between groups comparison was studied using Student’s t test for normally distributed data and Mann-Whitney’s U test.
Results and Discussion
Environmental Air Samples
Airborne benzene values measured are presented in Table 1. The average benzene concentration for the whole group of workers was 0.17 ppm. Compared with TLV-TWA recommended by the European Union (1 ppm), this value is relatively low. However, it’s close to TLV-TWA proposed by ACGIH (0.3 ppm) and almost twice as high as the NIOSH TLV-TWA recommended value (0.1 ppm). Concentrations were slightly higher among filling station attendants than tanker fillers (0.20 ppm and 0.16 ppm, respectively). The difference between these two groups was not statistically significant.
In previous studies, Ducos et al found lower levels of exposure for filling station attendants. Measured concentrations did not exceed 0.22 ppm (0.09 median for dosimeter samples). 11 In contrast, Javelaud et al found road tanker drivers to be exposed to 0.58 ppm (arithmetic mean) (0.21 ppm median). 12 The differences in the observed results are possibly because of the type and the volume of petrol loaded during the sampling. Benzene concentration is greater in unleaded petrol than in the others. 12 Levels of exposure depend also on climatic conditions. Machefer et al 13 reported higher filling station attendant’s exposure to benzene in winter than that measured in the same service-stations during autumn and summer. In the latter season, the authors registered the lowest benzene values.
Measurement of trans,trans,-Muconic Acid
t,t-MA values measured in controls and workers are presented in Table 2. In controls, the highest t,t-MAC measured concentration was 0.65 mg/L (0.39 mg/g creatinine). Arithmetic mean value was 0.18 mg/L (0,11 mg/g creatinine). These values agree well with the results obtained by Ducos et al who found a urinary t,t-MA arithmetic mean value of 0.13 mg/L in the nonoccupationally exposed group. Measured values did not exceed 0.7 mg/L (0,4 mg/g creatinine). 14 Similarly, Inoue et al 15 reported that in 64% of nonoccupationally exposed persons, urinary t,t-MA concentrations were lower than 0.1 mg/L (detection limit of the used method).
In our study, mean pre-shift t,t-MA concentrations (t,t-MAA) in exposed workers (0.30 mg/L; 0.19 mg/g creatinine), were significantly higher than in controls (P < 0.01) regardless of the expression mode (mg/L or mg/g creatinine). This is possibly because of previous day’s exposure as suggested by Qu et al 16 who estimated the half-life of t,t-MA to 13.7 hours.
At the end of the workday, mean urinary t,t-MA concentration shifted to 0.55 mg/L (0.32 mg/g creatinine). The difference between before and after-shift values was highly significant (P < 0.001) in both expression modes. Furthermore, the correlation of these difference values (Δ t,t-MA = t,t-MAB - t,t-MAA) corrected for creatinine with airborne benzene concentrations was: R = 0.69 (0.44 < R < 0.84 in confidence limit of 95%) (Fig. 1). Mean value of Δ t,t-MA was 0.16 ± 0.08 mg/g creatinine for an average exposure to 0,17 ± 0.11 ppm benzene.
In workers, post shift urinary t,t-MA values (t,t-MAB) considered as biological exposure indice (BEI) were relatively low. However, some of the measured values were close to the BEI corresponding to 1 ppm benzene exposure proposed by the German DFG hygienists (Deutsche Forschungsgemeinschaft) : 2 mg/L. 17 These t,t-MAB values (expressed in mg/l) correlated with airborne benzene concentrations : R = 0.47 (0.12 < R < 0.71 in 95% confidence range). Correlation was clearly better when t,t-MAB concentrations were corrected for creatinine : R = 0.76 (0.55 < R < 0.88 in 95% confidence range) (Fig. 2).
Similar correlation was obtained by Inoue et al (0.827), 15 though their study concerned workers exposed up to 210 ppm. Nevertheless, authors reported no changing in correlation when concentrations were not corrected for creatinine (R = 0.816).
t,t-MAB calculated by the regression equation for TWA of 0.5 and 1 ppm were 0.82 and 1.57 mg/g creatinine respectively, in good agreement with results obtained by Lawerys, 18 who proposed for a TWA of 1 ppm benzene, a corresponding t,t-MA value of 1.4 mg/g creatinine.
The effect of smoking on t,t-MA excretion was considered in several previous studies. The results obtained in some of these studies are summarized with ours in Table 3.
The mean urinary t,t-MA concentration among nonoccupationally exposed control group (t,t-MAC) was 0.10 mg/L for nonsmokers and 0.27 mg/L for smokers. These results are consistent with t,t-MAC mean concentrations reported by Lee et al 19 (0.13 and 0.25 mg/L, respectively). Geometric means were respectively 0.04 and 0.13 mg/g creatinine and medians 0.04 and 0.14 mg/g creatinine, in agreement with the data reported by Lauwerys et al 18 (0.06 and 0.13 mg/g creatinine geometric means) and by Ruppert (0.065 and 0.13 mg/g creatinine medians). 20
The difference between smokers and nonsmokers was significant enough (P < 0.02) as it has been reported by Lee et al 19 and Melikian et al 21 Moreover, Melikian et al found also a significant correlation (R = 0.55) between t,t-MA urinary excretion and cotinine, a major metabolite of nicotine. 21
However, if we consider individual values, Lauwerys et al 18 found that the highest values were observed in nonsmokers, whereas in our study, these concentrations did not exceed 0.09 mg/g creatinine (0.22 mg/L) for nonsmokers and reached 0.39 mg/g creatinine (0.65 mg/L) for smokers in agreement with results reported by Ducos et al. 14
These differences observed in the background t,t-MA concentrations are possibly because of the inter-individual variability of benzene metabolism as suggested by Johnson et al 22 who found that 5 to 13% of nonoccupationally exposed subjects excreted t,t-MA at levels similar to those of workers exposed to more than 1 ppm benzene. Gobba et al 23 also found that 22% of the 77 bus drivers studied had higher rate of benzene metabolism.
In exposed workers, t,t-MA excretion values among smokers and non-smokers are presented in Table 4. The concentrations of t,t-MA were higher among nonsmoking workers than those observed in nonsmoking controls. The difference is statistically significant (P < 0.01). In contrast, no significant difference was observed either between smoking and nonsmoking workers or between smoking workers and smoking controls. These results may suggest that occupational exposure to benzene would mask the effect of smoking on t,t-MA urinary excretion.
Alcohol intake influence on urinary t,t-MA excretion was also studied. Mean morning urine t,t-MA concentrations in alcohol consuming and nonalcohol consuming workers and controls are presented in Table 5.
In controls, t,t-MA level was greater among the alcohol-consuming group than that measured for the group of subjects who don’t consume alcohol. Conversely, in workers, t,t-MA mean concentrations measured for both groups were very close. The results observed for alcohol consuming and nonalcohol consuming groups were respectively similar to those reported above for smokers and nonsmokers (Table 3).
To check if the difference between alcohol consumers and nonalcohol consumers observed in controls was actually because of alcohol intake, we subdivided each category of controls into two subgroups considering smoking habits (Table 6).
In nonsmokers, the mean t,t-MA concentration was greater in alcohol consumers (0.07 ± 0.02) than in nonalcohol consumers (0.04 ± 0.01). Similarly, if we consider smokers, the t,t-MA mean value was also higher among alcohol consumers (0.19 ± 0.12) than in nonalcohol consumers (0.11 ± 0.08). These results should be confirmed statistically by a larger scale study allowing to compare nonsmoking alcohol consumers with nonsmoking nonalcohol consumers.
According to environmental monitoring results, the exposure of Tunisian tanker fillers and filling station attendants to benzene seem to be relatively low. Airborne benzene concentrations were nearly six times lower than the European Union TLV-TWA (1 ppm). However this exposure was not negligible as registered values were close to ACGIH TLV-TWA (0.3 ppm) and exceeded the limit value proposed by the NIOSH (0.1 ppm).
An excellent correlation was found between end-shift urinary t,t-MA levels and environmental exposure to benzene during the workday. Better correlation was obtained when urinary t,t-MA concentrations were corrected for creatinine. Variation of urinary t,t-MA excretion during work shift (Δ t,t-MA) was also well correlated with airborne benzene concentration. Even if some of the unexposed subjects had urinary t,t-MA levels comparable to those found in exposed persons at the end of the workday, the mean concentrations were significantly different.
According to the regression analysis of our results and data reported in cited papers, TWA of 0.5 to 1 ppm benzene exposure can be easily assessed by the evaluation of end shift urinary t,t-MA level (0.8 and 1.5 mg/g creatinine, respectively), significantly higher than t,t-MA values in nonoccupationally exposed subjects.
For TWA lower than 0.5 ppm, this bio-marker is still reliable if a statistically representative group of workers is monitored. In all cases, it is essential to correct t,t-MA concentration for creatinine and to consider smoking habits and alcohol intake in the interpretation of the results.
In Tunisia, we recommend the use of end shift urinary t,t-MA analysis for the benzene exposure bio-monitoring purpose as this method is relatively simple and reliable enough.
In unexposed subjects, all t,t-MA measured values were below 0.4 mg/g creatinine. A larger scale study should confirm the limit value of 0.5 mg/g creatinine in the general population proposed in other countries.
We are grateful for the participation of the workers and cooperation of the managements in ESSO-Standard Tunisie Service. We thank P. Goutet, A. Barot, P. Ducos and J Delcourt (INRS-France) for their technical assistance.
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