Document r62z3OjeRM0VXDa5MBX5eGMna

British Journal of Industrial Medicine 1986;43:692-697 Quantitative relation of urinary phenol levels to breathzone benzene concentrations: a factory survey O INOUE,'2 K SEIJI,'2 M KASAHARA,2 H NAKATSUKA,2 T WATANABE,2 S-G YIN,3 G-L LI,3 C JIN,3 S-X CAI,3 X-Z WANG,4 M IKEDA2 From the Center of Occupational Medicine,' Tohoku Rosai Hospital, and Department ofEnvironmental Health,2 Tohoku University School ofMedicine, Sendai 980, Japan, Institute ofHealth,3 China Centrefor Preventive Medicine, Beijing, and Anhui Province Institute for Prevention and Treatment of Occupational Diseases,4 Hefei, China ABSTRACT Urine samples were collected from 64 men and 88 women in shoe factories and printing plants at the end of a seven hour day shift in the latter half of a week in spring. Urine samples were also taken from 43 men and 88 women in the same factories but who were not exposed to solvents. Exposure to benzene during the shift was monitored by passive dosimeters. Both phenol in urine and benzene in activated carbon were analysed with FID gas chromatographs. The urinary concentrations of phenol were linearly related to the time weighted average concentrations of benzene in the breathzone air; the variation was so small that those exposed to 10 ppm benzene could be separated from the non-exposed at least on a group basis when the phenol concentration was corrected either for creatinine concentration or for specific gravity. The urinary phenol concentrations corresponding to lOppm benzene were 47T5mg/1 (as observed), 57-9mg/g creatinine, or 46-6 mg/l (specific gravity 1*016). Benzene is considered to be a cause of human leukaemia' and occupational as well as non- occupational exposure to this chemical has been a focus of keen attention. Among the biological indicators of exposure to benzene, urinary phenol has been most frequently used although it may be inferior to benzene in blood in its sensitivity and specificity,2 3 probably because urine is apparently much more readily available from factory workers than venous blood, and this is especially so in the case of the -general population. The existing data on the quantitative relation between exposure to benzene and urinary phenol excretion are, however, usually hampered by the technical limitations prevailing at the time of the study such as the low specificity of urinary phenol determination, the inability to measure average exposure during work, the small number of subjects, or a combination of these factors.2 The present study was initiated to establish the quantitative exposure excretion relation, using passive samplers to determine the time weighted average exposure intensity of exposure to benzene and FID gas chromatography for the specific mea- Accepted 21 January 1986 surement of phenol in urine. The results of studies with similar methodology have been described for toluene hippuric acid/o-cresol4 and tetrachloroethylene total trichloro compounds.5 Materials and methods EXAMINEES The factory survey was conducted in the latter half of a week in spring. The workers exposed to benzene were 64 men (aged 26-9 + 8-4 years as arithmetic mean + arithmetic standard deviation (AM + ASD)) and 88 women (29-9 + 8-5 years) in five workshops in two shoe factories and two small printing plants. Control subjects were 43 men (39 3 + 119 years) and 88 women (26 9 + 8-4 years) who worked in the same factories but had not been exposed to solvents. Those under medical treatment with drugs were excluded. COLLECTION AND ANALYSES OF URINE SAMPLES Each subject was asked to pass urine at about 1300. The urine sahilple for analysis was collected at about 1500 when the seven hour shift was over and the urinary phenol concentration was expected to reach a maximum; the urine was kept at - 80C before analy- 692 Benzene in air and phenol in urine sis. Phenol concentration was determined gas chromatographically by the method previously described for o-cresol4: 1 ml of urine was mixed with 0 5 ml 15% HCI and heated for one hour for acid hydrolysis. Then, 3,5-xylenol (either 1 25 or 5 pg) was added as an internal standard and the mixture was extracted with 2 ml of carbon disulphide. The organic phase was treated with sodium sulphate for desiccation. An aliquot of 1 ml was transferred to a test tube and the volume was reduced to about one twentieth or less under a stream of nitrogen. An aliquot of 1 to 5Spl was injected into a Hitachi FID gas chromatograph (model 163) equipped with 60-80 mesh KG-02 on Uniport HP glass columns (3 mm in inner diameter and 5m in length). The injection port and the oven were heated at 210C and 180C, respectively. The carrier gas (nitrogen) was allowed to flow at a rate of 30 ml/min and the supplies of hydrogen and air to the detectors were at 1 -0 kg/cm2 and 1 5 kg/cm2, respectively. Under such conditions phenol was clearly separated from o-, m-, and p-cresols on the chromatogram. The phenol concentration was expressed as a measured value, as the ratio to the creatinine concentration,6 or after correcting to the specific gravity of the urine of 1-016.7 The creatinine concentration was measured colorimetrically8 and the specific gravity by refractometry. DETERMINATION OF BENZENE CONCENTRATION IN BREATHZONE AIR Badge type passive samplers with K-filter 16009 were used for personal breathzone air sampling to measure the time weighted average exposure (from about 0800 to about 1500). The precise duration of sampling was recorded for each individual or workshop. After exposure, the activated carbon filters were brought to an analytical laboratory distant from the survey sites within two days and kept refrigerated before analysis (within two weeks).'0 An additional study disclosed that, when kept at 4C, there was no significant (p < 0-10) loss in benzene on the carbon over a six month period. The gas chromatrographic analysis of the carbon disulphide 693 extract showed that the major component of the solvent vapour in the air was benzene; other contaminants such as toluene, n-hexane, or ethyl acetate were detected at trace level (less than 5% of that of benzene at most) only occasionally. When the benzene concentrations were expressed on a group basis, a log normal distribution was assumed." Results PHENOL CONCENTRATIONS IN THE URINE OF THE NON-EXPOSED SUBJECTS Phenol concentrations in the control urine samples accumulated in the range of 0-10 mg/l in the cases of observed values and after correction for a specific gravity (1-016) or 1-10 mg/g when corrected for creatinine. A skew towards higher levels was observed suggesting a log normal rather than a normal distribution. Accordingly, geometric means (GMs) and geometric standard deviations (GSDs) were calculated with an assumption of a log normal distribution; the results are summarised in table 1. The difference in the GMs between the two sexes was small even though statistically significant in some cases. When the results for the two sexes were combined, the GMs were either below 10 mg/l (the observed value and the value corrected for a specific gravity) or 10mg/g (the value corrected for creatinine). CORRELATION BETWEEN BENZENE CONCENTRATIONS IN BREATHZONE AIR AND PHENOL CONCENTRATIONS IN URINE When the exposed workers were grouped by workshop and by sex, and benzene GM and phenol GM compared (table 2), the range of both breathzone benzene concentrations and urinary phenol concentrations was wide but the former were generally paralleled by the latter even though the number of workers in some workshops was small (workshops A and D, for example). It should also be noted that GSDs were greater than 2 in several cases, indicating that the exposures, even in a single workshop, might vary Table 1 Phenol concentration in urine samplesfrom non-exposed subjects Non-exposed subject Valuet correctedfor Observed valuet Creatinine4 No ofexaminees (mg/l) (mg/g) Specific (mg/l) gravity Men + women Men Women 131 691(2622) 863(1921) 43 7-65(2-861) 10-27(1-845)* 88 6-57(2 511) 7-93 (1-932) * and ** indicate that the difference between the two sexes is statistically significant (*p < 0-05, **p < 0 1). tGeometric mean (geometric standard deviation). $Phenol concentration divided by creatinine concentration. Phenol concentration adjusted to a specific gravity of 1-016. 7-10(1-983) 909(1 892)** 6-29 (1-968) 694 Inoue, Seiji, Kasahara, Nakatsuka, Watanabe, Yin, Li, Jin, Cai, Wang, Ikeda Table 2 Phenol concentration in the urine of workers exposed to benzene in various workshops Workshop No of workers Phenol concentration* Benzene concentration in breathzone air* Observed value (ppm) (mg/l) Value correctedfor Creatininet (mg/g) Specific gravity: (mg/i) Men: A B C D E Women: A B C D E 3 10(1000) 24 11-1(2 398) 20 32-6(1 421) 2 7, 76 15 60 2(1282) 1 1 19 181 (2-792) 37 42 4(2 485) 2 5,37 29 76-4(1-576) *Geometric mean (geometric standard deviation). tPhenol concentration divided by creatinine concentration. $Phenol concentration adjusted to a specific gravity of 1-016. Individual values shown. 12(2.798) 32(2-145) 106(1-784) 16,85 277(1657) 3 76(3-945) 134(2.235) 5,100 319(2178) 7(1-812) 42(2.125) 147(1.498) 15,160 313(1320) 5 102(3-041) 169(2-143) 6,165 427(1783) 8(1869) 34(2-321) 119(1-456) 16,136 206(2-259) 4 77 (2.818) 124(2-116) 4,114 315(1662) depending on the individual worker. Accordingly, further comparisons between the two measurements were made on an individual basis using scatter diagrams as shown in the figure. Despite the difference in the maximum exposure between the sexes (some of the female workers were exposed well above 100 ppm whereas exposure of male workers never exceeded this concentration), the correlation appeared to be similar for the two sexes. It should also be noted that the variation around the regression was smaller in every case when the phenol concentrations were corrected for creatinine concentration than when observed phenol concentrations or specific gravity corrected phenol concentrations were used. The correlation coefficients are summarised in table 3. The correlation was significant (p < 0-01) and the value of the coefficient was about 0-8 or higher in all cases; the largest coefficient was obtained when phenol concentrations were corrected for creatinine concentration. The intercepts on the phenol axis (table 3) were essentially the same as the GMs for the non-exposed subjects (table 1), and rather small by comparison with the corresponding slopes (equivalent to 1 to 3 ppm benzene exposure). As both the Table 3 Correlation between breathzone benzene concentration and urinary phenol concentration Measurement group No ofexaminees* At Bt Ct Observed value (mg/I): Men + women 283 3-816 94 0-822 Men 108 4-110 4-3 0-789 Womenll 175 3-763 10 6 0-828 Womenll 159 4-003 6-9 0 746 Value corrected for creatinine (mg/g):** Men + women 283 4451 13 4 0891 Men 108 4 097 156 0-858 Womenll 175 4-503 14 7 0-894 Womenl 159 4-784 8 0 0-893 Value corrected for specific gravity (mg/l):tt Men + women 283 3 129 15 3 0-881 Men 108 3-351 13 3 0 840 Womenll 175 3 099 14 5 0-890 Womenll 159 3-639 7-0 0 861 *Including non-exposed subjects (43 men and 88 women). tSlope (A) and the intercept on the Y axis (B) in the figure as: Y = AX + B, where Y is phenol concentration (unit; as described in the table) in urine and X is time weighted average benzene exposure concentration (ppm). "Correlation coefficient. p < 0-01 for all correlation coefficients. Exposed up to 92 ppm. liExposed up to 210 ppm. IWomen exposed to less than 100 ppm. **Phenol concentration divided by creatinine concentration. ttPhenol concentration adjusted for a specific gravity of 1-016. Men . women 0 100 7 Observed value (mg/0) 50 0./~~~ // // / I. / 7* '0 a / Men Women 1000 ._ 0. -6 Corrected for creatinine (mg/g creatinine) 500 0 Corrected for specific gravity (1.016) (mg/Il) 0 100 200 0 100 200 0 100 200 Benzene in air (ppm) Relation between benzene in breathzone air and phenol in urine. Points indicate individual values. Lines and curves are calculated regression line (solid line in centre), 95% confidence ranges ofsample means (thin curves), 95% confidence ranges of the regression line (the broken lines), and 95% confidence ranges of the individual samples (the outmost broken lines). 696 Inoue, Seiji, Kasahara, Nakatsuka, Watanabe, Yin, Li, Jin, Cai, Wang, Ikeda intercept and the slope were similar in men and women, it was clear that the regression lines did not vary between the two sexes. This was the case even when women exposed to less than 100 ppm were selected (taking the difference in maximum exposure levels between the sexes into consideration). Discussion The present study clearly demonstrates that the phenol concentration in urine (collected at around 1500 in the latter half of the week) is proportional to the benzene concentration in breathzone air (expressed as a time weighted average) at least up to 200 ppm. The slope of the regression line obtained in the present study-3 to 4 mg phenol/I urine (or g creatinine)/ppm benzene-was larger than the values reported in some previous studies. For example, a slope of 0 33 (unit: mg phenol/litre urine/ppm x hour benzene) was calculated by Lauwerys2 primarily from the data of Rainsford and Lloyd Davies,7 Berlin et al,'2 and Sherwood,'3 which is equivalent to 2-3 mg/l/ppm when a seven hour exposure is assumed. Most of the high values cited in the calculations of Lauwerys were, however, derived from workers who spent sub- stantial periods in uncontaminated air,7 whereas spot workroom air samples were used for benzene determination. Thus the intensity of exposure might have been overestimated. In another study in which phenol concentrations (measured colorimetrically by the Theis-Benedict method) were adjusted to a specific gravity of 1-024 a regression line with a slope estimated to be 7 0 mg/l/ppm was found.'4 Adjusting the specific gravity to 1 016 gives a slope of 4-7 mg/l/ppm, a value slightly higher than the present results. The data from Pagnotto and Bethlehem Steel as cited by the National Institute for Occupational Health and Safety gave a slope of 7-2 and 3 9 mg/l/ppm, respectively, assuming a linear regression.'5 Assuming that urinary phenol concentrations were adjusted to a specific gravity of 1 024 by Pagnotto, readjustment to 1 016 results in a slope of 4-8nmg/I/ppm. If cases with "normal" phenol concentrations are excluded from Bethlehem Steel data the slope becomes 3-8 mg/l/ppm. One of the critical points in evaluating urinary phenol as a biological estimator of benzene exposure is to determine whether the estimator can separate those exposed to benzene at a given occupational exposure limit such as 10ppm from those not exposed. Comparison of the lower 95% confidence range of the mean phenol concentrations at 10 ppm benzene (figure) and the upper 95% confidence range of the phenol concentrations in the non-exposed sub- jects as calculated by GM x (GSD)2 (table 1) shows that the former was larger than the latter in men, women, and men + women when phenol concentrations were corrected for either creatinine or a specific gravity, although this was not the case with observed (uncorrected) phenol concentrations. For example, the lower 95% confidence value (using phenol concentrations corrected for creatinine) for men + women was 45 1 mg/g creatinine: the corresponding upper 95% confidence limit for the non-exposed men + women was 31-9mg/g creatinine. Thus it is reasonable to conclude that the separation of those exposed to 10 ppm benzene from those not exposed is possible at least on a group basis, so far as the effects of medication" - 1 are excluded. It is clearly impos- sible to make the separation on an individual basis as the wide 95% confidence ranges indicate (figure). No follow up of phenol excretion was made in the present study to cover the entire period of phenol excretion. It is possible, however, to make a cross sectional balance study between the amount of benzene absorbed and the excretion of phenol in urine at the end of work with exposure to benzene at, say, 10ppm, using three assumptions; that about 50% of inhaled benzene is absorbed through the lungs (by analogy with toluene"9) and that the rates of respiration and urinary excretion under the conditions studied are 15 1/min and 1 ml/min, respectively. The input will be 10 ppm (= 31-9 mg/m3) x 15 x 10-3(m3/min) x 0-5 = 0-23925mg/m3 whereas the output into urine in the form of phenol is 38 16 mg/I x 1 x 10-3 1/min = 0 003816 mg/min or 0003167mg/min (0.03816mg/min x 78 11/94 11) as benzene, where 78 11 and 94-11 are molecular weight of benzene and phenol, respectively. Thus at the end of the workshift about 13% (=0-04755/023925 x 100) of benzene absorbed through the lungs will be converted to phenol and excreted into the urine. Further analyses of the urine samples are currently in progress for polyhydroxylated benzene metabolites such as catechol and hydroquinone, possible contributors to the development of benzene toxicity20-22 to correlate the urinary concentrations with the intensity of benzene exposure. We thank the Health Bureau of Hefei City and Dr S-L Fu, Ms R-G Zhang, Mr W-G Wu, Ms G-F Cui, Mr L-H Zai, Ms J-F Wan, and Ms L-S Hong for their supportive cooperation in the field investigation. Requests for reprints to: Professor M Ikeda. References I International Agency for Research on Cancer. Benzene. IARC monographs on the evaluation of the carcinogenic risk of chem- icals to humans 1982;29:99-148. 2 Lauwerys R. Industrial health and safety: human biological mon- Benzene in air and phenol in urine itoring of industrial chemicals 1. Benzene. Luxembourg: Commission of the European Communities, 1979:18-25. 3 Berlin RE, Logan DC. International seminar on the assessment of toxic agents at the workplace. Roles of ambient and biological monitoring, Luxembourg, 8-12 December, 1980. Int Arch Occup Environ Health 1982;50:197-207. 4 Hasegawa K, Shiojima S, Koizumi A, Ikeda M. Hippuric acid and o-cresol in the urine of workers exposed to toluene. Int Arch Occup Environ Health 1983;52:197-208. 5 Ohtsuki T, Sato K, Koizumi A, Kumai M, Ikeda M. Limited capacity of humans to metabolize tetrachloroethylene. Int Arch Occup Environ Health 1983;51:381-90. 6 Jackson S. Creatinine in urine as an index of urinary excretion rate. Health Phys 1966;12:843-50. 7 Rainsford SG, Lloyd Davies TA. Urinary excretion of phenol by men exposed to vapour of benzene: a screening test. Br J Ind Med 1965;22:21-6. 8 Ikeda M, Ohtsuji H. Hippuric acid, phenol and trichloroacetic acid levels in the urine of Japanese subjects with no known exposure to organic solvents. Br J Ind Med 1969;26:162-4. 9 Hirayama T, Ikeda M. Applicability of activated carbon felt to the dosimetry of solvent vapor mixture. Am Ind Hyg Assoc J 1979;40:1091-6. 10 Ikeda M, Kumai M, Aksoy M. Application of carbon felt dosimetry to field studies distant from analytical laboratory. Ind Health 1984;22:53-8. 11 Ikeda M, Ohtsuki T. Exposure concentration versus environmental concentration: a field survey in organic solvent workplaces. Tohoku J Exp Med 1985;146:225-35. 12 Berlin M, Fredga K, Gage JC, Lagesson V, Reitalu J, Tunek A. 1975. Cited from Lauwerys, 1979.2 697 13 Sherwood RJ. Evaluation of exposure to benzene vapour during the loading of petrol. Br J Ind Med 1972;29:65-9. 14 Walkley JE, Pagnotto LD, Elkins HB. The measurement of phe- nol in urine as an index of benzene exposure. Am Ind Hyg Assoc J 1961;22:362-7. 15 National Institute of Occupational Safety and Health. Criteriafor a recommended standard: occupational exposure to benzene. Washington: US Department of Health, Education and Welfare, 1974: tables XII-10 and 12, pp 131 and 133. 16 Fishbeck WA, Langer RR, Kociba RJ. Elevated urinary phenol levels not related to benzene exposure. Am Ind Hyg Assoc J 1 975;36:820-4. 17 Kociba RJ, Kalnins RV, Wade CE, Garfield EL, Fishbeck WA. Elevated phenol levels in beagle dogs treated with salol. Am Ind Hyg Assoc J 1976;37:183-91. 18 Eikmann T, Gabriel M, Lenaerts-Langanke M, Prajsnar D. Die Phenolauascheidung bei erwachsenen Menschen in Abhangigkeit von Medikamenteneinnahme und Krankheitsbild. Zentralbl Arbeitsmed 1983;33:40-8. 19 World Health Organisation. Recommended health-based limits in occupational exposure to selected organic solvents: toluene. Geneva: WHO, 1981:8. (WHO technical report series 664.) 20 Greenlee WF, Sun JD, Bus JS. A proposed mechanism of benzene toxicity: formation of reactive intermediates from polyphenol metabolites. Toxicol Appl Pharmacol 1981;59:187-95. 21 Bolcsak LE, Nerland DE. Inhibition of erythropoiesis by benzene and benzene metabolites. Toxicol Appl Pharmacol 1983; 69:363-8. 22 Gad-EI-Karim MM, Ramanujam S, Ahmed AE, Legator MS. Benzene myeloclastogenicity: a function of its metabolism. Am Ind Hyg Assoc J 1985;7:475-84.