Annals of Occupational Hygiene Advance Access originally published online on March 20, 2006
Annals of Occupational Hygiene 2006 50(6):593-598; doi:10.1093/annhyg/mel016
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Biological Monitoring for Trimethylbenzene Exposure: A Human Volunteer Study and a Practical Example in the Workplace
1 Health and Safety Laboratory, Harpur Hill Buxton SK17 9JN, UK
2 Health and Safety Executive, Stanley Precinct Bootle L41, UK
3 Health and Safety Executive Birmingham, UK
*Author to whom correspondence should be addressed. Tel: +44-1298-218435; fax: +44-1298-218470; e-mail: kate.jones{at}hsl.gov.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
|---|
This paper presents data from both a human volunteer study looking at exposure to 1,3,5-trimethylbenzene (TMB) and an occupational hygiene study of a printing firm using screen wash containing technical grade TMB. The biomarkers measured were TMB in blood and breath, and urinary dimethylbenzoic acids (DMBAs). The volunteer (N = 4) study showed that TMB was rapidly absorbed into the bloodstream reaching a mean level of 0.85 µmol l–1 during a 4 h exposure to 25 p.p.m. TMB. There was little decline 1 h post-exposure possibly indicating storage of TMB in adipose tissue. Breath TMB levels peaked within an hour of exposure commencing and averaged 137 nmol l–1 during exposure. Elimination of TMB in breath was biphasic with an initial half-life of 60 min. Peak excretion of urinary DMBA occurred 4–8 h after the end of exposure and averaged 40 mmol mol–1 creatinine. Elimination of DMBA in urine was biphasic with half-lives of 13 and 60 h indicating that accumulation of body burden throughout the working week is likely if exposure is repeated. The occupational hygiene study demonstrated an excellent correlation between personal air TMB levels and post-shift urinary DMBA levels (r = 0.997) collected on the third working day. The regression equation from this study indicates that 8 h exposure to 25 p.p.m. TMB would result in a urinary DMBA level of 206 mmol mol–1 creatinine. All workers showed pre-shift levels of DMBA from exposure to TMB on previous days. Both urinary DMBA and breath TMB levels can be used as biomarkers of TMB exposure. Urine samples should be taken post-shift towards the end of the working week as significant body burden accumulation throughout the working week can be expected. Breath sampling is more suited to task or single-shift monitoring.
Keywords: trimethylbenzene • biological monitoring • volunteer study • screen printing • urine • breath
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
|---|
Trimethylbenzenes (TMBs) occur naturally in petroleum deposits. In the UK the major usage of TMBs occurs in gasoline and mixed aromatic hydrocarbon solvents. The main end uses of the hydrocarbon solvents are surface coatings, adhesives, rubber and reaction solvents. TMBs form 1–20% of the total solvents used in different parts of the surface coating industry. The UK and US occupational exposure limits for TMBs are 25 p.p.m. [8 h Time Weighted Average (TWA)] to protect against the lead health effect of concern, reproductive toxicity (ACGIH, 2004; HSE, 1996, 2005)
Kostrzewski et al. (1997) found that 
70% of an inhalation exposure was retained in the lungs of human volunteers. Once absorbed into the blood, there is some partitioning into adipose tissue from where TMB may be leached slowly over time. TMB is initially metabolized by hydroxylation to form dimethylbenzyl alcohol; then after oxidation forms dimethylbenzoic acid (DMBA). The DMBA may then conjugate with glycine to form dimethylhippuric acid. Both DMBAs (Kostrzewski et al., 1997) and dimethylhippuric acids (Järnberg et al., 1997) have been used to determine exposure to TMB.
This paper presents data from both a human volunteer study looking at exposure to 1,3,5-TMB and an occupational hygiene study of a printing firm using screen wash containing technical grade TMB. The biomarkers measured were TMB in blood and breath, and urinary DMBAs.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
|---|
Volunteer study
Four volunteers (two males, two females) took part in the study, approved by the Health & Safety Executive's Research Ethics Committee, and were exposed for 4 h to 25 p.p.m. 1,3,5-TMB (>98% purity, Aldrich Chemical Company Ltd, Dorset, England). Exposures were performed in the Health and Safety Laboratory Controlled Atmosphere Facility, a room of
8 m3 volume built for this purpose. The methodology has been previously described by Brooke et al., 1998. Each volunteer provided a urine sample pre-exposure. All urine was collected (in timed samples) for 1 week and analysed for the urinary metabolites of 1,3,5-TMB i.e. 3,5-DMBA and/or its glycine conjugate (3,5-dimethylhippuric acid). The analytical method (Nutley, 1994) involves alkaline hydrolysis to convert conjugates back to the DMBA, followed by derivatization with N,N-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and detection using GCMS. The method is sensitive (detection limit is 0.1 µmol l–1), linear (up to 1000 µmol l–1) and reproducible (inter-assay coefficient of variation <10%). Creatinine concentration was measured using the method of Jaffe using an automated method based on Garde et al. (2003).
Venous blood samples were taken prior to, and at 1 h intervals from, the start of exposure until 1 h post-exposure. In order to minimize blood sampling, repeated samples were not taken post-exposure. Samples were analysed in duplicate for 1,3,5-TMB using headspace sampling followed by GCMS analysis based on previously described methodology (Brooke et al., 1998).
Prior to entering the chamber, and at timed intervals during (hourly) and after (0.03, 0.3, 1, 1.5, 5.5 and 18 h) exposure, breath samples were collected using portable prototype breath samplers from which the exhaled solvent vapours could be collected onto TenaxTM tubes for subsequent analysis by GCMS as previously described (Dyne et al., 1997).
Before and during exposure, volunteers completed a detailed questionnaire, developed by HSL, for recording subjective experiences of sensory irritation of the eyes, nose and throat.
A 2 cm2 gauze patch soaked with liquid 1,3,5-TMB was placed under an occluded patch on the dorsal skin of the hand of a further male volunteer in order to investigate dermal absorption. However, due to the rapid development of mild itching, erythema and oedema at the treatment site (within 30 min), the patch was removed and the skin was copiously rinsed with water. It was, therefore, not possible to proceed with this part of the investigation. The rapid onset of irritancy seen in this individual suggests a primary irritancy effect rather than an immunologically mediated response.
Workplace study
Twelve workers at a company that produced screen-print transfers for use on glassware provided breath and urine samples pre-shift and post-shift. Personal and static air samples were also taken. Six workers underwent concurrent biological and environmental monitoring.
Three workers printed sheets of transfers, which were then placed on drying trolleys in the open workspace. Printing and drying were conducted at a relative humidity of 55% (±3%) to regulate swelling or shrinkage of the paper. Transfers incorporating up to 12 different colours of ink, in addition to a clear or coloured covercoat, were produced routinely, requiring operators to change and clean screens with screen-wash solution after each colour application. After use screens were cleaned in an enclosed washroom area where a wall-mounted extractor fan provided general ventilation. The screen cleaner wore a PVC suit and a face shield, a respirator was provided. Completed transfers were applied to the glassware at the workstation at the rear of the production area. Operators soaked the transfers in water to detach them from the backing paper and applied them to the glass. The glassware was then re-packaged for despatch to have the transfers fixed by kiln firing at another site. Some test firings were done on site using a small electrically heated furnace. The firing process burns off the covercoat from the transfer, leaving the ceramic ink in situ. The three glassware decorators wore laboratory coats; none of the other employees (apart from the screen washer) wore overalls.
Air sampling (both static and personal samples) and analysis were undertaken in accordance with MDHS80. Sampling time varied from 118 to 440 min. All three isomers of TMB were measured (1,2,3-; 1,2,4- and 1,3,5-TMB). Results are expressed as p.p.m. concentrations for an 8 h TWA exposure.
Urine samples were analysed for total DMBAs according to the method above (Nutley, 1994). All six DMBAs were measured (2,3-; 2,4-; 2,5-; 2,6-;3,4- and 3,5-DMBA) as markers of exposure to the three isomers of TMB.
Breath samples were taken using the commercially available Bio-VOCTM breath sampler (Markes International, Pontyclun, Wales) and analysed for all three isomers of TMB as previously described (Dyne et al., 1997). The Bio-VOCTM sampler was developed from the prototype sampler used in the volunteer study and both devices give comparable results (HSL, unpublished data).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
|---|
Volunteer study
3,5-DMBA was excreted in urine with a peak concentration eliminated 4–8 h post-exposure (mean 40 mmol per mol creatinine, range 26–58 mmol per mol creatinine, see Fig. 1). Pre-exposure levels of 3,5-DMBA were <0.5 mmol per mol creatinine for all volunteers. The majority of the absorbed dose was excreted in the first 50 h post-exposure however levels of 3,5-DMBA were still detected after 160 h. As can be seen from Fig. 1, substantial levels of DMBA were still being excreted 24 (black arrow) and 48 (grey arrow) h after the initial exposure indicating that accumulation of TMB throughout the working week is likely.
|
The elimination of urinary 3,5-DMBA was biphasic (see Fig. 2 for an illustration) with an initial mean half-life of 13 h (range 9–18 h) and an estimated secondary half-life of 60 h (range 32–94 h), although this figure is a rough estimate because for two volunteers the urinary levels were below the detection limit by the end of the week.
|
An approximation of the retention of inhaled TMB was obtained by dividing the total cumulative excretion of TMB equivalents in the urine (to represent internal dose) by the calculated total external exposure (external dose). Significant excretion of the glycine conjugates via the bile and faeces was not anticipated in view of their low molecular weight. The external dose was calculated from the product of the airborne concentration (25 p.p.m. = 0.123 mg l–1), the exposure time (240 min) and the alveolar ventilation (0.269 x body weight0.7 l min–1; Sato et al., 1991). Using these assumptions, the percentage retention of the inhaled dose was estimated to be 52–56% for the two male volunteers and 23–39% for the two female volunteers.
The results of the blood measurements suggest that there was a rapid absorption of 1,3,5-TMB into the blood (Fig. 3). The data suggest that an overall steady state was reached within 1–2 h of exposure. The average blood concentration during the 4 h exposure was 0.85 µmol l–1 (21 p.p.m.). Little decline in blood levels was seen at 1 h post-exposure (0.65 µmol l–1 or 16 p.p.m.), which might reflect the release of stored 1,3,5-TMB from the fat tissues into the blood (TMB has been shown to have a high fat:blood partition coefficient; Zahlsen et al., 1990).
|
Figure 4 shows that breath levels of 1,3,5-TMB peaked rapidly on entering the chamber with little increase in concentration after the first hour (pre-exposure concentrations were <5 nmol l–1 for all volunteers). Concentrations of 1,3,5-TMB in breath during exposure ranged from 114 to 160 nmol l–1 (mean 137 nmol l–1, 3.3 p.p.m.). On leaving the chamber there was an initial rapid decline in breath levels (mean half-life 60 min). After
90 min post-exposure, a slower elimination occurred with a mean half-life of 600 min. Levels in the next morning sample had returned to those seen pre-exposure.
|
Very little sensory irritation was reported and the exposure was well tolerated by all the volunteers. The solvent odour was apparent but awareness of the odour reduced as the exposure time progressed.
Workplace study
The air monitoring results showed that all three isomers of TMB were present with 1,2,4-TMB dominating (70% of the total TMB detected). In total 24 personal and static air samples were taken and the total TMB levels ranged from none detected (detection limit 0.09 p.p.m.) to 25.3 p.p.m. (8 h TWA) with two results marginally exceeding the Occupational Exposure Standard (25 p.p.m., 8 h TWA).
Static air samples showed that the mixing of inks resulted in very low airborne TMB levels (<1 p.p.m., 8 h TWA) as this was done in a segregated area with local exhaust ventilation. The screen cleaning gave the highest airborne TMB levels (20 p.p.m., 8 h TWA) as screens were sprayed with cleaner and there was only a wall-mounted fan for extraction. Respiratory protection was provided for the worker involved although he admitted to not using it. Other work areas (screen preparation, printing, transfer application and proving) showed background TMB levels of between 3 and 14 p.p.m. (8 h TWA).
All workers showed some DMBAs detectable in their pre-shift sample, indicating exposure from the previous day (sampling was undertaken on the third working day).
It can be seem from Fig. 5 that both post-shift urine total DMBA and the increase-over-the-shift urine total DMBA (i.e. post-shift urine value minus pre-shift urine value) are very well correlated (r = 0.997 and 0.990, respectively) with the 8 h TWA personal air exposure to TMB. The least squares regression equation for post-shift urine sampling is as follows:
 |
 |
|
Breath samples showed a lower correlation than the urine samples with ambient air measurements (Fig. 6) and one sample in particular seemed anomalous. Removing this sample from the analysis gave a least squares regression coefficient of 0.69 and the correlation was represented by the following equation:
 |
|
|
DISCUSSION AND CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
|---|
Blood and breath samples from the volunteer study showed that 1,3,5-TMB was readily absorbed with a steady state being reached within an hour of the onset of exposure. Elimination from both breath and urine was biphasic, indicating that some TMB may be stored, probably in body fat due to the high fat:blood partition coefficient (Zahlsen et al., 1990), prior to being excreted. The long half-life for DMBA suggests that, if exposure is repeated, accumulation of TMB is likely throughout the working week.
The excellent correlation between airborne TMB in personal air samples and urinary DMBA levels in the workplace study suggests that absorption is largely by inhalation as any absorption through skin or ingestion would reduce the correlation. The irritation caused by liquid TMB in the volunteer study demonstrates that any occupational skin splashing is likely to be washed off rapidly, before significant absorption can occur.
The volunteer study gave a peak urinary DMBA level of 42 mmol per mol creatinine (range 30–58 mmol per mol creatinine) for a 4 h exposure to 25 p.p.m. 1,3,5-TMB. Using Kostrzewski et al.’s (1997) data, an 8 h constant exposure would result in an end-of-shift urine value 2.2 times greater than the level seen after 4 h, i.e. 93 mmol per mol creatinine from this volunteer study. The air/urine correlation from the workplace study estimated an ‘increase-over-the-shift' urine DMBA value of 112 mmol per mol creatinine after 8 h exposure to the Occupational Exposure Standard (25 p.p.m.), which would be equivalent to a single 8 h exposure. As the workplace study showed dominant exposure to 1,2,4-TMB (which leads to greater urinary excretion of DMBAs) and an increased work rate compared with the volunteer study, the two studies are in reasonable agreement.
The post-shift urine DMBA level estimated from the workplace study of 206 mmol per mol creatinine for samples taken on the third working day illustrate the impact of previous days' exposure on the body burden as this is nearly twice the expected level from a single shift (112 mmol per mol creatinine).
Because the breath and blood TMB levels reach steady state within 1 or 2 h, the volunteer study and the workplace study are more directly comparable than for urinary DMBA. End-of-shift sampling for breath analysis gives a post-shift TMB value of 159 nmol l–1 for the workplace study compared with 137 nmol l–1 during the volunteer study. Järnberg and Johanson (1999) hypothesized that breath TMB levels are influenced by recent exposure. This may explain the anomalous result in the workplace study when a worker had a low breath TMB value compared with his urinary DMBA level and his personal air exposure. If the worker's exposure to TMB at the end of the shift had been considerably lower than for the rest of the shift, his post-shift breath sample may be lower than expected for a constant 25 p.p.m. exposure. This worker was the screen cleaner and his exposure was likely to fluctuate considerably with exposure significantly higher during actual screen cleaning than during other tasks. Another possible explanation for the screen cleaner's low breath TMB level could be the use of the respiratory protective equipment provided. However, the worker's urine result was consistent with an 8 h exposure at 25 p.p.m. TMB. On questioning, the worker admitted that he did not wear the respiratory protection and that the mask was left out in the open workshop with the filter exposed. The condition of the mask and also the face shield (which was worn to protect against splashing) was poor.
This study has shown that urinary DMBA and breath TMB are suitable markers of TMB exposure. If exposures are repeated during the week and urine samples are taken towards the end of the working week, significant body burden accumulation and higher levels of metabolites can be expected. This study has shown that exposure to 25 p.p.m. for 8 h would result in an increase in DMBA over the shift of 100 mmol per mol creatinine. Breath sampling is more suited to task or single-shift monitoring and a value of 100–150 nmol l–1 is indicative of recent exposure to 25 p.p.m. TMB.
This study clearly shows a role for biological monitoring of TMB exposure and for assessing the use and effectiveness of respiratory protective equipment.
Received November 29, 2005; in final form February 14, 2006
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
|---|
ACGIH. (2004) TLVs and BEIs. Threshold limit values for chemical substances and physical agents and biological exposure indices. American Conference of Industrial HygienistsCincinnati, OH ISBN 1-882417-54-2.
Brooke I, Cocker J, Delic JI, et al. (1998) Dermal uptake of solvents from the vapour phase: an experimental study in humans. Ann Occup Hyg 42:531–40.
Dyne D, Cocker J, Wilson HK. (1997) A novel device for capturing breath samples for solvent analysis. Sci Total Environ 199:83–9.
Garde AH, Hansen AM, Kristiansen J. (2003) Evaluation, including effects of storage and repeated freezing and thawing, of a method for measurement of urinary creatinine. Scand J Clin Lab Invest 63:521–4.[CrossRef][Web of Science][Medline]
HSE. (1991) Occupational Hygiene Visit Report Database and National Exposure Database, Technology Division. (Health and Safety Executive., Bootle, UK).
HSE. (1996) EH64 Summary criteria for occupational exposure limits. (HSE Books, UK) ISBN 0-7176-1085-3.
HSE. (2005) EH40/2005 Occupational exposure limits 2005. (HSE Books, UK) ISBN 0-7176-2977-5.
Jarnberg J, Stahlbon B, Johanson G, et al. (1997) Urinary excretion of dimethylhippuric acids in humans after exposure to trimethylbenzenes. Int Arch Occup Environ Health 69:491–7.[CrossRef][Web of Science][Medline]
Jarnberg J and Johanson G. (1999) Physiologically based modelling of 1,2,4–trimethylbenzene inhalation toxicokinetics. Toxicol Appl Pharmacol 155:203–14.[CrossRef][Web of Science][Medline]
Kostrzewski P, Wiaderna-Brycht A, Czerski B. (1997) Biological monitoring of experimental human exposure to trimethylbenzene. Sci Total Environ 199:73–81.
Volatile organic compounds in air. MDHS 80. Methods for the determination of hazardous substances. ISBN 0-7176-0913-8. Available from: www.hse.gov.uk/pubs/mdhs.
Nutley BP. (1994) Analysis of urinary dimethylbenzoic acids for assessing exposure to trimethylbenzenes. Health and Safety Executive, Research and Laboratory Services Division, IR/L/OT/94/6.
Zahlsen K, Nilsen AM, Eide I, et al. (1990) Accumulation and distribution of aliphatic (n-nonane), aromatic (1,2,4–trimethylbenzene) and naphthenic (1,2,4–trimethylcyclohexane) hydrocarbons in the rat after repeated inhalation. Pharmacol Toxicol 67:436–40.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||