Int. J. Environment and Health, Vol. 4, Nos. 2/3, 2010
Using the exhaled breath condensate as a tool for non-invasive evaluation of pollutant exposure S. Marta Almeida*, Pedro M. Félix, Cristiana Franco, Maria do Carmo Freitas, Luís Cerqueira Alves and Teresa Pinheiro Instituto Tecnológico e Nuclear, Estrada Nacional 10, 2686-953 Sacavém, Portugal Email:
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Maria Alexandra Barreiros Laboratório Nacional de Energia e Geologia, Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal Email:
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Sílvia M. Garcia Institute of Welding and Quality, 2740-120 Porto Salvo, Portugal Email:
[email protected] Abstract: The aim of the present study was to investigate whether Exhaled Breath Condensate (EBC) can be employed for a better risk assessment among human exposure to heavy metals. The objective was to develop a human bioindicator to be applicable for professional exposure that, compared with other common indicators, presents important advantages: it is non-invasive and it is easily and quickly collected. EBC was sampled in industrial workers and in a control group consisting of healthy volunteers working in offices. Heavy metal concentrations in EBC were determined by Total Reflection X-Ray Fluorescence (TXRF) in parallel low-volume samplers, and the techniques Particle Induced X-ray Emission (PIXE) and Instrumental Neutron Activation Analysis (INAA) were used to characterise air particulate matter in the factory and in the offices. Results revealed that Pb and Cu were significantly higher in industrial workers EBC, raising the possibility of using this new matrix in occupational settings in order to assess the workers’ exposure to toxic agents. Keywords: EBC; exhaled breath condensate; APM; air particulate matter; heavy metals; lead; PM2.5; PM10; risk assessment; human biomonitoring; INAA; instrumental neutron activation analysis; PIXE; particle induced X-ray emission; TXRF; total reflection X-ray fluorescence.
Copyright © 2010 Inderscience Enterprises Ltd.
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S.M. Almeida et al. Reference to this paper should be made as follows: Almeida, S.M., Félix, P.M., Franco, C., Freitas, M.C., Alves, L.C., Pinheiro, T., Barreiros, M.A. and Garcia, S.M. (2010) ‘Using the exhaled breath condensate as a tool for non-invasive evaluation of pollutant exposure’, Int. J. Environment and Health, Vol. 4, Nos. 2/3, pp.293–304. Biographical notes: S. Marta Almeida has a degree in Environmental Engineering (1998, Universidade Nova de Lisboa) and a PhD in Environmental Sciences in (2004, Aveiro University). Since 1998, she has been developing work in research in environment sciences in several Portuguese research institutions – Technological and Nuclear Institute (ITN), Aveiro University (UA), National Institute of Engineer, Technology and Innovation (INETI) and the Welding and Quality Institute (ISQ). She has participated in several national and European R&D projects and is author or co-author of more than 20 scientific publications on atmospheric pollution, indoor air quality, epidemiology and risk assessment. Pedro M. Félix has a degree in Environmental Biology (2006, Faculty of Sciences, University of Lisbon) and acquired his Masters in 2008 in marine biology. He is developing research work in the Technological and Nuclear Institute (ITN) with the biomedical sciences group and participating in a cooperative project regarding occupational and environmental health, between several national institutions. His main interests include ecotoxicology and physiological responses to environmental exposure. His current work involves the use, improvement and validation of mass spectrometry technique on samples with biological matrices. Cristiana Franco has a degree in Chemical and Biological Engineering (2007, Instituto Superior de Engenharia de Lisboa, ISEL). In the same year, she was accepted in a Human Biology and Environment Masters Degree course (Faculdade de Ciências da Universidade de Lisboa, FCUL). She is doing her research work on occupational toxicology in the biomedical studies group at the Technological and Nuclear Institute (ITN), in cooperation with several national institutions. Occupational and environmental health, human biomonitoring and elemental analysis of biological samples (mass spectrometry) are the main topics of her present work. Maria do Carmo Freitas is a PhD Senior Researcher in the Unit of Reactors and Nuclear Safety of the Instituto Tecnológico e Nuclear (URSN-ITN; Nuclear and Technological Institute, Portugal) and Coordinator of the unit Neutron Activation in Environment, Nutrition and Epidemiology group. She is primarily involved in the determination of chemical elements in environmental and nutritional samples using nuclear analytical methods. Her current research interests include low-level counting of natural radioactivity, Comptonsuppression gamma-ray spectrometry, prompt-gamma radiation, and speciation in environmental and nutritional samples. Her interest in environmental aspects also concerns their potential associations to epidemiological risks. Luís Cerqueira Alves is a PhD Researcher at Nuclear and Technological Institute. He has been working in the development and application of ion beam analytical techniques with the 2.5-MV Van de Graaff accelerator installed at ITN, in particular using a nuclear microprobe. The main applications of the implemented techniques, either for trace element analysis or micron-scale structural characterisation, have been performed in the material science, biology/biomedicine, environmental (air particulate matter and biomonitors) and mineralogy fields. He has authored or co-authored more than 60 scientific articles in international journals.
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Teresa Pinheiro is a PhD Researcher in the Biomedical Studies Group, at the Nuclear and Technological Institute. Her main research field is biomedicine. The physiology of trace elements, biological imaging, micro-analytical techniques for elemental and molecular characterisation of cells and tissues have been the pivotal aspects used to study environmental health problems and human pathologies. She is author or co-author of more than 60 scientific publications, including book chapters and articles in biomedicine and human physiology. Maria Alexandra Barreiros is a PhD Physicist Researcher at Chemical Analysis Laboratory of the Portuguese National Energy and Geology Laboratory. Her field of scientific activity is X-ray spectrometry techniques (EDXRF, PIXE and TXRF) applied to elemental trace analysis in environmental and biological samples. Her present research interests are: metrology and quality assurance of X-ray spectrometry techniques for chemical analysis, mainly TXRF; traceability establishment and uncertainty evaluation of different analytical methodologies applying TXRF technique and development of new sample preparation methods. She is author or co-author of more than 50 scientific contributions. Sílvia Garcia is a Researcher in the Institute of Welding and Quality (ISQ). She graduated in Chemical Engineering, with a specialisation in Industrial Engineering and a PhD in Biotechnology. She was included in several international and national I&D projects in the chemical and environmental sector, with several scientific publications. She has a broaden experience in atmospheric pollution and in green chemistry. Her research fields are atmospheric pollution, health assessment, air pollution modelling, GIS applications and indoor air quality and pollution.
1
Introduction
A key issue for risk assessment in occupational health is the characterisation of dose at the target organ level. In the case of inhalable pneumotoxic metals, a non-invasive method for sampling from the lung would be extremely useful. Standard methods for the evaluation of lung pathobiology (bronchoscopy, induced sputum) have a high degree of invasiveness, which limits their applicability to occupational monitoring. Since the 1980s, with the development of new technologies, the characterisation of compounds in the exhaled air to evaluate the metabolism and to diagnose illness (diabetes and toxicology) has increased considerably. However, only some years later the scientific community recognised the properties of the Exhaled Breath Condensate (EBC) – obtained by cooling exhaled air under condition of spontaneous breathing – in the evaluation of the pulmonary function. Nowadays, the study of the exhaled air is considered one of the areas with higher interest in the respiratory health research because it gives important information about biochemical activity and inflammatory processes of the respiratory system. In the last years, an effort has been made to identify, in the exhaled air, bioindicators of respiratory system pathologies and response to toxic substances (Hunt, 2002). However, the elemental characterisation of the EBC for environmental health purposes is still in its infancy with lots of questions remaining unanswered. Caglieri et al. (2006) analysed the effect of inhaled chromium on different EBC biomarkers among chrome-plating workers. Goldoni et al. (2004) have shown that the EBC is a suitable matrix not only for assessing the biomarkers of oxidative stress in
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exposed workers [malondialdehyde (MDA)] but also for quantifying the levels of some pneumotoxic substances in the lung, in particular cobalt. In this work, EBC was used to assess lung dose and effects in workers exposed to cobalt and tungsten. EBC is a non-invasive and simple procedure, and portable devices have been developed; therefore, it has the potential for application in occupational settings (Antczak and Gorski, 2002; Lemiere, 2002). The objective of this work was to investigate whether EBC can be used for a better risk assessment among human exposure to heavy metals. For that, EBC collected in two groups of volunteers, exposed to different concentration of metals during their working activities, were compared.
2
Methodology
Two working places – located in the same urban industrialised area and characterised by significant different concentrations of heavy metals in the indoor air – were selected: a foundry factory which deals with Pb processing and the offices of a research institute. The factory operates 24 hours per day and personnel work on eight hours shifts, five days a week with two intercalary resting days. Distance between the factory and the offices is 30 km.
2.1 EBC 2.1.1 EBC sampling In this study, two groups of volunteers with no pathological history were selected: (a) one group constituted by 18 workers from the referred industry and (b) one group of 23 people not exposed to Pb or other metals (particulate or fumes) in their working activity. EBC was sampled for 15 min with an electric refrigerated system modified from a cold air challenge device (EcoScreen, Jager, Germany). Sampling occurred in two stages: A: first day of the five days working period – before the shift and B: last day of the five days working period – after the shift. The sampled EBC was stored at −20ºC in eppendorf tubes.
2.1.2 EBC chemical analysis EBC samples were analysed by Total Reflection X-Ray Fluorescence (TXRF) technique (Schwenke and Knoth, 1993; Barreiros et al., 2001) and quantitative calculations were performed by the internal standard method using QXAS software package (Van Espen et al., 1986). EBC samples were doped with a standard solution of gallium (Ga). The standard concentration in the sample was of 10 µg/l. Samples were homogenised by weak agitation. Blanks were prepared to control contamination and sample preparation, following the same procedure using Ultrapure Milli-Q water doped with the same solution of Ga. Five microlitre of the EBC samples was pipetted onto quartz sample carriers for TXRF measurements. From each sample solution at least two targets were analysed. To provide reliable measurement results for trace elements in biological samples, quality assurance schemes were carried out. Detection and quantification limits determination, and identification of uncertainty sources derived from sample preparation, calibration, spectra fitting, among others, which significantly contribute to the final uncertainty, have been established according to the requirements of NP EN ISO/IEC 17025. The evaluation
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programme on QC of TXRF showed that methods were traceable for a wide range of elements and for a wide range of sample matrices and enable the mathematical modelling of measurements and statistical control of methodologies.
2.2 Air Particulate Matter (APM) 2.2.1 APM sampling APM was sampled inside the factory, characterised by high loadings of metals, inside the offices and in the outdoor environment near these two sites. In each working place, particles were collected with four low-volume Gent samplers (Maenhaut, 1992) working in parallel. In the environment only one Gent sampler was used. Gent samplers were equipped with a PM10 pre-impactor stage and with a Stacked Filter Unit (SFU). The SFU carried, in two different stages, two 47-mm Nucleopore polycarbonate filters. Air was sampled at a rate of 15–16 l/min, which allowed the collection of particles with Aerodynamic Diameter (AD) between 2.5 and 10 µm in the first stage and particles with AD < 2.5 µm in the second stage.
2.2.2 APM gravimetric and chemical analysis The filter loads were measured by gravimetry in a controlled clean room (class 10,000). Nucleopore filters were weighed on a semi-micro balance. Filter mass before and after sampling was obtained as the average of three measurements, when observed variations were less than 5%. Each filter was divided in four parts. For chemical identification, one quarter was analysed by Particle Induced X-Ray Emission (PIXE) and another quarter by Instrumental Neutron Activation Analysis (INAA). PIXE (Johansson et al., 1995) analysis was carried out at a Van de Graaff accelerator, in vacuum and two X-ray spectra were taken for each of the samples; one with a 1.2 MeV proton beam and no absorber in front of the Si(Li) detector for low-energy X-ray elements and another with a 2.4 MeV proton beam and a 250 µm Mylar® filter to detect elements with atomic number higher than 20. The beam area at the target was 20 mm2. For INAA (Cornelis et al., 1976), the filter quarter was rolled up and put into a thin foil of aluminium and irradiated for 5 hours at a thermal neutron flux of 1.03 × 1013cm–2s–1 in the Portuguese Research Reactor. After irradiation the sample was removed from the aluminium foil and transferred to a polyethylene container. For each irradiated sample, two gamma spectra were measured with a hyperpure germanium detector: one spectrum three days after the irradiation and the other one after four weeks. The k0-INAA method (De Corte, 1987) was used and 0.1% Au–Al discs were co-irradiated as comparators. Blank Nucleopore filters were treated the same way as regular samples. All measured species were very homogeneously distributed; therefore, concentrations were corrected by subtracting the filter blank contents. In order to perform the quality control of the INAA process, each group of samples was irradiated with the National Institute of Standards and Technology (NIST) Standard Reference Material 1633a – Coal Fly Ash. Previous to the sampling campaign, tests of reproducibility within the filters and between filters were taken, using parallel sampling with two similar sampling units and measuring the particle species by INAA and PIXE. Results were reproducible to within 5–15%, providing strong support for the validity of
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the analytical techniques. The details of sampling and analytical control tests are given in Almeida et al. (2003a, 2003b). The accuracy of analytical methods was evaluated with the NIST Standard Reference Material 2783 (Air Particulate on Filter Media), revealing results with an agreement of ±10% (Almeida et al., 2006a).
3
Results and discussion
3.1 APM Figure 1 presents the total APM mass concentration measured inside the plant, inside the offices and in the local environment. It is clear that the average concentration in the industry was significantly higher than in the offices and in the local outdoor. PM10 and PM2.5 average concentrations measured in the industry were 1400 µg/m3 and 420 µg/m3, respectively. These concentrations did not exceed the limit value for respirable particles (3 mg/m3) established by the Portuguese NP1796 (Occupational Exposure Limits to Chemical Agents). PM2.5 and PM2.5–10 levels measured in the offices and in the environment did not present significant differences. This fact was expected because offices have natural ventilation made by the opened windows. The PM10 maximum concentration measured inside the offices (28 µg/m3) was lower than the limit value established by the Portuguese legislation (DL 79/2006 – 4th April) related to the indoor air quality which is 150 µg/m3. PM10 average concentration measured in the environment (30 µg/m3) was lower than the EU annual PM10 standard which is 40 µg/m3. PM2.5 average mass concentration was 14 µg/m3. This value did not exceed the PM2.5 limit value of 25 µg/m3 defined by the European Union Common Position No. 13/2007 of 25 June 2007.
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[PM] (μg/m )
Figure 1
Average PM mass concentration in the industry, offices and environment (values in µg/m3)
2000 1500 1000 500 50
PM2.5 PM2.5-10 PM10
40 30 20 10 0
Industry
Offices
Environment
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The ratio PM2.5/PM10 was lower in the industry (0.29) than in offices (0.44) and environment (0.45). This fact was expected as in the factory sampling was made in the emission source and, consequently, the deposition of the coarse and heavy particles have not occurred yet. Chemical analysis was performed in the particles collected in the industry, in the offices and in the environment. Figure 2 presents the element concentration for PM2.5 and PM2.5–10 measured in these three sampling points. The elements which occurred with higher concentrations (>10,000 ng/m3) in the industry were Pb, Sb, Cl and Na. These elements are associated with the process and materials used in the production. Pb average concentration in PM10 was 500 µg/m3. This value exceeded the limit value (50 µg/m3) established by the Portuguese NP1796. Sb average PM10 concentration was 100 µg/m3. The limit value established by the NP1796 is 500 µg/m3. In the offices the major elements (concentrations higher than 100 ng/m3) were Al, Ca, Si and Fe, which have a soil origin and Cl and Na which result from sea salts (Almeida et al., 2005; Almeida et al., 2006a; Almeida et al., 2006b). Significant higher element concentrations were measured inside the industry. Figure 3 shows that Pb, Sb and As presented very high industry/offices ratios (>1000 for As and >10,000 for Sb and Pb) revealing the importance of this industrial process in the emission of these elements (mainly fugitive emissions, since stack emissions are controlled). Figure 2 shows that concentrations in offices and outdoor local concentrations were not significantly different. Figure 2
Average element mass concentration in PM sampled in the industry, offices and environment (values in ng/m3)
Concentration (ng/m3)
6
10 5 10 4 10 3 10 2 10 1 10 0 10 -1 10
Industry
Offices
Environment PM2.5
Al As Br Ca Cl Cr Cu Fe K Mn Na Ni Pb Sb Si Ti V Zn
6
10 5 10 4 10 3 10 2 10 1 10 0 10 -1 10
PM2.5-10
Al As Br Ca Cl Cr Cu Fe K Mn Na Ni Pb Sb Si Ti V Zn
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Figure 3
Ratio between the element mass concentration in PM sampled in the industry and offices
100000
PM2.5
PM2.5-10
Industry/Offices
10000
1000
100
10
1
Al As Br Ca Cl Cr Cu Fe K Mn Na Ni Pb Sb Si Ti V Zn
Figure 4 presents the PM2.5/PM10 element concentration ratio for the three sampling points. The average ratios were 0.28 for the industry, 0.47 for the offices and 0.42 for the local outdoor air. Figure 4
Ratio between PM2.5 and PM10 for industry, offices and environment
1.0
Industry
Offices
Environment
PM2.5/PM10
0.8 0.6 0.4 0.2 0.0
Al As Br Ca Cl Cr Cu Fe K Mn Na Ni Pb Sb Si Ti V Zn
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The average PM2.5/PM10 element ratios for crustal elements like Al, Ca, Fe, Mn, Si and Ti were 0.21, 0.29 and 0.29 for industry, offices and local environment, respectively, indicating that they are primarily present as coarse particles. Higher PM2.5/PM10 ratios (0.31, 0.64 and 0.58 for the industry, offices and environment, respectively) were found for combustion-related elements such as Br, Cu, K, Ni, Pb, Sb, V and Zn. In the industry, coarse fraction is dominant even for these elements, whereas in the offices and environment PM2.5 is the main fraction for these anthropogenic elements.
3.2 EBC Figure 5 presents the EBC concentration for the elements measured by TXRF. Due to the characteristics of the data, two types of tests were applied to the logarithmic transformed data to assess the differences between the established groups. The Tukey test was applied to different sampling phases (A – before the first day of the working period; B – after the fifth day of the working period) and the Dunnett test was used to compare exposed and non-exposed individuals. Groups were considered significantly different for p < 0.05 (Table 1). Figure 5
Element concentration measured in EBC sampled in exposed workers (industry A – before the first day of the working period; industry B – after the fifth day of the working period) and non-exposed volunteers (control). Values in ng/ml
Industry-A
1000
Industry-B
Control
[EBC] (ng/ml)
100
10
1
0.1
Cr
Cu
Fe
Mn
Pb
Zn
A tendency for the increase of Cr, Cu, Mn and Pb concentrations after the fifth day of the working period seems to exist; however, no statistical differences were found. Fe concentrations in EBC presented a big variability both in controls and exposed individuals, maybe due to the role of this element in the biological processes of the lung. Zn levels in the EBC not only were lower throughout the exposed period of the individuals, increasing in the first day of the working period (A), but also showed the
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highest levels in the control group. Zn is a component of metalloproteins, having many physiological roles in metabolic and regulatory pathway. Also, Zn ions can compete for metal-specific target molecules and binding sites in membranes with other metals in the form of divalent cations (Güven, 1999; Herkovits et al., 2000; Boga et al., 2008). Thus, the decrease in the EBC levels of Zn observed to the end of the working shift suggests an imprisonment of this element in the respiratory tract that may possibly result from its biological affinity for target molecules in the respiratory system. Table 1
P-values of statistical tests used to apportion the difference between the sampling phases (A – before the first day of the working period; B – after the fifth day of the working period) and industrial workers and controls
Cr
A−B
A−CTR
B−CTR
0.13
0.72
0.47
Cu
0.93
0.10
0.02
Fe
0.99
0.75
0.80
Mn
0.80
0.79
0.21
Pb
0.73
0.00
0.00
Zn
0.34
0.87
0.72
Significant differences were clear between Pb measured in EBC collected in workers and non-exposed volunteers. This fact suggests that EBC can be used as a method for assessing Pb exposure. Cu showed a similar behaviour, presenting significant lower concentrations in the EBC sampled in the control group. Thus, Cu could also be regarded as a metal viable for assessment through EBC methodology in occupational exposure. For Cr and Mn, an increase of concentration in exposed individuals was observed. However, no statistical differences were found between exposed and non-exposed volunteers.
4
Conclusions
This preliminary study showed that significant higher concentrations were found for the Pb and Cu measured in EBC sampled in exposed volunteers. This fact may suggest that EBC method could be regarded as a viable tool for occupational exposure assessment. More work should be developed in individuals exposed to different levels of concentrations. Moreover, deep analysis of the particles granulometry should be performed in order to understand the level of penetration of the particles and their effect in the EBC.
Acknowledgements We gratefully acknowledge Fundação para a Ciência e Tecnologia (FCT) for funding S.M. Almeida by Ciencia 2007 programme and the project PTDC/AMB/65828/2006 – Exhaled breath condensate: a tool for noninvasive evaluation of pollutant exposition?
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References Almeida, S.M., Reis, M.A., Freitas, M.C. and Pio, C.A. (2003a) ‘Quality assurance in elemental analysis of airborne particles’, Nuclear Instruments and Methods in Physics Research B, Vol. 207, pp.434–446. Almeida, S.M., Freitas, M.C., Reis, M.A. and Pio, C.A. (2003b) ‘Quality assessment on airborne particulate matter of k0-INAA’, Journal of Radioanalytical and Nuclear Chemistry, Vol. 257, pp.609–613. Almeida, S.M., Pio, C.A., Freitas, M.C., Reis, M.A. and Trancoso, M.A. (2005) ‘Source apportionment of fine and coarse particulate matter in a sub-urban area at the Western European Coast’, Atmospheric Environment, Vol. 39, pp.3127–3138. Almeida, S.M., Freitas, M.C., Reis, M.A., Pio, C.A. and Trancoso, M.A. (2006a) ‘Combined application of multielement analysis – k0-INAA and PIXE – and classical techniques for source apportionment in aerosol studies’, Nuclear Instruments and Methods in Physics Research A, Vol. 564, pp.752–760. Almeida, S.M., Pio, C.A., Freitas, M.C., Reis, M.A. and Trancoso, M.A. (2006b) ‘Approaching PM2.5 and PM2.5−10 source apportionment by mass balance analysis, principal component analysis and particle size distribution’, Science of the Total Environment, Vol. 368, pp.663–674. Antczak, A. and Gorski, P. (2002) ‘Markers of pulmonary diseases in exhaled breath condensate’, International Journal of Occupational Medicine and Environmental Health, Vol. 15, pp.317–323. Barreiros, M.A., Pinheiro, T., Araújo, M.F., Costa, M.M., Palha, M. and Silva, R.C. (2001) ‘Quality assurance of X-ray spectrometry for chemical analysis’, Spectrochimica Acta, Part B, Vol. 56, pp.2095–2106. Boga, A., Erdogan S. and Sertdemir Y. (2008) ‘Effects of specific dosages of magnesium and zinc on the teratogenicity of cadmium, nickel, and cobalt in xenopus embryos, as assessed by the fetax test’, Dose-Response, Vol. 6, pp.16–29. Caglieri, A., Goldoni, M., Acampa, O., Andreoli, R., Vettori, M.V., Corradi, M., Apostoli, P. and Mutti, A. (2006) ‘The effect of inhaled chromium on different exhaled breath condensate biomarkers among chrome-plating workers’, Environmental Health Perspectives, Vol. 114, pp.542–546. Cornelis, R., Hoste, J., Speecke, A., Vandecasteele, C., Versieck, J. and Gijbels, R. (1976) ‘Activation analysis – Part 2’, in West, T.S (Ed.): Physical Chemistry, Series Two, Vol. 12, Analytical Chemistry – Part I, Butterworth, London, p.137. De Corte, F. (1987) The k0-Standardization Method – A Move to the Optimization of Neutron Activation Analysis, Agregé Thesis, Gent University, Belgium. Goldoni, M., Catalani, S., De Palma, G., Manini, P., Acampa, O., Corradi, M., Bergonzi, R., Apostoli, P. and Mutti, A. (2004) ‘Exhaled breath condensate as a suitable matrix to assess lung dose and effects in workers exposed to cobalt and tungsten’, Environmental Health Perspectives, Vol. 112, pp.1293–1298. Güven, K. (1999) Biochemical and Molecular Toxicology, University of Dicle press, Diyarbakir; Turkey, pp.1–188. Herkovits, J., Perez-Coll, C.S. and Herkovits, F.D. (2000) ‘Evaluation of nickel–zinc interactions by means of bioassays with amphibian embryos’, Ecotoxicology and Environmental Safety, Vol. 45, pp.266–273. Hunt, J. (2002) ‘Exhaled breath condensate: an evolving tool for non-invasive evaluation of lung disease’, Journal of Allergy and Clinical Immunology, Vol. 10, No. 1, pp.28–34. Johansson, S.A.E., Campbell, J.L. and Malmqvist, K.G. (1995) Particle-Induced X-ray Emission Spectrometry (PIXE), John Wiley & Sons, New York.
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Lemiere, C. (2002) ‘Non-invasive assessment of airway inflammation in occupational lung diseases’, Current Opinion in Allergy and Clinical Immunology, Vol. 2, pp.109–114. Maenhaut, W. (1992) Co-ordinated Research Program: CRP E4.10.08, IAEA, Belgium. Schwenke, H. and Knoth, J. (1993) ‘Total reflection XRF’, in Van Griecken, R.E. and Markowicz, A.A. (Eds): Handbook of X-Ray Spectrometry Methods and Techniques, Marcel Dekker Inc., New York, pp.453–489. Van Espen, P., Janssens, K. and Swenters, I. (1986) AXIL X-ray Analysis Software-Users Manual, Canberra Packard, Benelux, p.72.
