Air Quality in Metal Industries

Chapter 27

Air Quality in Metal Industries: Exhaled Breath Condensate, a Tool for Noninvasive Evaluation of Air Pollution Exposure T. Pinheiro,1, 2, * S.M. Almeida,1, 3 P.M. Fe´lix,1, 4 C. Franco,1 S.M. Garcia,5 C. Lopes6 and A. Bugalho de Almeida6 1

Instituto Superior Te´cnico, Universidade de Lisboa, Lisbon, Portugal; 2Instituto de Bioengenharia e Biocieˆncias; 3Centro de Cieˆncias e Tecnologias Nucleares; 4MARE - Marine and Environmental Sciences Centre, Universidade de Lisboa, Lisbon, Portugal; 5Instituto de Soldadura e Qualidade, Porto Salvo, Portugal; 6Hospital de Santa Maria, Centro Hospitalar de Lisboa Norte, Lisbon, Portugal *Corresponding author: E-mail: [email protected]

Chapter Outline 1. Introduction 1.1 Environmental Hazards and Health 1.2 Occupational Health and Air Quality 1.3 Human Biomonitoring for Health Assessment 1.4 Assessing Occupational Exposure to Metals 1.5 Biomarker of Exposure for the Respiratory System 1.6 Exhaled Breath Condensate, a Tool for Noninvasive Evaluation of Air Pollution 1.7 Collection of EBC 1.8 Validation of EBC Method

733 733 734 737 737 738

739 740 742

2. A Case Study: Using EBC to Assess Exposure in Workers of the Lead Industry 2.1 EBC a Tool for Noninvasive Evaluation of Pb Exposure 2.2 Study Design 2.3 Characterisation of Exposed and Nonexposed Workers 2.4 Work Environment Monitoring: Air Particulate Matter 2.4.1 PM Gravimetric and Chemical Analysis 2.4.2 PM Levels and Chemical Composition

Comprehensive Analytical Chemistry, Vol. 73. http://dx.doi.org/10.1016/bs.coac.2016.03.014 Copyright © 2016 Elsevier B.V. All rights reserved.

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749 749

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732 SECTION j III Real Scenarios 2.5 Biomonitoring With EBC 2.5.1 Preanalytical and Analytical Considerations in EBC Analysis 2.5.2 Optimisation of the EBC Sample Preparation and Validation of the Analytical Procedure 2.5.3 Pb Concentrations in EBC

752

752

753

2.5.4 Pb Concentrations in EBC Versus Pb Levels in the Work Environment 2.5.5 Pb Concentrations in EBC and in Blood in Exposed Workers 3. Advantages and Limitations of the Method 4. Future Perspectives Acknowledgements References

756

758 759 759 760 760

755

Acronyms, Symbols and Special Terms

ACGIH American Conference of Governmental Industrial Hygienists Al Chemical symbol of aluminium ALAD d-Aminolevulinic acid dehydratase As Arsenic BLV Biological limit values Cd Chemical symbol of cadmium Co Chemical symbol of cobalt CO Carbon monoxide CO2 Carbon dioxide Cr Chemical symbol of chromium Cu Chemical symbol of copper CRM Certified reference material EBC Exhaled breath condensate Fe Chemical symbol of iron FEV1 Volume exhaled during the first second of a forced expiratory manoeuvre started from the level of total lung capacity; lung function parameter measured in a spirometry test FVC Forced vital capacity determined from a maximally forced expiratory effort; lung function parameter measured in a spirometry test Hg Chemical symbol of mercury HNO3 Nitric acid ICP-MS Inductive coupled mass spectrometry

IL Interleukin; group of cytokines consisting of proteins secreted by cells and involved in cell communication INAA Instrumental neutron activation analysis Inhalable particle fraction ‘thoracic convention’ as defined in ISO 7708:1995: particles with a 50% efficiency cut-off at 10-mm aerodynamic diameter ISO International Organization for Standardization K Potassium Mn Manganese NIOSH National Institute of Occupational Safety and Health NIST National Institute of Standards and Technology Ni Nickel NO Nitric oxide NO2 Nitrogen dioxide O2 Molecular oxygen O3 Ozone OEL Occupational exposure limits OSHA Occupational Safety and Health Administration (United States) OSHA Occupational Safety and Health at Work (European Union) Pb Lead PIXE Particle-induced X-ray emission

Air Quality in Metal Industries Chapter j 27 PM Particulate matter; refers to an airsuspended mixture of solid and liquid particles PM2.5e10 Fraction of particulate matter with aerodynamic diameters between 2.5 and 10-mm cut points; this fraction is usually referred to as thoracic particle fraction PM2.5 Particulate matter fraction below 2.5-mm cut point; also referred to as fine fraction or respirable fraction; respirable particle fraction ‘highrisk respirable convention’ as defined in ISO 7708:1995, 7.1;

733

particles with a 50% efficiency cutoff at 2.5 mm-aerodynamic diameter Sb Antimony Se Selenium SFU Stacked filter unit SO2 Sulphur dioxide Sr Strontium TXRF Total reflection X-ray fluorescence V Vanadium W Tungsten Z Zinc ZPP complex Zinceprotoporphyrin complex

1. INTRODUCTION 1.1 Environmental Hazards and Health We are all exposed to thousands of natural and man-made chemicals every day, which are present in the air, ingested food and water and a number of consumer products [1e5]. Air pollution, outdoors and indoors, remains one of the major environmental problems in developed and developing regions of the world, affecting health and well-being of citizens. Key air pollutants in this respect are particulate matter (PM), ground-level ozone (O3), sulphur dioxide (SO2), nitrogen dioxide (NO2) and polycyclic hydrocarbons [6,7]. The health effects of exposure to air pollution probably exist since fire was discovered. Archaeological research on how ancient populations lived and died since 3000 BCE (before common era) suggests that ancient human beings perished to cardiovascular and respiratory diseases [8]. More recent history of air pollution and its effects on health dates back to 19th century with the introduction of bituminous coal as a form of energy fuelling the Industrial Revolution in addition to millions of domestic fires. Specific atmospheric conditions imprisoned coal burning products, such as CO2, SO2, smoke particles, organic acid compounds among others causing a dense fog that stayed for days and even weeks, causing devastating health effects in humans and animals. There are numerous reports on such episodes in the beginning of the 20th century. One of the worst man-made air pollution disaster occurred in London in 1952 blocking road, air and rail transport. In the following weeks, hospital statistics were startling: at least 4000 people died and more than 10,000 were heavily affected. A series of laws were brought in to avoid a repeat of the situation. This included the Clean Air Acts of 1956 and 1968, which banned emissions of

734 SECTION j III Real Scenarios

black smoke and decreed conversion to smokeless fuels. Several nations implemented environmental laws to regulate air pollution by controlling emissions from stationary and mobile sources. Over the past decade, the quality of the environment has improved considerably, but major challenges remain. Air pollution has declined, but not enough to ensure good air quality in all urban areas. Local and transboundary sources of pollution still raise concerns about long-term damage to human health [2,4]. Despite international efforts for abating the pollution of the air, still episodes of severe air pollution occur, which are leading some communities to take urgent action to lower air pollution and to adopt new regulatory measures. Nowadays environmental health has become a sensitive issue in society [5]. Since mid-1990s several research studies demonstrated that exposure to pollutants, especially PM, in many urban areas were linked to respiratory illness in children, premature cardiopulmonary death and disease in adults [9e11]. Evidence is also growing for a range of effects of prenatal exposure [12], and exposure during early childhood, on health in adult life. There is also increasing concern over the health impacts of prolonged low-dose exposures, long-term effects of endocrine-disrupting chemicals [13] and the emergence of new diseases in a changing environment [12]. A considerable number of regulatory measures have been taken in many countries to reduce potential risks for chemicals, which requires health risk assessment for workers and the general population. The potential health and economic benefits of mitigation measures are considerable and short- to medium-term impact in the economy recognised. Nevertheless, emission reductions do not always produce a corresponding drop in atmospheric concentrations, especially for PM, which is the most relevant pollutant so far linked to health problems.

1.2 Occupational Health and Air Quality In many occupational settings, especially in metal-processing industry and smelters, PM and metal-associated PM are relevant airborne hazards [13e15]. PM is an air-suspended mixture of solid and liquid particles that vary in number, size, shape, surface area, chemical composition, solubility and origin. In the workplace context, PM can be formed either during mineral ores processing by abrasion or smelting and burning processes by condensation [15e23]. Metal dust and fumes produced during metal processing, smelting or welding contain a wide range of biologically active substances [19,24,25]. Consequently, different size fractions of PM are formed, which can penetrate the thoracic region of the lung or be ingested [24]. PM is usually categorised in aerodynamic size ranges. The size distributions of these fractions were set in terms of establishing guidelines for acceptable levels of ambient and workplace air pollution by national and worldwide federations of standards bodies such as the American Conference

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of Governmental Industrial Hygienists (ACGIH), National Institute of Occupational Safety and Health (NIOSH), European and United States Agencies for Occupational Safety and Health at Work (OSHA) and International Organization for Standardization (ISO)/European Standardization Committee. The rationale behind is based on health and toxicological effects associated to exposure by inhalation resulting from occupational data and epidemiological studies [11,26,27]. Public health policy established fractions of airborne particles based on the principle of impaction. The inhalable or thoracic coarse particle (PM2.5e10) are defined as the aerodynamic diameters between 2.5 mm and 10 mm cut points, and fine particles (PM2.5) below 2.5 mm cut point, referred to as respiratory fraction which can be breathed more deeply into the lungs. Fine particles may play the largest role in affecting human health, as they remain suspended for longer periods of time [9] and associate with metals, which proved to be more toxic [28e30]. These studies generally observed that differences in the content and mixtures of metals had an important role in the biological effects of PM exposure. They also demonstrated that the biological effects observed under controlled PM exposures were consistent with specific epidemiological findings, paving the way to consolidate our understanding regarding biological implications and mechanisms. Legislation and international conventions regulate emissions, ambient and workplace air concentrations of many metals, such as, cadmium (Cd), chromium (Cr), nickel (Ni), copper (Cu), arsenic (As), antimony (Sb), manganese (Mn), mercury (Hg), vanadium (V) and lead (Pb). Some of these are known to be either teratogenic [31], carcinogenic [20], embryotoxic or mutagenic [31,32]. Evidences are also accumulating for increased risk among workers exposed to either metal dust or fumes of infertility and pregnancy adverse outcomes such as, premature or low-weight birth [12,33,34]. Country-based retrospective studies on occupational exposures to metals in airborne particulate and fumes had reported on quality of air issues aiming at identification of sources, methodologies and potential for workers’ exposure [35e39]. Regulatory measures to control exposure in industrial settings established occupational exposure limits (OELs) that means the limit of the time-weighted average of the concentration of a chemical agent in the air within the breathing zone of a worker in relation to a specified reference [40]. The air quality evaluation is then ensured by measuring the levels of relevant metals or substances in the workplace with stationary or personal samplers positioned at breath height. So far, the assessment of dangerous substances by worker’s biomonitoring is restricted to a few substances, such as Pb [41]. In the literature published in the last decade, as summarised in Table 1, the air quality in a wide range of industry categories where metal exposures are of concern have been addressed in combination with exposed worker’s biomonitoring, bringing the results of biomonitoring forward in terms of possible guidance values for the selected substances.

Particle Size/ Physical State

Sampling Method

PM2.5e10 PM2.5

Industry Category

Exposure/Measures

WBMd

Stationary

Coating [35] Foundry [19,35] Hard metal processing [18,24,35] Smelting [35,42]

Total mass Chemical elements (eg, Al, Be, Cr, Cr(VI), Mn, Fe, Ni, Cu, Zn, Rh, Pd, Cd, Sb, Ir, Pt, Pb)

[18, 19, 24]

Inhalablea

Personal sampler

Hard metal processing [24,44,45] Smelting [38] Welding [20,43]

Total mass Chemical elements (eg, B, Cr(VI), Co, Rh, Pd, W, Ir, Pt)

[20,24,43e45]

Respirableb

Personal sampler

Coating [22,47,49] Foundry [19,35] Hard metal processing [35,48,50,51] Smelting [35,51] Welding [21,35,46]

Total mass Chemical elements (eg, Si, Cr, Cr (VI), Mn, Fe, Co, Ni, Se, W, Pb) [19]

[19,21,35,46e51]

Ultrafine (10e100 nm)

SMPS

Coating [17] Foundry [16,17] Hard metal processing [17] Refining [17] Smelting [17] Welding [17]

Number of particles and surface area

Gasc

Stationary/ Personal

Foundry [19] Refining [25,52] Welding [21]

PAHs Aldehydes CO2, CO, O3, NO2 Hg

SMPS, Stationary scanning mobility particle sizer; PAHs, Polycyclic aromatic hydrocarbons. a Inhalable e particles with a 50% efficiency cut-off at 10-mm aerodynamic diameter. b Respirable e particles with a 50% efficiency cut-off at 2.5-mm aerodynamic diameter. c Solid sorbent systems or automatic analysers. d Matrices:, blood, exhaled breath condensate, hair, semen and urine.

[19,21,52]

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TABLE 1 General Characteristics of Air Quality Assessment in the Industry Where Metal Exposures Are of Concern, Extracted From the Published Literature in the Last 10 Years. The Studies Involving Worker’s Biomonitoring (WBM) Are Also Indicated

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Therefore exposure assessment is of paramount importance in Environmental and Health and Safety research area. Relevant research issues are: l

l

l

the characterisation of exposures among workers, other populations and environment; the measurement of exposure levels by monitoring indicators of biological responses; the development of models to predict those exposures.

Both at collective and at individual level, information on exposure and burden of disease is still required. Also more research is needed on new ways of identifying substances, strengthening epidemiological research and improving risk assessment methodologies.

1.3 Human Biomonitoring for Health Assessment Human biomonitoring is a growing discipline used for exposure and risk assessment in environmental and occupational health [53]. Human Biomonitoring is the sampling and measurement of specific chemicals in biological tissues. The presence of chemicals in the body is seized by the body burden concept. Such body burdens can be assessed through human biomonitoring which integrates information on exposure to potentially toxic chemical elements and substances as well as bioavailability, toxicokinetics and metabolism [54]. Thus, biomonitoring has advantages on environment monitoring because it covers the internal dose of a compound, the toxic effect and the individual susceptibility. Most of our knowledge concerning the health effects of toxic metals largely stems from studies conducted on populations with relatively high exposure in industry or in heavily polluted environments. Workers who are occupationally exposed to chemicals, such as metals, are exposed to levels that are much higher than the levels to which the general population is exposed. Studies on these workers and the levels of chemicals found in their bodies enabled to determine increased health risks. For example, substances such as Pb and Hg, measured in the blood and urine of factory workers, helped monitoring their exposure and assessing the body burden and health risks of exposure. To reduce potential risks, a considerable number of regulatory measures were implemented in many countries in particular for chemicals, which requires health risk assessment for workers and the general population. A better understanding of determinants of health is also required to improve effective health promotion and disease preventive policies and to reduce public health costs.

1.4 Assessing Occupational Exposure to Metals Human exposure in the workplace is dangerous as pollutant concentrations are higher compared to outdoors and as the workers are closer and stay longer near

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the emission sources. Airborne particles or their chemical compounds may interact with the cells and tissues of exposed organs causing a range of various deteriorations. For risk assessment and occupational toxicology, the monitoring of toxicants, such as metals at the target organ level is extremely important. The classical approaches to assess exposure are indirect. They rely either on measures of air quality in the workplace by measuring chemicals (organics and inorganics) in PM or on gases. The blood levels or urinary excretion of certain metals such as Pb, Hg, Co and Cr, among others have been proposed as biomarkers of exposure because of their correlation with airborne concentrations [55]. Biological limit values (BLV), which are reference values for evaluating potential health risk for occupational health, have been introduced in national legislations. So far, the sole specific mandatory policy on biomonitoring is on Pb [41]. Measures of biological indicators in conservative media, such as blood and urine can only give information concerning systemic levels. In addition, the concentrations in blood of a specific substance of concern reflect only exposure to soluble fraction of that substance, which is influenced by absorption from all routes, including dermal exposure. Similar information can be obtained from urine, where certain substances are reduced to specific chemical states or degradation compounds.

1.5 Biomarker of Exposure for the Respiratory System In occupational settings, exposure to metal dusts and fumes produced during metal processing, smelting or welding mainly occurs as a result of inhalation. The metals associated to airborne PM usually have chemical forms that are poorly absorbed and may persist in the lung, where they can cause deleterious effects by interacting with cells. Thus, the lung is the key organ in the front line of defence against insidious substances. However, at present, there is no direct biological indicator to assess exposure by inhalation, which is by far the major route for airborne pollutants’ entrance into the body. Standard methods for the evaluation of lung pathology (tissue biopsy, bronchoscopy, bronchoalveolar lavage and induced sputum) are highly invasive, and difficulties in obtaining adequate samples are considerable, which restricts their applicability to occupational monitoring. A noninvasive method for sampling from the lung would be extremely useful. One possibility of directly assessing hazardous substances, which interact with respiratory tissues, is the prospect held out by ‘exhaled breath’ as a biomarker of exposure for the respiratory system.

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1.6 Exhaled Breath Condensate, a Tool for Noninvasive Evaluation of Air Pollution One of the first uses of exhaled breath dates back to 1930. Exhaled breath proved to be useful to estimate an individual’s alcohol level content of blood. The noninvasive test offering immediate results provided law enforcement. The technological advances during the last 40 years paved the way to the development of methods that enable assessing metabolism and diagnose illness. Paradigmatic examples are the urea and NO tests. The urea breath test has been in clinical practice for a considerable period of time as one of the most important noninvasive methods for detecting Helicobacter pylori infection [56], whereas abnormalities in exhaled nitric oxide (NO) have been documented in several lung diseases, particularly asthma [57]. However, only some years later the scientific community recognised the ability of exhaled breath as a reporter of the respiratory system. We breathe continuously in and out, and a fraction of the inhaled air remains in the conducting airways. Eventually, after a certain number of inhalation/exhalation cycles this air volume is fully recycled. This characteristic is not only beneficial in many aspects of the ventilation (humidification, temperature equilibrium and gas exchange) but also enables a longer period of time for inhaled pollutants to interact with the epithelial lining fluid inside the lungs. If exhaled breath under conditions of spontaneous breathing passes through a cold trap, which is kept at a temperature below the saturation temperature of the vapour, an exhaled breath condensate (EBC) can be obtained. The EBC is then an aqueous suspension containing condensed volatile substances from breath and semivolatile and nonvolatile substances, which are exhaled as small droplets that leave the airway lining fluid as a result of convective processes of the turbulent air flow [58,59] (Fig. 1). Therefore, gases such as O2, CO2, CO, NO and other molecules existing in both sol- and gel-phase in the respiratory lining fluid, such as ethane, pentane, hydrogen peroxide, ammonia, small peptides, proteins, salts and ions, are present in EBC although in significantly lower concentrations. Whether the composition of EBC reflects lower (smaller) [60] or central airways [59] is still a matter of debate. Significant efforts have been made to qualitatively and quantitatively characterise EBC. The procedure was initially demonstrated to be useful in assessing pulmonary pathophysiological conditions as EBC and bronchoscopy features correlated [61,62]. Mass spectrometry techniques, immunoassay and proteomic approaches are providing information on a large number of small molecules and proteins [63,64], although the significance and the clinical application of these discoveries often lack clarification [65e67].

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FIGURE 1 Formation of exhaled breath: Droplets of respiratory lining fluid (DLF) are released from the surfaces of the airways during tidal breathing. Pollutants may provoke an irritating effect in the airways, tissues and cells, which eventually trigger cell activation and inflammatory stimulus with the release of many peptides and by-products of metabolism (eg, leukotrienes, prostaglandins, interleukins and reactive oxygen species).

Ambient air contains particles and molecules, which may influence EBC composition. Atmospheric compounds can directly contribute to EBC composition and analyte levels. These compounds may also react with molecules trapped in EBC and interact with epithelial lining fluid and cells of the respiratory airways leading to inflammatory and biochemical changes that are subsequently reflected by changes in EBC composition. One of the consequences of these processes is cell permeability increase. Hence, by-products of the cellular activity may pass from interstitial and from intracellular spaces to the extracellular space, and they can potentially be detected in the exhaled air (Fig. 1). Although the properties of EBC point to a high potential for multiple substance determination, the use of EBC as a biomarker of exposure to pollutants in environmental health and occupational studies has been considerably modest. Very few researchers performed an ionic and/or elemental characterisation of the EBC. This characterisation could give important information for environmental and occupational health studies. Mutti et al. [68] reported on 33 metals measured in EBC, which included, Pb, Cd, Ni, Al, Cu, Se, Fe and Mn. Studies conducted on smelters and plating industry showed that EBC metal concentrations, such as Co, Cr, Cu, Ni, Mn, Pb, Sb and W [43,48,69] reflected workplace levels. In a few case studies, bioindicators of effect were also tested, such as oxidative stress markers [25,47]. These studies have shown that the EBC is a suitable matrix not only for assessing the biomarkers of response in exposed workers but also for quantifying the levels of some pneumotoxic substances in the lung, in particular, metals.

1.7 Collection of EBC The most common sampling procedure for EBC collection involves normal tidal breathing through a collection device (Fig. 2). EBC is usually collected

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FIGURE 2 Schematic of EBC collection: The subject breathes normally and inhales room air through a two-way nonrebreathing valve (a). The droplets of epithelial lining fluid diluted by water vapour of exhaled air enter a condenser region (b) cooled at temperatures, usually < 15 C. The EBC is then collected in a container (c). The two-way valve prevents the mixture of inhaled and exhaled air and also serve as a saliva trap.

with portable equipment commercially available or custom-made condensers. Therefore, EBC collection can be performed in any setting, such as workplace, home and so on. Usually, 10 min in adults and up to 15e20 min in children, yields about 2e3 mL of condensate, which is a sufficient sample for multiple biomarkers analysis [58,69e71]. EBC collection is entirely noninvasive. The procedure is easy and does not cause discomfort or risk for the individual, such as inflammatory reaction. Minimal technical skills are required, allowing for repeated sampling from both children and adults alike. Therefore, EBC collection is especially suited for the sequential and longitudinal sampling of individuals at any age. Collectors for EBC sampling are available from various manufacturers, all based on condensation of exhaled breath at low temperatures. ECoScreen (Jaeger, Germany) and RTube (Respiratory Research Inc, Charlottesville, USA) are the most used apparatus for EBC collection. Both systems are similar as they have unidirectional valves and saliva trap, but the cooling conditions are different [67,72]. The RTube device is a disposable system cooled by a cooling sleeve. The temperature inside the cooling sleeve increases from 20 C (initial condition) to 20 C in 10 min [73], which is the typical EBC collection period of time. The ECoScreen is equipped with an electrical condenser. The temperature profile, measured with a general-purpose thermocouple, for sequential operating cycles of 10 min, showed that the temperature inside the cooling sleeve during sampling was maintained at 32  2 C, under controlled ambient conditions (19 C and 48% relative humidity). Various inert materials can be used for the condensing surface, for example, polyethylene, glass, aluminum and Teflon, depending on the analytical requirements.

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Relative humidity of ambient air, condensation temperature and ventilation rate may influence the EBC volume, pH, droplet size and condensation. As long as the respiratory rate is kept constant, the EBC volume linearly increases with the time of collection, as it depends on the respiratory rate, ventilation volumes and lung function parameters [74]. Increases in condensing temperature, ambient temperature or relative humidity cause a decrease in EBC pH and an increase of droplet size in the exhaled air [75,76]. Keeping temperature at about 25 C during EBC collection was shown to improve protein and lipid mediator recovery [74]. The collection conditions were also studied in the context of metal analysis. Temperature and relative humidity during collection were shown to influence Cr, Mn, Ni and Cd concentrations in EBC [77]. The value of EBC in occupational exposure assessments and in clinical applications is still not consensual. Guidelines for EBC collection and measurement of response biomarkers were proposed and adopted by American Thoracic Society (ATS)/European Respiratory Society (ERS) Task Force on recommendations for EBC [71] However, divergence of published results persist, which may be due as previously referred by the Task Force to the lack of standardisation in the collection and analysis of EBC.

1.8 Validation of EBC Method One characteristic of EBC matrix is its heterogeneity, as the condensed droplets of lining fluid may contain miscellaneous particles in a variety of dimensions [78] (Fig. 3). These features may become relevant in occupational assessments, where workers are exposed to metal dust [18,79]. Therefore, the collection conditions and representativeness of the sample will inevitably influence analytical results [77]. Guideline directives for the analysis of metals in EBC have not yet been developed. Depending on the analytical technique applied to metal concentration measurements results produced can diverge. Fe´lix et al. [69] showed that direct analysis of EBC samples taken from workers exposed to fumes and metal dust in a smelter industry, by Total Reflection X-ray Fluorescence (TXRF) and Inductive Coupled Plasma Mass Spectrometry (ICP-MS), did not provide coherent results being unrepresentative concerning the total EBC sample. These findings call the attention for homogenisation requirements of EBC matrix, as aliquots taken for analysis may arbitrarily contain PM in suspension [78] as depicted in Fig. 3. A systematic study of the EBC matrix and of the analytical procedures should precede any occupational application. Validation of the methodology can provide confidence of the results and indicate the validity of the measurements. Therefore, not only procedures for EBC collection should be established but also analytical figures of merit, such as trueness and reproducibility should be studied for the chemical of interest.

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FIGURE 3 Images of the dry deposit of EBC collected from workers of a smelter industry. The distribution maps of Cl, Ti, Fe and Pb showing particles likely inhaled PM. Cl and Pb correspond to the same scanned area and show that three large Pb-containing particles also associate with Cl. Ti and Fe maps show individual particles. The elemental maps were obtained by particle-induced X-ray (PIXE) technique in a nuclear microscopy setup. The EBC deposit was scanned with a 2-MeV proton beam of 3-mm dimension. Content levels from minimum (black/blue) to maximum (grey/red). For further details refer to T. Pinheiro, M.A. Barreiros, L.C. Alves, P.M. Fe´lix, C. Franco, J. Sousa, S.M. Almeida, Nucl. Instrum. Methods B 269 (2011) 2404e2408.

Unresolved issues identified by the American Thoracic Society/European Respiratory Society Task Force on EBC [71] are the variation in the method of collection, as discussed previously and in the approach for quality control. Quality Control methods, currently used in analytical chemistry [80,81] can be used to determine the overall uncertainty of EBC analysis. These tasks can be carried out using appropriate certified reference materials (CRMs) and a pool of EBC samples [77,82]. Trueness can be estimated in terms of overall recovery obtained from spiking aliquots of the EBC pool at different concentrations for elements of interest. Precision can be estimated in terms of repeatability using the native

744 SECTION j III Real Scenarios

EBC sample pool and trueness in terms of recovery obtained from spiking aliquots of the EBC pool with the elements of interest at different concentrations (see Section 2.5.2.). The development of such practices will assure the implementation of appropriate methodology to discriminate exposure levels (eg, sample preparation and analytical technique), individual variability and the differentiation of groups of individuals exposed to different levels of contaminants. The methods to determine some analytes in EBC, such as cytokines, which are indicators of biological response to pollutants and other aggressors, require (1) quick storage at 80 C as some constituents may be unstable at room temperature and (2) preconcentration to increase analytical sensitivity. Lyophilisation is the recommended procedure by reducing sample loss [83,84], although solid phase and solvent extraction methods were also described [85]. Major analytical difficulties derive from insufficient sample volume due to the low overall protein abundance. Other factors influencing analyte recovery are its stability during EBC collection, apparatus surface coating and the adhesion properties of the analyte to these surfaces [67]. Preliminary results using flow cytometry detection of multiple cytokines in EBC 30-fold concentrated yielded relative consistent results for only two cytokines, IL-2 and IL-4. Results obtained for healthy individuals nonexposed and occupationally exposed to metals are listed in Table 2. The results highlight the difficulty in detecting these mediators in EBC even for samples highly preconcentrated. Similar analytical inconsistencies have been described in literature [63,64,67,68,83e85], although published data are difficult to compare as analytical procedures, recovery indexes or concentration values are rarely indicated [67,86]. The large body of literature currently available unequivocally reflects the potential of EBC as a reporter of the respiratory system in health and disease. Being a noninvasive and undemanding method of sampling from the lungs, EBC becomes an attractive bioindicator of exposure especially fitted to occupational assessments. The study of the exhaled air is currently 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. These characteristics position EBC in the forefront of collection methods for biomarker discovery.

2. A CASE STUDY: USING EBC TO ASSESS EXPOSURE IN WORKERS OF THE LEAD INDUSTRY The Pb-processing industry was taken as paradigmatic application to demonstrate the value of EBC in risk assessment among exposed workers. The exploitation of Pb is an important activity wherever they may be located in the world. Industrial activities involving Pb consist of mining, smelting, refining and recycling [37,39,87].

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TABLE 2 Flow Cytometry Analysis of 11 Interleukins in EBC of 16 Healthy Individuals: Eight Controls (Occupationally Nonexposed) and Eight Workers (Smelter). Concentration Values Are Means and Standard Deviations. The Number of Valid Results for Each Analyte and Each Group is Indicated. The EBC Samples Were Lyophilised and d Resolved in the Assay Buffer of the Commercial Kit (FlowCytomix Human Th1/Th2, Bender Medsystems, Austria), Achieving a 30-Fold Preconcentration (See Text for Details). The FACSCalibur, BD Cytometer, Was Used Analyte

Detection Limit (pg/mL)

Valid N

Controls (pg/mL)

Valid N

Workers (pg/mL)

45.3; 14.3a

4

15.8  5.5

IL12p70

1.5

2

IFN-g

1.6

0

IL2

16.4

3

IL10

1.9

0

0

IL8

0.5

0

0

IL6

1.2

0

0

IL4

20.8

4

IL5

1.6

0

0

IL-1b

4.2

0

0

TNF-a

3.2

0

TNF-b

2.4

2

0 82.2  7.4

62.8  11.4

4

5

1 a

2681; 775

82.6  6.8

60.7  4.3

10.8

0

a

Individual values of concentration.

Nowadays, global human activities are dependent on Pb processing, batteries being one of the most known and common item containing Pb. Since mid-1970s, global Pb production duplicated [88] primarily due to the increase in demand for Pb-based batteries, which accounts for more than 90% of the entire Pb market. The high density of Pb is also appropriate for several product categories including weighting applications, and shielding against sound, vibration and radiation. The variety of Pb uses naturally reflects a diversity of working conditions where Pb represents an occupational risk. Human exposure to Pb sources whether through inhalation or ingestion may cause detrimental health effects. Exposure hazards attributed to Pb has been substantially documented and recognised enforcing specific legislation worldwide. Protection of workers in the industry does, and always will, require close attention.

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2.1 EBC a Tool for Noninvasive Evaluation of Pb Exposure Occupational and environmental exposures to Pb continue to be a public health concern due to its adverse effects on many biological functions [89]. There is any known physiological role for Pb. Therefore any quantity that is taken up by the organism can trigger toxic effects. The health and environmental impacts of Pb have been recognised for many centuries [90]. In fact, historical practices of the uses of Pb have left their legacy. Pb poisoning was fully recognised in late Antiquity, by Greeks and Romans. In 16th century Paracelsus described the health effects of Pb as the ‘miners disease’. During the following centuries, neuronal and haematological effects of Pb, among other outcomes were methodically and incessantly described. The technological boom and the related scientific advances that took place during the second half of the 20th century brought new evidences for the toxicity of Pb. The absorption of Pb by the body can follow the inhalation, ingestion, dermal contact or placental routes. The blood circulation rapidly mobilises Pb to soft tissues, including liver, lung, spleen, kidney and bone marrow. Blood represents a conservative biological pool, with a half-life period for Pb of about 16e40 days. Eventually Pb is metabolised in the tissues, with distribution mainly to the skeleton, which can be taken as a slow turnover pool, the biological half-life of Pb in bones being 17e27 years [55]. Many of the biochemical pathways involved in Pb toxicity are not still elucidated, but cytotoxic and genotoxic effects of Pb associate with oxidative processes. The high affinity of Pb for protein thiol groups, impairing protein function, is responsible for its tremendous impact on health. Among other actions, Pb inhibits key enzymes of the haem synthesis in the erythrocyte cell, such as, the d-aminolevulinic acid dehydratase (ALAD) and the ferrochelatase [91]. Consequences are dramatic, as haem is essential for cell metabolism and oxygen transport. Regulations impose strict rules, both to the industry and to the workers, to reduce the Pb levels in the work environment and the dose received by the worker. The occupational exposure to Pb is usually monitored with indirect bioindicators of exposure and effect. The concentration of Pb in blood serves as a biomonitor of exposure. The Pb levels in blood reflect the dose and the exposure of the last week-period. Enzyme activities (eg, ALAD) and specific protein concentrations (eg, zinceprotoporphyrin (ZPP) complex, a by-product of ferrochelatase inactivation) represent effect indicators. These indicators reflect the past action of Pb in a 3-month timescale, which is related to erythrocytes turn over [55,91]. Thus, the variations of Pb in blood of exposed individuals are subjected to a high time lag. According to US Environmental Protection Agency [92] the amount of airborne Pb that can be absorbed through the intestine in adults is about 10% whereas 30e50% can be deposited and absorbed in the lungs. In

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occupational settings where inhaled particles may vary considerably in size and composition [18,30,32,78], absorption routes may contribute unevenly to Pb concentrations in blood. The proportion of particles of different size fractions that are inhaled and that interact with the lung cannot be easily estimated, as the primary inhaled particles or their constituents, which eventually reach circulation, follow different pathways at different times. These aspects prevent the true estimation of dose, and limit biomonitoring through blood for long-term assessment of Pb exposure. The possibility of sampling noninvasively from the lung, the target organ as far as occupational exposure to Pb is concerned, is the prospect held out by EBC. Repeated sampling during the working day and the working week time periods is possible and EBC may provide unique information on the amount of metal pollutants inhaled and their changes in the lung. Therefore EBC may become a human bioindicator that could be applicable for professional exposition to Pb.

2.2 Study Design Two types of Pb-processing industries located in the same urban industrialised area were selected, a Pb-recycling industry (smelter) and a battery-assembling industry. Due to the production characteristics of each factory, the air extraction requirements, architectonics, number of employees and tasks performed, differ [69]. In the recycling factory there are no fixed work-posts. In the batteryassembling factory two main production areas were considered: (1) lead plates cut (automatic and manual) and (2) assembling lines. Workers alternate frequently in their workplace between automatic and manual cuts and within the several posts in the assembling line, but not between the two main categories of manufacturing. National regulations (transcript from European Union regulations, National Decree 274/89, 1989) are strictly applied, for both air quality and personnel safety. The Pb-recycling factory (Ind1) operates 24 h per day and personnel work on 8-h shifts, five days a week with two intercalary resting days. The battery assembly factory (Ind2) operates only on two 8-h shifts 5 days a week with two intercalary resting days. The study design accounted for: l

l

working environment monitoring to characterise air particulate matter, identify the industry signatures and estimate the magnitude of the exposure; workers biomonitoring to estimate exposure to Pb and effects, using EBC.

A nonexposed group of individuals (CTR) working in offices, distant 30 km from both factories, not exposed to gases, dusts or fumes in their working activity were also enrolled. The working week consisted of 35 h, from

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Monday to Friday. This group provided a baseline for Pb biomonitoring of exposed workers. The workplace monitoring consisted of PM measures and chemical characterisation of different working sites in the factory. The biomonitoring assessment had into account daily and working week variations. This was possible by using EBC to measure Pb concentrations through time.

2.3 Characterisation of Exposed and Nonexposed Workers All subjects gave their informed consent to participate in the study and filled a questionnaire reporting on age, smoking habits, gender and past respiratory diseases. The Pb-exposed workers were clinically evaluated. Respiratory complaints, such as allergy, occasional cough, wheezing and sputum production, were registered and their respiratory function was assessed by spirometry (Vitalograph Compact II spirometer, Ennis, Ireland). The most useful volumes to diagnose airway obstruction or restriction are the forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC), aside with the FEV1/ FVC ratio (Tiffeneau index). To assess possible airway inflammation response, the exhaled NO was measured using a portable device NIOX MINO (Aerocrine, Solna, Sweden). The workers selected did not report any history of respiratory pathology, and respiratory function tests were all normal. The characterisation of the three studied groups is summarised in Table 3. TABLE 3 Workers Characterisation: Demographics and Clinical Assessment Values Include Mean  SD (When Applicable) CTR (N ¼ 52)

Ind1 (N ¼ 15)

Ind2 (N ¼ 15)

Sex (m/f)

29/23

15/0

64/17a

Age (years)

34  8

40  8

42  10

Working years

e

13  8

14  9

Smokers (%)

24

29

38

Allergic complaints (%)

e

12

31

Respiratory symptoms (%)

e

35

16a

FVC (%)

e

103  16

89  13a

FEV1 (%)

e

103  18

90  14a

Tiffeneau index

e

0.99  0.06

1.0  0.1

NO (ppb)

20  14

18  13

15  11

FEV1, Forced Expiratory Volume in the First Second; FVC, Forced Vital Capacity; Tiffeneau Index, (FEV1/FVC); NO, Exhaled Nitric Oxide a Significant differences between industries (ManneWhitney results).

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2.4 Work Environment Monitoring: Air Particulate Matter For work environment, monitoring was based on PM characterisation. PM was collected with low volume Gent samplers [18,93,94]. The samplers were equipped with a PM10 preimpactor stage and with a Stacked Filter Unit (SFU) of two stages, carrying 47-mm Nuclepore polycarbonate filters. Air was sampled at 15e16 L/min, which allowed the collection of particles with aerodynamic diameter (AD) between 2.5 and 10 mm (PM2.5e10) in the first stage and particles with AD <2.5 mm (PM2.5) in the second stage. Sampling was carried out in factories using several Gent samples working in parallel. Samplers were operated during labouring period at 1.6-m height, which corresponds to the breathing height of workers, thereby ensuring the best representativeness of working conditions. In Ind1, four samplers were used in parallel distributed throughout the factory, in representation of the whole labouring area. In Ind2, where activities were divided in different rooms and/or areas, four samplers were distributed along each manufacturing line (manual and automatic plates cut and assembly).

2.4.1 PM Gravimetric and Chemical Analysis The filter loads were measured by gravimetry in a controlled clean room (ISO 7). Nucleopore filters were weighed using a 0.1 mg sensitivity balance (Mettler Toledo UMT5). Filter mass before and after sampling was obtained as the average of three measurements, assuring that the variation coefficient was <5%. Each filter was divided in sections for chemical analysis by two techniques, particle-induced X-ray emission (PIXE) and instrumental neutron activation analysis (INAA) [95e98]. Blank nucleopore filters were treated the same way as regular samples. All measured elements were homogeneously distributed; therefore, concentrations were corrected by subtracting the filter blank contents. 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 e Coal Fly Ash. Tests of reproducibility within filters and between filters were taken using parallel sampling with two similar sampling units and measuring the particle chemical elements by INAA and PIXE. Results were reproducible to within 5e15%, providing strong support for the validity of the analytical techniques [97,98]. 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%. 2.4.2 PM Levels and Chemical Composition PM levels in both industrial sites were significantly higher than in offices. As shown in Fig. 4, Ind1 showed significantly higher PM2.5 and PM2.5e10 mass concentrations than Ind2 (p ¼ 0.0001). In Ind1, no significant differences in total mass concentration were observed between the four sampling locations. However in Ind2, there were

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FIGURE 4 Particulate matter concentration (mg/m3) in the three sampling sites for both fractions (fine and coarse). The total length of the bars represents PM10 or total mass. For better visualisation scale-breaks to each fraction were made in the same percentage, so the proportion of the PM fractions is maintained. Adapted from Int. J. Hyg. Environ. Health 216(1), P.M. Fe´lix, S.M. Almeida, T. Pinheiro, J. Sousa, C. Franco, H.Th. Wolterbeek, Assessment of exposure to metals in lead processing industries, 17e24 (2013) with permission from Elsevier.

significant differences between the two main production areas, that is, Pb plates cut and assembling lines. The PM2.5e10 mass concentrations were higher in the cut section than in the assembling line sections (p ¼ 0.05) whereas the PM2.5 mass concentrations were higher at the assembling line sections than at the cut sections (p ¼ 0.04). The average elemental concentrations measured in the two PM size fractions, show that the most representative elements with a contribution higher than 1% to the total mass, besides Pb, in Ind1 and Ind2 varied. As anticipated by the differences encountered between the PM mass concentrations measured in the industries and in offices, levels of elemental concentrations in offices were remarkably low when compared to both factories. Fig. 5 and Fig. 6 depict 17 selected elements whose concentrations were measured in PM2.5 and PM2.5e10 fractions in the two factories (Ind1 and Ind2), in offices where the control group subjects worked and in the outdoor environment. Overall, PM concentrations in offices do not show any unusual enrichment, resembling those measured in the outdoor environment. With respect to Ind1, Na, Cl, Sb and Pb concentrations were meaningful in both PM2.5 and fractions PM2.5e10. Also, Fe had an expression higher than 1%

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FIGURE 5 Average elemental mass concentrations (ng/m3) for 17 selected elements in PM2.5 fraction sampled in Industry 1, Industry 2, offices and outdoor environment. The elements most representative for the total PM2.5 fraction mass concentration are above 1%. See text for details. Adapted from Int. J. Hyg. Environ. Health 216(1), P.M. Fe´lix, S.M. Almeida, T. Pinheiro, J. Sousa, C. Franco, H.Th. Wolterbeek, Assessment of exposure to metals in lead processing industries, 17e24 (2013) with permission from Elsevier.

in the coarse fraction. In Ind2 the most representative elements in PM2.5 fraction were Al, Si, Ca, Br and Pb whereas Na, Al, Si, Cl, Ca and Pb were present in PM2.5e10 in concentrations above 1%. The Pb concentrations measured in both industries, which range from 100e500 mg/m3 in Ind1 and 5e10 mg/m3 in Ind2, were in accordance with published data (eg, [37], [42]). The Pb concentrations in the workplace have been reviewed by Koh et al. in 2015 [37]. In this retrospective study, reported Pb concentration in total suspended and inhalable particles collected with personal air samplers in smelters was 100e3100 mg/m3, whereas in battery assembly industries it was 2e1980 mg/m3. In both industries, the most represented element in PM2.5 and PM2.5e10 was Pb. The relative contribution of this element in Ind1 was 41% in PM2.5 and 32% in PM2.5e10. In Ind2, the relative contribution of Pb for both fractions was 8% and 19%, respectively. Therefore, Pb is more associated with the coarse fraction in Ind1 than in Ind2. This can be justified, as Pb processing in battery fabrication is mostly

FIGURE 6 Average elemental mass concentrations (ng/m3) for 17 selected elements in PM2.5e10 fraction sampled in the Industry 1, Industry 2, offices and outdoor environment. The elements most representative for the total PM2.5e10 fraction mass concentration are above 1%. See text for details. Adapted from Int. J. Hyg. Environ. Health 216(1), P.M. Fe´lix, S.M. Almeida, T. Pinheiro, J. Sousa, C. Franco, H.Th. Wolterbeek, Assessment of exposure to metals in lead processing industries, 17e24 (2013) with permission from Elsevier.

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mechanical (eg, abrasion, cut). In Ind1, smelting processes dominate. Pb fumes are generated in the combustion, which can originate secondary fine particles. In Ind1, the major contributions for PM levels of Sb associate with Pb ore constituents, while Na and Cl associate with additives used for Pb refinement in the crucibles. Noteworthy, a strong correlation between Cl and Pb in PM was observed (r ¼ 0.96; p < 0.0001 for PM2.5). This finding can also be inferred from Fig. 3, where a good correlation of Pb- and Cl-containing particles is observed.

2.5 Biomonitoring With EBC The workers’ biomonitoring was performed with EBC method. Sampling frequency was adjusted to timeeactivity patterns of the exposed individuals. The EBC was collected using commercial equipment (EcoScreen, Jager, Germany). EBC was sampled breathing tidally for 15 min [69,94]. The volume of condensate obtained was of about 2 mL, which was appropriate for analysis [69]. The EBC samples were collected in controlled environmental conditions of temperature and relative humidity, differing from the daily working conditions. For the exposed groups, EBC was collected at the occupational health unit located in an isolated building within the factory complex, at a distance of 50 m from the workplace. For the nonexposed group (CTR), the EBC was collected in a clean room (ISO 7). Two modalities were used for EBC sampling from exposed workers (Ind1 and Ind2). One modality was designed to account for the fluctuations of Pb during the working day and working week. Sampling occurred in four stages: A e at the beginning of the shift, EBC was collected before starting work; B e at the end of the 8-h shift, EBC was collected immediately after stopping work and before hygiene requirements at the end of the work shift; C and D e the procedure was repeated in the last day of the working week. In the second modality, EBC sampling occurred in two stages: A e 1st day of the 5 days working period, before the shift; and D e last day of the 5 days working period, after the shift. For CTR group, EBC was only collected in one occasion.

2.5.1 Preanalytical and Analytical Considerations in EBC Analysis The concept implemented aimed at minimising handling and preanalytical manipulation of the EBC sample. The main objective was to simplify procedures while guaranteeing good practices for chemical analysis of EBC matrix. EBC is a very diluted matrix, where elemental concentrations are expected to be in a very low level. Therefore rigorous methodologies should be implemented to assure good quality of results. In most environmental health assessments, conclusions are based on the analysis of large number of samples and therefore undemanding analytical procedures are encouraged. The optimisation of the methodologies and the validation of the analytical procedure to assess metal concentrations in whole EBC samples were

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carried out to estimate the reliability of results produced. Two techniques were used: inductively coupled plasma mass spectrometry (ICP-MS) and total reflection X-ray fluorescence (TXRF) [69,82]. The ICP-MS equipment, ELAN DRCe (PerkinElmer, SCIEX, Waltham, MA, USA), was operated at 1100 W, with argon gas flow of 15 L/min and 0.85 aerosol L/min gas carrier using a Peltier-cooled cyclonic spray chamber. Data were collected, processed and analysed with ELAN 3.4 software. A TXRF EXTRA II-A spectrometer (Atomika, Munich, Germany) equipped with two 2-kW fine focus X-ray tubes (Mo and W anodes) was used as a reference technique (accredited for water analysis by the Portuguese Quality System, ISO/IEC/17025) [99]. For TXRF analysis, EBC samples were doped with Ga as an internal standard, at a concentration of 100 mg/L. EBC samples were analysed directly by pipetting 20 mL into appropriate quartz sample carriers. From each sample at least two replicates were analysed. For ICP-MS analysis, 500 mL of EBC samples were doped with Y as an internal standard at a concentration of 10 mg/L and diluted fivefold with acidified 1% (v/v) HNO3 ultrapure water. A total volume of 2.5 mL is the minimum required to perform an ICP-MS granting adequate conditions of sample introduction and analysis. Sample handling and preparation was performed in a clean room (ISO 7). Teflon containers thoroughly cleaned to avoid metal contaminations were used in all steps of the procedure. All reagents used were high-purity grade. Ultrapure water of 18 MU cm (Milli-Q Element; Millipore, Billerica, MA, USA) was used for dilution of stock solutions and to prepare blank solutions. Concentrated Suprapure nitric acid (HNO3) high-purity grade was obtained from Merck (Darmstadt, Germany). Multielement atomic spectroscopy standard solution Fluka 70,008 (SigmaeAldrich, St. Louis, MO, USA) was used for calibration. Stock solutions of 1000  10 mg/L of Y (AAS Specpure Y solution; Alpha Aesar, Ward Hill, MA, USA) and Ga (AAS Specpure Ga solution; Alpha Aesar) were used for internal standardisation and preparation of spiked samples.

2.5.2 Optimisation of the EBC Sample Preparation and Validation of the Analytical Procedure The sample preparation was minimised. Immediately after collection (not exceeding 2 h), the EBC samples were acidified with 3% (v/v) HNO3 and sonicated for 10 min at room temperature. Samples were stored at 80 C until analysis and thawed only once. This step assures that the EBC sample has a representative analysis of the elemental concentrations in the total sample. The procedure was validated using two different techniques, TXRF and ICP-MS, based on different approaches as described earlier. These techniques were chosen, as both are multielemental, require small sample volumes and have appropriate sensitivity.

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The analytical performance was carried out for selected elements which may provide exposure indication and biological information: (1) Pb which is relevant in the studied occupational context; (2) Cr, Mn, Cu, Cd and Sb, which are pollutants with toxic potential and present in the work environment and (3) K and Zn and to a certain extent Sr, which are essential elements and may be indicators of physiological response to inhaled toxicants. The quality control assessment had into account the validation of the techniques used and the estimation of the overall uncertainty achieved in the analysis of EBC. The validation protocol consisted of analysis of certified reference materials (CRMs) with an aqueous matrix and elemental concentration levels similar to those of EBC. Two CRMs, NIST 1643e and NIST 1640, were analysed by ICP-MS and TXRF. The elements selected for control were K, Cr, Mn, Cu, Zn, Sr, Cd, Sb and Pb. The precision is given by the relative standard deviation of the mean concentration values. Precision was below 5% for all of analysed metals by both techniques. The trueness calculated as the relative difference to the certified value was also below 5% for most of the elements determined. Deviations above 5%, as verified Mn, Zn and Pb determination by TXRF, were within the 10% acceptable performance of this technique [69]. The estimation of the overall uncertainty was performed using a pool of EBC collected from a group of workers. This method enables combining all different sources of error derived from sample preparation and analysis. The method applied precision (repeatability) and trueness (recovery) studies for the elements referred earlier. Precision was studied using the native EBC sample pool and trueness obtained from spiking aliquots of the EBC pool with monoelemental standard solutions of K, Mn, Cu, Cd, Sb and Pb (Certipure Merck) at different concentrations [99]. To guarantee realistic uncertainty estimation with both TXRF and ICP-MS a preestablished amount of each element was added to the EBC pool according to the concentration level in the native pool. The contribution to the combined uncertainty from precision was 5% for both techniques for all elements, excluding Mn determination by TXRF, which is between the detection limit and quantification limit (2.9 mg/L). The combined uncertainty calculated in the spike study, varied between 8% and 22% for ICP-MS and between 10% and 22% for TXRF. The expanded uncertainties at 95% confidence (coverage factor of 2) were <25% for the majority of elements [98]. As the concentration levels in EBC are low, the estimated uncertainties in EBC analysis by TXRF were somewhat larger than those obtained with ICP-MS, due to the lower sensitivity of TXRF. As an example, the overall uncertainty estimation for Pb determination using ICP-MS had a contribution from precision of 2% and trueness of 8%, which yields an expanded uncertainty of the order of 16%. This methodology enables to detect the major source of uncertainty, which was recovery and therefore to identify which analytical step can be improved. The relative large values for recovery

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suggest that additional efforts to improve sample homogeneity should be carried out. Nevertheless, the methodology used can discriminate between individuals exposed to different levels of metals, as will be shown later.

2.5.3 Pb Concentrations in EBC The Pb concentrations measured in EBC remarkably distinguish the three groups studied: workers of Ind1, Ind 2 and individuals working in offices (CTR group). The contrast between groups can be depicted in Fig. 7. Workers of Ind1 showed the highest concentrations of Pb (mean  SE: 33.9  3.4) whereas workers of Ind 2 had EBC levels of Pb closer to the CTR group, though still significantly higher (mean  SE: 2.2  0.3 vs mean  SE: 1.0  0.2; p < 0.01). The study of Pb concentration changes in EBC over the working week showed steady Pb concentrations in individuals exposed to high Pb levels. The Pb concentrations in EBC of workers were assessed through two sampling modalities: EBC collection in 4 time points and 2 time points over the week period of time, as previously mentioned (see Section 2.5). In Ind 1, where exposure levels were high, workers were assessed in 4 time points to assess first day variations and week variations. The changes of EBC Pb concentrations were unimportant during the week working period (Fig. 8A), although the pause of two days between shifts may lead to a tentative diminishing of Pb levels in EBC that did not reach statistical significance. In Ind2, where workers were exposed to lower levels of Pb than in Ind1, the concentrations of Pb in EBC were significantly lower in the beginning of the shift relative to the end of the working week (Fig. 8B). Therefore, a significant amount of Pb could be

FIGURE 7 Boxplots of Pb concentrations in EBC for the three groups studied, Pb-recycling factory e Ind1; battery assemblage e Ind2; offices e CTR. The bottom and top of the box represent the lower (25%) and upper (75%) quartile, respectively; the horizontal line represents the median (50%); the vertical line represents the data minimum and maximum values; the square inside the box represents the mean value. The significant differences are indicated.

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FIGURE 8 Boxplots of Pb concentration in EBC over time. Graph A: Ind1; Graph B: Ind2. A e beginning of the shift first day of the week; B e end of the 8-h shift (first day of work); C e beginning of the shift last day of the week and D e end of the 8-h shift (last day of work). The bottom and top of the box represent the lower (25%) and upper (75%) quartile, respectively, the horizontal line represents the median (50%), the vertical line represents the data minimum and maximum values; the dots are outliers (1.5  the upper quartile). Significant differences are indicated.

washed out from lungs during the two-day period of pause between shifts in Ind2 workers. This was not verified in Ind1 workers.

2.5.4 Pb Concentrations in EBC Versus Pb Levels in the Work Environment When the concentrations of Pb in EBC were related to the concentrations of Pb measured in total PM (weighed for PM2.5 and PM2.5e10fractions) in the working environments studied, a nonlinear relationship was found (Fig. 9).

FIGURE 9 Rise in mean Pb concentrations of EBC (Pb-EBC) in the three groups of exposure according to Pb concentration in total mass of PM in the workplace.

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Biokinetic models foresee this type of relationship indicating a deviation from linearity for high doses of Pb [100,101]. Current occupational health models can provide an explanation for the steady Pb concentrations in EBC of workers exposed to high Pb levels (Ind1). These models are usually applied to predict the decline of Pb in the organism [102]. They indicate that a continued exposure to occupational levels of Pb shows a slower rate of Pb concentration increase in the organism over 2-year time, which eventually reaches saturation level to 5-year exposure period. The workers enrolled in this case study were all performing the same working activity for more than 2 years. Therefore, the minor Pb changes in EBC observed may reflect the long exposure period of the workers and Pb toxicokinetic findings. Other interesting feature of EBC was its potential to distinguish workers according to prevalent PM size in the work area. In Ind2 two working activities, diverging in the characteristics of the work and Pb exposure of the employees, that is, Pb plates cut section and batteryassembling lines, enabled assessing the signatures of the workplace emissions and better estimate their representativeness in EBC. Two groups of workers could be studied which clearly differed in EBC Pb concentrations (Fig. 10). Felix et al. [94] showed that the highest concentrations of Pb in EBC were mainly derived from the PM2.5e10 fraction (coarse particles), and that this PM fraction was strongly correlated with the cut section. In fact these workers showed also higher Pb concentrations in EBC than those working in the battery-assembling line, as referred previously. Therefore, these findings suggest that EBC besides reflecting the exposure level also expresses the factory fingerprint that in this case is described by the amount of Pb that is associated to the PM2.5e10 size fraction.

FIGURE 10 Graphical distribution (boxplots) of Pb concentration in EBC of workers of Ind2 performing different activities: Battery assembly line (Line) and Pb plate cut (Cut). Significant differences are indicated.

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2.5.5 Pb Concentrations in EBC and in Blood in Exposed Workers The relationship observed between Pb concentrations in EBC and in blood follows a logelog model as represented in Fig. 11. However, the correlation between Pb in EBC and in blood was feeble (r ¼ 0.65). This may be due to differences in the sequestration time of Pb in the two body compartments. The EBC Pb content may be a fast-term indicator expressing the internal lung dose almost in real time, whereas blood is a medium-term indicator, as Pb in blood has a long half-life (about 120 days) and accounts for several contributions (eg, different routes of exposure, mobilisation from accumulation organs). Nevertheless the relationship observed for Pb concentrations in both compartments call the attention for the deviations of linearity observed in models of blood Pbeair Pb relationship at very low and high air Pb levels. This may also derive from incomplete apportion of sources for the amount of Pb measured in blood. Efforts have been done to estimate the importance of inhaled PM size fractions in blood Pb [102] to better estimate Pb changes in blood. In fact, environmental health studies conducted in nonexposed population, consequently showing low Pb blood levels, demonstrated that PM originated from dust-sweepings outdoor and indoor cannot be considered a negligible predictor for Pb in blood [103]. Therefore the deviation from linearity observed when the two pools are compared can be considered an additional argument for the value of EBC as a reporter of direct exposure.

FIGURE 11 Logelog representation of Pb concentrations in EBC (Pb-EBC) and in blood (Pb-blood). The line represents the linear fit function (y ¼ 11.5 þ 0.92x; R ¼ 0.653) and the dotted lines the 95% CI.

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3. ADVANTAGES AND LIMITATIONS OF THE METHOD As far as Pb is concerned, and as demonstrated by the case study presented, Pb concentration changes in EBC are dose dependent. EBC proved to be adequate to assess Pb occupational exposure and the methodology was successfully validated. In addition, EBC may serve to identify exposure characteristics, such as the Pb-associated size fraction, which currently is an important issue in environmental health hazards assessment and in physiological models optimisation. The clear advantage of EBC as a noninvasive method for sampling from the lung is the possibility it offers to assess dose at the target organ. EBC can be easily obtained without discomfort and allows repeated sampling. These features position EBC in the forefront of collection methods for a better risk assessment of exposed workers. The major limitations of the method are inevitably associated to analytical problems. Although technological advances especially in the sphere of mass spectrometry-based techniques enable excellent limits of detection, the EBC is a diluted sample, what always brings analytical challenges. This limitation is particularly relevant in small peptides and protein measures or whenever metal speciation becomes pertinent [104,105].

4. FUTURE PERSPECTIVES Remarkable progresses have been made since mid-2000s in exploring EBC characteristics and capabilities as a potential source of respiratory biomarkers. However, efforts are still needed to increase knowledge on important areas of research such as origin of EBC formation, methodological developments, individual variability and the stability of EBC matrix, among others. The stability and recovery of EBC constituents carried by exhaled droplets during the condensation phase is indeed an unravelled territory. Although, some studies called the attention for the importance of collection conditions and materials used in EBC collection [64,106], there is still a considerable lack of data on this subject. This certainly limits methodological developments towards the determination of respiratory biomarkers whether they are metals, small molecules or peptides. This research will allow the validation of sampling methodologies and help establish figures of merit and adequate conditions for each biomarker.Overall these studies would significantly contribute to the identification and validation of new biomarkers that could contain exposure, clinical and diagnostic significance. This would be of the utmost importance to estimate health risks. Knowledge of both exposure to PM and the potential hazards they may induce allows for evaluating risk and establishing appropriate measures to mitigate risk. Addressing gaps in our knowledge of exposure to metals, such as detailed characterisation of the source of exposure and long-term evaluation of

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workers including both biomarkers of exposure and effect will definitely help to ensure the safety and health of people and the environment.

ACKNOWLEDGEMENTS The authors gratefully acknowledge Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT) for funding S.M. Almeida contract (IF/01078/2013) and the projects, PTDC/AMB/65828/2006, UID/Multi/04349/2013 and UID/BIO/04565/2013.

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