Characterizing metal(loid) solubility in airborne PM10, PM2.5 and PM1 in Frankfurt, Germany using simulated lung fluids

Atmospheric Environment 89 (2014) 282e289

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Atmospheric Environment journal homepage: www.elsevier.com/locate/atmosenv

Characterizing metal(loid) solubility in airborne PM10, PM2.5 and PM1 in Frankfurt, Germany using simulated lung fluids Clare L.S. Wiseman a, *, Fathi Zereini b a

School of the Environment, University of Toronto, 33 Willcocks St., Suite 1016V, Toronto, ON M5S 3E8, Canada Institute for Atmospheric and Environmental Sciences, Department of Environmental Analytical Chemistry, J.W. Goethe University Frankfurt, Frankfurt am Main, Germany b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Copper was found to be the most abundant element in all PM size fractions.
Many toxic elements measured in airborne PM are soluble in simulated lung fluids.
Copper, As, V and Sb were the most soluble in both simulated lung fluids.
Metal(loid) mobility is strongly pH dependent, with higher solubility in ALF.

V, Pb, Cu, As & Sb solubility (%) in PM10, PM2.5 and PM1 extracted with artificial lysosomal fluid (ALF) and Gamble’s solution (24 h) (n ¼ 36).

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 September 2013 Received in revised form 22 February 2014 Accepted 24 February 2014 Available online 25 February 2014

The purpose of this study is to assess the solubility of traffic-related metal(loid)s associated with airborne PM of human health concern, employing a physiologically-based extraction test with simulated lung fluids (artificial lysosomal fluid (ALF) and Gamble’s solution). Airborne PM (PM10, PM2.5 and PM1) samples were collected in Frankfurt am Main, Germany, using a high volume sampler. Following extraction of the soluble metal(loid) fractions, sample filters were digested with a high pressure asher. Metal(loid) concentrations (As, Ce, Co, Cr, Cu, Mn, Ni, Pb, Sb, Ti and V) were determined in extracts and digests per ICP-Q-MS. All metal(loid)s occurred at detectable concentrations in the three airborne PM fractions. Copper was the most abundant element in mass terms, with mean concentrations of 105 and 53 ng/m3 in PM10 and PM2.5, respectively. Many of the metal(loid)s were observed to be soluble in simulated lung fluids, with Cu, As, V and Sb demonstrating the highest overall mobility in airborne PM. For instance, all four elements associated with PM10 had a solubility of >80% in ALF (24 h). Clearly, solubility is strongly pH dependent, as reflected by the higher relative mobility of samples extracted with the acidic ALF. Given their demonstrated solubility, this study provides indirect evidence that a number of toxic metal(loid)s are likely to possess an enhanced pulmonary toxic potential upon their inhalation. The copresence of many toxic elements of concern in airborne PM suggests an assessment of health risk must consider the possible interactive impacts of multi-element exposures. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Airborne PM Metal bioaccessibility Metal solubility Simulated lung fluids Artificial lysosomal fluid Gamble’s solution

* Corresponding author. E-mail address: [email protected] (C.L.S. Wiseman). http://dx.doi.org/10.1016/j.atmosenv.2014.02.055 1352-2310/Ó 2014 Elsevier Ltd. All rights reserved.

C.L.S. Wiseman, F. Zereini / Atmospheric Environment 89 (2014) 282e289

1. Introduction Epidemiological studies have consistently demonstrated a relationship between increases in cardio-respiratory related mortality and morbidity and air particulate pollution among exposed urban populations (Dockery et al., 1993; Burnett et al., 1999). In particular, exposures to fine and ultrafine fractions of airborne PM, notably PM2.5 and PM0.1, have been associated with a number of negative cardio-respiratory health outcomes such as ischemic heart disease, ischemic stroke, vascular diseases and hypertension (Brook et al., 2010). Ambient PM is a complex mixture of different organic and inorganic constituents, including various transition elements such as vanadium (V), nickel (Ni) copper (Cu), which are suspected to illicit airway hyperresponsiveness, pulmonary inflammation and altered respiratory immune responses in exposed individuals, due to their ability to produce reactive oxygen species (ROS) (Carter et al., 1997; Costa and Dreher, 1997). Although significant advances have been made in recent years in characterizing the composition of airborne PM, there remain large gaps in knowledge regarding the chemical characteristics and hazard potential of associated metal(loid) constituents. As pointed out recently by Barrett et al. (2008) in their review of atmospheric levels of As, Cd, Ni and Pb regulated as part of the European Union’s Fourth Daughter Directive (4DD) 2004/107/EC (EU, 2004), the limited availability of data on metal concentrations in airborne PM is a major barrier in terms of assessing human exposures and related health risks. While data on total metal(loid) concentrations in airborne PM is important, we particularly require more knowledge regarding the solubility of metal(loid) fractions, as is the evidence suggests that it is this fraction which is most likely to be bioaccessible and capable of inducing pulmonary toxicity (Costa and Dreher, 1997; McNeilly et al., 2004). This highlights the importance of not only assessing the metal(loid) composition of airborne PM of relevance to human health but of determining the soluble fractions which may have the greatest toxic potential. In recent years, attention has increasingly shifted to the identification and measurement of various metal(loid)s believed to play a role in the cardio-respiratory effects observed in epidemiological investigations, including the determination of soluble, bioaccessible metal(loid) fractions (e.g. von Schneidemesser et al., 2010; Puls et al., 2012; Wang et al., 2013). For the most part, studies which have assessed solubility have done so using water as the leaching agent. While these studies have yielded important information regarding metal(loid) bioaccessibility, the use of simulated biological fluids would be preferable, as these are more likely to reflect the true conditions that exist in the human body (Mukhtar and Limbeck, 2013). To date, few studies have employed the use of simulated biological fluids to specifically asses metal(loid) solubility in airborne PM. Puls et al. (2012) used synthetic gastric juice to measure the solubility of platinum group elements (PGE) in airborne PM (PM10 and PM2.5) collected in Vienna, Austria, based on the assumption that a significant fraction of inhaled PM will end up in the gastrointestinal tract. Colombo et al. (2008) used the simulated lung fluids, artificial lysosomal fluid (ALF) and Gamble’s solution, to measure the soluble fractions of PGE in road dust and milled auto catalyst. Zereini et al. (2012b) employed the same simulated fluids to assess the solubility of PGE associated with airborne PM collected in Frankfurt, which is the only known study to date to have employed such a physiologically-based extraction test on field collected airborne PM samples. The lack of data for field samples highlights the need to employ such tests to further examine metal(loid)s solubility in airborne PM, as a proxy for bioaccessibility, to help elucidate which constituents may play a role in eliciting negative health outcomes in exposed populations.

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To contribute to an enhanced understanding of airborne PM and its toxic potential following inhalation, this study aims to assess the bioaccessibility of metal(loid)s commonly associated with airborne PM10, PM2.5 and PM1 (As, Ce, Co, Cr, Cu, Mn. Ni, Pb, Sb, Ti and V) collected in Frankfurt am Main, Germany. To achieve this, the soluble or bioaccessible metal fractions are determined using simulated lung fluids, artificial lysosomal fluid (ALF) and Gamble’s solution. 2. Material and methods 2.1. Sample collection and preparation Airborne PM (PM10, PM2.5 and PM1) samples were collected between June 2009 and November 2010 at a height of 4 m using a high volume sampler (Digitel DHA-80) at a monitoring station of the Hessisches Landesamt für Umwelt und Geologie (Hessian Ministry for Environment and Geology) in Frankfurt am Main, Germany (Zereini et al., 2012a,b). This station is located directly along a four lane road (coordinates: 50 070 28.5300 N; 8 41030.8800 E), with an approximate volume of 32,500 vehicles/day (speed limit: 50 km/h). Airborne PM10 samples were collected on cellulose nitrate membrane filters (pore size: 3 mm, filter diameter: 150 mm), while airborne PM2.5 and PM1 samples were collected on quartz microfiber filters (filter diameter: 150 mm). The sampling times for PM10, PM2.5 and PM1 samples were 48 h, 72 h and 96 h per filter, respectively. The filtered volume of air per sample was ca. 1350 m3 for PM10, 2150 m3 for PM2.5 and 2070 m3 for PM1. Airborne PM2.5 and PM1 samples were collected once per week, while PM10 was sampled every two days during the collection period. A total of 36 samples taken over a 17-month period were analyzed as part of this study. The sample filters were conditioned in a climate-controlled room at a constant temperature of 22  C and a humidity level of 54% for ca. 48 h before being weighed. Empty and sample filters were weighed 3 to 5 times, depending on the stability of the analytical balance (Sartorius BP 210S). A mean weight value was calculated per sample. Sample mass was determined as the difference in filter weight pre- and post-sample collection, as a function of the total amount of air filtered (Zereini et al., 2012a). 2.2. Sample extraction Artificial lysosomal fluid and Gamble’s solution, commonly employed in physiologically based extraction tests to simulate conditions in the human lung (Stopford et al., 2003; Midander et al., 2007; Colombo et al., 2008), were freshly prepared prior to sample extraction. Compared to Gamble’s solution, ALF is more acidic (pH 4.5), representing the cellular conditions that exist following an immune response in the lung and associated macrophage activity. Gamble’s solution has a pH of 7.4 and simulates the neutral conditions of interstitial fluid between lung cells. Solutions were prepared according to Colombo et al.’s (2008) adaptation of the original fluids reported elsewhere (Stopford et al., 2003; Midander et al., 2007). The pH of the solutions was measured following the dissolution of the chemical constituents with distilled water in a 1 L flask. The initial pH of the ALF solution was 4.3, which remained stable following a repeat measurement after 24 h. With the addition of sample filters, the pH levels generally remained the same, with the exception of some samples where the pH climbed to 4.4. The acidic pH of the lysosome may vary to a limited extent, as demonstrated by that measured for lysosomal fluid from rabbits, which ranged from 4.5 to 5.0 (Collier et al., 1992). As the ALF solution was very close to the desired pH of 4.5, it was decided not to

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use NaOH to adjust the pH to avoid altering the chemical composition (i.e. salt ratios) of the original solution. Sample filters were leached with ALF and Gamble’s for 24 h and 30 day periods. For this, each sample was placed in a 250 mL amber, nontransparent, high density polyethylene bottles containing 50 mL of solution (Zereini et al., 2012b). On average, the filters contained 43 mg of airborne PM sample material (determined as the difference between filters pre- and post-sampling). A maximum of 100 mg of sample material per filter was determined for collected PM. Samples were placed in an incubation oven at a temperature of 37  C and consistently shaken several times every day over the length of the extraction period. Samples were then filtered using polypropylene membrane filters (pore size: 0.2 mm) to separate the soluble from the solid (insoluble) fraction. Parallel extractions involving three filters were conducted for each solubility test (i.e. as a function of time and solution used). It should be noted that each filter is unique and represents a distinct sample collected during a particular time period. As the chemical composition of airborne PM can be highly variable over time and space as a function of many factors, including meteorological conditions, variability in sample results are to be expected. 2.3. Analytical determination The insoluble, solid sample fractions (i.e. filter residues) were digested with 1.5 mL HNO3 (69%, Suprapur, Merck) and 4.5 mL HCl (37%) (Suprapur, Merck) in quartz glass tubes placed in a high pressure asher (Anton Paar, Austria) for one hour at 320  C and a pressure of maximum 130 bar. This method has been demonstrated elsewhere to be highly effective in achieving the full digestion of complex environmental matrices, including those containing difficult to digest mineral oxides, such as airborne PM, road dust and soils (e.g. Messerschmidt et al., 2000; Alsenz et al., 2009; Zereini et al., 2012a,b). Sample digests were filtered onto Teflon dishes using quartz microfiber filters. The filters were rinsed several times with purified water to minimize sample loss through adsorption onto filter surfaces. All samples, including those extracted with ALF and Gamble’s solution (i.e. the soluble fraction) and the insoluble fraction of samples digested using the high pressure asher, were heated in Teflon dishes with 2 mL HNO3 (69%) (Suprapur, Merck) at 120  C to near dryness. This step, involving sample evaporation with concentrated HNO3, was repeated another 2e3 times. Sample solutions were then transferred to conical tubes and filled to the 10 mL mark with dilute HNO3 (0.5%) for the direct determination of As, Ce, Co, Cr, Cu, Mn, Ni, Pb, Sb, Ti and V using a quadrupole ICP-MS. The isotopes measured were: 51V, 52Cr, 53Cr, 55Mn, 59Co, 60Ni, 63Cu, 65 Cu, 75As, 121Sb, 141Ce, 206Pb, 207Pb, 208Pb, 49Ti. The ICP-Q-MS settings used were: plasma flow at 17.7 L/min, auxiliary flow at 1.68 L/ min, sheath gas flow at 0.25 L/min, nebulizer flow at 0.94 L/min, ICP RF power at 1.40 kW. The method detection limits, calculated as three times the standard deviation of the blank value, were as follows: 0.27 mg Ti/L, 0.28 mg V/L, 1.46 mg Cr/L, 0.24 mg Mn/L, 0.02 mg Co/L, 0.24 mg Ni/L, 0.47 mg Cu/L, 0.01 mg As/L, 0.01 mg Cd/L, 0.05 mg Sb/L, 0.03 mg Pb/L and 0.32 mg Ce/L. The instrumental detection limits were as follows: 0.67 ng Ti/L, 0.68 ng V/L, 3.40 ng Cr/L, 0.58 ng Mn/L, 0.04 ng Co/L, 0.56 ng Ni/L, 1.14 ng Cu/L, 0.02 ng As/L, 0.02 ng Cd/L, 0.11 ng Sb/L, 0.08 ng Pb/L and 0.77 ng Ce/L. 2.4. Quality assurance/Quality control All labware was carefully pre-cleaned in an acid bath (ca. 20% HNO3), followed by rinsing several times with purified water, prior to use. All of the chemicals that were used were of trace metal grade (Suprapur grade, Merck). Filters were conditioned in a climate-

controlled environment (22  C; 54% humidity) for 48 h and then weighed three to five times. Weights were recorded when readings remained stable following successive weigh-ins. Two internal standards were used (115In and 169Tm (both at 4 mg/L (Merck))) to correct for spectral drift and an external standard solution (ICP-MS Multielement Standard Solution VI (Merck)) was freshly prepared to calibrate the instrument prior to analysis. In contrast to Sector Field ICP-MS (ICP-SFMS), quadrupole-based ICP-MS instruments are unable to resolve for spectral interferences of <1 nominal mass units. Certain spectral interferences, notably 35 16 Cl O, 35Cl16O1Hþ or 40Ar12Cþ and 40Ar35Cl are well-known to potentially bias the measurement of 51V, 52Cr and 75As, respectively (Falciani et al., 2000). The application of an acid evaporation step with HNO3 following acid digestion, as done here, helps to eliminate residual chloride in the sample digests, which can be a major contributor to the formation of Cl-interfering spectra (Wang et al., 1995). The standard reference material BCR 713 (wastewater), containing certified values for As, Cu, Cr, Mn, Ni and Pb, was treated in a similar fashion as the soluble sample fractions extracted with ALF and Gamble’s solution and measured with each sample series. The following metal concentrations were determined for BCR 713 (in mg/L): Cr: 27  4.1 (certified value 22  2.4), Mn: 50  3.8 (certified value 43  3.0), Ni: 29  8.1 (certified value 30  5.0), Cu: 72  11 (certified value 69  4.0); As: 12  0.5 (certified value 9.7  1.1), Pb: 52  3.7 (certified value 47  4.0). The simulated lung fluids, ALF and Gamble’s solution, were also measured as blanks. For ALF, the absolute concentrations of Ti, V, Cr, Mn, Co, Ni, Ce, Pb, Cu, As and Sb varied between 0.002 and 1.2 mg/L. Chromium had the highest measured concentration, with an absolute mean of 1.2 mg/L, while Sb had the lowest level of 0.002 mg/L. For Gamble’s solution, the absolute metal(loid) concentrations ranged between 0.001 and 0.04 mg/L. 3. Results 3.1. Absolute metal(loid) concentrations Total metal concentrations in PM10, PM2.5 and PM1 were determined additively from the results obtained for the mobilized elemental (in solution) and filtered, nonsoluble residue fractions of each sample (Table 1). A high degree of variability in total metal(loid) concentrations were observed for between PM size fractions. Metal(loid) concentrations generally tended to decrease with increasingly smaller particle size fractions. In PM10, the five metals which were most abundant in mass terms, ranked from highest to lowest, were Cu > Mn > Ti ¼ Cr > Pb (mean levels of 105 ng Cu/m3, 20 ng Mn/m3, 17 ng Ti/m3, 17 ng Cr/m3 and 11 ng Pb/m3). The

Table 1 Absolute mean metal(loid) concentrations (minemax) (ng/m3) in PM10, PM2.5 and PM1 collected in Frankfurt am Main. Element

PM10 in ng/m3 (n ¼ 12)

PM2.5 in ng/m3 (n ¼ 12)

PM1 in ng/m3 (n ¼ 12)

Ti V Cr Mn Co Ni Ce Pb Cu As Sb

17 3.3 17 20 0.4 5.2 1.5 11 105 1.7 7.7

24 2.5 9.7 19 0.4 5.0 0.9 13 53 1.0 2.7

5.0 1.2 4.0 3.0 0.06 2.2 0.6 4.8 11 0.6 0.5

(7.6e36) (0.8e5.1) (9.4e32) (4.5e50) (0.2e0.7) (2.1e9.3) (0.4e6.5) (1.1e54) (15e307) (0.8e4.4) (2.6e13)

(2.5e67) (0.6e3.9) (4.4e17) (4.6e40) (0.1e0.8) (2.3e10) (0.5e1.6) (0.6e46) (13e121) (0.4e1.8) (0.9e6.6)

(1.9e17) (0.5e2.2) (1.4e7.6) (0.2e9.2) (0.03e0.14) (0.3e4.1) (0.1e2.1) (1.9e20) (3.1e35) (0.3e1.5) (0.3e1.7)

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variability in Cu levels was particularly high, with concentrations ranging between 15 and 307 ng/m3. Antimony and Ni concentrations in PM10 are also comparatively elevated, with means of 7.7 and 5.2 ng/m3, respectively. The levels of V, Ce and As in PM10 were relatively low, with concentrations of 3.3 ng V/m3, 5.2 ng Ni/m3, 1.7 ng As/m3 and 1.5 ng Ce/m3. The lowest levels PM10 were observed for Co (mean: 0.4 ng/m3). Deviations from the overall trend of increasingly lower concentrations with smaller particle size fractions (e.g. higher mean Ti and Pb concentrations in PM2.5 vs. PM10) may be attributed, in part, to the fact that samples were collected over different time frames, which can expectedly result in a high degree of chemical heterogeneity. Similarly, the five elements with the highest concentrations in PM2.5 were (from most to least abundant): Cu > Ti > Mn > Pb > Cr. The mean concentrations of these elements were 53 ng Cu/m3, 24 ng Ti/m3, 19 ng Mn/m3, 13 ng Pb/m3 and 9.7 ng Cr/m3. Concentrations of Cu in PM2.5 relative to PM10, which are ca. 50% lower in the finer PM fraction, indicate that this metal is comparatively more abundant in the coarse particle fraction. Lower levels of V, Ni and As were measured, with means of 2.5 ng V/m3, 5.0 ng Ni/m3 and 1.0 ng As/m3. Again, Co had the relative lowest concentrations with 0.4 ng/m3. In the finest PM size fraction examined here, PM1, Cu, Ti, Pb, Cr and Mn had the highest mean concentrations, with 11 ng Cu/m3, 5.0 ng Ti/m3 4.8 ng Pb/m3 and 4.0 ng Cr/m3 and 3.0 ng Mn/m3. The levels of Ce, As and Sb were lower, with 0.6 ng Ce/m3, 0.6 ng As/m3 and 0.5 ng Sb/m3. Cobalt had the lowest levels, with a mean of 0.06 ng/m3.

3.2. Artificial lysosomal fluid (ALF) The extraction tests with ALF solution indicate that metal(loid) mobility (As, Ce, Co, Cr, Cu, Mn, Ni, Pb, Sb, Ti and V) is strongly dependent on pH for all PM size fractions (Tables 2e4). For each element, the mobilized fractions in the respective airborne PM fractions demonstrated a high degree of variability. High interelemental variability was also observed between size fractions. For instance, a mean of about 25% of Ti present in PM10 was determined to be mobile after 24 h in ALF, while the mobilized fraction of As was determined to be 89% on average. The soluble metal(loid) fractions measured for PM10 in ALF after 24 h ranged from highest to lowest as follows (Table 2): Pb > As > Cu > Sb > V > Co > Mn > Ni > Ce > Cr > Ti. The metal(loid)s with the largest soluble fractions were Pb, As, Cu, Sb and V, with means of 96, 83, 82 and 81, respectively. Cobalt, Mn, Ni and Ce had smaller soluble fractions, with 43e60% of the total amount present in PM10. Chromium and Ti in PM10 were the least Table 2 Mean soluble fractions (minemax) (%) in PM10 extracted with ALF and Gamble’s solution after 24 h and 30 days (n ¼ 12).

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Table 3 Mean soluble fractions (minemax) (%) in PM2.5 extracted with ALF and Gamble’s solution after 24 h and 30 days (n ¼ 12). PM2.5 Artificial lysosomal Artificial lysosomal Gamble’s element fluid (ALF) (24 h) fluid (ALF) (30 d) solution (24 h)

Gamble’s solution (30 d)

Ti V Cr Mn Co Ni Ce Pb Cu As Sb

7.0 47 20 6.0 12 27 2.0 6.0 53 80 63

18 61 31 52 52 25 29 84 80 81 50

(6e26) (58e66) (19e43) (30e75) (4061) (14e39) (18e43) (77e91) (77e84) (75e85) (18e85)

29 63 53 70 61 44 52 60 77 80 89

(16e37) (52e74) (41e68) (58e79) (47e77) (25e62) (31e92) (10e98) (55e95) (58e95) (82e96)

2.0 33 9.0 9.0 35 16 17 4.0 31 64 31

(1.0e3.0) (16e47) (8.0e10) (5.0e14) (17e60) (8.0e25) (1.0e46) (1.0e9.0) (21e42) (62e65) (10e46)

soluble, with fractions of 37 and 25%, respectively. Following a reaction time of 30 days in ALF, As, Sb, Cu, Pb and V were observed to have the greatest solubility, while Ce was the least soluble of all elements (Table 2). The metals Ni, Co, Cr and Mn all had soluble mean fractions which ranged between 45 and 65%. Overall, no definitive trends in solubility could be established as a function of time of extraction, given the lack of a significant relationship between these two variables for most elements. The soluble metal fractions present in PM2.5 demonstrated similar patterns to that measured for PM10 exposed to ALF after 24 h (Tables 2 and 3). The highest soluble fraction in PM2.5 was again determined to be Pb, with a mean of 84%. This was followed by As, Cu and V, which had mean soluble fractions of 81, 80 and 61%, respectively. Antimony and Co associated with PM2.5 were also observed to be relatively soluble, with fractions of 50 and 52%, respectively. Similar to PM10, Ti was the least soluble element in PM2.5 following extraction with ALF after 24 h (mean: 18%). With the exception of Pb, Cu and As, metal(loid) solubility was observed to increase in ALF with the length of reaction time. This trend was more apparent for PM2.5 relative to that observed for PM10. This could be an indication that Pb, Cu and As are present mainly in a form that is initially mobilized at a rapid pace, with solubility decreasing as a function of time. However, a reduction in mobility as a function of time may also be due to the sorptive dynamics of particles during the extraction period, resulting in sample loss. As was discussed previously by Zereini et al. (2012b), metal(loid)s may be readsorbed to particle surfaces following an initial mobilization by the extractant, which are then later isolated as part of the insoluble, residual fraction. Metal(loid)-associated particles may also be lost by being sorped to labware surfaces over time, which highlights the need to pay close attention to the potential artefacts that may result as a function of the extraction time employed. Table 4 Mean soluble fractions (minemax) (%) in PM1 extracted with ALF and Gamble’s solution after 24 h and 30 days (n ¼ 12).

PM10 Artificial lysosomal Artificial lysosomal Gamble’s element fluid (ALF) (24 h) fluid (ALF) (30 d) solution (24 h)

Gamble’s solution (30 d)

PM1 Artificial lysosomal Artificial lysosomal Gamble’s element fluid (ALF) (24 h) fluid (ALF) (30 d) solution (24 h)

Ti V Cr Mn Co Ni Ce Pb Cu As Sb

4.0 50 14 5.0 16 43 4.0 7.0 25 77 70

Ti V Cr Mn Co Ni Ce Pb Cu As Sb

25 81 37 57 60 52 43 96 83 89 82

(22e26) (77e86) (14e56) (29e80) (53e66) (29e69) (19e59) (9e98) (82e87) (85e93) (81e84)

33 74 61 65 56 45 21 75 80 90 83

(18e41) (71e79) (57e67) (48e86) (31e73) (44e46) (14e29) (61e92) (66e98) (89e90) (76e94)

10 66 21 27 38 17 20 26 40 57 52

(2.0e26) (51e79) (5.0e48) (9.0e61) (19e67) (1.0e33) (2.0e57) (2.0e67) (23e72) (27e73) (16e86)

(2.0e6.0) (43e63) (6.0e20) (2.0e8.0) (7.0e28) (29e50) (3.0e5.0) (4.0e10) (10e49) (59e87) (62e85)

(1.0e16) (29e72) (7.0e41) (5.0e6.0) (2.0e26) (12e42) (1.0e3.0) (1.0e16) (51e55) (60e92) (41e78)

12 82 61 63 60 35 42 78 87 82 72

(2.0e21) (78e90) (56e64) (39e79) (38e76) (10e55) (5e69) (65e92) (82e93) (77e86) (64e86)

29 88 63 77 66 41 76 67 56 90 83

(17e47) (77e95) (38e85) (75e82) (59e73) (36e46) (46e93) (35e96) (12e79) (89e90) (76e94)

6.0 66 21 8.0 41 12 5.0 5.0 48 80 59

Gamble’s solution (30 d)

(2.0e12) 9.0 (2.0e19) (44e80) 59 (51e75) (15e27) 30 (22e44) (8.0e9.0) 10 (5.0e13) (38e45) 39 (27e45) (6.0e20) 33 (30e36) (1.0e16) 6.0 (2.0e9.0) (2.0e7.0) 6.0 (1.0e11) (33e61) 42 (2e67) (69e95) 77 (59e87) (35e75) 70 (62e85)

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In PM1, V, Cu and As were observed to have the highest solubility in ALF (24 h), with mobile fractions of >80% of the total elemental concentrations (Table 4). Lead, Sb, Cr, Mn and Co associated with PM1 were also found to be comparably soluble, with means ranging between 60 and 78%. Again, Ti was determined to have the lowest solubility of all elements in ALF, with a soluble fraction of 12% (24 h). Again intersample variability for the measured soluble fractions was high, especially for Mn (39e79%) and Co (38e76%). The elements V, Cr, Mn, Cu associated with PM1 were more soluble in ALF (24 h) compared to coarser size fractions (Table 4). Following a reaction time of 30 days in ALF, the solubility of Ti and Ce was observed to increase notably, with means increasing from 12 to 29% and 42 to 76%, respectively. The solubility of other metal(loids) such as V, Mn, Co, Ni, As and Sb were found to increase slightly over time. A decrease in solubility with time was observed for Pb (from 78 to 67%) and Cu (from 87 to 56%). Overall, a high intersample variability in observed results for all airborne PM fractions is to be expected, as PM is typically heterogeneous in terms of its chemical composition as a result of various factors such as time of day, traffic characteristics and driving behavior, meteorological conditions, etc. In all PM fractions, the elements Pb, Cu, As and Sb were observed to have the highest solubility in ALF, while Ti had the lowest. 3.3. Gamble’s solution In the near neutral pH of Gamble’s solution, metal(loid) mobility for all airborne PM size fractions was found to be notably lower compared to samples extracted with ALF (Tables 2e4). For instance, Pb associated with PM10 was determined to have a mean soluble fraction of 26% (24 h) in Gamble’s solution, which was more than 3.5 times lower compared to PM10 samples extracted with ALF. After 24 h, V (mean: 66%), As (mean: 57%) and Sb (mean: 52%) were observed to be the most soluble in Gamble’s solution (Table 2). The intersample variability should, however, be noted with measured soluble fractions ranging from 27 to 73% for As, 16 to 86% for Sb and 51 to 79% for V. Cobalt (38%) and Cu (40%) had also relatively large soluble fractions. The elements Pb, Ce, Ni and Cr associated with PM10 all had mean mobile fractions of <26%, demonstrating a lower solubility in Gamble’s solution. Similar to ALF, Ti was observed to be the least soluble of all elements, with a mean mobile fraction of 10%. After an extraction period of 30 days in Gamble’s solution, the mean mobile fraction of Ni, As and Sb associated with PM10 increased to 43, 77 and 70%, respectively. The measured mean soluble fractions for all other elements declined over time. For instance, the mean soluble Pb fraction declined from 26% after 24 h to 7% after 30 days. For PM2.5 samples extracted with Gamble’s solution, As had the highest solubility, with a mean mobile fraction of 64% after 24 h. Vanadium, Co, Cu and Sb had mean soluble fractions of between 31 and 35%. Cr, Mn, Pb and Ti associated with PM2.5 were observed to be the least soluble elements in Gamble’s solution (mean: <9%). Following an extraction period of 30 days with Gamble’s solution, the mean solubility increased for As (80%), Sb (63%), V (47%), Cu (53%), Ni (27%), Cr (20%) and Ti (7%). Similar to PM2.5, As was observed to have the highest solubility in PM1 samples extracted with Gamble’s solution (mean solubility: 80% (24 h) and 77% (30 d)). V, Sb, Cu and Co also had relatively large mobile fractions, with means of 66, 59, 48 and 41% after 24 h, respectively. The elements with the smallest soluble fractions in Gamble’s solution after 24 h were Pb, Ce, Ti and Mn, all with a mean mobile fraction of <8%. An extraction period of 30 days did not serve to impact the size of the soluble fractions for most elements, with similar or slightly lower mean mobile fractions for Ti, V, Mn, Co, Ce, Pb, Cu and Ni. Comparatively higher mobile fractions were

measured for Sb, Ni and Cr after 30 days, with a mean solubility of 70, 33 and 30%, respectively. In sum, a particle-size dependent relationship, with an increase in metal(loid) solubility being observed for smaller particle size fractions, was generally not discernable for samples extracted with Gamble’s solution. The most notable exception was As, which was observed to demonstrate an inverse relationship with particle size following an extraction period of 24 h, with a measured mean soluble fraction of 80% in PM1 (compared to 57% for PM10). Some elements appeared to even demonstrate a positive relationship with PM size, with a reduced solubility for PM1 compared to PM10. For instance, Pb associated with PM10 exposed to Gamble’s solution (24 h) had a mean soluble fraction of 26%, while Pb in PM1 had a solubility of only 5%. 4. Discussion The concentrations of several metal(loid)s in airborne PM collected as part of this study are comparable to that reported for other urban areas in several recent studies (Querol et al., 2007; Mugica-Álvarez et al., 2012; Roig et al., 2013). Querol et al. (2007) reported similar mean concentrations of V, Mn, Co, As and Pb for PM10 collected at an urban background site in a ceramic tile cluster in Modena, Italy, with levels of 3.0, 14, 0.3, 1.8 and 15 ng/m3, respectively. Concentrations of Cu and Sb, with 30 ng Cu/m3 and 3.9 ng Sb/m3, for PM10 at the same site were, however, lower compared to mean levels determined here. In another study conducted in Mexico City, ambient PM10 had concentrations of Cu (median: 110 ng/m3) and Ni (median: 4.32 ng/m3) (Mugica-Álvarez et al., 2012), levels which are comparable to that determined for PM10 samples collected in Frankfurt. Concentrations of Co (median: 2.4 ng/m3) and Pb (median: 84 ng/m3) were, however, reported to be significantly higher in PM10. In the same study, PM2.5 had comparable or lower concentrations of most elements, with the exception of V (median: 20 ng/m3) and Pb (median: 46 ng/m3). Roig et al. (2013) reported urban concentrations of 1.2 ng As/m3, 4.5 ng Ni/m3 and 15 ng Pb/m3 in PM10 collected in NE Spain, levels which were comparable to those determined as part of this study. Concentrations of Cr (1.1 ng/m3), Cu (52 ng/m3), Sb (2.3 ng/m3) and V (8.5 ng/m3) were, however, lower relative to levels examined for PM10 samples collected in Frankfurt. While other urban studies have reported comparable levels of several metal(loid)s, the existing data demonstrates that elemental levels of airborne PM are highly variable. A large number of factors including anthropogenic activities, meteorological conditions and natural perturbation (e.g. forest fires) can strongly influence the metal(loid) composition of airborne PM, resulting in a site and time-specific elemental fingerprint at a local and regional level. The absolute mean concentrations of Pb, Ni and As in PM collected in Frankfurt as part of this study fall below the levels set by the European Commission for member states of the EU (EU, 2004). The air quality regulations that exist for the EU establish mean concentrations for Pb, Ni and As as an annual average, in consideration of the variability that may exist at different time and spatial scales due to many different factors. Samples were collected from June 2009 to November 2010 as part of this study and, as such, are considered to be representative of airborne levels which are expected to fluctuate over the course of a year. With respect to solubility, the elements Pb, Cu, As, V and Sb associated with all examined airborne PM size fractions were found to be the most soluble in simulated lung solutions. Their toxic potential is, therefore, likely to be enhanced in exposed individuals, especially for those elements which occur in higher concentrations in airborne PM. One metal of concern is Cu, which has been consistently demonstrated to occur at some of the highest relative

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metal concentrations in the atmosphere of Frankfurt in recent years (see also Zereini et al., 2005; Zereini, 2010). The site of the monitoring station where the airborne PM samples were collected as part of this study is known to be especially impacted by Cu source emissions. From 2008 to 2010, a mean of 66 ng Cu/m3 was determined for this site (Zereini, 2010). An earlier study which assessed metal concentrations in total suspended particulates in samples collected at the same monitoring station in 2001/2002 reported a mean Cu level of 102 ng/m3 (range: 21e264 ng/m3) (Zereini et al., 2005). The reported solubility of Cu associated with PM10 and PM2.5 in the Mugica-Álvarez et al. (2012) was comparable to that determined as part of this study. Although they used water for the extraction tests, their results may be compared to those measured here using the more neutral Gamble’s solution. In this study, the soluble Cu fraction ranged from 31 to 40% for PM10 and PM2.5 (24 h), while the Mugica-Álvarez et al. (2012) study reported a soluble Cu fraction of between 30 and 35% for the same size fractions. Generally speaking, the solubility of Cu associated with airborne PM would be expected to be highly variable, as there are commonly a variety of different emission sources for this metal (Hildemann et al., 1991; Golwer and Zereini, 1998). Little is known regarding the chemical species of Cu in the atmosphere as a function of source emissions, along with other influential factors in the local environment, which hampers an assessment of associated health risks. Redox active elements such as Cu, however, have been shown in other studies to have the greatest potential to induce the formation of reactive oxygen species (ROS), which likely play a major role in PM-associated impacts on pulmonary health (Becker et al., 2005). The comparatively high concentrations of Cu associated with all airborne PM fractions, in combination with a determined solubility of 87% in PM1, 80% in PM2.5 and 83% in PM10 for Cu in ALF, in this study should serve as a red flag as to the potential hazard this metal may pose. The risks associated with Cu exposures in urban populations, where attrition from brake linings has been identified as a major contributor to Cu concentrations in roadside environments (Sternbeck et al., 2002; Wiseman et al., 2013), warrants particular attention in light of this. Although Pb and As are not as abundant in mass terms, they are of concern given their toxicity and potential to elicit negative health responses in exposed individuals (Iavicoli et al., 2011; Klumpp and Ro-Poulsen, 2011). In addition to Sb, Pb and As were observed to be the most soluble in all airborne PM fractions. After 24 h in ALF, Pb had a mean soluble fraction of 96% in PM10, 84% in PM2.5 and 78% in PM1, an indication that this metal is largely present in a form which is potentially reactive upon exposure to biological fluids. Despite the fact that atmospheric levels of Pb have been dramatically reduced through the introduction of Pb-free gasoline in the 1980s in Europe, this metal remains present at detectable levels in airborne PM. In addition to the resuspension of historically contaminated soil, the continued use of Pb in various applications such as wheel weights (Root, 2000) and road paint (Wiseman et al., 2013) are potential source emissions. Environmental concentrations of Sb, also demonstrated here to be present in airborne PM in a form that is highly soluble, have increased significantly in recent years due to its use in brake linings (Kupiainen and Pirjold, 2011). The demonstrated high solubility of Sb, together with its increased presence in the environment in recent years, suggest that greater attention needs to be paid to this metal and its potential to impact the health of exposed individuals. Arsenic in PM10 was determined to have a mean soluble fraction of 89% after 24 h in ALF. The high solubility of As is of concern given the toxicity of this metalloid, which is recognized as a human carcinogen (IARC, 1987). In a recent study, a significant association between As in airborne PM collected in NE Spain and cytotoxicity was reported in A549 lung carcinoma cell lines (ATCC, CCL-185),

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using a MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Roig et al., 2013). Levels of As, which were also examined in a previous study of airborne PM collected in Frankfurt (Zereini et al., 2005), are relatively low. As such, current exposures to As are likely to be of low concern compared to exposures to other more abundant elements. Nonetheless, there is a need to monitor airborne concentrations of this toxic metalloid in the future, especially in light of its solubility. In addition to such factors as the presence of cations, particle size has been demonstrated in various experimental studies to strongly influence solubility, with fine and ultrafine PM more likely to be mobilized in solution compared to coarse particles (Artelt et al., 1999). As such, finer PM is often predicted to have a greater relative toxic potential. In this study, a relationship between particle size and the mean solubility of most metal(loid)s was not detected, as suggested by the absence of a significant difference in the mean solubility of the various size fractions. This should not be interpreted to mean that a relationship does not exist between metal(loid) solubility and particle size per se. The importance of particle size has been demonstrated elsewhere. For instance, Mugica-Álvarez et al. (2012) observed a slight particle size dependence on solubility, with increases in solubility for V, Mn, Cu, Cr, Co and Pb associated with PM2.5 relative to PM10. Similar to the absolute metal(loid) concentrations determined as part of this study, metal(loid) solubility in ALF and Gamble’s solution was observed to be highly variable both within and between PM size fractions. For instance, the soluble Ni fraction ranged from 20 to 69% for PM10, 14 to 39% in PM2.5 and 10 to 55% in PM1. This variability is not unexpected, as airborne PM is known to be highly heterogenous. As such, airborne PM samples collected at the same location are likely to be highly variable in terms of their chemical composition and characteristics, due to such things as sample timing (e.g. time of day, day of the week, meteorological conditions) (Zereini et al., 2012b). In this study, a separate sample filter was used for each respective extraction experiment, representing a sampling period with its own unique physico-chemical signature shaped by traffic density and composition and distinct meteorological characteristics. Given the potential for such factors to contribute to variability in results, it would be recommended that more samples be analyzed over a longer time period to be more statistically robust, as discussed in Zereini et al. (2012b). Although the evidence suggests that particle size is a primary factor determining the toxic potential of airborne PM, other factors must be considered in the interpretation of results, especially where different simulated biological fluids are employed to assess bioaccessiblity. In addition to particle size, elemental solubility is known to be strongly influenced by the pH of biological fluids (Stopford et al., 2003; Midander et al., 2007). In light of this, it is important to conduct solubility studies using different simulated fluids, reflective of the biological conditions that exist in vitro, with variable pH levels of biological relevance. The importance of pH was demonstrated by Midander et al. (2007), for instance, who reported that the mobility of Cu powder (artificial patina) was higher in ALF relative to Gamble’s solution. In this study, an enhanced solubility was observed for Cu, Cr and Mn in PM1 and Pb, Cu and As in PM2.5 following an extraction period of 24 h with the acidic ALF (pH 4.3), compared to samples exposed to the more neutral Gamble’s solution (pH 7.8) (Tables 2 and 3). The elements Co, Mn, Ni and Ce associated with PM10 were determined to have a mean solubility of between 43 and 60% in ALF after 24 h (Table 2). Although their solubility is not as high compared to Pb, Cu, As, V and Sb, the mean size of the soluble fractions for these elements remains relatively large. In light of this, the potential human health implications of exposures to Co, Mn, Ni and Ce also need to be assessed. Nickel, for instance, which tends to

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accumulate in the lung following exposures via inhalation, ingestion and dermal absorption, can induce a variety of pulmonary toxic responses including bronchitis, asthma, and alveolar epithelium hyperplasia (Forti et al., 2011). Nickel-induced damage to the lung epithelial cell layer may also cause an increase in permeability in this highly important mechanical barrier to toxic substances. The associated disruption of immune defenses can also result in an enhanced susceptibility to pathogen-induced respiratory diseases such as pneumonia (Strengert and Knaus, 2011). Exposures to both soluble and insoluble forms of Ni have been associated with various negative pulmonary endpoints. However, soluble forms of Ni (e.g. NiCl2) have been demonstrated to generate higher levels of oxidative stress and ROS in human bronchial cell lines, attributed, in part, to greater subsequent intracellular concentrations of the highly bioavailable form of Ni (i.e. Ni2þ) (Forti et al., 2011). Similarly, Co and Mn have also been associated with various negative human health effects such as interstitial lung disease (Co) (Araya et al., 2002) and neurotoxicity resulting in parkinsonism-like effects (Mn) (Aschner et al., 2006). The potential health impacts of Ce exposures via inhalation of airborne PM are not clear, given the paucity of data, especially in the case of chronic human inhalation exposures (US EPA, 2009). Environmental levels of Ce reported to be elevated along roads have been attributed to the use of catalysts to reduce harmful vehicular emissions, which commonly contain this element together with other metals which are embedded in the washcoat of catalytic converters (Zereini et al., 2005; Zereini, 2010). In recent years, however, engineered nanoparticles of Ce (CeO2) have been increasingly used in other traffic-related applications such as a catalyst in diesel to reduce particulate emissions (Health Effects Institute, 2001). As nanoparticles have been demonstrated to be more toxic and capable of inducing pulmonary oxidative stress in human lung cancer cells (e.g. Lin et al., 2006), this raises questions regarding the potential health risks of possible environmental exposures to engineered forms of Ce. In the end, it is important to emphasize the significance of employing different biological fluids to represent varying conditions in the human lung to assess the toxic potential of not only metal(loid)s associated with fine and ultrafine PM but also for PM that falls in the coarse particle size range. Airborne PM of variable sizes is likely to have different cellular targets with varying associated toxic responses (Becker et al., 2003; Amatullah et al., 2012). Attention has been increasingly paid to the health effects of ultrafine particles and their role in cardiovascular health of exposed individuals, due to their ability to penetrate deep into the alveoli where they may bypass the mucosal (epithelial) barrier and negatively impact cardiac function. Biomaterial (endotoxins, exotoxins and fungal spores), which is likely to elicit an immune response upon contact at the site of deposition in the human lung, is often associated with the coarser fractions of airborne PM (Becker et al., 2003). As a consequence, metal(loid)s present in this fraction may be more likely to be mobilized into solution by the more acidic conditions that will result from associated respiratory immune defence mechanisms, as indicated by the results for ALF solution in this study. This highlights the need to consider the complex mixture of various PM constituents and their possible interactive effects, not just for metal(loid)s but of other commonly present components found in airborne PM of biological relevance. 5. Conclusions In sum, this study demonstrates the presence of a wide variety of metal(loid)s with different levels of solubility in simulated lung fluids. The results show that all examined elements associated with various field-collected airborne PM fractions have some degree of

solubility in simulated lung fluids, ALF and Gamble’s solution. As such, the examined metals and metalloids associated with airborne PM collected in Frankfurt are a potential human health concern. Certain elements, however, are of greater relative concern given their solubility. Notably, the elements Pb, Cu, As, V and Sb were observed to have the highest solubility in simulated lung fluids. As such, they possess an elevated potential to induce pulmonary toxicity. Of course, the toxic potential of these elements must also consider their relative abundance in mass terms, as well as their demonstrated toxicity in cell lines and animal models. For instance, although As is highly toxic, its high solubility in lung fluids may be considered to be not as alarming considering the relatively lower abundance in mass terms. Although comparatively not as soluble, other elements such as Ni and Co are also of concern due to their elevated presence in airborne PM, combined with their demonstrated toxicity in cell lines and animal models and potential to be soluble in biological fluids. Ultimately, the human health effects of exposures to airborne PM cannot be assessed on a single constituent basis, given the variety of potentially toxic metal(loid)s and their demonstrated solubility in simulated lung fluids. The chemical complexity of airborne PM and its variability as a function of time and space highlights the need to assess the impacts of exposures to metal(loid) mixtures, as well as other constituents of PM such as biomaterial, and any possible synergistic and/or additive effects.

Acknowledgments We would like to thank K. Liebl and his working group from the Hessisches Landesamt für Umwelt und Geologie for their help and support in the set-up and collection of PM sampling at their monitoring station at Friedberger Landstraße in Frankfurt am Main. We also thank W. Haunold and R. Sitals from the Institute of Atmospheric and Environmental Sciences at the Goethe University of Frankfurt for their assistance during sample collection.

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