Loss of Surface Reactivity upon Heating Amphibole Asbestos

Asbestos is a general term that encompasses a group of fibrous silicates of the .... of X-ray diffraction using a Philips diffractometer PW1830 and Co...
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Langmuir 2002, 18, 4345-4350

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Loss of Surface Reactivity upon Heating Amphibole Asbestos Maura Tomatis,† Laura Prandi,† Silvia Bodoardo,‡ and Bice Fubini*,† Dipartimento di Chimica IFM and Interdepartmental Center “G. Scansetti” for Studies on Asbestos and other Toxic Particulates, Universita` degli Studi di Torino, via P. Giuria 7, 10125 Torino, Italy, and Dipartimento Scienza dei Materiali e Ingegneria Chimica, Politecnico di Torino, corso Duca degli Abruzzi, 24, 10141 Torino, Italy Received October 29, 2001. In Final Form: February 20, 2002 Two amphibole asbestos, crocidolite and amosite, have been heated at 400 and 800 °C in order to examine the variations in some surface properties relevant to asbestos toxicity, such as iron mobility, free radical generation, and hydrophilicity. At 400 °C, only the surface is modified, while at 800 °C the crystalline structure partially collapses (X-ray diffraction) with loss in specific surface (Brunauer-Emmett-Teller). Two chelators, ferrozine and desferrioxamine, were employed to evaluate Fe(II) and Fe(III), respectively. More iron was extracted by deferoxamine from crocidolite than from amosite, but when compared per unit surface, the extent of iron removed from the two asbestos was the same. Ferrozine removed more iron from unheated amosite than from crocidolite. Upon heating, because of the oxidation of Fe(II) to Fe(III), less (400 °C) or no (800 °C) iron is removed by ferrozine. The amount of iron removed by deferoxamine is virtually unaffected by heating at 400 °C but dramatically decreases after the 800 °C treatment on both asbestos. Cyclic voltammetry evidences more redox active ions at low and neutral pH in the unheated crocidolite than in the 800 °C heated crocidolite. Both unheated asbestos generate HO• from H2O2 and carbon-centered radicals from the formate ion. The HO• yield is unaffected following the 400 °C treatment but suppressed by heating at 800 °C. Carbon-centered radicals are not generated by the heated fibers, but ascorbic acid restores radical activity on the 400 °C heated fibers but not on the 800 °C heated fibers. Water vapor is strongly and irreversibly adsorbed on 400 °C heated crocidolite but more weakly and only reversibly adsorbed on the 800 °C heated one, because of the stabilization of siloxane bridges and the embedding of surface-exposed cations. When crocidolite mixed with kaolin is heated at 800 °C, the above effects of the thermal treatment are further enhanced. In conclusion, at 800 °C amphibole asbestos lose most surface characteristics implied in their health effects, suggesting possible ways to inactivate asbestos in milder conditions than those currently employed, for example, with a plasma torch.

Introduction Asbestos is a general term that encompasses a group of fibrous silicates of the serpentine and amphibole families, widely used in the past in several applications but now banned in most Western countries, because of their severe health effects. Indeed, exposure to any form of airborne asbestos fibers causes asbestosis, a nonmalignant debilitating fibrosis, as well as lung cancer and pleural mesothelioma.1-3 Once an occupational issue, asbestos has become nowadays an environmental one, caused by the huge amount of asbestos in buildings, in dismissed industrial areas, and in asbestos-cement materials.4 Asbestos removal from buildings was the subject of a passionate debate some 10 years ago.2,5 The removal versus in situ inactivation is still a much debated question nowadays, as well as the choice of the way to dispose of removed materials. In some cases, very expensive and drastic procedures, such as plasma ashing, have been proposed and employed.6,7 The molecular basis * To whom correspondence should be addressed. Phone: +39011 670 7566. Fax: +39-011 6707855. E-mail: [email protected]. † Universita ` degli Studi di Torino. ‡ Politecnico di Torino. (1) Mossman, B. T.; Gee, J. B. L. N. Engl. J. Med. 1989, 320, 17211730. (2) Mossman, B. T.; Bignon, J.; Corn, M.; Seaton, A.; Gee, J. B. Science 1990, 247, 294-301. (3) McDonald, J. C.; McDonald, A. D. Ann. Occup. Hyg. 1997, 41, 699-705. (4) Ryan, G.; Buchan, R. M.; Keefe, T. J.; McCammon, C. S. Appl. Occup. Environ. Hyg. 1996, 11, 1417-1421. (5) Abelson, P. H. Science 1990, 247, 1017-1021.

of asbestos toxicity, in particular the reactions taking place at the fiber surface, is still partially unclear.8 Once these issues have been clarified, new, easier, and cheaper inactivation ways could be imagined, simply by modifying the chemical properties that impart their pathogenic potential to asbestos. It is generally accepted, however, that iron-generated reactive oxygen species (ROS)9-11 and cell-derived ROS and RNS (reactive nitrogen species) following contact with asbestos are implied in the mechanism whereby asbestos activates cell signaling pathways and damages DNA.8,9,11,12 Damage may originate both from iron mobilized from the fibers by endogenous chelators13 attaining DNA and from radical species, whose formation is catalyzed by the fiber surface itself. Damage may take place either from direct action of the fiber on the target cell or through recruitment and activation of immune cells (macrophages, PMN) that release inflammatory products (oxidants, cytokines, growth factors) acting on target cells. Macrophages engulf and attempt to get rid of the fibers through a clearance mechanism, which is inhibited by the fibrous habit and other factors. (6) Inaba, T.; Nagano, M.; Endo, M. Electr. Eng. Jpn. 1999, 126, 73-82. (7) Inaba, T.; Iwao, I. IEEE Trans. Dielectr. Electr. Insul. 2000, 7, 684-692. (8) Fubini, B.; Otero-Are´an, C. Chem. Soc. Rev. 1999, 28, 373-381. (9) Kamp, D. W.; Graceffa, P.; Pryor, W. A.; Weitzman, S. A. Free Radical Biol. Med. 1192, 12, 293-315. (10) Hardy, J. A.; Aust, A. E. Chem. Rev. 1995, 95, 97-118. (11) Kamp, D. W.; Weitzman, S. A Thorax 1999, 54, 638-652. (12) Mossman, B. T.; Churg, A. Am. J. Respir. Crit. Care Med. 1998, 157, 1666-1680. (13) Lund, L. G.; Aust, A. E. Carcinogenesis 1992, 13, 637-642.

10.1021/la011609w CCC: $22.00 © 2002 American Chemical Society Published on Web 05/01/2002

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Any study aimed to investigate new ways to modify the asbestos surface, to obtain a material where the above adverse features are eliminated, will contribute to establishing new and sound procedures for asbestos inactivation and disposal. In this respect, the present paper investigates the processes associated with the thermal treatment of asbestos in order to identify whether at a temperature much lower than that attained in plasma ashing, irreversible modifications in the nature of the asbestos fibers may be attained. Serpentine asbestos, namely, chrysotile, fully decomposes in a temperature range (450-700 °C) in which the amphiboles are substantially stable. As the various asbestos have been often employed in a mixed state, any heating procedure, to be generally applied, must be effective also on amphiboles. We have therefore investigated the modifications brought about by heating in air the two most commonly employed amphibole asbestos: crocidolite [Na2FeIII2(FeII,Mg)3Si8O22(OH)2], a fibrous form of riebeckite, commonly known as blue asbestos; and amosite [(FeII,Mg)7Si8O22(OH)2], a fibrous form of grunerite, commonly known as brown asbestos. Both are abundant in South Africa and Australia and were sold all over the world in the past century. The fibers have been treated in air at 400 °C in order to examine surface modifications taking place and at 800 °C where the onset of the bulk reaction of transformation of the asbestos minerals has already taken place. We have investigated how two of the properties associated with asbestos toxicity, namely, extraction of iron by chelators and generation of free radicals,10,14,15 are modified by the thermal treatments. Iron extraction was investigated by incubation of an aqueous suspension of the fibers with ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′disulfonic acid) that selectively binds Fe(II) and with deferoxamine that favors oxidation and substantially binds any removable iron ion, following a technique described by Aust and associates.16,17 Free radical release has been measured, by us and other authors,18-22 by means of the spin trapping technique (electron paramagnetic resonance, EPR) applied to two different free radical generation mechanisms usually investigated in our laboratory: detection of HO• in the presence of H2O2, mimicking contact of the fibers with H2O2-rich lysosomal fluids, following phagocytosis by alveolar macrophages and recruited PMN; and detection of CO2•- from the formate ion, used as a “model” target molecule for homolytic cleavage of a carbon-hydrogen bond. This mechanism may be triggered either directly by active sites at the surface of the particles, upon contact with the formate (14) Kane, A. B. In Mechanisms of Fibre Carcinogenesis; Kane, A. B., Boffetta, P., Saracci, R., Wilburn, J. D., Eds.; IARC Scientific Publication No. 140; International Agency for Research on Cancer: Lyon, France, 1996; p 11. (15) Fubini, B.; Aust, A. E.; Bolton, R. E.; Borm, P. J. A.; Bruch, J.; Ciapetti, G.; Donaldson, K.; Elias, Z.; Gold, J.; Jaurand, M. C.; Kane, A. B.; Lison, D.; Muhle, H. ATLA, Altern. Lab. Anim. 1998, 26, 579617. (16) Lund, L. G.; Aust, A. E. Arch. Biochem. Biophys. 1990, 278, 60-64. (17) Chao, C. C.; Aust, A. E. Arch. Biochem. Biophys. 1994, 308, 64-69. (18) Fubini, B.; Mollo, L.; Giamello, E. Free Radical Res. 1995, 23, 593-614. (19) Fenoglio, I.; Prandi, I.; Tomatis, M.; Fubini, B. Redox Rep. 2001, 6 (4), 235-241. (20) Vallyathan, V.; Mega, J. F.; Shi, X.; Dalal, N. S. Am. J. Respir. Cell Mol. Biol. 1992, 6, 404-413. (21) Zalma, R.; Guignard, J.; Pezerat, H.; Jaurand, M. C. In Effects of Mineral Dust on Cells; Mossman, B. T., Begin, R., Eds.; NATO ASI Series H Cell Biology; Springer-Verlag: Berlin, 1989; Vol. 30, p 257. (22) Fubini, B.; Giamello, E.; Mollo, L.; Zanetti, G.; Eborn, S. K.; Aust, A. E. Res. Chem. Intermed. 1999, 25, 95-109.

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ion, or by a short-lived radical in solution, which in turn reacts with formate as a primary trap. This latter mechanism takes place with several other target molecules with “loose” C-H bonds, such as amino acids or small peptides,22 and consequently will occur with proteins and lipids. Experimental Section Materials. The fibers of the two amphibole asbestos, crocidolite and amosite, from the same batch employed in previous research23 were from UICC (Union International Contre le Cancer). Thermal treatments were performed by heating the fibers in air at 400 and 800 °C for 2 h. In one case, crocidolite fibers were initially mixed with equal amounts in weight of kaolin and then ground for 10 min in an agate jar. The mixed product obtained was subsequently heated in air for 2 h at 800 °C. Abbreviations used are as follows: unheated crocidolite, CROC; crocidolite heated in air at 400 °C, CROC-400; crocidolite heated in air at 800 °C, CROC-800; unheated amosite, AMO; amosite heated in air at 400 °C, AMO-400; amosite heated in air at 800 °C, AMO-800. Chemicals. The spin trapping agent DMPO (5,5′-dimethyl1-pyrroline-N-oxide), supplied by Fluka, was purified by filtration through activated charcoal, according to the method proposed by Buettner and Oberley.24 Sodium formate, hydrogen peroxide (30%), and ascorbic acid were from Sigma. Phosphate buffer (mono and disodium phosphate) was from Merck. Deferoxamine mesylate (desferioxamine B) and ′ferrozine were from Sigma. Methods. X-ray Diffraction (XRD). The crystallinity of the original and thermally modified fibers was controlled by means of X-ray diffraction using a Philips diffractometer PW1830 and Co KR radiation. Iron Release. Crocidolite and amosite fibers (1 mg/mL) were suspended (up to a final volume of 200 mL) in a 0.15 M NaCl solution containing 1 mM deferoxamine or 3 mM ferrozine, pH 7.4, for 3 days at 37 °C, continuously shaken, and kept in the dark. In the case of crocidolite preheated at 800 °C with kaolin, the amount employed was 2 mg/mL in order to keep the same amount of asbestos in all tests. The pH was readjusted at regular time intervals, throughout the incubation period, to prevent alteration of the rates of iron mobilization, due to the changes in acidity caused by surface reactions. At regular time intervals, 2.50 mL samples were taken and centrifuged at 10 000 rpm for 20 min to remove the asbestos. The total amount of iron present in the supernatant was determined spectrophotometrically on a Uvikon 930 dual beam spectrophotometer. The complex Fe3+deferoxamine was determined by measuring the absorbance at 428 nm (EmM ) 2.8 mM-1 cm-1). The complex Fe2+-ferrozine was determined by measuring the absorbance at 562 nm (EmM ) 27.9 mM-1 cm-1). Spin Trapping. The radical release upon incubation of 45 mg of fibers with a 0.078 mM H2O2 solution or a 1.0 M solution of sodium formate was detected using the spin trapping technique (′DMPO as trapping agent), as described in previous papers.18,22,23 The radical adducts formed were monitored by EPR spectroscopy (PS100.X Adani EPR spectrometer). The number of radicals released is proportional to the intensity of the EPR signal measured by double integration. Kinetics of free radical yield with the spin trapping technique was followed for at least 1 h. The extraction of one hydrogen atom from the formate ion was performed both in the absence and in the presence of ascorbic acid in the medium. In the latter case, the pH of the solution (1.5 mM in ascorbic acid) was set to 6.6 with hydrochloric acid. The conditions were close to those employed when investigating DNA damage from the same samples.25 The sequence of operations was the same as that performed in the absence of the ascorbic acid. Blanks were made by operating in the same way except that no solid particulates were introduced into the solution. Cyclic Voltammetry. To carry out electrochemical measurements with asbestos fibers that are nonconductive, a very close (23) Martra, G.; Chiardola, E.; Coluccia, S.; Marchese, L.; Tomatis, M.; Fubini, B. Langmuir 1999, 15, 5742-5752. (24) Buettner, G. R.; Oberley, L. W. Biochem. Biophys. Res. Commun. 1978, 83, 69-74. (25) Otero Are`an, C.; Barcelo`, F.; Fubini, B.; Fenoglio, I.; Llabre`s i Xamena, F. X.; Tomatis, M. J. Inorg. Biochem. 2001, 83, 211-216.

Surface Reactivity of Amphibole Asbestos contact between the sample and an electronic conductor was attained with a procedure reported by El Murr et al.,26 modified by a preliminary step in which the solid sample was intimately mixed with graphite in an inert solvent (acetone) and sonicated for 15 min (Liarre starsonic 35 80-180W).27 The asbestos/graphite weight ratio was 1:4. The solvent was then removed from the paste by heating at 473 K for 2 h, and some drops of 0.2 M K2SO4 solution and dodecane were added to the mixture, just to ensure conductivity. The working electrode, specifically designed, consisted of a poly(vinyl chloride) body containing a platinum disk, supporting the asbestos/graphite paste. The electrolytic cell contained a Pt counter electrode and a Hg/Hg2SO4 reference electrode, indicated as SSE. All reported potential values are referred to this SSE electrode, the potential of which is 615 mV with respect to the standard hydrogen electrode. All solutions were kept at constant ionic strength. The required pH values were obtained as follows: pH 7 by means of a 0.2 M K2SO4 solution and pH 0.5 with a 0.2 M sulfuric acid solution. All electrolytic solutions were prepared using analytical grade reagents and bidistilled water. Electrochemical measurements were run at ambient temperature by means of an AMEL system 5000 potentiostat and consisted of subsequent voltammetric cycles at a sweep rate of 5 mV s-1. This value was the best compromise between an appreciable intensity of the signal and a good peak resolution. The potential was scanned linearly (first in the negative then in the positive direction) between -1000 and +1000 mV/SSE. The redox cycle was repeated several times in order to investigate the shape of the curves and variations in peak intensity during cycling. Heat of Adsorption of Water. The heat of adsorption was measured, as reported in previous papers,28 by means of a TianCalvet microcalorimeter (Setaram) connected to a volumetric apparatus, which allows simultaneous measurement of uptake (na), heat released (Q), and equilibrium pressure (p) for small increments of water vapor contacted with the asbestos fibers. Subsequent doses of the adsorptive were admitted onto the sample, and adsorbed amounts, released heat, and equilibrium pressure were measured for each dose when the thermodynamical equilibrium was attained. Original and heat-treated asbestos fibers were directly outgassed in a vacuum in the calorimetric cell for 2 h at 400 °C, to remove from the surface any contaminants adsorbed from the environment, and subsequently located into a calorimetric vessel without exposure to the atmosphere. The temperature of the calorimeter was maintained at 30 °C throughout the adsorption experiment. A typical adsorption sequence comprised three subsequent runs, with the following procedure: (i) dosing successive amounts of water vapor onto the sample up to a defined equilibrium pressure, typically 10 Torr (Ads I), (ii) desorption at 30 °C under vacuum, and (iii) readsorption of similar doses up to the same pressure, to evaluate the fraction of adsorbate which is reversibly held at the surface (Ads II). Specific Surface. The surface area of the crocidolite and amosite fibers employed was measured by means of the BrunauerEmmett-Teller (BET) method (nitrogen adsorption at -196 °C, “Quantasorb” Quantacrome).

Results Effect of the Thermal Treatments on the Structure of the Fibers. Both asbestos fibers, as observed from XRD diffraction patterns (not reported for brevity), do not change their crystalline structure following thermal treatments at 400 °C. Conversely, a significant transformation of crocidolite and amosite takes place upon heating at 800 °C.25 This observation is consistent with the evolution of the surface area, determined by nitrogen adsorption (BET (26) El Murr, N.; Kekerni, M.; Sellami, A.; Ben Taarit, Y. J. Electroanal. Chem. 1988, 246, 1021-1026. (27) Prandi, L.; Bodoardo, S.; Penazzi, N.; Fubini, B. J. Mater. Chem. 2001, 11, 1495-1501. (28) Fubini, B.; Bolis, V.; Cavenago, A.; Garrone, E.; Ugliengo, P. Langmuir 1993, 9, 2712-2721.

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Figure 1. Effect of the heating on the specific surface area of (b) crocidolite and (9) amosite. Table 1. Iron Released after Incubation in Deferoxamine B and Ferrozine Solution from Both Crocidolite and Amositea

crocidolite CROC-400 CROC-800 amosite AMO-400 AMO-800

deferoxamine iron removed

ferrozine iron(II) removed

155 146 37 105 105 30

1.30 1.10 n.d. 5 2 n.d.

a Data are expressed as nmol of iron mobilized/milligram of solid. n.d. ) not detectable.

method). As shown in Figure 1, the specific surface of crocidolite slightly decreases upon heating at 400 °C (from 7.9 to 7 m2/g) while it is reduced to 5 m2/g upon heating at 800 °C (≈40% reduction). The amosite fibers appear more resistant to the thermal treatment, and the specific surface only decreases from 4.8 to 4.4 m2/g after heating at 800 °C (≈10% reduction). Therefore, both fibers undergo progressive surface and bulk modifications upon heating in air and these are more dramatic with crocidolite than with amosite. Iron Release. The effect of the thermal treatments on iron mobilization have been examined following a technique previously employed by Aust and co-workers.16,17 Two different chelators have been employed: ferrozine, which only extracts Fe2+, and desferioxamine, which binds Fe3+ and also favors oxidation of iron to Fe3+. The amount of iron mobilized from unheated asbestos fibers (both crocidolite and amosite) by ferrozine and deferoxamine was compared with that from fibers heated in air. The results, obtained after incubation for 3 days, are reported in Table 1 and show that the amount of total iron mobilized by deferoxamine is the same from the unheated and 400 °C heated fibers. With deferoxamine, iron was mobilized to a greater extent from crocidolite than from amosite. As crocidolite has a surface area higher than that of amosite (Figure 1), the two values are very similar (20 µmol/m2 in the case of crocidolite and 21 µmol/m2 in the case of amosite) when normalized per unit surface. As expected, in the presence of ferrozine more iron was mobilized from amosite, which has only Fe2+ in its structure, than from crocidolite. Heating at 400 °C modifies the amount of iron mobilized by ferrozine from both amosite and crocidolite, in agreement with the fact that most of the Fe2+ ions at the surface of the fibers are oxidized to Fe3+ after heating in air at 400 °C. The effect is more pronounced on amosite. The treatment at 800 °C causes a dramatic decrease in the amount of iron mobilized by deferoxamine from the fibers. Only 30% of the iron removed on untreated fibers

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absence of Fe2+. Exposed iron ions at the surface of mineral dusts can generate HO•, in the presence of H2O2, via the Haber-Weiss cycle30,31

Fe3+ + H2O2 f O2-• + 2H+ + Fe2+ H2O2 + O2-• f O2 + OH- + HO• The superoxide may also reduce Fe3+ to Fe2+, thus providing few Fe2+ ions for the Fenton reaction.

Fe3+ + O2-• f Fe2+ + O2 Fe2+ + H2O2 f Fe3++ OH- + HO• (Fenton reaction)

Figure 2. Free radical release from aqueous suspensions of heated asbestos fibers. EPR spectra of the [DMPO-HO•] adduct of (A) crocidolite and (A′) amosite and EPR spectra of the [DMPO-CO2•-] adduct of (B) crocidolite and (B′) amosite: (a) original sample, (b) sample heated in air at 400 °C, (c) sample heated in air at 800 °C.

Figure 3. Effect of ascorbic acid on the homolytic rupture of the carbon-hydrogen bond of crocidolite fibers. EPR spectra of the [DMPO-CO2•-] adduct of (a) the original sample, (b) the sample heated in air at 400 °C, and (c) the sample heated in air at 800 °C.

is mobilized from both asbestos heated at 800 °C. This decrease much exceeds that expected from the reduction in specific surface and indicates that with the onset of the phase transitions at 800 °C, iron ions become less accessible to the chelators. Free Radical Release. The EPR spectra obtained for both original and heated asbestos in contact with H2O2 are shown in Figure 2. They show the four-line spectra, with intensity ratio 1:2:2:1, hyperfine constants aN ) aH ) 15G, and a g value of 2.00, typical of the DMPO-OH• adducts. EPR patterns obtained on amosite and on crocidolite show an overall similar behavior. Both original fibers generate HO•, as previously reported.18 The thermal treatment at 400 °C (Figure 2, spectrum b) does not significantly reduce the intensity of the EPR signal, while after the 800 °C treatment (Figure 2, spectrum c), only a very weak signal is observed. Considering that after heating at 400 °C most of the Fe2+ ions at the surface of the fibers will be oxidized to Fe3+, the result reported in Figure 2 indicates that HO• may be generated even in the

Following incubation of crocidolite and amosite with the formate ion, the EPR spectra reported in Figure 2A′,B′ were obtained. The six-line spectra reported, with hyperfine constants aN ) 15.6G and aH ) 19G and a g value of 2.0055, are consistent with the DMPO-COO° adduct. Such spectra highlight hydrogen extraction by a homolytic rupture of the carbon-hydrogen bond. The original sample (Figure 2a′) is active, but if the samples are heated in air at a temperature of 400 °C or above (Figure 3, spectra b′ and c′) no radical yield is detectable, indicating that all radical-generating sites are destroyed following the surface modification taking place at a relatively low heating temperature, probably due to the mere oxidation of surface Fe2+ to Fe3+. The test with formate (cleavage of the hydrogen-carbon bond) has been also carried out on the same fibers in the presence of ascorbic acid. Significant differences in this case may be observed between the results from fibers heated at 400 and at 800 °C (Figure 3). The fibers heated only at 400 °C are reactivated in the presence of ascorbic acid, while those heated at 800 °C are still inactive. Note that the intensity of the EPR spectrum of the sample heated at 400 °C (Figure 3b) is similar to the original one (Figure 3a), indicating that reactive sites are fully regenerated by ascorbate. The additional two-line signal that appears in all spectra recorded in the presence of ascorbic acid is due, as expected, to the ascorbyl radical.19 Cyclic Voltammetry. Cyclic voltammetry was successfully employed27,32 to investigate the oxidation state and mobility of iron ions on crocidolite and amosite as a function of pH. Figure 4a reports the result (dotted line) obtained at neutral pH for a crocidolite-modified carbon paste electrode. Three couples of anodic and cathodic peaks (indicated in the figure by (a), (b), and (c)) are visible. Couple a (centered at -0.25 V) is ascribed to the reduction/ oxidation of iron ions in the asbestos structure which are progressively released in subsequent cycles. As the number of subsequent cycles increases, the current intensity associated with these peaks decreases, while a new couple of peaks (couple b), centered at 0.0 V, appears. Both the increase in intensity with the subsequent cycles and the redox potential of these peaks suggest that they are related to a redox process involving mobilized iron ions, whereby iron progressively leaves the solid and is released into the solution. When compared to the results obtained with crocidolite preheated at 800 °C, peak couples a and b are (29) Rouxhet, P. G.; Gillard, J. L.; Fripiat, J. J. Mineral. Mag. 1972, 38, 583-592. (30) Halliwell, B.; Gutteridge, J. M. C. Arch. Biochem. Biophys. 1986, 246, 501-514. (31) Halliwell, B.; Gutteridge, J. M. C. Free. Radical Biol. Med. 1995, 18, 125-126. (32) Shen, Z. H.; Parker, V. D.; Aust, A. E. Anal. Chem. 1995, 15, 307-311.

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Table 2. Water Vapor Adsorption on Variously Heated Crocidolite Fibers: Values taken at an equilibrium pressure of Water Vapor of 5 Torr and at 30 °C water vapor uptake (µmol/m2) crocidolite CROC-400 CROC-800

heat released (J/m2)

molar enthalpy of adsorption (kJ/mol)

total

reversible

irreversible

total

reversible

irreversible

total

reversible

irreversible

12.5 12.3 4.3

6.4 6.4 4.3

6.1 5.9

1.1 1.0 0.25

0.5 0.5 0.25

0.6 0.5

88 84 56

78 78 56

90 86

Figure 4. Cyclic voltammograms of a crocidolite-modified electrode (dotted line) and of a heated (at 800 °C) crocidolitemodified electrode (solid line) (a) at pH 7.0 and (b) at pH 0.5.

still present but we note that the cyclic voltammograms show a very different behavior, particularly as far as the intensity of the peaks is concerned. Heated crocidolite is less prone to release iron ions, as detectable from the voltammograms obtained after the same number of cycles. The same experiment on the two samples carried out at pH 0.5 is illustrated in Figure 4b. In both cases, iron mobilization is higher than at neutral pH and only the redox couples b and c can be detected. Here again, however, heated crocidolite differs from the unheated one. The peak due to iron mobilization on untreated fibers reaches a steady state in the first cycle, while with heated crocidolite, a steady state is reached only after 7-8 cycles, confirming that on the thermally modified fibers iron ions are more concealed and less accessible than on the original fibers. Heat of Adsorption of Water. Table 2 compares the amount of water vapor adsorbed, the released heat, and the energy of interaction on the original fibers and on the fibers heated in air at 400 and 800 °C. Data have been taken at an equilibrium pressure of water vapor of 5 Torr. The extent of reversible processes is measured by the second adsorption run (Ads II), while irreversible processes are obtained by subtracting data of adsorption II from the correspondent ones for total adsorption (Ads I). Figure 5

shows the volumetric isotherms (uptake vs equilibrium pressure) and the differential heat of adsorption of water vapor (heat vs uptake) on CROC, CROC-400, and CROC800 for Ads I, while Figure 6 compares the volumetric isotherms for the first and the second run (Ads I and Ads II) for CROC (Figure 6a) and CROC-800 (Figure 6b). The amount of water vapor adsorbed onto CROC and CROC400 is very similar, indicating that the thermal treatment at 400 °C in air, prior to outgassing in a vacuum at the same temperature, does not alter the degree of hydrophilicity. The treatment at 800 °C, conversely, decreases the amount of water vapor adsorbed down to less than 1/3 of that adsorbed at the same pressure values on unheated crocidolite (Table 2 and Figure 5a). Furthermore, upon heating at 800 °C, Ads I and Ads II coincide, indicating that no irreversible uptake of H2O takes place on such modified fibers (Figure 6b and Table 2), while the irreversible amount of water vapor fixed onto the surface of unheated crocidolite is about half of the total adsorption (Figure 6a and Table 2). On all samples, the energy of interaction decreases upon increasing coverage down to a plateau value: this behavior indicates an heterogeneity in the surface sites that interact with water vapor (Figure 5b). The higher the heating temperature, the lower the energy of adsorption: the plateau value attained at high coverage by the energy of interaction of water is 75 kJ/ mol on CROC but only 50 kJ/mol on CROC-800. Furthermore, the energy of interaction attains the plateau value at a much lower coverage (2.9 µ mol/m2) on CROC800 than on CROC (3.8 µ mol/m2), indicating that the hydrophilic sites which strongly interact with water on the surface of CROC-800 (heat > 44 kJ/mol) are much fewer than those of unheated crocidolite. The adsorption of water on a heated silicate surface is due to the superposition of several processes: (i) dissociation of water onto siloxane bridges (Si-O-Si) with formation of two adjacent silanols, as happens with pure silica surfaces;28 (ii) coordination of water molecules on the metal cations exposed; (iii) adsorption of water via H-bonding onto the surface silanols. The first is irreversible under the experimental condition adopted for adsorption (30 °C); that is, all siloxanes converted into silanols remain in this form if the sample is not heated again. In contrast, H-bonding is nearly fully reversible, that is, by evacuation of the gas phase the adsorbed water is mostly desorbed. Coordination of water on poorly coordinated cations may be either reversible or irreversible, depending on the charge density on the cation. The adsorption of water on heated samples is mostly determined by the effect of the progressive condensation of silanols into siloxanes and the embedding of metal ions during the thermal treatment. At 400 °C, some of the silanols are condensed into siloxanes but this transformation is largely reversible. Upon contact with the surface, water molecules are dissociated into silanols (Figure 5a). In contrast, on CROC800 most of the siloxane bridges are stabilized and remain in this form upon contact with substantial amounts of water and some metal ions are embedded in the silicate matrix, with loss of coordination valencies available for water adsorption. The total amount of water vapor adsorbed thus decreases and no irreversible processes take place (Figure 6).

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Figure 5. Adsorption of water vapor on crocidolite: (a) amount of water adsorbed; (b) energy of adsorption as a function of uptake in the first adsorption run; (9) crocidolite, (b) crocidolite heated in air at 400 °C, (2) crocidolite heated in air at 800 °C.

Figure 6. Adsorption isotherms of water vapor on crocidolite asbestos: (a) crocidolite; (b) crocidolite heated in air at 800 °C; full symbols, total adsorption (Ads I); empty symbols, reversible adsorption (Ads II).

Crocidolite Heated at 800 °C with Kaolin. The treatment with kaolin at 800 °C makes the surface of the sample even less reactive than the simple heating at 800 °C: the amount of iron released is reduced to half (20 nmol/mg) of that found on CROC-800. Nearly no HO• yield was obtained with crocidolite mixed with kaolin and heated in air at 800 °C. The radical activity of the fibers heated at 800 °C is however so low that in the present conditions, any further inactivation caused by the presence of kaolin cannot be clearly evidenced. Discussion The modifications taking place at the surface of amphibole asbestos fibers heated in air at 800 °C have been examined with various techniques, all of which indicate a dramatic loss of reactivity with respect to the unheated fibers or those only heated at 400 °C, that is, below the temperature where the onset of the phase transition is expected. The specific surface decreases, suggesting a sort of shrinkage of the surface around the fibers, but most of the phenomena reported much exceed what is expected by the simple reduction in surface area. The mobility of iron is seriously affected. Fewer ions are mobilized by chelators, and even under a variable electric potential, the iron ions on the 800 °C heated samples are concealed and do not contribute to the voltammograms recorded. The heated surface shows a much lower affinity to water adsorption. This is partly due to a sort of embedding of metal ions that, under the thermal treatment and the consequent structural modifications, saturate their coordination valencies within the silica framework, thus losing the potential for strong water coordination. Extraframe formation of iron oxyhydroxide, if any, would also contribute to this effect. The irreversible conversion of silanols into siloxane bridges at the silica sites also

causes a progressive hydrophobization of the heated surface, as reported for pure silica.33 Contrary to silica, the overall surface of CROC-800 is still hydrophilic, probably because of the alternate presence of silica tetrahedra and metal ions, but the absence of any irreversible uptake of water indicates that no dissociation may take place on siloxane bridges and no iron ions are available at the surface for a strong coordination of water. Surface iron ions in a poor coordination state constitute the metal centers where NO is adsorbed and where free radical reactions are catalyzed.23 They are the first ions to be removed by chelators. After the 800 °C thermal treatment, indeed, both reactions are fully suppressed and cannot be regenerated, confirming the fully irreversible modification of the active iron sites. Accordingly, the DNA damage caused by CROC-800 and AMO-800 was much less than that caused by unheated or less heated fibers.25 In conclusion, a relatively short (2 h) thermal treatment at 800 °C in air of amphibole asbestos fibers modifies surface reactivity by reducing the chelator-assisted release in solution of iron ions and suppressing the generation of free radicals at the fiber surface, which are both regarded as the physicochemical reactions implied in the mechanisms of asbestos carcinogenicity. Cellular tests and an appropriate in vivo validation are required in order to confirm that the heated fibers have indeed lost their carcinogenic potential. The present data, however, are quite encouraging in seeking in thermal treatments in mild conditions of the asbestos fibers (possibly mixed with other materials such as kaolin) new ways to inactivate asbestos, milder and easier than those currently in use. LA011609W (33) Fubini, B. Ann. Occup. Hyg. 1998, 42, 521-530.