Chem. Res. Toxicol. 1994, 7,451-457
451
Factors That Influence the Formation and Stability of Hydrated Ferrous Sulfate in Coal Dusts. Possible Relation to the Emphysema of Coal Miners Xi Huang,? Roger Zalma, and Henri Pezerat* Laboratoire de Rkactivitk de Surface et Structure, Universitk P. et M . Curie, 4, Place Jussieu, 75252 Paris, Ckdex, France Received August 30,1993g
Epidemiological studies have shown that a causal relationship may exist between coal dust exposure and emphysema in coal miners. Emphysema can be considered as one of the human pathologies associated with oxidative stress, resulting from oxidant-induced al-antitrypsin (a1AT) inactivation and uncontrolled proteolysis of lung tissue. We have previously reported that certain coal dusts contained hydrated ferrous sulfate (FeS04) that inactivated al-AT [Huang et al. (1993) Chem. Res. Toxicol. 6,452-4581. In the present study, we have shown that the FeS04 originated from oxidation of pyrite (FeSz), which is a typical contaminant of coal dusts. The relative humidity and microenvironment around individual pyrite particles influence the formation of FeSO4 in the coal. However, the subsequent human exposure to coal dust containing FeS04 depends on the stability of the formed FeS04. We found that pH played the most important role in stabilizing the FeS04, such that a final pH < 4.5 after oxidation of pyrite stabilized FeSO4, whereas at high pH the conversion of reactive Fe2+to Fe3+was immediate. Sulfuric acid (HzSOd), which is also produced by the oxidation of pyrite, can lower the pH, but it can also be neutralized by other minerals in coal dusts, such as calcite (CaC03). The stability of FeSO4 in coal dust can also be influenced by the length of exposure to air. Our studies demonstrated that coal samples differed in their capacity to stabilize FeSO4. This current study strengthens our previous reported hypothesis that emphysema, which occurs irregularly in coal miners, could be directly related to exposure to coal dust containing FeS04.
Introduction Epidemiological studies of mortality and morbidity in coal miners have identified three pathologies that are related to coal dust inhalation: pneumoconiosis, chronic obstructive bronchopneumopathy such as emphysema and/or chronic bronchitis, and stomach cancer (1-3).These three pathologies are irregularly distributed among coal miners who worked in different coal basins, with little knowledge of the reasons for the differences in disease incidence (4). Quartz, which is often considered to be important in coal miners’ disease,plays only an aggravating role in pneumoconiosis (5). The coal dust features that cause these varied pathologies have not yet been identified. We have found that certain coal filtrates contained FeSO4 that was capable of oxygen radical formation and inactivation of al-antitrypsin (al-AT)l(6, 7). This effect of these coal filtrates is similar to that of cigarette smoke which may also trigger emphysema (8-11). Although hydrated ferrous sulfate (FeSO4)may originallybe present in some coal mines, the majority of the FeS04 is more likely to be formed by the oxidation of pyrite (FeS2)during mining operations at high relative humidity according to the reaction: FeS,
+ 0, + H,O
-
FeSO,
+ H2S04
(1)
It is well-known that pyrite is a typical contaminant of coal dusts (12). Previous studies have shown that pyrite-
* To whom correspondence should be addressed. t Present address:
Nelson Institute of Environmental Medicine,New York UniversityMedical Center,Long MeadowRoad, Tuxedo,NY 10987. *Abstract published in Aduance ACS Abstracts, April 15, 1994.
rich coals tend to undergo low-temperature oxidation. This oxidation may give rise to mining complications including acid mine drainage from abandoned mine faces and coalrefuse piles, roof weaknesses, pillar collapses in underground mines, and spontaneous combustion of stock piles (13-15). Multiple physicochemical factors, such as oxygen partial pressure, temperature, and relative humidity play a role in pyrite oxidation (12,16,17), and these conditions are likely to vary in different coal mines. Our initial studies were based on the hypothesis that hydrated ferrous sulfate,principally FeSO4-7H20,resulting from the oxidation of pyrite in certain coals, may be inhaled in coal dust where it is converted to Fe3+ in lung tissue, resulting in the transitory appearance of reactive oxygen species (ROS) (6,7).These ROS can inactivate al-AT as we have previously shown (6) and may be a potential trigger of emphysema in coal miners. However, the capacity for inhaled coal dusts to produce ROS in lung tissue depends on the content of formed FeSO4 in the dusts. The scope of our previous investigations was extended in the current study to examine the influences of pyrite particle size, humidity, pH, and air exposure on the formation and stability of FeS04 in coal dusts. We began with pure pyrite and then mixed various coal samples with the pyrite to study the formation of FeSO4 as indicated by the evolution of oxidizing activity under controlled experimental conditions. Using ESR,ROS generated by pyrite or the coalpyrite mixtures were detected by measuring their ability to react with formate as we have previously described (6). ~
~~~
Abbreviations: al-AT, al-antitrypsin;a.u., arbitrary unit; DMPO, 5,5-dimethyl-l-pyrrolineN-oxide; HBL, HouilBres du Baesin de Lorraine; ROS,reactive oxygen species.
0893-228~/94/2707-0451$04.50/0 0 1994 American Chemical Society
452 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
The stability of FeS04 in coal dusts was evaluated with bipyridine, which forms a complex detectable by spectrophotometer. Our studies demonstrated that the coals from different mines had variable capacities to stabilize FeS04 as a function of pH.
Materials and Methods Chemicals. 2,2'-Bipyridine, o-phenanthroline monohydrate, potassium thiocyanate, and sodium formate were purchased from Merck (Paris, France). 5,5-Dimethyl-l-pyrrohe N-oxide (DMPO) was obtained from Sigma (Paris, France). Pyrite was obtained from the gold mine of Salsigne (France) and was characterized by X-ray diffraction. This pyrite contained only traces of quartz (18).
Four different French coal samples obtained from Gardanne, MBricourt, Escarpelles, and La Mure mines were provided by the Centre #Etudes de Recherche8des Charbonnages de France (as described in ref 6) and were stored under argon. Pyrite was present in small quantities in these coal samples (1%). Detailed physicochemical characteristics of these coal samples were previously described (6, 19). Sixteen other coal samples, in block form, were kindly provided by Dr. Mahieu, Chief of Medicine of the coal mines of Houillares du Bassin de Lorraine (HBL). Two samples from each of 8 basins a t these mines were randomly extracted. These blocks of coal were broken in air and then immediately stored under argon. Evolution of Oxidizing Activity of Pyrite under Controlled Conditions. The influence of pyrite particle size and relative humidity on pyrite oxidation to FeSO4 was studied as follows: Ten grams of fresh pyrite was respectively ground for 15 and 30 min, in air, in a mechanical mill. The powder was then divided, placed in the upper part of three desiccators containing saturated aqueous solutions of NaOH, MnC12.4H20, or BaC1.j2H20, and left to age (to be oxidized by oxygen). These saturated solutions were allowed to establish relative desiccator humidities of 5.7%, 57.6%, and 89.8%, respectively. The oxidizing activity of aged pyrite was periodically examined as previously reported (6,20). In brief, 45 mg of the particles or 0.5 mL of coal filtrates recovered from a suspension of 45 mg of solid in 1mL of distilled water was added to a reaction (total volume of 2 mL) containing 1M sodium formate and 50 mM DMPO in 250 mM phosphate buffer (pH 7.4). Aliquots of this suspension were withdrawn 5 min after the addition of all reagents and were filtered through 0.65-pm filters (Cellulose Acetate, Prolabo, Paris, France). Controls contained 0.5 mL of distilled water instead of particles or filtrates. The detection of the radical adduct DMPO,C02'was performed by ESR as previously described (6,20). The active compound(s) in the aged pyrite were extracted by distilled water and then crystallized and analyzed by IR and X-ray diffraction according to standard protocols. Evolutionof Oxidizing Activity of Coal-Pyrite Mixture. The coal samples we studied were originally obtained for coal conversion studies, and the coal samples were withrawn from the center of only one seam in each mine. Hence, the pyrite content is very weak and not representative of the content in the entirety of each coal mine. In order to study the differences in the physicochemical properties of pyrite oxidation in the coals, we used a predetermined percentage of pyrite without taking into account the original pyrite content of each coal sample. Three coal samples from the Gardanne, Escarpelles, and La Mure mines were separately ground for 30 min in air. Coal particles (1.8 g each) were mixed with 200 mg of freshly ground pyrite (10% by weight) in a dish and were then left to age in a relative humidity of 89.8%. The oxidizing activities of the coal-pyrite mixtures were periodically examined by ESR, and the pH of these dry mixtures was measured after suspension in distilled water. Studies on the Oxidizing Activity of the Coals Enriched with Aqueous Extracts of Aged Pyrite. To mimic certain mining conditions such as the presence of water in the coal mines, the four coal samples were also mixed with aqueous extracts of
Huang et al. aged pyrite which had formed some FeSO,. Five grams of each of the four ground coal samples were suspended in 10 mL of an aqueous extract recovered from 250 mg of pyrite powder that was aged in air for one and a half years. The suspension was evaporated to dryness and then was left to age in air. The oxidizing activities of these enriched coals were periodically examined by ESR. The water-soluble Fe2+ in these samples was extracted by distilled water a t 37 "C for 15 min and then filtered twice through 0.65-pm filters (Cellulose Acetate, Prolabo, Paris, France). The aqueous filtrate containing Fez+ was detected by the formation of 2,2'-bipyridine-Fe2+ complexes which can be analyzed by spectrophotometer (520 nm). The quantity of Fe2+ was determined by comparison with the standard OD curve obtained using commercial FeS04.7HzO (Merck, Paris, France). Acidification of the Coals by Addition of HISO,. It was previously reported that pH 4.5 was the critical pH for oxidation of Fe2+(21). Since the oxidation of pyrite also produces the acid toconsume HdOd, it wasinterestingtostudythecapacityofcoals H2SO4 to yield a lower pH. Ground coal samples (2.7 g each) were suspended in 30 mL of distilled water, and the necessary volume of 1M H2SO4 was added to lower the pH to 4.5. Studies on the Evolution of Oxidizing Activity of HBL Coals. To verify our findings on the aging process and the importance of the pH which can allow coal samples to gain oxidizing activity during a period of time, eight of the sixteen coal samples from the HBL coal mines were also studied. The iron content in these coal samples was analyzed by atomic absorption, and the pHs of aqueous suspension of these coals were measured. The divalent iron (% FeO) in the coal dusts was obtained by volumetric redox reaction with potassium dichromate after a sulfo-fluohydric attack in an inert atmosphere. The total iron (% Fe203) was analyzed in hydrochloric acid after a fluosulfuric oxidation. The iron in pyrite was not specifically measured and included only in total iron. Four pairs of these coals were selected for the aging studies. To mimic real mining conditions, the coal samples were coarsely ground in air and then left to age under ambient atmospheric conditions. After 56 days, the oxidizing activities of these coal samples were studied using ESR with DMPO as a spin-trapping agent.
Results Evolution of Oxidizing Activity of Pyrite under Controlled Conditions. The effect of relative humidity on the oxidizing activity of pyrite is presented in Figure 1. The oxidizing activity was evaluated by DMPO spin trapping of reactive oxygen radicals. Freshly ground pyrite is not capable of oxidizing formate, but its oxidizing activities were increased as a function of relative humidity and aging. When pyrite was aged in a relative humidity of 57.6 5% or 89.85% , it became very active by the end of 2 months. In contrast, when the same pyrite was aged in a low relative humidity (5.8%1, it did not begin to acquire activity before 6 months. Figure 2 shows the effect of grinding on the oxidizing activity of pyrite. Pyrite that was ground for 30 min exhibited higher oxidative activity than pyrite that was ground for only 15 min. Identification of the Active Compound in the Filtrate of Aged Pyrite. A powder was obtained after evaporation of each of the aged pyrite filtrates. By comparison of IR spectra, this powder was shown to be identical to commercial ferrous sulfate (6). X-ray diffraction also yielded a pattern similar to that of FeS0q7H20;however, an additional phase was also noted. This is probably due to the presence of a small quantity of another hydrated sulfate. oBhenanthroline and potassium thiocyanate studies confirmed the presence of an abundant quantity of Fe2+and showed only small quanti-
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 453
FeSOl in Coals and Its Relevance t o Emphysema
Table 1. Evolution of Oxidizing Activity and pH of CoalPyrite Mixture
A 70004
d
~_____
coal sample Gardanne suspension filtrate
P /
i
/
initial final PHO pHb 8.17 7.19 d
-
Escarpelles suspension 6.53 3.04 filtrate La Mure suspension 7.18 3.05 filtrate -
________~
DMP0,COp intensity (a.u.)c 0 P
8 c
-50
-
e
-
-
27 65
130 -
920 -
c
c
1550
91 120 c
970 2000 70 1730
pH of aqueous coal suspensions before aging (45mg in 2 mL of distilled water). pH of suspensionsof the coal-pyrite mixturesafter 91 days of aging (45 mg in 2 mL of distilled water). C Intensity of the DMPO,C02'- signal shown by suspensions of pyrite-coal mixtures (45 mg) or by filtrates (equivalent to 22.5 mg) after 0,8,27, and 91 days of aging. *Undetectable. e Not tested. (I
*
Time (months)
Figure 1. Evolution of oxidizing activity of pyrite as function of relativehumidity and time. The pyrite was mechanicallyground in air for 15min and then left to age in differentrelative humidities of 89.8% (O),57.6% (m) and 5.7% (A). The oxidizing activity was measured by spin trapping of DMPO,COt*-viaESR (Varian CSE instrument). The constants for DMPO,C02*- were as follows: g = 2.0055, aN = 15.6G, aH = 19.OG. The ESR specifications were as follows: field set 3380 G, scan range 100 G, microwave power level 10 mW, time constant 1s. lo00 a.u. corresponds to 4.14 X 10'8 radicals/L.
a
t
Q-
JI,
Time (days)
7000
Figure 3. Evolutionof the oxidizingactivityof the coals enriched with aqueous extract of aged pyrite as a function of time of exposure to air. Five gramsof eachof the four groundcoalsamples was suspended in 10 mL of an aqueous extract recovered from 250 mg of pyrite powder that was aged in air for one and a half years. The coal mixture suspension was evaporated to dry and then was left to age in air. The oxidizing activity of these enriched coals was periodically examined by ESR. (m) Escarpelles, (A) MBricourt, (0) La Mure, ( 0 )Gardanne.
Time (months)
Figure 2. Effect of the grinding time on the evolution of the oxidizing activity of pyrite in a relative humidity of 89.8%.( 0 ) Ground for 30 min and (0) ground for 15 min. ties of Fe3+. Therefore, the active compound in the filtrate of aged pyrite was primarily F e S 0 ~ 7 H z 0with minor contamination by Fez(SO4)s . Oxidizing Activity of Coal-Pyrite Mixtures. The oxidizing activity of suspensions or filtrates of three coals (Gardanne, MBricourt, and La Mure) mixed with 10% of pyrite was measured after 0,8,27, and 91 days. For the Escarpelles and La Mure coal samples that did not originally exhibit oxidizing activity, strong oxidizing activity was acquired (Table 1). Interestingly, the initial pH values of these two coal suspensions were 6.53 and 7.18, respectively, but were lowered (pH < 3.1) after 91 days of aging with pyrite. In contrast, the pH of aqueous suspension of coal from Gardanne, which was 8.17 prior to pyrite addition, maintained a pH greater than 7. This
Gardanne coal in suspension or in filtrate form did not show a significant formate-oxidizing activity (Table 1). We also noted a large difference in the oxidizing activities of the filtrates and suspensions from the La Mure coal mine. Interestingly, when La Mure coal particles without prior aging with pyrite were added to the above filtrate, the oxidizing activity in this filtrate disappeared. The same result was noted to a lesser extent with coal from Escarpelles, although the quantity of coal in the filtrate assays is equivalent to only half of that in the suspension assays. The data suggest that the coal itself may prevent ROS formation or destroy the preserved short-lived ROS. Oxidizing Activity of the Coals Enriched with Aqueous Extracts of Aged Pyrite. The oxidizing activity of aqueous suspensions of various coals enriched with aqueous extracts of aged pyrite is shown in Figure 3. The coal from Escarpelles presented strong oxidizing activity (1760 a.u. at 5 days) that slowly decreased as a function of duration of exposure to air. The coal from MBricourt exhibited moderate oxidizing activity (525 a.u. at 3 days) that also slowly declined. The coal from La Mure showed a low level of activity 3 days after enrichment; however, this activity completely disappeared. The
454 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Huang et al.
Table 2. Stability of F$+ in the Coal Samples Enriched with Aqueous Extracts of Aged Pyrite and the Final pH of the Coal Suspensions. aging time Fez+ Fez+ final ( % of coal)b (% of total Fe2+)c pHd (days) f 7.61 Gardanne 56 fe 8.6 4.61 MBricourt 46 0.030 15.3 3.51 La Mure 47 0.053 Escarpelles 3 0.19 53.5 f 57 0.13 37.7 3.56 a Five grams of each coal were suspended in 10 mL of aqueous extract from 250 mg of aged pyrite which contained 17.5mg of soluble Fez+as measured by bipyridine. b Percentage of available Fez+after coal enrichment by aqueous extract of aged pyrite per 100 g of coal. Percentage of available Fez+ after coal enrichment per total Fez+ (17.5 mg). pH of the aqueous coal suspensions at the indicated time. e Undetectable. f Not tested.
seams. It is also noteworthy that the oxidizing activities of these coals were not directly correlated with their percentage of Fez+ (as presented in % of FeO) in these coal samples. This can be explained by the fact that most of the Fez+was present in phyllosilicates in the inorganic portion of the coal dusts and was not accessible to oxidation. The oxidizing activity that we measured after aging was dependent on FeS04 content which resulted from pyrite oxidation. We also note that the coals that had an initial pH less than 4.9 showed oxidizing activity after aging. The low initial pH can favor the appearance of oxidizing activity, but this is not essential since the pH can also be decreased by the HzSO4 produced during pyrite oxidation.
Table 3. Necessary Volume of 1 M HzSO, To Reduce the pH of Aqueous Coal Suspensions to pH 4.5. DMPO,C02'coal samples 1M (NL) intensity (a.u.) fb Gardanne 3340 220 200 MBricourt La Mure 170 160 70 Escarpelles 30
Epidemiologistshave observed that the incidence of coal miners' pneumoconiosis varies for different mines and that the relationships between pneumoconiosis and exposure to dusts from coals of different rank have been determined (1,2,22,23). The risk of a miner's death as certifiably due to emphysema is directly related to his cumulative lifetime exposure to respirable coal dust (24). Since coal mine dust is a complex mixture of minerals and organic macerals, it is difficult to identify these components that play a role in the development of emphysema in coal miners. Emphysema is a human pathology that can be considered to be associated with biological mechanisms of oxidative stress. Oxidative inactivation of q - A T by various oxidants may lead to uncontrolled proteolysis of lung tissue, hence emphysematous lesions (9-11). Our previously reported results strongly suggest that emphysema, which occurs irregularly in coal miners in different mines, could be directly related to exposure to coal dusts that are capable of releasing FeS04 in the pulmonary medium (6). We have shown in this study that FeS04 originated from pyrite oxidation in the presence of 02 and HzO. After inhalation of coal dust containing FeS04, conversion of reactive Fe2+to Fe3+ (by reducing oxygen in the pulmonary medium) may result in the transitory appearance of ROS that can inactivate q - A T in extracellular medium and damage lung tissue. Overall, the probability of developing coal-induced emphysema depends on the probability of inhaling coal dust containing FeS04 and also depends upon the stability of the formed FeS04 in the coal dust. Coal is well-known as a sedimentary rock composed principally of macerals and silicates, carbonates, sulfides, sulfates, and other mineral materials. The inorganic portion of coal can range from a few percent to more than 50% (by weight) and can vary from one location to another (25). Although some FeS04 may exist since the beginning of coal formation, most of the FeSO4is likely to be obtained from pyrite oxidation during mining operations (12). Our first approach was to study the factors that influence the formation of FeS04 in coal. We began with pure pyrite, and mixed different coals with this pyrite to study the evolution of oxidizing activity of aged pyrite in the presence of these coals. The studies with pure pyrite show that relative humidity can greatly influence the kinetics of FeS04 formation and the oxidizing activity of pyrite. We also observed that the particle size of pyrite may affect its oxidative activity as detected by ESR DMPO,CO2- signals. The oxidizing activity resulted from the formation of a coating of hydrated FeS04, which was identified by IR
Coal samples (2.7 g) were suspended in 30 mL of distilled water, and then 1M HzS04 was added until the pH was reduced to pH 4.5. Undetectable. @
*
oxidizing activity of the coal from Gardanne was the same as that of the control. The quantity of water-soluble Fe2+in these enriched coals was measured by adding bipyridine to their aqueous filtrates. Table 2 shows the percentage of Fez+stabilized in these enriched coals and the pH of the aqueous coal suspensions after various days of exposure. The available Fe2+in the coal samples can be logically classified in the same order as the oxidizing activity shown in Figure 3, except for the coal from La Mure. The data suggest that this coal can effectively stabilize Fe2+and either prevent oxidant formation or destroy the formed oxidants. Quantity of HzSO4 Needed To Decrease the p H of Coal Suspensions to 4.5. We have found that the formation of HzS04 by pyrite oxidation may influence the life time of FeS04. Table 3 shows the necessary volume of 1 M HzS04 required to decrease the pH of the coal suspensions to pH 4.5. Again, the coals are ordered by this classification in the same rank that was found for their capacity to stabilize FeS04 (Table 2). Moreover, all filtrates from these acidified coal suspensions, except that from Gardanne coal, presented oxidizing activity (Table 3). This oxidizing activity disappeared in the presence of o-phenanthroline (10 mM) or desferol (6.75 g/L, Ciba, Paris). These results suggest that H2S04 produced by pyrite oxidation not only decreases the pH and stabilizes the formed FeS04, but also releases the acid-soluble Fez+ from the coal dusts. Studies on the Evolution of Oxidizing Activity of HBL Coals. Table 4 shows that initially all of the studied coal samples did not have oxidizing activity, but after 56 days of exposure to air, some oxidizing capacity could be acquired by some of these coals. In all cases where two coal samples were extracted from the same coal basin, we have noted that these samples exhibit different characteristics, such as pH and percentage of Fe2+ or Fe3+. This is a clear demonstration of the heterogeneity within coal
Discussion
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 455
FeSOd in Coals and Its Relevance to Emphysema
Table 4. Evolution of Oxidizing Activity of HBL Coals and Their pHs DMPO,C02*-intensity (a.u.)c initial D H ~ 0 56 coals FeO (5%)" Fez03 (%la 0.38 f 0.13 0.42 0.17 6.81 t Louise 1 0.42 f 0.31 Louise 2 5.89 110 0.31 h 0.23 2.62 1.18 1.67 f 0.36 4.83 190 FD Etage 950 1 t 0.79 0.37 0.93 0.43 6.62 FD Etage 950 2 155 2.85 0.58 4.08 1.52 4.82 La Houve 1 t 1.96 0.79 3.71 1.75 5.78 La Houve 2 t 0.55 0.18 0.70 0.24 7.19 Puit Nord 1 3.24f 1.14 60 1.53 h 0.08 Puit Nord 2 4.90 a The analyses of iron were performed three times and are given in divalent iron (% FeO) and total iron (5% Fez03). The iron of pyrite was included in total iron. b pH of aqueous coal suspensions before aging. Intensity of the DMPO,C02'- signal shown by suspension of coal after 0 and 56 days of aging. d Undetectable.
*
* * *
and X-ray diffraction (18). This confirmed our previous reports (6). In previous studies (26,27),pyrite, chalcopyrite, and pyrrhotite were reported to be the most readily oxidized minerals, forming a variety of iron (ferrous and ferric) sulfates that eventually form goethite. The oxidizing activity of aged pyrite was also studied in relation to the incidence of lung cancers among metallic miners (28). The oxidation of mixtures of pyrite and different coal samples under controlled conditions showed behavior that differed in several respects. For example, FeS04 was absent in the coal from Gardanne, and no significant oxidizing activity was observed even after mixing with 10% pyrite and 3 months' aging (Table 1). During this time, the pH of this mixture remained high (pH >7). In contrast, the coal samples from MBricourt, Escarpelles, and La Mure showed strong oxidizing activity a few days after mixing with pyrite, and the corresponding pHs were seen to be lowered. Regarding the highest ranked coal in our studies, that from the La Mure mine, a 0.5-mL filtrate from 22.5 mg of the coal presented a much higher oxidizingactivity than that produced by 45 mg of this coal in suspension (Table 1). This indicates that this highly ranked coal may either prevent the formation of the oxidizing species, or may destroy the formed oxidizing species and the free radical adduct DMPO,C02'-. This effect may explain the overall weak oxidizing activity of coal from La Mure, as seen in Figure 3. This effect of high-rank coals may be due to their reducing properties as previously observed by Nickel et al. (29). Our second approach was to study the stability of the formed FeS04 in coal dusts. Our results have shown that the pH of the coal dusts was the most important stabilizing factor. In fact, it has been shown by others that the rate of oxidation of Fe2+ by oxygen in abiotic systems is a function of pH (17, 21). At pH values greater than 4.5, the kinetics of oxidation of Fe2+were as follows (21): d[Fe2+l/dt = k[Fe2+l[021 [OH-12
(2)
where k = 8.0 X 1013L2 mol-2 atm-I min-l a t 25 "C. At pH values below 3.5, the reaction proceeds at a rate independent of pH, that is d[Fe2+l/dt= k'[Fe2+l[021
(3)
where K' = 1.0 X lo-' atm-l min-I at 25 OC. Our results show that the lifetime of formed FeS04 greatly depends on the pH and ranges from several days to 10 days or longer, according to the ambient conditions (pH,the accumulated quantityof Fe2+,etc.). This finding was strengthened by our studies on the coals enriched
with aqueous extracts of aged pyrite and on the eight coal samples from HBL mine (Tables 2 and 4). In this study, we showed that only the coals with low pH after pyrite oxidation could stabilize the formed FeS04 and hence oxidize formate. The initial low pH can favor the stabilization of FeS04, but is not a determinant factor (Table 4, i.e., Louise 2). In general,two factors are essential for oxidizing activity to develop after aging of coal dusts. First, the coal dusts must contain pyrite. Second, the buffering capacity of the coal dusts must be low in order that HzS04 produced by pyrite oxidation can sufficiently lower the pH to 4.5 and stabilize FeS04. Finally, the produced HzS04 can further release acid-solubleFe2+from the coal dusts and enhance their oxidizing potential. In summary, the damaging effect of coal dusts depends on many factors related to the physicochemical properties of the coals. The formation and stabilization of FeS04 were seen to vary from one place to another due to the heterogeneity of coal samples taken from different areas of certain mines. The pH of a particular coal depends not only upon its origin, but also upon its geological environment (water, bacteria, rock, etc.) during coalification. The systematic asociation of mineral compounds with organic compounds also plays an important role. Coals rich in calcite (CaC03) have high pHs, whereas coals rich in kaolinite and/or illite have rather low pHs. The functional chemical groups (carboxyl, phenol, or quinone) on the coal's surface may also influence the pH of a particuliar coal. These functional groups are also related to the coal rank, which is defined as the ratio of C/H (25). Our studies have shown that the acidity of a particuliar coal is not constant and can vary for different coal mines over a period of time. This can be explained by the ongoing oxidation of coal and its accompanying minerals, such as pyrite. The four coal samples studied here showed large differences in their consumption of 1 M HzSO4 in order to reach the critical pH 4.5 required for oxidation of Fe2+ (Table 3 ) (21). This indicates that the buffering capacities of these coal samples are very different. The coal sample from Gardanne contained a large quantity of calcite (CaC03) which can neutralize the H2SO4 resulting from pyrite oxidation. This yields a less harmful salt, calcium sulfate. When the pH remained high (pH >4.5), the oxidation of Fe2+to Fe3+was immediate. Consequently, there is no accumulation of FeS04 in the coal. On the other hand, coal samples with a low pH after pyrite oxidation were capable of stabilizing FeSOl and releasing acid-soluble Fez+ in coal dusts and, hence, were capable of producing ROS that could inactivate q - A T (6).
456
Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Our studies show that factors other than pH can influence the stability of FeS04 in coal. The oxidizing activity of coal samples mixed with pyrite, or enriched with aqueous extracts of aged pyrite, decreased as a function of the duration of exposure to air. This indicates that the formation of FeS04 is a dynamic process and that the oxidizing activity generated by coal samples can vary from the time when the coal dust is extracted to the time when it is tested in biological studies. Overall, the FeS04stabilizing capacity of the four French coal samples examined in this study can be classified in increasing order as the following: Gardanne
