Two years’ air mutagenesis monitoring in a northwestern rural area of Italy with an industrial plant

Mutation Research, 319 (1993) 293-301 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00

293

MUTGEN 01939

T w o years' air m u t a g e n e s i s m o n i t o r i n g in a n o r t h w e s t e r n rural area o f Italy with an industrial plant R. Scarpato a, F. Di Marino a, A. Strano a, A. Curti c, R. Campagna d, N. Loprieno a, I. Barrai b and R. Barale b "Dipartimento di Scienze dell'Ambiente e del Territorio, Univ. Pisa, Italy, b Dipartimento di Biologia Evolutiva, Univ. Ferrara, Italy, c Ecologia, Protezione Ambientale e Sicurezza, ACNA Chimica Organica, Cengio, Italy and d Ambiente, Istituto Guido Donegani, Novara, Italy

(Received 27 January 1993) (Revision received 19 May 1993) (Accepted 11 June 1993)

Keywords: Airborne particulate mutagenicity; Ames test; Urban, industrial and rural areas; Chemical plant; Vehicular traffic; Italy

Summary The mutagenicity of organic extracts from inhalable airborne particles, collected in a northwestern rural area of Italy in which an industrial plant producing chemical intermediates is present, was assessed during the years 1989 and 1990. The Ames plate test with Salmonella strains TA98 and TA100 with and without metabolic activation was used. Eight sites in the first and three sites in the second year were monitored once and twice a month respectively. Results show that the mutagenicity of air particulate matter reaches maximum values in the cold months and is not dependent on plant activities. In addition, a correlation analysis between mutagenicity data and number of vehicles seems to indicate traffic emissions as the main source of mutagens.

Particulate matter from polluted air contains several classes of known mutagens and carcinogens such as mineral fibers, polycyclic aromatic hydrocarbons, the latter's nitro and oxygenate derivatives and nitrosamines (Crebelli et al., 1991). Mutagenic activity of many chemical compounds has been found to correlate with carcinogenicity data (Ashby and Tennant, 1991); therefore, mutagenic assays, in particular the Ames

Correspondence: Dr. R. Barale, Dipartimento di Scienze dell'Ambiente e del Territorio, Genetica, Universit~ di Pisa, Via S. Guiseppe 22, 1-56100 Pisa, Italy.

test, are widely employed in detecting potential genotoxic effects of complex environmental mixtures (Krewski et al., 1992). To date, few mutagenicity studies have been performed on airborne particles from rural areas (Alink et al., 1983; Takeda et al., 1984). High mutagenicity levels of organic extracts have been found in air monitoring from a rural area in Italy over a period of a year, whereas other investigations of this type have reported negative or only slightly positive results (Pitts et al., 1977; Alfheim and MOiler, 1979; Reali et al., 1984). Several studies carried out on urban and industrial areas have indicated that airborne mutagens originate from combus-

294 tion processes related to different sources (domestic heating, electric power production, industrial plant emissions and vehicular traffic) (Motykiewicz et al., 1988; Barale et al., 1989; Miguel et al., 1990). However, these compounds released into the atmosphere may undergo transformations due to physical agents; furthermore, there may be interactions between the compounds. Organic pollutants adsorbed on particulate matter are present in aerosolic form: particles of size > 10 /xm are removed effectively from the upper respiratory tract, whereas particles of size < 10/xm accumulate into the alveoli. Since a person usually inhales 10,000 to 20,000 liters of air daily, even very low concentrations of pollutants may prove to be biologically effective. In the light of these considerations, the aim of this study has been to assess the air quality, from a mutagenetic point of view, of a sparsely populated rural area in northwestern Italy in which an industrial plant is present. This factory is one of the most important world producers of several chemical compounds such as phthalocyanines and naphthalene, benzene and anthraquinone intermediates which are mainly utilized in agrochemistry and pharmaceutics. Material and methods

Sampling sites The plant, ACNA Chimica Organica, S.p.A., Cengio (SV), is near the top of the Bormida valley. It represents an emission point of dusts whose fallout has been evaluated by mathematical models in order to characterize airborne particulate sampling sites. Two areas were identified as follows: Area of estimated maximum fallout (zone 1) Site A: within the plant. Site B: 0.8 km SSE of the plant, with very little traffic. Site C: 2.3 km NNW of the plant, with very little traffic. Site D: 1.8 k m E of the plant, village with heavy traffic. Area without fallout (zone 2) Site E: 15 km NW of the plant, rural area. Site F: 21 km N of the plant, village with less traffic than site D.

Site G: 18 km SSW of the plant, village with very little traffic. Site H: 36.5 km NW of the plant, small town with very heavy traffic. The second group of places represents the reference sites: site E is considered an unpolluted area whereas the others are considered affected by normal human activities.

Sampling schedule During the year 1989, air monitoring was performed by collecting a single sample every month in all places, except in the case of site F (January-April) and site E (June-December), for a total of 83 samples. Since production cycles of the plant stopped from June to December 1989, in order to evaluate a possible influence of industrial emissions on ambient air, mutagenetic study continued in the corresponding months of the year 1990. Only three sites were monitored, namely site A which is within the plant and sites B and C, the locations most severely affected by dust fallout but not by vehicular traffic. To obtain more relevant information, collections were performed fortnightly in each site for a total of 42 samples. Inhalable particulate matter, size < 10 /xm, was continuously sampled for 24 h on glass microfiber filters (Whatman Ltd, England, 20.3 x 25.4 cm) by High Volume PM10 Sampler Model 1200 (Andersen Samplers, Inc., Atlanta, GA, USA). For each sample air volume was about 1300 m 3, while particulate matter ranged from 26 to 343 /~g/m 3. For possible correlation studies, temperature, pressure, wind strength and direction, humidity and number of motor vehicles (by observation from personnel devoted to control of the air samplers) were recorded during each air sampling.

Extraction procedure After sampling, organic material was acetone extracted by sonication (20 min) and Soxhlet extraction for 16 h. The solvent was evaporated in a Rotavapor under a nitrogen stream and resuspended in an appropriate volume of dimethyl sulfoxide (DMSO). From trials previously performed in our laboratory, acetone seems to be the most effective solvent in extraction of mutagens from airborne

295

particulate samples, as confirmed by other authors (Lee et al., 1991).

Mutagenicity assay The standard Ames plate test (Maron and Ames, 1983) was applied mainly by using the Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation ($9, at 10% in the mix, obtained from Sprague-Dawley male rats pretreated with Aroclor 1254). The indirect mutagen 2-aminofluorene (2AF) was used at a concentration of 5.0 ~ g / p l a t e for checking both strain sensitivity and $9 efficiency. Five doses (three plates per dose) of each organic extract were tested in both strains and all experiments were duplicated. At each sampling a unexposed filter was processed, as described above, and tested for control of the extraction procedure. Statistical analysis Results of the mutagenicity assay are expressed as number of revertants per plate; for each dose the mean + standard deviation was calculated and the dose-effect relationship was evaluated by the statistical method of Margolin et al. (1981). This method is based on a simple equation which allows simultaneous estimation of mutagenic and toxicity effects by the following model: Y = (a + bX) exp(-c~X) where Y is the frequency of the observed effect, X is the dose, a and b are the linear regression coefficient and ~b is the toxicity factor. The parameter utilized was

the specific effect calculated at the average dose, namely the specific average reversion activity (SARA) expressed as revertants per m 3 of sampled air or per mg of collected particulate (Barale et al., 1991a,b). The A N O V A test was used to compare mutagenicity data among the sites and between the two years in the same period; correlation and regression analysis (linear and multiple) were applied to assess effect of the other variables on mutagenicity. Finally, the canonic correlation was used to assess possible correlations between two sets of variables. In this case the first set consisted of mutagenic activity assessed by both strains + $9 (four variables) and the second set consisted of temperature, amount of particulate matter and number of vehicles (three variables). All statistical analyses and graphics were carried out with use of the S T A T G R A P H I C S package, a PLUS W A R E product. Results

Salmonella typhimurium strain TA98 proved to be the most effective to assess mutagenic activity of organic extracts from airborne particulate as also reported by others (Barale et al., 1991; Crebelli et al., 1991). For this reason, both statistical elaboration and graphic display of data refer to the TA98 strain only. Regarding the year 1989, values from each sample are expressed as arithmetical mean between the two trials, and in the TAgB+s9

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296

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297 TABLE 1

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RESULTS OF T H E C A N O N I C A L C O R R E L A T I O N ANALYSIS WHICH IS THE LINEAR CORRELATION BETWEEN THE TWO AXES OF TWO MULTIDIMENSIONAL SWARMS OR SETS

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Correlation coefficient

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following year also between the two fortnightly samplings.

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298

mutagenicity levels of the industrial area (sites A, B, C, D), in the absence of metabolic activation, are significantly lower (ANOVA test) than those observed in the sites of zone 2 (positive reference sites: F, G, H). In the case of site E (rural area), mean SARA (12.16 + 5.21) is closer to those of sites A, B, C and D. However, in the presence of $9, there are no significant differences between the two groups of sites. In addition, by statistical analysis (ANOVA test) no significant differences were found among the sampling sites of zone 1 during the year 1989 either in the presence or in the absence of $9. The results of the study expressed as SARA per mg of collected particulate are reported in Fig. 2: boxes and whiskers represent the annual dispersion of mutagenicity values of each site. Both in the presence and in the absence of metabolic activation, the genotoxic response is very close among all locations. The annual trend of temperature and SARAs (rev/m 3) is shown in Fig. 3; boxes and whiskers represent the monthly dispersion of temperature

and mutagenicity values of the sites as a whole. SARAs are the arithmetical mean between TA98 without $9 and TA98 with $9. As expected, the genotoxic response is highest in the cold months, displaying a typical parabolic trend inversely correlated with temperature. The canonical correlation analysis showed a general significant association between physical and mutagenicity variables, except in the case of site A. The association, however, was studied using a relatively small number of samples/ station. Furthermore, although significant, it has considerable standard errors (see Table 1). Year 1990 Mutagenicity results from the sites of the industrial area (A, B, C) with and without metabolic activation, expressed as SARA (rev/m3), are displayed in Fig. 4; vertical bars represent the mean + SE. No significant differences (ANOVA test, method of Bonferroni), either in the absence (F = 2.841, p = 0.064) and in the presence of $9 YEAR 1990

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Canonical analysis applied to the three stations gave similar results to those observed in the previous year. The same considerations made for this technique for 1989 apply to 1990. Since the number of observations was increased, correlation became significant also in the case of site A (see Table 1).

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( F = 2.230, p = 0.114), were found among the three locations jointly considered, and between each group of two sites (A vs. B, A vs. C, B vs. C). Mutagenic activity ranged from 7.54 + 0.66 (site A) to 13.95 + 2.67 (site C) and from 8.84 + 1.11 (site A) to 17.95 + 4.98 (site C) without and with metabolic activation respectively. The genotoxic response of the TA98 strain both in the presence and in the absence of $9, expressed as a function of collected particulate, is shown in Fig. 5 and proves to be similar among the locations. The seasonal trends of temperature and mutagenicity, expressed as SARA (rev/m3), for the three sites during the 7 months of sampling, shown in Fig. 6, were found to be very like those of 1989 (see Fig. 3). Fig. 7 shows the correlations between the mutagenicity basal levels and the annual amount of vehicular traffic, and between the traffic-induced monthly mutagenicity and temperature.

Mutagenic effects of airborne particulate matter from a rural area, in which industrial activities are present, were evaluated in a 2-year study. The results, when expressed as revertants per m 3 with TA98 without $9, show that during 1989 the sites of maximum dust fallout (zone 1) presented mutagenicity levels significantly lower than the reference sites (zone 2), although this finding did not hold in the presence of $9. In particular, the contribution of indirect-acting frameshift mutagens proved to be significantly higher during the months of February and November for the sites of both zones. A number of indirect mutagens may also be released into the atmosphere by wood combustion from stoves and fireplaces (van Houdt et al., 1986), which are commonly used for heating and cooking in zone 1 sites (typical rural area). This could explain the homogeneous response with metabolic activation of these locations. The results expressed as mutagenicity per mg of collected particulate showed that there were no appreciable differences in mutagenicity potency among sampling sites. This means that quality of airborne particles is almost the same among all localities suggesting the presence of one sole major source of mutagens in the monitored areas. The parabolic trend of mutagenicity observed during 1989 shows an inverse correlation with temperature. Indeed, temperature plays an important role in mutagenicity of particulate matter and its effects have been carefully evaluated (Barale et al., 1989). In other works, genotoxic effects of organic extracts from airborne particles have likewise been found to significantly increase as a function of temperature reduction (M011er et al., 1982; van Houdt et al., 1987; Barale et al., 1991). This can be explained by several facts such as a more rapid photochemical inactivation and increased volatilization of mutagens

300 during the summer as well as thermic inversion and increased deposition rates of vapor-phase mutagens onto airborne particulate matter in winter (Atherholt et al., 1985; Hiramatsu et al., 1986; De Flora et al., 1989). Since plant production cycles stopped from June to December 1989, air monitoring was repeated for sites A, B and C in the corresponding period of the following year. Temperature and mutagenicity showed a relationship similar to that described above. With respect to genotoxic response, no significant differences were detected among the three sampling sites, as in the previous year. In a further statistical analysis, performed in sequential steps, sites A, B and C were jointly considered. No appreciable variations (ANOVA test) in levels of mutagenic activity with TA98 with $9 were found during the period J u n e - D e c e m b e r comparing the 2 years of study ( F = 0.031, p = 0.862). On the other hand, a significant difference was obtained by comparing the results for TA98 without $9 ( F = 4.449, p = 0.037). In order to elucidate this finding, a comparison between the 2 years for each site gave the following results. Site A, with SARAs ( r e v / m 3) of 9.86 _+ 2.72 and 6.93 _+ 1.63 for 1989 and 1990 respectively, was not found to significantly differ between the two years ( F = 2.314, p = 0.136); a similar result ( F = 3.241, p -0.084) was obtained in the case of site C, the SARAs ( r e v / m 3) of which were 7.54 _+ 0.66 and 13.95 _+ 2.67 for 1989 and 1990 respectively. Only site B, with SARAs ( r e v / m 3) of 4.96 _+ 0.93 and 11.09 + 1.82 for 1989 and 1990 respectively, showed a statistically significant difference ( F - 5.252, p = 0.027). Therefore, air mutagenicity as presently evaluated seemed to vary through the years regardless of plant activity. In the light of these considerations, it can be argued that plant emissions do not seem to influence significantly the mutagenicity of the monitored area. By contrast, in other studies, it has been found that the biological activity of inhalable particulate from industrial zones was always higher than from other places (Takeda et al., 1984; Motykiewicz et al., 1988); however, the quality of emissions was considerably different from the present case. In order to identify the source of pollution which affects mutagenicity levels in the examined region, we assessed the possible influence of both

temperature and number of vehicles passing through the area on SARA ( r e v / m 3) by multiple regression analysis. Fig. 7a shows the significant relationship (r = 0.985, p < 0.001) found between the constants of multiple regression, taken as a reliable index of basal mutagenicity, and the annual average number of vehicles passing through each site (since it was not possible to sample throughout the year, site E and site F were not considered in this analysis). It underlines that there were no appreciable variations in the amount of vehicular traffic during the day of sampling from one month to another. Other works attribute a substantial role to vehicular traffic in determining air mutagenicity (M¢ller et al., 1982). In the present study traffic emissions, accounting for almost the total mutagenicity (r 2 - 0.970) detected in the monitored areas, are to be considered the main source of air pollution. This is also in agreement with the hypothesis suggested by mutagenicity data expressed as mg of collected particulate. Moreover, Fig. 7b shows the significant correlation between the traffic-induced monthly average revertants of all the sites (estimated by linear regression) and the corresponding temperature values (r = 0.885, p < 0.001). Once again, it should be noted that mutagenicity levels are highest in the cold months. Therefore, it may be established that temperature modulates effects of the sources of atmospheric pollution. By contrast, the other registered meteorological parameters, such as humidity, rain, wind strength and direction, were found not to correlate with genotoxicity, as confirmed by other works (Barale et al., 1989). Conclusions Air mutagenesis monitoring was performed to assess whether atmospheric pollution of a region in northwest Italy might be due to usual human activities a n d / o r to the presence of a chemical plant. From our data, it emerges that the genotoxic response of organic particulate matter sampled in this region is fundamentally caused by traffic emissions, on which temperature acts as modulating agent. Atmospheric emissions from the industrial plant do not seem to perturb these mutagenicity levels. This could be due to the fact

301

that this industrial activity is not characterized by any combustion for processing or energy production. A further, by no means secondary, reason could be the extensive application of advanced technology in the production process and in controlling emissions. However, it is possible that a certain contribution to air mutagenicity might also be due to domestic heating by wood, especially in winter. Because the goals of this study were different, we did not quantify the contribution of this source and therefore it could not be taken into account for statistical elaboration. At the present state of knowledge, this paper represents the first work in which it has been possible to statistically estimate the effects of vehicular traffic on air mutagenicity on a large scale. The experimental approach to this project has supplied some useful information relating to the mutagenicity of moderately polluted areas and may be of general use for comparison with results obtained from other environments. References Alfheim, I., and M. M~ller (1979) Mutagenicity of long-range transported atmospheric aerosols, Sci. Total Environ., 13, 275 -278. Alink, G.M., H.A. Smit, J.J. van Houdt, J.R. Kolkman and J.S.M. Boleij (1983) Mutagenic activity of airborne particulates at non-industrial locations, Mutation Res., 116, 21-34. Ashby, J., and R.W. Tennant (1991) Definitive relationship among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by U.S. NTP, Mutation Res., 257, 229-306. Atherholt, T.B., G.J. McGarrity, J.B. Louis, L.J. McGeorge, P.J. Lioy, J.M. Daisey, A. Greenberg and F. Darack (1985) Mutagenicity studies of New Jersey ambient air particulate extracts, in: M.D. Waters, S.S. Sandhu, J. Lewtas, L. Claxton, G. Strauss and S. Nesnow (Eds.), Short-Term Bioassay in the Analysis of Complex Environmental Mixtures IV, Plenum, New York, pp. 211-231. Barale, R., D. Zucconi, F. Giorgelli, A.L. Carducci, M. Tonelli and N. Loprieno (1989) Mutagenicity of airborne particles from a nonindustrial town in Italy, Environ. Mol. Mutagen,, 13, 227-233. Barale, R., F. Giorgelli, R. Scarpato, C. Scapoli, N. Loprieno and I. Barrai (1991a) Correlation between mutagenicity of airborne particles and air pollution parameters in eleven Italian towns, Int. J. Environ. Health Res., 1, 37-53. Barale, R., L. Giromini, G. Ghelardini, C. Scapoli, N. Loprieno, M. Pala, F. Valerio and I. Barrai (1991b) Correla-

tion between 15 polycyclic aromatic hydrocarbons (PAH) and the mutagenicity of the total PAll fraction in ambient air particles in La Spezia (Italy), Mutation Res., 249, 227-241. Crebelli, R., S. Fuselli, G. Conti, L. Conti and A. Carere (1991) Mutagenicity spectra in bacterial strains of airborne and engine exhaust particulate extracts, Mutation Res., 261, 237-248. De Flora, S., A. Camoirano, A. Izzotti, F. D'Agostini and C. Bennicelli (1989) Photoactivation of mutagens, Carcinogenesis, 10, 1089-1097. Hiramatsu, Y., T. Nishimura, K. Tanabe and H. Matsushita (1986) Mutagenicity of the photochemical reaction products of pyrene with nitrogen dioxide, Mutation Res., 172, 19-27. Krewski, D., B.G. Leroux, J. I(reason and L. Claxton (1992) Sources of variation in the mutagenic potency of complex chemical mixtures based on the Salmonella/microsome assay, Mutation Res., 276, 33-59. Lee, H., S.M. Law and S.T. Lin (1991) The effect of extraction solvent on the mutagenicity of airborne particles, Toxicol. Lett., 58, 59-67. Margolin, H.B., N. Kaplan and E. Zeiger (1981) Statistical analysis of the Ames Salmonella/microsome test, Proc. Natl. Acad. Sci. USA, 78, 3779-3783. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Miguel, A.G., J.M. Daisey and J.A. Sousa (1990) Comparative study of the mutagenic and genotoxic activity associated with inhalable particulate matter in Rio de Janeiro air, Environ. Mol. Mutagen., 15, 36-43. M011er, M., J. Alfheim, S. Larssen and A. Mikalsen (1982) Mutagenicity of airborne particles in relation to traffic and air pollution parameters, Environ. Sci. Technol., 16, 221225. Motykiewicz, G., J. Michalska, J. Szeliga and B. Cimander (1988) Mutagenic and clastogenic activity of direct-acting component from air pollutants of the Silesian industrial region, Mutation Res., 204, 289-296. Pitts, J.N. Jr., D. Grosjeau, T.M. Mischke, W.F. Simmon and D. Poole (1977) Mutagenic activity of airborne particulate organic pollutants, Toxicol. Lett., 1, 65-70. Reali, D., H. Schlitt, C. Lohse, R. Barale and N. Loprieno (1984) Mutagenicity and chemical analysis of airborne particulates from a rural area in Italy, Environ. Mutagen., 6, 813-823. Takeda, N., K. Teranishi and K. Hamada (1984) Mutagenicity of air pollutants collected at industrial, urban-residential and rural area, Bull. Environ. Contam. Toxicol., 32, 688692. van Houdt, J.J., C.M.J. Dainen, J.S.M. Boleij and G.M. Alink (1986) Contribution of wood stoves and fire places to mutagenic activity of airborne particulate matter inside homes, Mutation Res., 171, 91-98. van Houdt, J.J., G.M. Alink and J.S.M. Boleij (1987) Mutagenicity of airborne particles related to meteorological and air pollution parameters, Sci. Total Environ., 61, 23-36.