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Research Article

A Preliminary Characterization of the Mutagenicity ofAtmospheric Particulate Matter Collected During Sugar

Cane Harvesting Using the Salmonella/MicrosomeMicrosuspension Assay

Gisela de Arag~ao Umbuzeiro,1* Alexandre Franco,2 Dulce Magalh~aes,2

Francisco Jose Viana de Castro,1 Fabio Kummrow,1,3 Celia Maria Rech,1

Lilian Rothschild Franco de Carvalho,2 and Perola de Castro Vasconcellos2

1CETESB-Cia de Tecnologia de Saneamento Ambiental, Av. Prof. Frederico Hermann Jr., 345, 05459-900, S ~ao Paulo, SP, Brazil

2Instituto de Quı mica da Universidade de S ~ao Paulo. Av. Prof. Lineu Prestes,748, 05599-970, S ~ao Paulo, SP, Brazil

3Departamento de Analises Clı nicas e Toxicolo ´ gicas da Universidade Federal de Alfenas, R. Gabriel Monteiro da Silva, 714, 37130-000, Alfenas, MG, Brazil

During sugar cane harvesting season, whichoccurs from May to November of each year, thecrops are burnt, cut, and transported to the mills.There are reports showing that mutagenic activityand PAH content increase during harvesting sea-son in some areas of S~ao Paulo State in compari-son with nonharvesting periods. The objective of this work was to preliminarily characterize the mu-tagenic activity of the total organic extracts aswell as corresponding organic fractions of air-

borne particulate matter (PM) collected twice fromtwo cities, Araraquara (ARQ) and Piracicaba(PRB), during sugar cane harvesting season usingthe Salmonella/microsome microssuspensionassay. One sample collected in S~ao Paulo metro-politan area was also included. The mutagenicityof the total extracts ranged from 55 to 320 rever-

tants per cubic meter without the addition of S9and from not detected to 57 revertants per cubicmeter in the presence of S9 in areas with sugarcane plantations. Of the three fractions analyzed,the most polar ones (nitro and oxy) were the mostpotent. A comparison of the response of TA98with YG1041 and the increased potencies withoutS9 indicated that nitro compounds are causingthe observed effect. More studies are necessary toverify the sources of the mutagenic activity such as

burning of vegetal biomass and combustion of heavy duty vehicles used to transport the sugarcane to the mills. The Salmonella/microsomeassay can be an important tool to monitor theatmosphere for mutagenicity during sugar caneharvesting season. Environ. Mol. Mutagen.49:249–255, 2008. VVC 2008 Wiley-Liss, Inc.

Key words: Salmonella/microsome; Ames test; sugar caneharvesting; mutagenicity; air pollution; nitro-aromatics

INTRODUCTION

In Brazil, diesel, ethanol, and gasohol (a mixture of gasoline with anhydrous ethanol) are used as automotive

fuels in light vehicles and more recently in heavy duty

cars. Biofuels are a very interesting alternative for coun-

tries like Brazil because of its agricultural capability, but

some problems related to the sugar cane alcohol produc-

tion can occur. At harvesting season, from May to No-

vember, crops are burned to make the process of manual

harvesting easier, to protect rural workers from sharp

leaves, insects and poisonous snakes, and to increase the

sugar content by weight due to water evaporation [Godoi

et al., 2004]. Burning of biomass in tropical areas may be

an important global source of atmospheric particles [Lara

et al., 2005]. In S~ao Paulo State, one of the biggest sugar

cane producers, there are regulations that aim to reduce,and in the near future, to avoid sugar cane burning;

*Correspondence to: Gisela de Arag~ao Umbuzeiro, Divis~ao de Toxicolo-

gia, Genotoxicidade e Microbiologia Ambiental, CETESB, Av. Prof.

Frederico Hermann Jr., 345, 05459-900, S~ao Paulo, SP, Brazil. E-mail:

[email protected]

Received 24 October 2007; provisionally accepted 7 January 2008; and

in final form 9 January 2008

DOI 10.1002/em.20378

Published online 20 February 2008 in Wiley InterScience (www.interscience.

wiley.com).

VVC 2008Wiley-Liss, Inc.

Environmental and Molecular Mutagenesis 49:249^255 (2008)



however, in some areas this activity is still performed.

Also during sugar cane harvesting season, large increases

in heavy duty vehicle traffic occur as a result of the need

to transport the sugar cane. Andrade [2004] observed

increases in the mutagenic activity and PAHs levels in

the atmosphere during sugar cane harvesting season incomparison to the nonharvesting period in Araraquara

city, Brazil as well as increases in the concentration of

atmospheric particulate matter (PM10). Although muta-

genic PAHs are of great importance they could not

account for the majority of mutagenic activity detected

in highly urbanized areas in Brazil [Sato et al., 1995;

Vargas, 2003; Andrade, 2004]. According to Claxton

et al. [2004], the mutagenicity of airborne particulate can

result from at least 500 identified compounds from vari-

ous chemical classes. Mutagenic nonsubstituted PAHs are

the most studied and well known compounds; however,

more recently, nitro- and oxy-PAHs have been shown to

be very important because of their strong biological activ-ity [Enya et al., 1997; WHO, 2003].

The Salmonella/microsome microsuspension assay has

been used for large, multisite, and/or time series studies,

for bioassay-directed fractionation studies, for identifying

the presence of specific classes of mutagens, and for site-

or source-comparisons of relative levels of airborne muta-

gens [Claxton et al., 2004]. In Brazil, this assay has been

used to evaluate the mutagenic activity of airborne partic-

ulate matter collected in the S~ao Paulo metropolitan

region with strains TA98 and TA100 among others [Sato

et al., 1995; DeMartinis et al., 1999] and in the metropoli-

tan region of Porto Alegre (RS) [Ducatti and Vargas,

2003; Vargas, 2003]. The use of strains of Salmonella

typhimurium with different metabolic capacities can indi-

cate the class or classes of compounds present in an envi-

ronmental sample [DeMarini et al., 2004; Roubicek and

Umbuzeiro, 2006; Marvin and Hewitt, 2007].

The objective of this work was to preliminarily character-

ize the mutagenic activity of total organic extracts and frac-

tions of airborne particulate matter (PM) from two cities,

Araraquara (ARQ) and Piracicaba (PRB), during sugar cane

harvesting season using Salmonella strains TA98, YG1041,

and YG1042 in the Salmonella/microsome microsuspension

assay. One sample collected in S~ao Paulo metropolitan area

was included in the study for comparison.

MATERIALS AND METHODS

Sampling Sites

Two sites, one in Araraquara city (ARQ) and other in Piracicaba city

(PRB) were selected because of the intense sugar cane production in the

surrounding areas. During harvesting season, plant burning and sugar

cane transportation to the sugar cane mills are very frequent activities.

At the ARQ site, sample collection was performed at the campus of the

State University of S~ao Paulo (UNESP) located in Araraquara City,

300 km from S~ao Paulo city, where Andrade [2004] observed an

increase in particulate matter and mutagenicity during this period. It is

an urban site surrounded by sugar cane plantations. The atmospheric par-

ticulate matter Hi-vol sampler was located over 4 m high, and 50 m

from a highway with traffic that includes heavy duty vehicles used to

transport sugar cane.

At the PRB site, sample collection was performed at the campus of

the Escola Superior de Agricultura Luis de Queiroz (ESALQ) of theUniversity of S~ao Paulo located in Piracicaba city in a rural/urban area,

200 km from S~ao Paulo city. The Hi-vol sampler was placed in an

open area, 1 km from the sugar cane plantations. This site is impacted

by sugar cane burning and is under some influence of light and heavy

duty vehicles from the city.

A site located in the West area of S~ao Paulo City (SPA) was also

included. S~ao Paulo is the largest city of Brazil and considered the

‘‘worst-case scenario’’ in terms of air pollution in the country [CETESB,

2006]. The sampling site is impacted by heavy traffic including light and

heavy vehicles as well as industrial emission but is not directly affected

by sugar cane activities. The PM Hi-vol sampler is located in a green

area, 2 km far from a major highway in S~ao Paulo city. At this site,

the sampler was placed in an open area on the roof of the Department of

Atmospheric Sciences, located on the campus of the University of S~ao

Paulo (USP), 20 m above ground level.

Sampling Procedures

The PM samples were collected for 24-hr periods in quartz fiber filters

(20 cm 3 25 cm and 47 mm) in July of 2003. Two samples of each site

related to sugar cane activities were tested for mutagenicity (ARQ6 and

13, PRB9 and 15). In S~ao Paulo city (SPA), only one sample was col-

lected in July of the same year and analyzed. July corresponds to winter

in Brazil. Airborne particles were collected at SPA and ARQ using a

High-vol sampler device equipped with a size-selective inlet (Andersen)

for the collection of particles smaller than 10 lm (i.e., PM10) and a sens-

ing device for measuring the flow rate through the filter. At PRB, sam-

ples were collected using a High-vol sampler device for total particulate

matter (PM) collection. At PRB, the mutagenic potency may be underes-

timated because it is known that the organic components are preferen-

tially attached to the smaller particles [Ohura et al., 2004; Ji et al.,

2007]. Comparisons of the results obtained at PRB with the other two

sites must therefore be interpreted with caution.

Preparation of the Samples and Organic

Extraction Procedures

Prior to the sampling activities the filters were precleaned by heating

in oven at 8008C for 8 hr then inserted in the sampler device. Filters

were weighed before and after sampling for mass determination of the

total particulate matter (PM or PM10). The filters were then wrapped in

aluminum foil and stored in a freezer at 2208C until they were

extracted. Organic extraction of the filters was performed with 200 ml of

methylene chloride (DCM) in a soxhlet apparatus at temperatures below

408C for 20 hr with a flow rate of 40 min. The volume of the obtainedextract was reduced to 3 ml in a rotary evaporator with vacuum and fil-

tered using Teflon membranes (NucleoporeTM) for the removal of par-

ticles. Half of the material was dried with a gentle stream of pure nitro-

gen gas, dissolved in Dimethylsulfoxide (DMSO) and tested as a total

extract. The other half was dried using the same procedure and resus-

pended in 50 ll of DCM for fractionation.

The fractionation procedure was performed using High Performance

Liquid Chromatography (HPLC) with a silica column (nucleosil 100-10,

Macherey-Nagel). Mobile phase was initially 100% hexane for the first

10 minutes and then increasing quantities of DCM were added until it

reached 100% DCM after 40 min. The flow rate was 1 ml per minute

[Franco, 2006]. Three different fractions were collected. The recovery of

first fraction was from 7 to 17.5 min (PAH fraction), the second from

Environmental and Molecular Mutagenesis. DOI 10.1002/em

250 Umbuzeiro et al.



17.5 to 28 min (nitro-PAH fraction), and the third was collected from

28 to 45 min (oxy-PAH fraction). The initial 50 ll injected generated a

final volume of 20 ll [Franco, 2006]. According to Brack [2003], the

loss during fractionation cannot be avoided, and to compare the muta-

genic potency of the total organic extract with the obtained fractions, aproper adjustment was necessary. Each fraction was dried as described

earlier and dissolved in DMSO for testing.

Salmonella/Microsome Microsuspension Assay

The total extract as well as the PAH, nitro-PAH, and oxy-PAH frac-

tions were assayed for mutagenicity using the Salmonella typhimurium

strains TA98 (hisD3052, rfa, Dbio, Duvr B, pKM101) [Maron and Ames,

1983], YG1041 (a derivative of the TA98, able to produce high levels of

nitroreductase and O-acetyltransferase), and YG1042 (a derivative of

TA100, able to produce high levels of nitroreductase and O-acetyltrans-

ferase) [Hagiwara et al., 1993] with and without S9 metabolic activation,

in the Salmonella/microsome microsuspension assay [Kado et al., 1983].

YG1042 ( HisG46, rfa, Dbio, Duvr B, pKM101, pYG233) was included

because it is a derivative of the TA100 which is considered more sensi-tive to PAHs (e.g., benzo(a)pyrene) than the other two strains [DeMarini

et al., 2004] and it was used to test only some samples. Not all the sam-

ples were tested with all the cited strains due to limited extract quanti-

ties. The strains were kindly provided by Dr. Larry D. Claxton from the

US Environmental Protection Agency, NC. Overnight cultures of the

strains (109 cells/ml), 5-fold concentrated by centrifugation (10,000 g

at 48C, for 10 min), were resuspended into 0.015 M sodium phosphate

buffer. Fifty microliters of cell suspension, 50 ll of 0.015 M sodium

phosphate buffer or S9 mix, and 5 ll of the sample were added to a tube

and incubated at 378C for 90 min without shaking. After incubation,

2 ml of molten agar was added, and the mixture was poured onto a mini-

mal agar plate (90 mm diameter). Colonies were counted after 66 hr of

incubation at 378C using an automatic colony counter. Metabolic activa-

tion was provided by Aroclor 1254-induced Sprague Dawley rat liver S9

mix (MolTox, Boone, NC), which was prepared at a concentration of 4% v/v. For TA98, the positive controls were 0.125 lg/plate of 4-nitro-

quinoline-oxide (4NQO) (Acros), and 0.625 lg/plate of 2-aminoanthra-

cene (2AA) (Sigma-Aldrich), both dissolved in DMSO. For YG1041, the

positive controls were 5 lg/plate of 4-nitro-O-phenylenediamine (4NOP)

(ICN Biomedicals), and 0.0312 lg/plate of 2AA. For YG1042, positive

controls were 2.5 lg/plate of 2-nitrofluorene (Sigma- Aldrich) and 0.625

lg/plate of 2AA. The doses of the extracts varied from 0.05 to 500 lg

equivalent of airborne particulate matter per plate depending on the

strain and condition tested. The assay was performed using duplicates

of each dose tested, except for the negative control that was tested in 5

replicates.

The data were analyzed with the Salanal computer program using the

Bernstein model [Bernstein et al., 1982]. Samples were considered

positive when a significant difference among the tested doses and the

negative control (ANOVA) and a significant positive dose response were

observed.

RESULTS AND DISCUSSION

Andrade [2004] monitored the mutagenic activity of organic extracts of atmospheric particulate matter samples

(PM10) using the Salmonella/microsome assay in Arara-

quara city at the same site where this study was con-

ducted (ARQ). The author collected samples during

harvesting and nonharvesting seasons. The standard plate

incorporation method of the assay was used with YG1024

in the presence and absence of S9. Fourteen samples col-

lected during sugar cane harvesting season (August 2002,

November 2002, and September 2003), and 11 samples

were collected during the months in which no sugar cane

harvesting activity occurs (March 2003 and January 2004)

were analyzed. The average of the particulate matter con-

centration was approximately three times higher during

harvesting vs. nonharvesting season. The results of the

mutagenicity evaluation are presented in Table I. The

samples collected during sugar cane harvesting season

were on average 7.7 times more mutagenic without S9

and 3.7 times more with S9 than the samples collected

during nonharvesting season. During sugar cane harvest-

ing season the average of the mutagenic potencies without

S9 was 2.3 times higher compared with the results

obtained in the presence of S9 suggesting the presence of

nitro-compounds. Samples collected during the nonhar-

vesting season showed similar responses with and without

S9 (Table I). These results clearly show that the muta-genic activity is much higher during sugar cane harvesting

season and the use of specific strains can provide impor-

tant information about the mutagenic compounds present

in the samples.

The mass, volume, and concentration of the airborne

particulate matter collected at each site analyzed in this

study are presented in Table II. Although at PRB, the air-

borne particulate was total PM and at the other sites

(ARQ and SPA), the particles collected were smaller than

10 lm, all the filters contained similar concentrations

except the SPA sample that was less concentrated. The


TABLE I. Mean and Standard Deviation of the MutagenicActivity (revertants per m3) Obtained with YG1024 with andwithout S9 for the Airborne Particulate Matter (PM10)Samples Obtained by Andrade [2004] in Araraquara city(ARQ site)

Season YG10242S9 YG10241S9

Harvesting (2002–2003) 10.12 (4.93)a 4.66b (2.86)

n 5 14 n 5 11

Nonharvesting (2003–2004) 1.31 (0.41) 1.26 (0.51)

n 5 9 n 5 10

aStandard Deviation.bn 5 number of samples with a calculated potency; according to the

author, some dose response data did not fit the Bernstein model; there-

fore, the potencies were not calculated.

TABLE II. Total Mass of Airborne Particulate Matter (PM10)Collected in Quartz Fiber Filters, the Volume of AirSampled, and the Concentration of Each Filter in Each SiteAnalyzed in this Study

Site Mass (mg) Volume (m3) Concentration (lg/m

3)

ARQ6 231.0 1,610 143.4

ARQ13 190.3 1,604 118.6

PRB9a

211.0 1,616 130.6

PRB15a 232.3 1,615 143.8

SPA 141.6 1,606 88.2

aTotal Particulate Matter (PM).

SugarCaneHarvesting Atmospheric Mutagenicity 251



concentrations measured on the filters analyzed in this

study were in compliance with the 24 hr-Brazilian pri-

mary standards: 240 lg/m3 for PM and 150 lg/m3 for

PM10 [Brazil, 1990].

The Salmonella/microsome microsuspension assay

results were expressed in mutagenic potency as revertants

per lg equivalent of the collected mass. The doses of the

fractions were adjusted based on the loss during the frac-

tionation procedures (Section ‘‘Preparation of the Samples

and Organic Extraction Procedures’’), therefore a direct

comparison of the potencies of the total extract and

related fractions was possible. As aforementioned, not all

extracts were tested with all strains due to mass extract

limitations. The mean of the average of revertants per

plate obtained for the positive controls in independent

experiments (n) were as follows: TA98-S9: 420.7 (n 5

3); TA981S9: 698.0 (n 5 3); YG10412S9: 933.9 (n 5

4); YG10411

S9: 872.3 (n5

3); YG10422

S9: 695.25 (n5 4); and YG10421S9: 1220.5 (n 5 4).

Mutagenicity of the Total Organic Extracts

The total organic extract mutagenicity of the four air-

borne particulate matter samples collected at the cities

during sugar cane harvesting season (ARQ and PRB) var-

ied from 55 to 320 revertants per cubic meter for TA98

without S9 and from not detected to 57 revertants per

cubic meter with S9 (Table III). In S~ao Paulo city (SPA),

88 revertants per cubic meter were observed for TA98

without S9 and 19 revertants per cubic meter with S9.

We selected potencies from the literature for airborne par-

ticulate matter samples obtained with TA98 from studies

that employed the same protocol used in this study, the

microsuspension Salmonella/microsome assay. The results

of these comparisons are also presented in Table III. We

observed that the potencies obtained for ARQ and PRB

are of the same order of magnitude as the ones observed

for the pooled samples from S~ao Paulo city (1990/91), for

Martinez, CA, USA and were significantly higher than

Porto Alegre, RS, Brazil and Brescia, Italy (Table III).

We also observed that the mutagenicity of all samples

collected in S~ao Paulo State (this study and from [Sato,

1995]) remarkably decreased with the addition of S9; this

differs from the results of Martinez, USA and Brescia,

Italy. This decrease with S9 is a typical response of fra-

meshift nitroaromatic compounds according to Claxton

et al. [2004].To evaluate the contribution of the nitroaromatics to

the total mutagenic activity we employed Salmonella

strain YG1041 in addition to TA98. Figure 1 shows the

relative percentage of the mutagenic activity detected by

TA98 in relation to YG1041. The strong increase in the

mutagenic activity with YG1041 and decrease of the

response when S9 was added (Table III) corroborate that

nitroaromatics are the major contributors to the mutage-

nicity detected in the extracts analyzed in this study.

From the obtained data, it is not possible to directly cor-

relate the increase of the mutagenic activity with the


TABLE III. Mutagenic Activity Expressed in Number of revertants per m3 for the SitesAnalyzed in this Study and Comparison with Other Sites that Used the MicrosuspensionProtocol (Kado Modification) to Evaluate Mutagenic Activity

Site Month of the year TA982S9 TA981S9

Cities under the influence of sugar cane activities

ARQ6 Winter, 2003 (this study) 170 32ARQ13 320 57

PRB9 55 14

PRB15 220 Not detected

S~ao Paulo city, SP state, Brazil

SPA Winter, 2003 (this study) 88 19

Pinheirosa

Winter, 1990 31 11

Spring, 1990 193 16

Summer, 1990–1991 94 17

Fall, 1991 228 45

Parque Dom Pedroa Winter, 1990 33 46

Spring, 1990 390 140

Summer, 1990–1991 450 114

Fall, 1991 270 74

Other cities

Porto Alegre, RS, Brazilb

Spring, 1997 to Summer, 1998 0.8–17.3Brescia, Italy

c2.0 7.6

Martinez, CA, USAc 85–296 304–727

aPooled samples from each season, data from Sato [1995].bData from Ducatti and Vargas [2003].cData from Claxton et al. [2001].




compounds derived from the sugar cane burning becausethe Salmonella/microsome assay detects only the effect of

the substances present in the mixture. To verify the origin

of the mutagenic compounds, a source apportionment

study should be performed [Lewtas and Gallagher, 1990].

The identities of the mutagenic compounds attributable to

diesel and gasoline emissions have been quite well docu-

mented, unlike sugar cane burning emissions. Therefore,

it will be necessary to characterize the mutagenic com-

pounds that are formed during cane burning as well as

their atmospheric transformation products before conduct-

ing the cited source apportionment study. Vasconcellos

et al. [in press] found remarkably high levels of several

mutagenic Nitrated Polycyclic Aromatic Hydrocarbons

(NPAH) in the nitro fraction of samples from the same

sites analyzed in this study which could be responsible

for much of the mutagenic activity. The majority of the

NPAH found were related to vehicle (i.e., diesel-powered)

emissions, with two important exceptions, 2-nitrofluoran-

thene and 2-nitropyrene, which are formed in the atmos-

phere. Mutagenicity testing of the chemical species found

using the same method/strains as well as their quantifica-

tion should be carried out in new extracts to determine

which compounds are contributing to the mutagenic effect

of the nitro fraction. Further chemical characterization is

still required for the oxy PAH fraction.Some of the samples were also analyzed with YG1042;

this strain is derived from TA100, which is known to be

very sensitive to nonsubstituted PAHs such as benzo(a)-

pyrene and other PAHs of the same class [DeMarini

et al., 2004]. This strain is also particularly sensitive to

nitroaromatics and aromatic amines that cause base sub-

stitution mutations [Hagiwara et al., 1993]. However, for

the samples analyzed here, the total extract exhibited low

mutagenic activity in YG1042 when compared to

YG1041 (data not shown), indicating that this class of

mutagens is not present in sufficient amounts in the

extracts to revert the Salmonella mutations under the

tested conditions.

Mutagenicity of the Fractions

The sums of the potencies obtained with YG1041 with-

out S9 of each fraction are similar to the potencies

obtained for the total extract except for PRB9 (Fig. 2).

With S9, only the PRB9 and ARQ6 samples presented

similar potencies (i.e., the sum of potencies of the frac-tions were comparable to that of the total extract)

(Fig. 3). For PRB9 without S9 and for ARQ13 and SPA

with S9, the sums of the potencies of the fractions were

lower than the potency of the total extract indicating a

possible loss of compounds during the fractionation pro-

cedure or interactions among the mutagenic compounds.

Despite these differences, it is possible to observe that the

nitro and oxy fractions were the most potent ones, repre-

senting each one, around half of the total mutagenic activ-

ity detected with YG1041 in the absence of S9 (Fig. 3).

The mutagenic activity detected in the nitro-fractions


Fig. 1. Relative percentage of mutagenic activity detected with TA98 in

relation to YG1041 without and with S9 during sugar cane harvesting

season (ARQ and PRB sites) in comparison to S~ao Paulo city, without

direct influence of sugar cane activities.

Fig. 2. Comparison of the mutagenic activity of the total extract with

the sum of the PAH, Nitro, and Oxy fractions. The results of PRB15

were not presented because the PAH fraction of this sample was not

analyzed.

Fig. 3. Mutagenic activity of each fraction obtained with the YG1041

in the presence and absence of S9. The results of PRB15 were not pre-

sented because the PAH fraction of this sample was not analyzed.

SugarCaneHarvesting Atmospheric Mutagenicity 253



could be explained by the high levels of NPAH detected

in samples of particulate matter collected at the same

sites [Vasconcellos et al., in press]. In the presence of S9

the oxy fractions, which contain more polar compounds,

seem to be even more important than the nitro com-

pounds. The importance of the most polar compounds inthe mutagenic activity was also observed by DeMartinis

et al. [2002] when evaluating the mutagenic activity of

S~ao Paulo city atmospheric particulate matter. Umbuzeiro

et al. [in press] showed that the oxy fractions of two air-

borne particulate samples, collected in a Sao Paulo metro-

politan area, were able to induce more DNA adducts in

reductive conditions in comparison to the nitro fractions.

FINAL CONSIDERATIONS

Despite the differences in sample collection procedures,

it was possible to observe that the mutagenic potenciesobtained with the microsuspension Samonella/microsome

assay during sugar cane harvesting season are related to

the presence of NPAHs and can achieve the same order

of magnitude of metropolitan areas like S~ao Paulo. The

results indicate that further studies are necessary to iden-

tify the compounds generated during sugar cane burning

and to verify which compounds could be contributing to

the total observed mutagenicity. Also, the contributions of

the heavy duty vehicles combustion as another potential

source will need to be evaluated. Nevertheless, a monitor-

ing program using the Salmonella/microsome assay to

assess the mutagenic activity in areas where sugar cane is

burned and transported to the mills along with studies tocharacterize the chemicals present in those samples could

give important information about the potential adverse

effects of these complex mixtures to humans. Another

advantage of this monitoring program would be to verify

the effects of the reduction of sugar cane burning on the

levels of the detected mutagenic activity in these regions.

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Accepted by—

E. Zeiger


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a preliminary characterization of themutagenicity of atmospheric pm

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