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In Situ Monitoring of the Mutagenic Effects of the
Gaseous Emissions of a Solid Waste Incinerator
in Metropolitan São Paulo, Brazil, Using the
Tradescantia Stamen-Hair Assay
Maria Izildinha Ferreira a , Helena Petrenko b , Debora Jâ de Araujo Lobo b , Geraldo
Stachetti Rodrigues c , Andreia Moreira c & Paulo Hilario Nascimento Saldiva d
a Secretary of Green and Environment of the Municipality of São Paulo , Brazil
b Laboratory of Experimental Air Pollution, Department of Pathology, School of
Medicine , University of São Paulo , Brazil
c Environment Unity of the Brazilian Company of Agriculture Studies , Jaguariúna ,
Brazil
d Department of Pathology, School of Medicine , University of São Paulo , Brazil
Published online: 27 Dec 2011.
To cite this article: Maria Izildinha Ferreira , Helena Petrenko , Debora Jâ de Araujo Lobo , Geraldo Stachetti
Rodrigues , Andreia Moreira & Paulo Hilario Nascimento Saldiva (2000) In Situ Monitoring of the Mutagenic Effects of the
Gaseous Emissions of a Solid Waste Incinerator in Metropolitan São Paulo, Brazil, Using the Tradescantia Stamen-Hair
Assay, Journal of the Air & Waste Management Association, 50:10, 1852-1856, DOI: 10.1080/10473289.2000.10464217
To link to this article: http://dx.doi.org/10.1080/10473289.2000.10464217
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Ferreira et al.
TECHNICAL PAPER
ISSN 1047-3289 J. Air & Waste Manage. Assoc. 50:1852-1856
Copyright 2000 Air & Waste Management Association
In Situ Monitoring of the Mutagenic Effects of the Gaseous
Emissions of a Solid Waste Incinerator in Metropolitan São
Paulo, Brazil, Using the Tradescantia Stamen-Hair Assay
Maria Izildinha Ferreira
Secretary of Green and Environment of the Municipality of São Paulo, Brazil
Helena Petrenko and Debora Jã de Araujo Lobo
Laboratory of Experimental Air Pollution, Department of Pathology, School of Medicine, University of São
Paulo, Brazil
Geraldo Stachetti Rodrigues and Andreia Moreira
Environment Unity of the Brazilian Company of Agriculture Studies, Jaguariúna, Brazil
Paulo Hilario Nascimento Saldiva
Department of Pathology, School of Medicine, University of São Paulo, Brazil
ABSTRACT
school (3.70 1.36) was significantly lower than that mea-
sured at both the museum (4.89 1.12) and the incinera-
The present work was designed to determine the potential
genotoxicity at the vicinity of a solid waste incinerator in
the metropolitan area of São Paulo, using the Tradescantia
stamen-hair bioassay. Experiments were carried out between
December 1998 and April 1999 in four regions (40 pots of
plants per site) selected on the basis of their pollution lev-
els predicted by theoretical modeling of the dispersion of
the incinerator’s plume. The exposure sites were defined as
follows: highest level (incinerator); a high level (museum)
located 1.5 km from the emission point; a moderate level
(school, at a distance of 3.5 km from the incinerator); and
a control (at Jaguariúna countryside). The difference in
genotoxicity among the groups was statistically significant
(p < 0.001). The frequency of mutations observed in the
countryside was significantly lower [2.25 1.55, mean
SD (standard deviation)] than that of the sites close to the
incinerator. The frequency of mutations measured at the
tor (5.69
1.34). In conclusion, we found a positive
correlation between the spatial distribution of the emis-
sions of the incinerator located in an urban area and the
mutagenic events measured by the Tradescantia stamen-
hair assay. The in situ approach employed in this study
was simple, efficient, and of low cost. No air or chemical
extraction of pollutants was necessary for genotoxicity test-
ing as required by other assays.
INTRODUCTION
Solid waste management is a major problem in large cit-
ies, and incineration is routinely used to eliminate waste.
Incinerator emissions, however, can deliver potentially
toxic organic and inorganic compounds to the exposed
population. Gaseous emissions of incinerators have been
clearly demonstrated to induce mutation in different bio-
assays, including bacteria and plants.1-3 The main pollut-
ants derived from incinerator emissions vary according
to the kind of material incinerated and the technical speci-
fications of the incinerator.
The Vergueiro incinerator of the city of São Paulo is
mainly dedicated to the elimination of solid waste from
hospitals. This facility was built in the 1950s and, because
of progressive expansion of the town in past decades, it is
presently situated in a densely populated urban area. Since
the beginning of its operation, the Vergueiro incinerator
IMPLICATIONS
The finding of a positive association between the emissions
of a waste incinerator situated in a densely populated area
and mutations in a sentinel system indicates that the pres-
ence of a stationary source may cause harmful effects on
the exposed population. These results indicate that control
measures should be implemented to improve the air quality
in São Paulo by controlling incinerator emissions in an area
already contaminated by millions of vehicles.
1852 Journal of the Air & Waste Management Association
Volume 50 October 2000

Ferreira et al.
did not receive any important technological upgrades,
even those necessary to control its emissions. In addition,
no systematic procedure of pollution monitoring of its
vicinity was ever performed.
between December 1998 and April 1999, using 40 flower
pots per site. The points of exposure were selected on
the basis of the influence of incinerator emissions, tak-
ing into account wind direction and dispersion of the
incinerator’s plume:
During 1995–1997, the Secretary of Green and Envi-
ronment of the Municipality of São Paulo formulated a
theoretical dispersion model of the plume emitted by the
Vergueiro incinerator chimney and predicted the concen-
tration of dioxins and heavy metals around its vicinity.
The results, based on the industrial source complex long-
term model,4 indicated that the levels of those contami-
nants might greatly surpass those prescribed by the
national air quality standards, and hundreds of thousands
of people might be constantly exposed to hazardous pol-
lutants. Although the aforementioned scenario of distri-
bution of pollutants was never measured or confirmed, it
is mandatory to look for some indicators of biological
damage in the areas under risk delineated by the theo-
retical projections, in order to get useful information to
preserve public health.
•
Vergueiro incinerator, 15 m from the emission
point. This site corresponds to the highest expo-
sure level.
•
Lasar Segall Museum, an art museum with a large
garden 1.5 km from the incinerator. The museum
location is in an area under great influence from
incinerator emissions.
•
Caranda school, 3.5 km from the incinerator.
According to the modeling projections, this lo-
cation is situated at a point of intermediate ex-
posure.
During the months of January and April, samples of Tra-
descantia were collected at the EMBRAPA facilities in or-
der to determine the baseline rate of mutations in the
same period of the study.
The present work was designed to determine
genotoxicity around the vicinity of the Vergueiro incin-
erator. Considering the potential severity of the situation,
we decided to perform in situ short-term studies to gather
information in a timely manner to be used to implement
policies aimed at protecting the exposed population. The
Tradescantia stamen-hair bioassay, which is a sensitive tool
to detect airborne genotoxic agents,5 was chosen for this
study.
Flower pots were kept in wood stands with a semi-
transparent cover. The stands were open at their sides.
Plants were arranged inside the stands to protect them
from rain and terrestrial predators. The investigators
enrolled in the study were responsible for maintaining
the Tradescantia specimens during the period, with vis-
its at least once a week. Daily data of temperature and
relative humidity were obtained from the Institute of
Astronomy and Geophysics of the University of São
Paulo. No climatic data were available for the region of
Jaguariúna.
MATERIAL AND METHODS
Exposure Protocol
The exposure protocol was based on the idea of keep-
ing Tradescantia specimens in field conditions in the
vicinity of the Vergueiro incinerator and performing
repeated samplings to determine the frequency of mu-
tations (based on the stamen-hair assay). Such an ap-
proach was previously employed for studying pollution
in the surroundings of nuclear power plants,6 oil refin-
eries,7 solid waste incinerators,2 mine dumps,8 and the
rubber industry,9 as well as other pollution sources.10
During the same period, the spontaneous rate of muta-
tions of Tradescantia samples was determined in a con-
trol region in the countryside.
Tradescantia plants clone 4430 were obtained from
the Environment Unity of the Brazilian Company of Ag-
riculture Studies (EMBRAPA, Jaguariúna, Brazil), which is
located in the countryside of the state of São Paulo. The
installations of EMBRAPA are situated at a distance of
150 km from São Paulo city in an area devoid of signifi-
cant atmospheric pollution sources.
Tradescantia Stamen-Hair Assay
The Tradescantia stamen-hair assay was described by Spar-
row et al.11 and is based on the fact that the stamen-hair
cells of this plant are heterozygous for flower color, mak-
ing it possible to detect mutations on the basis of their
pigmentation change from blue (dominant) to pink (re-
cessive). A mutant (pink cell) may continue to divide, giv-
ing rise to a contiguous series of pink cells. This condition
is considered as a single mutant event. Two pink cells sepa-
rated by blue cells are considered as two mutation events.
Ten flowers were collected at each sampling. The av-
erage number of stamen hairs per flower was determined
at the beginning of the experiment.5 The six stamens from
each flower were placed in a drop of glycerol-distilled water
on a microslide for manually untangling stamen hairs.
After this procedure, the stamen hairs were observed un-
der a dissecting microscope, and the number of pink
events was determined in each flower. The series of 10
flowers was scored and averaged to provide a single data
point for each sampling. The results were expressed in
terms of pink events/1000 hairs.5
Three exposure sites in the area of the incinerator
were selected, and the experiments were carried out
Volume 50 October 2000
Journal of the Air & Waste Management Association 1853

Ferreira et al.
Table 1. Descriptive statistics of the frequency of mutation events/1000 stamen
Statistical Analyses
hairs obtained in the four places of study.
The comparison of the frequency of mutations among
the groups was done by means of analysis of variance.
After testing the significance of the results, post-hoc
comparisons were performed using the Student-Neuman-
Keuls test. The possible influence of temperature and rela-
tive humidity on the frequency of mutations was
evaluated by verifying the cross-correlation among these
parameters considering phase lags up to 9 days. All the
statistical computations were done using the SPSS statis-
tical software (version 9.0). The level of significance was
set at 5%.
Places of Exposure (Distance from the Source)
Jaguariúna
(150 km)
School
Museum
(1.5 km)
Incinerator
(15 m)
(3.5 km)
Count
Mean
18
6
8
10
2.25
1.55
0.36
0.40
7.41
2.39
3.48
1.29
0.52
2.20
5.80
1.65
4.89
1.12
0.40
3.30
6.60
1.25
5.69
1.34
0.42
3.30
7.50
1.79
SD
SE
Minimum
Maximum
Variance
RESULTS
Table 1 shows the frequencies of mutation events of each
monitoring site. The difference among the groups was
statistically significant (p < 0.0001). Student-Neuman-
Keuls post-hoc analysis showed that the frequency of
mutations observed in Jaguariúna (control) was signifi-
cantly lower than that of the remaining groups. The fre-
quency of mutations measured at the school (moderate)
was significantly lower than that at the museum (high)
and the incinerator (highest). The museum and the in-
cinerator areas presented a higher frequency of mutations
in comparison with the other areas.
relationship between frequency of mutations and prox-
imity to the incinerator can be observed.
DISCUSSION
The present study was designed to evaluate the mutage-
nicity of the gaseous emissions of the Vergueiro incin-
erator located in the metropolitan area of São Paulo.
Results of the 5-month in situ monitoring using the
Tradescantia stamen-hair mutation assay showed a sig-
nificant correlation between levels of mutagenicity and
distance from this stationary source. However, due to
the limitations of an ecological study like ours, it is not
possible to ascribe the obtained results to the incinerator
Table 2 depicts the values of temperature and relative
humidity measured in São Paulo during the period of study.
The lagged correlation analysis did not reveal any influ-
ence of temperature on the rate of mu-
tations, showing that our system is quite
Table 2. Descriptive statistics of the climatic variables during the period of study.
stable under the variations of tempera-
ture experienced by São Paulo during
our exposure period. A small negative
effect was observed between the relative
humidity of the sixth day prior to flower
sampling, exhibiting a coefficient of
correlation of –0.449 (p = 0.028).
Month of Exposure
Count
Mean
Minimum
Maximum
December 1998
Humidity
TempMax
TempMed
TempMin
Humidity
TempMax
TempMed
TempMin
Humidity
TempMax
TempMed
TempMin
Humidity
TempMax
TempMed
TempMin
Humidity
TempMax
TempMed
TempMin
31
31
31
31
31
31
31
31
28
28
28
29
31
31
31
31
30
30
30
30
80.58
27.67
21.99
17.76
61.06
28.59
23.07
19.60
59.74
28.83
22.94
19.40
55.26
28.39
22.40
18.39
52.95
25.33
19.32
15.31
65.50
20.20
18.18
14.20
33.00
20.90
19.36
17.60
44.00
26.40
20.79
17.90
38.00
20.60
18.15
14.90
29.00
20.70
13.07
5.20
95.40
32.90
25.52
20.70
95.00
35.40
27.52
21.00
74.00
32.30
25.33
20.20
77.00
32.70
26.10
22.30
82.00
32.00
24.78
20.70
Figure 1 shows the individual data
January 1999
February 1999
March 1999
April 1999
of the frequency of mutations mea-
sured at each of the three sites located
in the vicinity of the Vergueiro incin-
erator and the mean and correspond-
ing standard error (SE) of the
spontaneous frequency of mutations of
the control area. In general, the fre-
quency of mutations measured at the
points with high (museum and incin-
erator) and moderate (school) levels of
exposure presented values well above
the control. The differences among the
groups could be better identified when
aggregate means of all points were
used, as depicted in Figure 2. A clear
1854 Journal of the Air & Waste Management Association
Volume 50 October 2000

Ferreira et al.
emissions alone, since most of the
pollution contained in the atmo-
sphere of São Paulo is produced by
automotive emissions. This pollu-
tion scenario is quite complex, and
the possibility of synergic interac-
tions between the emissions of the
incinerator and those derived from
auto engines may have contributed
to our results. In fact, the soluble
fraction of the inhalable particulate
matter collected from the atmo-
sphere of São Paulo was mutagenic
when tested with the Tradescantia
micronucleus assay.12 Nevertheless,
the dose-response relationship be-
tween the distance of the source of
emissions (see Figure 1) and mutage-
nicity is a good indication that the
pollution derived from the incinera-
tor superimposes the effect of auto-
motive pollution.
Plants are known to be sensi-
tive bioindicators of mutagenesis, dis-
closing positive results with very low
levels of contamination.5 Although
the positive response in a plant sys-
tem cannot be directly equated to ge-
netic damage in humans, it
nevertheless serves as a first alert of
the potential hazard to human health.
We think that the result of this
eucariotic plant assay is perhaps more
creditable than that of the microbial
tests. Based on the current findings,
the emission of mutagenic pollutants
in a densely populated area deserves
an effective control policy. It would
be possible to corroborate the valid-
ity of these genetic data in conjunc-
tion with human health data taken
from epidemiologic surveys around
the area of these four sites. However,
to undertake this conservative ap-
proach—collecting the evidence of
cancer—would cost lives and time.
The Vergueiro incinerator is consid-
ered to be one of the essential station-
ary pollution sources in São Paulo and
its control may help to improve health
in a city in which air quality has al-
ready been deteriorated by millions of
cars running on its jammed streets.
Figure 1. Individual data points of the three sites of exposure in the vicinity of the Vergueiro
incinerator. The horizontal reference line represents the mean and the corresponding SEs (dotted
line) of the frequency of spontaneous mutations detected in the control region (Jaguariúna).
Figure 2. Mean ( SE) values of the frequency of mutations measured in the experimental sites.
The reference horizontal lines represent the mean (dashed) and the SE (dots) of the control region
(Jaguariúna).
Volume 50 October 2000
Journal of the Air & Waste Management Association 1855

Ferreira et al.
This is not the first time that mutagenic effects have
São Paulo and Dr. Ana Julia F.C. Lichtenfels from the Labo-
ratory of Experimental Air Pollution in the School of
Medicine at the University of São Paulo for their help and
suggestions during the study.
been detected in the vicinity of waste incinerators. Also
using Tradescantia assays (stamen-hair mutation and mi-
cronucleus), Ma et al.2 found a positive association between
mutagenesis and the distance from an incinerator in the
United States. Similar findings (using the Tradescantia mi-
cronucleus test on emissions of municipal incinerators)
were reported by Fomin and Hafner3 in Germany. Watts
et al.,1 using the Ames test, also found a positive mutagenic
effect of compounds extracted from the particulate matter
derived from a solid waste incinerator. The foregoing data
give support to our findings, mainly if one considers that
the Vergueiro incinerator is not equipped with systems of
filtration similar to those present in the facility investigated
by Watts et al.1
REFERENCES
1. Watts, R.; Fitzgerald, B.; Hell, G.; Garabedian, H.; Williams, R.; War-
ren, S.; Franklin, L.; Lewtas, J. Use of Bioassay Methods to Evaluate
Mutagenicity of Ambient Air Collected near a Municipal Waste Com-
bustor; J. Air Pollut. Control Assoc. 1989, 39, 1436-1439.
2. Ma, T.H.; Xu, C.; Liao, S.; McConnell, H.; Jeong, B.S.; Won, C.D. In
Situ Monitoring with the Tradescantia Bioassays on the Genotoxicity
of Gaseous Emissions from a Closed Landfill Site and an Incinerator;
Mutat. Res. 1996, 359, 39-52.
3. Fomin, A.; Hafner, C. Evaluation of Genotoxicity of Emissions from
Municipal Waste Incinerators with Tradescantia Micronucleus Bioas-
say (Trad-MCN); Mutat. Res. 1998, 414, 139-148.
4. Hanna, S.R.; Egan, B.; Vando, C.; Curreti, J. A Complex Terrain Dis-
persion Model for Regulatory Applications at the Westvaco Luke Mill;
Atmos. Environ. 1984, L8, 685-699.
5. Ma, T.H.; Cabrera, G.L.; Cebulska-Wasilewska, A.; Chen, R.; Loarca,
F.; Vendenberg, A.L.; Salamone, M.F. Tradescantia Stamen Hair Muta-
tion Bioassay; Mutat. Res. 1994, 310, 211-220.
It is important to note that climatic variables, such
as temperature and ambient relative humidity, may in-
fluence the frequency of mutations in an experiment like
ours, where plants were maintained in the field. In fact,
we detected a small negative association between fre-
quency of mutations and the values of relative humidity
on the sixth day prior to flower sampling. However, it is
hard to imagine that the differences among the places
observed in our work are derived from weather, since the
three sites located at the vicinity of the incinerator have
the same climate properties. Unfortunately, we do not
have climatic data for the region of Jaguariúna, but the
information available from the nearest metereologic base
(Campinas, 30 km from Jaguariúna) indicates similar
weather in comparison to São Paulo. Thus, it is difficult
to ascribe to weather the observed differences between
Jaguariúna and the three sites of exposure in São Paulo.
In conclusion, we found a positive correlation be-
tween the spatial contribution of the emissions of a waste
incinerator located inside an urban area and mutagenic
events measured by the Tradescantia stamen-hair assay.
The in situ approach employed in this study was simple,
efficient, and of low cost. According to the present find-
ings, an effective pollution control measure at the incin-
erator should be adopted by authorities of São Paulo in
order to prevent additional hazards to the health of the
exposed population.
6. Ichikawa, S. In Situ Monitoring with Tradescantia around Nuclear
Power Plants; Environ. Health Perspect. 1981, 37, 145-164.
7. Lower, W.R.; Drobney, V.K.; Aholt, B.J.; Politte, R. Mutagenicity of
the Environments in the Vicinity of an Oil Refinery and Petrochemi-
cal Complex; Teratog. Carcinog. Mutagen. 1983, 3, 65-73.
8. Fomin, A.; Paschke, S.; Arndt, U. Assessment of the Genotoxicity of
Mine-Dump Material Using the Tradescantia-Stamen Hair (Trad-SHM)
and the Tradescantia-Micronucleus (Trad-MCN) Bioassays; Mutat. Res.
1999, 426, 173-181.
9. Arutyunyan, R.M.; Pogosyan, V.S.; Simonyan, E.H.; Atoyants, A.L. In
Situ Monitoring of the Ambient Air around the Chloroprene Rubber
Industrial Plant Using the Tradescantia Stamen-Hair Mutation Assay;
Mutat. Res. 1999, 426, 117-120.
10. Van’t Hof, J.; Schairer, L.A. Tradescantia Assay System for Gaseous
Mutagens. A Report of the U.S. Environmental Protection Agency
Gene-Tox Program; Mutat. Res. 1982, 99, 303-315.
11. Sparrow, A.H.; Underbrink, A.G.; Rossi, H.H. Mutation Induced in
Tradescantia by Small Doses of X-Rays and Neutrons: Analysis of Dose-
Response Curves; Science 1972, 176, 916-918.
12. Batalha, J.R.; Guimaraes, E.T.; Lobo, D.J.; Lichtenfels, A.J.; Deur, T.;
Carvalho, J.A.; Alves, E.S.; Domingos, M.; Rodrigues, G.S.; Saldiva,
P.H. Exploring the Clastogenic Effects of Air Pollutants in São Paulo
(Brazil) Using the Tradescantia Micronucleus Assay; Mutat. Res. 1999,
426, 229-232.
ACKNOWLEDGMENTS
About the Authors
This work was supported by grants from São Paulo State
Research Support Foundation (FAPESP), National Coun-
cil for Scientific and Technological Development (CNPq),
Programs of Centers of Excellence (PRONEX), and Labo-
ratory of Medical Investigation, Faculty of Medicine, Uni-
versity of São Paulo (LIM-HCFMUSP). We gratefully
acknowledge the Secretaria Municipal do Verde e Meio
Ambiente of São Paulo. We thank Drs. Marisa Domingos
and Edenise Segalla Alves from the Botanical Institute of
Maria Izildinha Ferreira, Helena Petrenko, and Debora Jã
de Araujo Lobo are biologists whose main interest is in en-
vironmental mutagenesis. Paulo Saldiva is a pathologist in-
terested in environmental toxicology and biomonitoring of
air pollution. Andrea Moreira and Geraldo Stachetti
Rodrigues are ecologists interested in developing new strat-
egies of monitoring environmental mutagenic effects of toxic
substances using plant bioassays. Address correspondence
to Saldiva at pepino@usp.br.
1856 Journal of the Air & Waste Management Association
Volume 50 October 2000