This article appeared in a journal published by Elsevier.

The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elseviers archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright

Author's personal copy

Aquatic Toxicology 88 (2008) 214219

Contents lists available at ScienceDirect

Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Action mechanisms of petroleum hydrocarbons present in waters impacted

by an oil spill on the genetic material of Allium cepa root cells
Daniela Morais Leme a , Dejanira de Franceschi de Angelis b , Maria Aparecida Marin-Morales a,
a
b

Departamento de Biologia, Instituto de Biociencias, Universidade Estadual Paulista (UNESP), Rio Claro, SP, Brazil
Departamento de Microbiologia, Instituto de Biociencias, Universidade Estadual Paulista (UNESP), Av. 24A, 1515, 13506-900, Rio Claro, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 28 January 2008
Received in revised form 17 April 2008
Accepted 29 April 2008
Keywords:
Allium cepa
Chromosomal aberrations
Aneugenic and clastogenic agents
Cell death
Polluted waters
Petroleum hydrocarbons

a b s t r a c t
Chromosomal aberration (CA) assays have been widely used, not only to assess the genotoxic effects
of chemical agents, but also to evaluate their action mechanisms on the genetic material of exposed
organisms. This is of particular interest, since such analyses provide a better knowledge related to the
action of these agents on DNA. Among test organisms, Allium cepa is an outstanding species due to its
sensitivity and suitable chromosomal features, which are essential for studies on chromosomal damage
or disturbances in cell cycle. The goal of the present study was to analyze the action mechanisms of
chemical agents present in petroleum polluted waters. Therefore, CA assay was carried out in A. cepa
meristematic cells exposed to the Guaeca river waters, located in the city of Sao Sebastiao, SP, Brazil, which
had its waters impacted by an oil pipeline leak. Analyses of the aberration types showed clastogenic and
aneugenic effects for the roots exposed to the polluted waters from Guaeca river, besides the induction of
cell death. Probably all the observed effects were induced by the petroleum hydrocarbons derived from
the oil leakage.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Chromosomal aberrations (CA) are characterized by changes in
either chromosomal structure or in the total number of chromosomes, which may occur both spontaneously and as a result from
the exposure to physical or chemical agents (Russel, 2002). Physical
and chemical agents can induce CA through different mechanisms,
involving clastogenic and aneugenic actions. Clastogenic action is
characterized by the induction of chromosomal breakage during
cell division, while aneugenic action comprises the inactivation of
a cell structure, such as the mitotic spindle, leading to chromosomal
losses (Fenech, 2000).
Several CA types are derived from nuclear abnormalities, such
as nuclear buds, micronuclei (MN), mini cells, lobated nuclei and
polinucleated cells. The MN can result from acentric fragments
(aneugenic agent) or whole chromosomes (clastogenic agent) that
were not incorporated to the main nucleus during the cell cycle
(Fenech, 2000). According to Fernandes et al. (2007), nuclear buds
are indicative of an initial process of releasing the excedding nuclear
material and, consequently, they can also be related to the MN formation, which might be further eliminated from cytoplasm as mini

Corresponding author. Tel.: +55 19 3526 4143, fax: +55 19 3536 0009.
E-mail address: mamm@rc.unesp.br (M.A. Marin-Morales).
0166-445X/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2008.04.012

cells. Lobate nuclei and polynucleated cells are resultant of CA, as a

consequence of multipolar anaphases, which are associated or not
with chromosomal adherence, making the cells inviable (Fernandes
et al., 2007).
Because of the increasing chemical dumping in the environment, several bioassays have been carried out in order to evaluate
the effects induced by these agents. The CA assay, one of the oldest
and most used tests, has been considered as one of the few direct
methods capable of measuring mutations in systems exposed to
putative mutagenic or carcinogenic substances (Rank et al., 2002).
Moreover, such test, based on classical cytogenetics, is easily carried
out in a great number of organisms, making it useful for environmental monitoring.
Among the test organisms used in this assay, higher plants,
including Allium cepa, are acknowledged as excellent bioindicators of genotoxic and mutagenic effects of environmental chemicals
(Grant, 1994, 1999). Thus, CA test in A. cepa has been considered as
an efcient short-term assay for environmental pollutant evalua 1985, 1988; Cotelle et al., 1999) and, in particular, for
tion (Fiskesjo,
water pollutants (Smaka-Kincl et al., 1996; Rank and Nielsen, 1993,
1994; Matsumoto et al., 2006; Fatima and Ahmad, 2006; Migid et
al., 2007, Leme and Marin-Morales, 2008). Furthermore, the chromosomal features of this species favor carrying out the CA test,
not only for genotoxic effect assessment, but also for understand 1985;
ing the action mechanisms of the tested chemicals (Fiskesjo,
Rank and Nielsen, 1997).

Author's personal copy

D.M. Leme et al. / Aquatic Toxicology 88 (2008) 214219

215

Besides its sensitivity, some researchers have also selected the

Allium test because of the high correlation between it and other test
systems. These features are essential to an accurate assessment of
environmental risks, as well as to a successful extrapolation of test
results obtained in exposed organisms to other species. Regarding these characteristics, Fiskesjo (1985) showed that the Allium
test presents a similar sensitivity to that of some algal and human
lymphocyte test systems. Rank and Nielsen (1994) showed a correlation of 82% between Allium test and carcinogenicity assays in
rodents, besides demonstrating that the former was more sensitive
than Ames or microscreen tests. Furthermore, studies of sensitivity
amongst higher plants have also showed that A. cepa is more sensitive than some other species, such as Vicia faba (Ma et al., 1995;
Migid et al., 2007).
Taking into account that petroleum hydrocarbons can cause a
harmful effect on the genetic material of exposed organisms and
that their action mechanisms on DNA need to be understood, the
aim of the present study was to analyze the action mechanisms of
petroleum hydrocarbon polluted waters impacted by an oil pipeline
leak.
2. Material and methods
2.1. Material
Water sample from the Guaeca river, located in the Serra do Mar
State Park, next to the city of Sao Sebastiao, SP, Brazil, was used
as a tested material due to petroleum hydrocarbon contamination
derived from an oil pipeline leakage, in February 2004 (Fig. 1). This
river runs through a permanent preservation area, being characterized by a high surrounding biodiversity and by the proximity to the
city water reservoir. The oil leakage was a result of a crack in the
pipeline, which is located underground. Thus, the leaked oil rst
affected the underground waters and later has arisen in the Guaeca
river spring, affecting the waters along its entire system. However,
the total volume of oil leaked was not estimated, since the day the
leakage started could not be accurately determined.
The water sampling was carried out in July 2005 at the spring
of the Guaeca river (Fig. 1). This was the oil leak surface area and
the only site where the presence of petroleum hydrocarbons was
detected through previous Total Petroleum Hydrocarbons (TPHs)
and Polycyclic Aromatic Hydrocarbons (PAHs) chemical analyses
(Table 1). For the water sampling procedure, the method of supercial water collection was used, according to CETESB protocol (1987).
A brand new 20 l plastic container was used for the water sampling.
The container was rst submerged in the water where sampling
was going to take place for a initial rinse. Afterwards, the container
was submerged again in the collection site, but this time in the middle of the river, until it was completely lled up. Then, the container
was immediately sealed up and cooled at 4 2 C to be transported
to the laboratory where the bioassay was carried out.
2.2. Test organism
Seeds of A. cepa were used as test organism because they
are genetically and physiologically homogeneous, besides being
available all year roundfeatures that ensured a reliable assay performance.
2.3. Test procedure
The chromosomal aberration assay using A. cepa meristematic
cells was carried out according to a modied version of Grants
protocol (1982).

Fig. 1. Location of Sao Sebastiao city, collection site (S1) in the Guaeca river and the
pipeline responsible for the oil impact.

Onion seeds were germinated, at room temperature (20 5 C),

in several Petri dishes, each dish covered with lter paper and individually wetted with the Guaeca river water and the negative and
positive control treatments. Ultra pure water (Milli-Q) was used
as a negative control, and 4 104 M of methyl methanesulfonate
(MMS, SigmaAldrich, CAS 66-27-3) was used as positive control.
When the roots reached about 2.0 cm in length, approximately 5
days after the beginning of the assay, they were xed in alcoholacetic acid (3:1-v/v) for 24 h. Then, the xed roots were stained
with Schiff reagent. To prepare the slides, the meristematic regions
were coated with coverslips and carefully squashed into a drop of 2%
acetic carmine solution. The coverslips were removed using liquid
nitrogen and the slides were mounted in synthetic resin (Mounting
Media, Permount , Fisher Scientic) to further analysis.
Several types of CA were analyzed within different cell division
stages (prophase, metaphase, anaphase and telophase) and classied according to the action mechanism (aneugenic or clastogenic
effects). The chromosomal breaks and bridges were considered to
result from clastogenic effect, whereas the chromosomal losses,
laggards, adherence, multipolarity and c-metaphases were considered to result from aneugenic effect, since the latter aberrations
derive from disturbances in the mitotic spindle. Nuclear abnormalities, such as the presence of lobated nuclei and polynucleated cells,
were used as indicators of cell death processes. The micronucleus
(MN) incidence, as well as its size, has also been visually analyzed
to evaluate clastogenic and aneugenic effects.
The analysis was done by scoring 5000 cells per treatment, 500
cells per slide, comprising a total of 10 slides. Statistical analysis was

Author's personal copy

216

D.M. Leme et al. / Aquatic Toxicology 88 (2008) 214219

Table 1
TPHs and PAHs detected through chemical analysis in Guaeca river water sample
Sampling

Site

Chemical analysis

Types of hydrocarbons

July 2005

S1

TPHs

C12
C13
C14
C15
C16
C17
C18

Total of n-alcanos

MCNR

PAHs

Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Pyrene
Benzo(a)anthracene
Chrysene

Total of PAHs

Concentration (g/l)

Detection limit (g/l)

148.34
148.34
310.40
271.53
261.53
966.30
509.27

50.00
50.00
50.00
50.00
50.00
50.00
50.00

2615.71

79896.62

7.08
4.56
1.91
15.12
78.78
9.43
3.82
18.88

0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25

139.58

MCNR: Complex mixture not resolved.

performed using the KruskalWallis test, accepting the probability

of 0.05 to show a signicant effect.

Table 2
CA and nuclear abnormalities in Allium cepa meristematic cells after exposure to
the polluted waters of Guaeca river and the negative (Milli-Q water) and positive
control (MMS) treatments

2.4. Chemical analysis

The analyses of the Total Petroleum Hydrocarbons and the Polycyclic Aromatic Hydrocarbons (PAHs) in the Guaeca river water
sample were conducted by the Analytical Technology Company, Sao
Paulo, SP, Brazil.
The TPHs analyses were performed according to the U.S.
EPA 8015 method, involving gase chromatography with ame
ionization detector (Gcd). The PAHs analyses followed the
U.S. EPA 8270 method, using also the gas chromatography,
but with mass detector (Gcms). This approach was able to
detect the following PAHs: naphthalene, acenaphthylene, acenaphthene, uorene, phenanthrene, anthracene, uoranthene,
pyrene, benzo(a)anthracene, chrysene, benzo(b)uoranthene,
benzo(k)uoranthene, benzo(a)pyrene, indene(123-cd)pyrene,
dibenzo(a,h)anthracene, and benzo(g,h,i)perilene.

Milli-Q water

MMS

Guaeca river water

sample

Chromosomal aberrations
Chromosome breaks
Chromosome bridges
Chromosome losses
Laggards
Adherence
Multipolarity
C-metaphases

0.01
0.10
0.01
0.01
0.40
0.01
0.08

0.31
0.84
0.31
0.31
1.33
0.31
0.70

0.16
0.50
0.14
0.07
1.20
0.02
0.07

0.87
1.99*
1.13
0.51
4.22*
0.31
0.51

0.05
0.38
0.31
0.10
0.47
0.20
0.38

0.67
2.45
1.45*
0.70
2.64
0.70
2.82

Nuclear abnormalities
Lobated nuclei
Polynucleated cells
Nuclear buds
Micronucleus
Mini cells

0
0
0,07
0,12
0

0
0
0.96
0.95
0

0
0.29
0.48
0.53
0.02

0
3.37
1.71
2.28
0.31

1.05
0.01
1.86
2.68
1.96

6.65*
0.31
8.37*
9.10*
8.13*

5000 cells analyzed per treatment. Mean S.D.

*
Signicantly different from negative control
KruskalWallis test.

(p < 0.05),

according

to

3. Results
The results from CA assay in A. cepa root cells exposed to polluted
water of Guaeca river are shown below in Table 2 and Fig. 2.
The CA and nuclear abnormalities observed in the present study
were visualized in all stages of the cell cycle: interphase, prophase,
metaphase, anaphase, and telophase (Fig. 2).
The cells in interphase showed the following abnormalities:
nuclear buds, MN, mini cells, lobate nuclei and polynucleated
cells (Fig. 2a1 a6 ). In prophase, MN was the only type of abnormality observed (Fig. 2b1 b3 ). In metaphase, we observed cells
with MN, chromosomal breaks, chromosomal losses, chromosomal adherence, and C-metaphases (Fig. 2c1 c3 ). Cells in anaphase
showed chromosomal bridges, chromosomal losses, multipolarity
and laggards in the chromosome segregation (Fig. 2d1 d3 ). Buds,
MN, chromosomal bridges and multipolarity were also observed
in telophase cells (Fig. 2e1 e3 ). Moreover, the MN observed in the
roots exposed to Guaeca river water showed different sizes, ranging
from small to large MN (Fig. 2a2 , a5 , a6 , b1 b3 , c3 , e2 , and e3 ).
In the meristematic cells of A. cepa exposed to Guaeca river
waters, all the CA and nuclear abnormality frequencies were found

to be higher than those found in negative control, although not all

the differences were statistically signicant. Only the frequencies
of chromosome breaks, lobated nuclei, nuclear buds, MN and mini
cells showed statistically signicant differences to the negative control (Table 2).
4. Discussion
The present data showed a signicant MN number in the roots
exposed to the polluted waters of Guaeca river (Table 2). Furthermore, MN present in these roots showed different sizes (Fig. 2a2 , a5 ,
a6 , b1 b3 , c3 , e2 , and e3 ). This result, suggests that the petroleum,
composed by a complex mixture of hydrocarbons, can have both
clastogenic and aneugenic actions. Moreover, these actions can be
related to the PAHs present in the water sample, since their toxic,
mutagenic and carcinogenic effects have already been documented
(Aina et al., 2006). However, other techniques, such as chromosomal bandings (C-banding, NOR and base-specic uorochromes)

Author's personal copy

D.M. Leme et al. / Aquatic Toxicology 88 (2008) 214219

217

Fig. 2. Meristematic cells of Allium cepa exposed to polluted water of Guaeca river. (a) Normal interphasic nucleus; (a1 ) nuclear bud; (a2 ) MN; (a3 ) mini cell; (a4 ) lobated
nucleus; (a5 and a6 ) polynucleated cells; (b) normal prophase; (b1 ) mini cellbeginning of the formation; (b2 ) prophase with large MN; (b3 ) prophase with a small MN; (c)
normal metaphase; (c1 ) metaphase with chromosomal break; (c2 ) metaphase with chromosomal loss; (c3 ) metaphase with MN; (d) normal anaphase; (d1 ) anaphase with
chromosomal bridge; (d2 ) anaphase with chromosomal losses; (d3 ) multipolar anaphase with bridge and chromosomal loss; (e) normal telophase; (e1 ) telophase with bud;
(e2 ) telophase with MN; (e3 ) telophase with MN and bridge.

and uorescent in situ hybridization (FISH), must be carried out to

corroborate the proposed hypothesis.
According to Fenech (2002), MN are determined from acentric
fragments (clastogenic agent) or whole chromosomes (aneugenic
agent) that were not incorporated to the main nucleus during the
cell division cycle. Yamamoto and Kikuchi (1980) showed that,
through measuring the MN diameter, it would be possible to determine if the agent tested was clastogenic or aneugenic. The authors
showed that the MN derived from clastogenic agents were, in general, smaller than MN derived from aneugenic action. Although this
method has been satisfactory for a great number of MN, it does not
show reliable evidence in many other cases. However, other authors
(Combes et al., 1995; Fenech, 2000) concluded that the utilization
of MN size analysis as a parameter to determine xenobiotic clastogenic or aneugenic actions would not be reliable enough, since,
depending on the species, there are differences in relation to the
chromosome size in karyotypes, e.g. in human.
Opposite to man, some higher plant species, such as A. cepa, have
a symmetric karyotype, which is homogeneous in relation to chromosomal size, with large and few chromosomes (2n = 16) (Grant,
1985). Thus, the method proposed by Yamamoto and
1982; Fiskesjo,
Kikuchi (1980), which considers the MN size to determine whether
an agent is clastogenic and/or aneugenic, can be effective for the A.
cepa test system.
Chromosomal fragments can be derived from chromosomal
breaks in anaphase bridges, which can originate from cohesive

1993). Our data showed no

chromosomal translocations (Fiskesjo,
signicant value for chromosomal breaks in A. cepa meristematic
cells exposed to Guaeca river waters. However, they showed, as
mentioned above, signicant value for MN with both different sizes
and condensation levels (Table 2 and Fig. 2a2 , a5 , a6 , b1 b3 , c3 , e2 ,
and e3 ). Thus, the smaller MN can indicate the occurrence of chromosomal breaks, due to the clastogenic action of the petroleum
hydrocarbons present in the sample tested.
According to Fiskesjo and Levan (1993), Marcano and Del Campo

(1995), Marcano et al. (1999) and Turkoglu

(2007), chromosomal
adherence is a common sign of toxic effects on the genetic material and may cause irreversible effects on the cell, triggering the cell
death process. Marcano et al. (2004) showed that the chromosomal
adherence can lead to chromosomal bridges and, thereby, chromosomal breaks. The chromosomal bridges derived from adherence
can be multiple and can persist until telophase (Giacomelli, 1999).
Our data showed no signicant chromosomal adherence value for
the roots exposed to Guaeca river waters (Table 2). However, the
presence of this abnormality can explain the presence of chromosomal bridges and breaks in these roots, as well as the aneugenic
effects of petroleum hydrocarbons.
C-metaphases are evidence of aneugenic agents, since they pro
vide the complete inactivation of the cell mitotic spindle (Fiskesjo,
1985, 1993). The spindles are inactivated when no equatorial
plate is organized and, consequently, the centromere division is
blocked. According to Krisch-Volders et al. (2002) and Fernandes

Author's personal copy

218

D.M. Leme et al. / Aquatic Toxicology 88 (2008) 214219

et al. (2007), the presence of C-metaphases can result in multinuclear cells, although the most frequent result is the induction
of a great amount of MN. The present data showed no signicant
C-metaphase value in A. cepa meristematic cells exposed to the
Guaeca river water sample (Table 2). However, these results are
consistent with the data of Krisch-Volders et al. (2002), since a
great number of MN on the cells exposed to polluted waters was
observed, indicating the aneugenic effect induced by the presence
of petroleum hydrocarbons.
According to Rank and Nielsen (1998), multipolar anaphases
result from a misfunction of the mitotic spindle, which leads to
unbalanced chromosome distribution, heading them for more than
two poles onto the cells, opposite to what occurs in the normal
division cycle. In the present study, no signicant multipolarity
value was observed for the A. cepa cells exposed to the water tested
(Table 2, Fig. 2a4 ).
Fernades (2005) showed that lobated nuclei can result from
multipolar anaphases with chromosomal bridges. According to this
author, the presence of multipolarity during the nuclear division
seems not to avoid the reorganization of the nuclear envelope and
the membrane would follow the unbalanced distribution of the
genetic material within the cell, resulting in the lobated nuclei. Our
data showed a signicant number of lobated nuclei in the roots
exposed to the Guaeca river waters (Table 2, Fig. 2a4 ). We conclude that the presence of such nuclear abnormality leads to the
induction of cell death process, once they were not observed in
the F1 cells (non-meristematic region) of A. cepa roots (Leme and
Marin-Morales, 2008).
Some authors report that chromosomal losses and breaks, as
well as the excess material, promoted by the DNA replication, can
induce MN, which can be eliminated from the cell in the form of
mini cells, i.e., small cytoplasm portions with a reduced fraction
of nuclear material (Fernandes et al., 2007). In the present study,
we observed signicant numbers of mini cells in the meristematic
cells of A. cepa exposed to the Guaeca river water sample (Table 2
and Fig. 2a3 ). The presence of these mini cells seems to be a result
from the MN elimination of the multimicronuclei cells, which is
consistent with the data of Fernandes et al. (2007), studying effects
of aneugenic agents.
Taking into account that the A. cepa test system evaluates the
environmental risks present due to its high sensitivity and good
1985; Rank
correlation with tests using other organisms (Fiskesjo,
and Nielsen, 1994; Fatima and Ahmad, 2006), we can infer that
the pollution caused by the oil leakage still affected Guaeca river
waters, even one year after the accident, revealing that the presence
of the petroleum hydrocarbons, although at low concentrations, can
cause harmful effects in the exposed organisms. However, although

A. cepa has a good correlation with other test systems (Fiskesjo,

1985; Rank and Nielsen, 1994; Ma et al., 1995; Migid et al., 2007),
the extrapolation of the present results requires caution, since living
organisms might have many biological differences, such as distinct
metabolic systems, which can lead to a differential action response
to chemical agents.
Therefore, we can conclude that petroleum hydrocarbons can
show both clastogenic and aneugenic effects, as well as induce cell
death process, on the genetic material of exposed organisms. In
addition, we can also conclude that these effects are probably a
result from the mixture of PAHs detected by the chemical analysis
of the Guaeca river water sample.
In summary, we suggest that the CA assay in A. cepa, associated with the MN size analysis, can be an efcient test to
assess the action mechanisms of different chemical agents, including petroleum hydrocarbons. However, it is important to carry
out other cytogenetic techniques to accomplish more reliable
results.

Acknowledgements
We would like to thank Dr. Joao Carlos, C. Milanelli and Instituto
Florestal, Sao Sebastiao, SP, Brazil, for his collaboration during eld
works, and the Programa de Recursos Humanos ANP/FINEP/MCTCTPETRO, PRH-05 of Universidade Estadual Paulista (UNESP), Rio
Claro, SP, Brazil, for the nancial support.
References
Aina, R., Palin, L., Citterio, S., 2006. Molecular evidence for benzo[a]pyrene and
naphthalene genotoxicity in Trifolium repens L. Chemosphere 65, 666673.
Companhia de Tecnologia e Saneamento Ambiental (CETESB). Guia de coleta e

preservaca o de amostras de agua,

1987. Coord. Edmundo Garcia Agudo (et al.)
Sao Paulo.
Combes, R.D., Stopper, H., Caspary, W.J., 1995. The use of the L5178Y mouse
lymphoma cells to assess mutagenic, clastogenic and aneugenic activity of
chemicals. Mutagenesis 10, 403408.

Cotelle, S., Masfaraud, J.-F., Ferard,

J.-F., 1999. Assessment of the genotoxicity
of contaminated soil with the Allium/Vicia-micronucleus and Tradescantiamicronucleus assays. Mutat. Res. 426, 167171.
Fatima, R.A., Ahmad, M., 2006. Genotoxicity of industrial wastewaters obtained from
two different pollution sources in northern India: a comparison of three bioassays. Mutat. Res. 609, 8191.
Fenech, M., 2000. The in vitro micronucleus technique. Mutat. Res. 455, 8195.
Fenech, M., 2002. Biomarkers of genetic damage for cancer epidemiology. Toxicology,
4114116.

Fernades, T.C.C., 2005. Investigaca o dos efeitos toxicos,

mutagenicos e genotoxicos
do herbicida triuralina, utilizando Allium cepa e Oreochromis niloticus como sistemas-teste. Dissertaca o (Mestrado em Biologia Celular e
Molecular)Universidade Estadual Paulista, Rio Claro/SP, 212pp.
Fernandes, T.C.C., Mazzeo, D.E.C., Marin-Morales, M.A., 2007. Mechanism of
micronuclei formation in polyploidizated cells of Allium cepa exposed to triuralin herbicide. Pestic. Biochem. Physiol. 88, 252259.
G., 1985. The Allium test as a standard in environmental monitoring. HeredFiskesjo,
itas 102, 99112.
G., 1988. The Allium testan alternative in environmental studies: the relFiskesjo,
ative toxicity of metal ions. Mutat. Res. 197, 243260.
G., 1993. The Allium cepa test in wastewater monitoring. Environ. Toxicol.
Fiskesjo,
Water Qual. 8, 291298.
G., Levan, A., 1993. Evaluation of the rst ten MEIC chemicals in the Allium
Fiskesjo,
test. Atla 21, 139149.
Grant, W.F., 1982. Chromosome aberration assays in Allium. Mutat. Res. 99, 273291.
Grant, W.F., 1994. The present status of higher plant bioassays for detection of environmental mutagens. Mutat. Res. 310, 175185.
Grant, W.F., 1999. Higher plant assays for the detection of chromosomal aberrations
and gene mutationa brief historical background on their use for screening and
monitoring environmental chemicals. Mutat. Res. 426, 107112.

Giacomelli, F.R.B., 1999. Avaliaca o do comportamento meiotico

em variedades de
aveia (Avena sativa) recomendadas para regiao sul. Dissertaca o (Mestrado em
Maringa/PR,

131pp.
Genetica)Universidade
Estadual de Maringa,
Krisch-Volders, M., Vanhauwaert, A., De Boeck, M., Decordier, I., 2002. Importance
of detecting numerical versus structural chromosome aberrations. Mutat. Res.
504, 137148.
Leme, D.M., Marin-Morales, M.A., 2008. Chromosome aberration and micronucleus
frequencies in Allium cepa cells exposed to petroleum polluted watera case
study. Mutat. Res. 650, 8086.
Ma, T.-H., Xu, Z., Xu, C., McConnell, H., Rabago, E.V., Arreola, G.A., Zhang, H., 1995. The
improved Allium/Vicia root tip micronucleus assay for clastogenicity of environmetal pollutants. Mutat. Res. 334, 185195.

Marcano, L., Del Campo, A., 1995. Estudio ultraestructural del nucleolo
en pobla
ciones meristematicas
de cebolla Allium cepa L. tratadas com inhibitores

metabolicos.
Ciencia 3, 7382.
de la actividad
Marcano, L., Carruyo, I., Montiel, X., Moreno, P., 1999. Inhibicion

biosintetica
nucleolar inducidas por el plomo en meristemos radiculares de
cebolla (Allium cepa). Rev. Fac. Agron. Univ. Zulia. 16, 476487.
Marcano, L., Carruyo, I., Del Campo, A., Montiel, X., 2004. Cytotoxicity and mode
of action of maleic hydrazide in root tips of Allium cepa L. Environ. Res. 94,
221226.
Matsumoto, S.T., Mantovani, M.S., Malaguttii, M.I.A., Dias, A.L., Fonseca, I.C., MarinMorales, M.A., 2006. Genotoxicity and mutagenicity of water contaminated with
tannery efuents, as evaluated by the micronucleus test and comet assay using
the sh Oreochromis niloticus and chromosome aberrations in onion root-tips.
Genet. Mol. Biol. 29, 148158.
Migid, A.H.M., Azab, Y.A., Ibrahim, W.M., 2007. Use of plant genotoxicity bioassay for
the evaluation of efciency of algal biolters in bioremediation of toxic industrial
efuent. Ecotox. Environ. Saf. 66, 5764.
Rank, J., Nielsen, M.H., 1993. A modied Allium test as a tool in the screening of the
genotoxicity of complex mixtures. Hereditas 18, 4953.
Rank, J., Nielsen, M.H., 1994. Evaluation of the Allium anaphase-telophase test in
relation to genotoxicity screening of industrial wastewater. Mutat. Res. 312,
1724.

Author's personal copy

D.M. Leme et al. / Aquatic Toxicology 88 (2008) 214219

Rank, J., Nielsen, M.H., 1997. Allium cepa anaphase-telophase root tip chromosome
aberration assay on N-methyl-N-nitrosourea, maleic hydrazide, sodium azide,
and ethyl methanesulfonate. Mutat. Res. 390, 121127.
Rank, J., Nielsen, M.H., 1998. Genotoxicity testing of wastewater sludge using the
Allium cepa anaphase-telophase chromosome aberration assay. Mutat. Res. 418,
113119.
Rank, J., Lopez, L.C., Nielsen, M.H., Moretton, J., 2002. Genotoxicity of maleic
hydrazide, acredine and DEHP in Allium cepa root cells performed by two different laboratories. Hereditas 36, 1318.

219

Russel, P.J., 2002. Chromosomal mutation. In: Cummings, B. (Ed.), Genetics. Pearson
Education Inc., San Francisco, pp. 595621.
Smaka-Kincl, V., Stegnar, P., Lovka, M., Toman, M.J., 1996. The evaluation of waste,
surface and ground water quality using the Allium test procedure. Mutat. Res.
368, 171179.

Turkoglu,
S., 2007. Genotoxicity of ve food preservatives tested on root tips of Allium
cepa L. Mutat. Res. 626, 414.
Yamamoto, K.I., Kikuchi, Y., 1980. A comparison of diameters of micronuclei induced
by clastogens and by spindle poisons. Mutat. Res. 71, 127131.