The evaluation mutagenic potential of pesticides most used in southeastern Brazil, through bioassays with Allium cepa, it was an important study to understand harmful action of two classes of pesticides widely used in Brazil. The effects significant of cytotoxic and genotoxic in Allium cepa were evaluated in the following concentrations: 10 μL/mL, 25 μL/mL and 50 μL/mL, for Pyraclostrobin and Iminoctadine being in all cases compared to the negative and positive controls. With the results obtained it was possible to verify that the two pesticides have the ability to promote genetic changes. This study is a consequence of numerous complaints about harmful effects these substance, since small changes in DNA can cause irreversible problems the human health.

1. Evaluation mutagenic potential of pesticides through bioassays with Allium cepa
WJCBG
Evaluation mutagenic potential of pesticides through
bioassays with Allium cepa
Rodrigo Pratte-Santos1*
, Diego Roncete Ramos2
, Thelry Leite Becalle3
, Jairo Pinto De
Oliveira4
and Adilson Ribeiro Prado5
1*,2,3
Pio XII College, Bolivar de Abreu St, 29146-330, Cariacica, ES, Brazil.
1,4
Federal University of Espirito Santo, Mal. Campos Av., 29043-900, Vitória, ES, Brazil.
5
Federal Institute of Espirito Santo, ES-010 - Km 6,5, 29173-087, Serra, ES, Brazil.
The evaluation mutagenic potential of pesticides most used in southeastern Brazil, through
bioassays with Allium cepa, it was an important study to understand harmful action of two
classes of pesticides widely used in Brazil. The effects significant of cytotoxic and
genotoxic in Allium cepa were evaluated in the following concentrations: 10 μL/mL, 25
μL/mL and 50 μL/mL, for Pyraclostrobin and Iminoctadine being in all cases compared to the
negative and positive controls. With the results obtained it was possible to verify that the
two pesticides have the ability to promote genetic changes. This study is a consequence of
numerous complaints about harmful effects these substance, since small changes in DNA
can cause irreversible problems the human health.
Keyword: Bioassays, Allium cepa, pesticides, mutagenic potential, organophosphate insecticides.
INTRODUCTION
The pesticides has being used in large scale in pest
control in modern agriculture, the reason of this fact is
the increase in productivity obtained with these
substances. But indiscriminate use of these agents may
bring harm to the environment and human health. In
2010 the Brazilian National Agency for Sanitary
Surveillance conducted study in 26 states, noting high
indices of pesticides in various types of foods marketed
(Freire et al. 2013; Faria et al. 2014). As an example
the state of Espírito Santo, southeastern Brazil, showed
dissatisfaction in 26.47% of the samples on the level of
pesticide. Organophosphate pesticides appear as the
main chemical group responsible for this
unsatisfactoriness (Chrisman et al. 2009; Boccolini et
al. 2013).
It is believed that with the growth of agriculture in the
state, the need for higher yields, fewer losses for pests
and good results with positive balance motivates the
irregular use of these substances. Thus a farmer
resorts to using products and techniques to improve
their production. Therefore makes use of pesticides in
order to reduce costs, not checking the risks and
operational security required for this type of work
(Chrisman et al. 2009).
Organophosphates are chemicals that having the
element phosphorus in its formation. Being widely used
in agriculture as insecticides, pesticides and herbicides,
with highlight between the pesticides that have the
ability to react with Acetylcholinesterase. Causing
decomposition of acetylcholine after transmission of
nerve impulses from one neuron to another, causing
the accumulation of acetylcholine in the synaptic
receptors blocking the nerve transmissions (Tus̆ arová
et al. 1999; Smulders et al. 2003). In 1989 was enacted
law nº 7802 which provides for research,
experimentation and monitoring of pesticides, their
components and related products. Law that guides and
classifies all the harmful actions of these substances to
human life, this being a major reference used in Brazil
(Recena et al. 2006; Soares and Porto, 2009).
*Corresponding author: Rodrigo Pratte-Santos,
3
Pio XII
College, Bolivar de Abreu St, 29146-330, Cariacica,
ES, Brazil, E-mail: rodrigopratte@hotmail.com Tel.: +55
27 3421-2563
World Journal of Cell Biology and Genetics
Vol. 2(1), pp. 005-010, March, 2015. © www.premierpublishers.org, ISSN: 2167-0409x
Research Article

2. Evaluation mutagenic potential of pesticides through bioassays with Allium cepa
Pratte-Santos et al. 005
Table 1. Pesticides used for assessment of cytotoxicity and genotoxicity in Allium cepa.
Active
Ingredient
Trade
name
Chemical
Group
Class Toxicological
class
chemical
composition
Iminoctadin
(Albesilate)
Bellkute Guanidines Contact
fungicide
Class I Iminoctadina Tris
Albesilato: 315g/L
(31.5%m/v)
Inert ingredients:
735g/L (73.5%m/v)
Piraclostrobin Comet Estrobirulines Systemic
fungicide
Class II Piraclostrobina: 250
g/L ou 25.0% m/v
Inert ingredients: 802
g/L ou 80.2% m/v
Due to various problems caused by incorrect use of
pesticides, several tests being conducted with the goal
of check structural DNA damage (Burrack and
Chapman, 2013; Sparks, 2013), among them is the test
of micronucleus (Mn) (Rodríguez et al. 2015). This test
consists in the investigation of cells previously exposed
to chemical agents, aiming to detect possible
chromosomal aberrations, micronuclei seen that are
formed by the extrusion of whole chromosomes or from
fragments during cell division (Ginzkey et al. 2014;
Sanada et al. 2014; Rodríguez et al. 2015). An
important feature of the micronucleus test is the ability
to express chromosome damage when exposed to
chemical agents indicating loss, breakage or presence
of micronuclei. In several studies the test proved great
effectiveness and reliability, besides presenting some
advantages over other tests such as low cost and
speed of analysis (Ayed-Boussema et al. 2011). Thus,
the aim of this study was to evaluate the mutagenic
potential of pesticides in use in southeastern Brazil
through bioassay with Allium cepa to check the
mutagenic effect of different concentrations of
pesticides on the cells and what the possible
relationship of concentration of the pesticide to
abnormalities in cell division.
MATERIALS AND METHODS
For the tests were selected two types of very pesticides
currently marketed. Table 1 shows the main features of
these substances used. The bulbs of Allium cepa were
obtained commercially and in all bioassays biotypes of
the same species and origin were used. The
Iminoctadine (Albesilato) is a contact fungicide
chemical group of the guanidines which operates
mainly in the biosynthesis of cell membrane lipids.
Composed by: 1,1-iminiodi(octamethylene)
diguanidinium tris (alkylbenzenesulfonate). The
Pyraclostrobin acts as an inhibitor of electron transport
in the mitochondria of the cells of fungi by inhibiting the
formation of ATP. Is essentially to metabolic processes
in fungi. Composed by: Methyl N-(2-{[1-(4-
chlorophenyl)-1H-pyrazol-3-yl] oxymethyl}phenyl)N-
methoxycarbamate.
The present study, a bioassay with Allium cepa, in
which 16 examples were placed in distilled water for 24
hours at room temperature, to stimulate the
development of root meristem was performed. After this
time, the bulbs were placed in test solutions for a period
of 48 hours. It is noteworthy that all the experiments
were done in duplicates and the blades of analysis
were examined in blind test. For each slide 2000 cells
were counted by identifying changes mitotic and
micronuclei according to the criteria proposed by
Titenko Holland et al. (1997) (Titenko-Hollandd et al.
1997). For positive control, Allium cepa biotypes were
exposed to the copper sulphate (CuSO4), since it has a
mutagenic potential proven in the literature and as
negative control, biotypes were exposed to distilled
water. The roots length was used as an assessment of
mitotic index as the reference standard, these case
more precisely three roots were used. For each bulb,
the length of the three longest roots was estimated with
a ruler. In each case treatment was compared with
negative control. Moreover the toxicity (growth
inhibition) was defined as the difference between
treatment and negative control was statistically
significant. Data were statistically analyzed using the
software ANOVA with significance level of 5%. Was
used for perform linear regression the software
BioEstat, ver. 5.0, being possible to confirm the results
obtained from the ANOVA.
Methodology used to assess the formation of
micronuclei is described below, being similar the
proposed in another papers (Garriott et al. 2002; Kato
et al. 2011):
1. An onion was placed in contact with distilled
water so that the region where there was the formation
of onion roots of ringtone the solution;

3. Evaluation mutagenic potential of pesticides through bioassays with Allium cepa
World J. Cell Biol. Gen. 006
Figure 1. Chromosomal abnormalities observed in meristematic cells of Allium cepa exposed to pesticides,
photos taken at the time of analysis of biotypes (Viewed in 40x). A) Prophase, Anaphase and Telophase,
normal processes. B) Chromosomes Laggards. C) Chromatid loose. D) Accessory Chromosome
(micronucleus). E) Chromosome bridge. F) Chromatid and loose nuclear Defragmentation
Table 2. Analysis of root length Pyraclostrobin treated.
Parameters C- C+1 C+2 10 µl/ml 25 µl/ml 50 µl/ml
root length (cm) 4.0 ± 0.7 1.8 ± 0.4* 1.6 ± 1.3* 2.0 ± 0.7* 1.1 ± 0.6* 1.8 ± 0.4*
% growth 100 45* 40* 50* 28* 45*
*Significantly different value of the negative control. (p<0.05)
Legend: C-: Negative Control; C+1: Positive Control 1; C+2, Negative Control 2.
2. Roots grew to reach 2 cm and cut into sizes of
1 to 2 cm of the apical and transferred to a vessel
containing aceto-orcein hydrochloride;
3. The system was heated to the boiling point;
4. Was left to cool for 5 minutes;
5. This procedure was repeated for two more
times and after the 3rd heating, was left repose for 15
minutes;
6. One of the roots was put on a clean and
separates the apical 2-3 mm sheet, disregarding the
rest of the structure;
7. A drop of orcein was added on the meristem
sectioned by total immersion;
8. A small stirring was applied to obtain a single
cell extension;
9. The excess dye was removed;
10. A compression between the coverslip covered
with paper drying was applied;
11. Then the samples were observed with an
optical microscope Zeiss, at 10x and 40x.
RESULTS AND DISCUSSION
The tests of cytotoxicity and genotoxicity were
performed by the Allium cepa test based on some
parameters of analysis, such as atypical chromosomal
patterns, which consist of fragmentation, retardation
and chromosomal bridge. Another aspect studied was
the appearance of micronuclei formed by result of
chromosomal breakage, clearly indicating the
disturbances in the mitotic process. We also evaluated
the appearance of the root length, biotypes established
as control parameter, thus demonstrating the action of
pesticides on cell mitosis.
Of the 16 analyzed biotypes, abnormalities were found
in 94.4%. Root length showed significant differences
compared to the negative control. The fact proves
effective action of the pesticide in the phase of cell
proliferation. Each sample to be analyzed was prepared
in two blades each having a root. Thus it was possible
to estimate the number of micronuclei and
abnormalities (laggard chromosomes, chromosome
bridges and fragments). This experiment was
conducted in 2,000 cells, in Figure 1 it is possible to
verify some of obtained results, a fact that
demonstrates the feasibility of the study.
Bioassays with Pyraclostrobin concentrations of 10
μL/mL, 25 μL/mL and 50 μL/mL were used there was a
significant reduction in root growth (Table 2) (p <0.05).

4. Evaluation mutagenic potential of pesticides through bioassays with Allium cepa
Pratte-Santos et al. 007
Table 3. Analysis of abnormalities visualized in anaphases / telophases roots treated with Pyraclostrobin.
Parameters
C- C+1 C+2 10 µ/mL 25 µ/mL 50 µ/mL
% Fragment 0 0 0 0 0 0
% Latecomer 0 46 27 1 86 27
% Bridge 0 0 13 0 0 0
% Micronuclei 0 10* 12* 0.5 0.4 1.8
*Significantly different value of the negative control. (p<0.05)
Legend: C-: Negative Control; C+1: Positeive Control 1; C+2, Negative Control 2.
Table 4 - Analysis of root length treated with Iminoctadine Tris (Albesilato).
Parameters C- C+1 C+2 10 µ/ml 25 µ/ml 50 µ/ml
Root length (cm) 4.0 ± 0.7 1.8 ± 0.4* 1.6 ± 1.3* 1 ± 0.6* 0.6 ± 0.1* 0.4*
% Growth 100 45* 40* 25* 15* 10*
*Significantly different value of the negative control. (p<0.05)
Legend: C-: Negative Control; C+1: Positive Control 1; C+2, Negative Control 2.
Table 5. Analysis of abnormalities seen on the roots telophases anaphases and treated with Iminoctadine tris (Albesilato).
Parameters C- C+1 C+2 10 µ/ml 25 µ/ml 50 µ/ml
% Fragment 0 0 0 17 20 30
% Latecomer 0 46 27 0 0 0
% Bridge 0 0 13 5 0 8
% Micronuclei 0 10* 12* 0.6 1.7 3.6
*Significantly different value of the negative control. (p<0.05)
Legend: C-: Negative Control; C+1: Positive Control 1; C+2, Negative Control 2.
The analysis of root length showed a reduction of 50%,
72.5% and 55% at concentrations exposed 10 μL/mL,
25 μL/mL and 50 μL/mL respectively compared to the
negative control. The statistical test showed significant
differences at all concentrations tested.
The analysis of the abnormalities observed in dividing
cells revealed a high percentage of laggard
chromosomes (Table 3), more precisely in
anaphase/telophase in the 25 μL/mL.
The presence of forty-four micronuclei in the
concentration of 10 μL/mL, thirty-one micronucleus
concentration of 25 μL/mL and center and forty-three in
the concentration of 50 μL/mL were quantified.
In tests conducted with Iminoctadine Tris (Albesilate)
concentrations of 10 μL/mL, 25 μL/mL and 50 μL/mL
were used. All concentrations tested had reduced root
growth (Table 4) in the rates of 75%, 85% and 90%
lower than the negative control.
The analysis of abnormalities showed a significant
increase in micronuclei concentration of 50 μL/mL
(Table 5). It was also noted the presence of
chromosomal fragmentation proportionally increased
concentration: 17%, 20% and 30% for the
concentration of 10 μL/mL, 25 μL/mL and 50 μL/mL.
The result of the micronucleus test samples were made

5. Evaluation mutagenic potential of pesticides through bioassays with Allium cepa
World J. Cell Biol. Gen. 008
Figure 2. Micronuclei observed in bioassays of Iminoctadine and Pyraclostrobin.
from the bioassays with Pyraclostrobin and
Iminoctadine Tris (Albesilato). These tests were
significantly different from the negative control
regarding the amount of micronuclei. In addition, the
other changes in the genetic material can be see in the
Figure 2.
The morphological changes occurred in chromosomal
level, as well as degradation of the nucleus. This study
showed that different dosage in exposure periods
miscellaneous can promote great changes in the DNA
structure, therefore can lead to mutagenic effects in live
organisms. Similar study were conducted with rodents
and obtained similar results (Kligerman et al. 1993;
Taneja, 2000; Oliveira et al. 2012). With morphological
changes in chromosomes due to action of the
pesticides it is can say that incorrect exposure promote
DNA mutations. Issues that may affect the person who
comes into direct contact with this product and your
successors. The root irregular growth was altered with
exposure dose of the pesticide being inversely
proportional, that is, the higher the dose, the lower were
growth. Thus, it is believed that exposure to pesticides
also generates impractical economic effects, because
the root growth of cells demonstrates the difficulty in
reproducing biotype and also alter your growth.
The analyzes of changes in the mitotic index proved the
formation of micronuclei becoming evident that the
results are according to dosage applied. However, we
note that in the second addition the process grows cell
defragmentation until it enters the third phase dosage
(higher concentration). In the higher concentration there
is a decrease in defragmentation process, perhaps by a
possible attempt to repair. Higher doses showed large
amounts of micronuclei formed, as well as
chromosomal fragments, compared as negative
controls. There by it was possible to check the action of
pesticides on mitotic rate, formation of nuclei, as well as
the size of the root. Thereby significant differences
were observed between the systems studied relative
the control group, thus indicating that workers exposed
to pesticides are subject to the actions these chemical
agents. Such behavior have been highlighted by other
studies, showing the harmful effects of pesticides.
The Pyraclostrobin (N-methoxy carbamate) is an
effective systemic fungicide which acts as an inhibitor
of electron transport in the mitochondria of the cells of
fungi inhibiting the formation of ATP. In general, the
known data show a low acute and chronic toxicity of
pyraclostrobin and your concentration normally it is
within the standards set by Brazilian law. According to
Ministry of Agriculture, the systemic fungicide, which is
the active ingredient pyraclostrobin, it has genotoxic
effects in some fish species. In this research the
pyraclostrobin significantly different compared to the
negative control (p) <0.05, and present an increased
mitotic index and laggard chromosomes that were not
observed in the positive controls (Table 2).
The Iminoctadine Tris (Albesilato) (C72H131N7O9S3)
belonging to the chemical group of guanidine, is a
fungicide used in crops of coffee, peach and potato.
The Iminoctadine Tris (Albesilato) was significantly
different compared to the negative control p<0.05,
therefore, higher potential for formation of micronuclei
present in 67% of analyzed slides (Table 4), in addition
to causing a decrease in mitotic index, which was
observed in 33% of the blades.
The increased use of agrochemicals has caused
environmental and health impacts of the population,
mainly professionals working directly with these
products (Recena et al. 2006; Soares and Porto 2009).
How much of the agriculture in the state comes from
family culture, where children still undergoing training
assist parents, this problem becomes even more
serious because children are more sensitive to genetic
changes. This problem becomes more serious
considering that treats irreversible problem and which
may be passed to the descendants (Fujii and Inoue,
1983; Giria et al. 2002; Benedetti et al. 2013).

6. Evaluation mutagenic potential of pesticides through bioassays with Allium cepa
Pratte-Santos et al. 009
CONCLUSION
After adapting and improvement of techniques already
published, the bioassays revealed that the Allium cepa
was affected by the action of pesticides containing
pyraclostrobin and iminoctadine. The effects significant
of cytotoxic and genotoxic were evaluated in the
following concentrations: 10 μL/mL, 25 μL/mL and 50
μL/mL, for Pyraclostrobin and Iminoctadine being in all
cases compared to the negative control. With the
results obtained it was possible to verify that the two
pesticides have the ability to promote genetic changes.
Thus any manipulation of these substances must be
performed with adequate safety equipment. This study
besides proving the harmful potential of these
substances warn the serious problems that these
substances can generate.
ACKNOWLEDGMENTS
This work was supported by institutional research
funding from the PIO XII College, Cariacica, Espirito
Santo, Brazil.
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Accepted March 2, 2015.
Citation: Pratte-Santos R, Ramos DR, Becalle TL, De
Oliveira JP, Prado AP (2015). Evaluation mutagenic
potential of pesticides through bioassays with Allium
cepa. World Journal of Cell Biology and Genetics 2(1):
005-010.
Copyright: © 2015 Pratte-Santos et al. This is an
open-access article distributed under the terms of the
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unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
cited.