Heliyon 6 (2020) e04846

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Heliyon
journal homepage: www.cell.com/heliyon

Research article

Determination of malathion's toxic effect on Lens culinaris Medik cell cycle

~o b, **
Seir Antonio Salazar Mercado a, *, Jesús David Quintero Calen
a
Department of Biology, Universidad Francisco de Paula Santander, Avenida Gran, Colombia No. 12E-96B Colsag, San Jose de Cúcuta, Colombia
b
Department of Agricultural Sciences, Universidad Francisco de Paula Santander, Colombia

A R T I C L E I N F O A B S T R A C T

Keywords: The present study aimed to determine the toxic effect of malathion pesticide on root growth, cell division and the
Ecology chromosomal abnormalities frequency using the L. culinaris test. Initially, the lentil seeds were subjected to
Environmental chemistry different doses of malathion (0.0 0.5, 1, 2.5, 5, 10, 15, 20, 25 and 30 mgL-1) and during 24, 48, and 72 h, the root
Environmental engineering
length was measured. Subsequently, at 72h, the mitotic index, mitotic inhibition, and cellular abnormalities were
Environmental toxicology
Plant biology
calculated for all treatments. According to the obtained results, it was visualized that the root growth was
Systems biology inversely proportional to the concentration of malathion at all times of exposure. After 72h of exposure, the lowest
Mitotic index values of the mitotic index and inhibition were presented at malathion concentrations 20, 25 and 30 mgL-1.
Lentil Additionally, micronuclei cell abnormalities, metaphase sticky chromosomes, split chromosomes, nuclear lesions,
Cytotoxic irregular anaphase, anaphase bridges, binucleated cells, absence of nucleus and telophase bridge were observed.
Genotoxic Finally, Malathion induced mitodepressive and cytotoxic effects in the meristematic cells of the L. culinaris root
Relative abnormality rate tip. A high frequency of abnormality was found in the micronuclei, which represented an indicator of a high
degree of toxicity at the cellular level.

1. Introduction organs such as kidneys and heart. In rats, the mean lethal dose is 1200
mg/kg, via oral (Gallo and Lawryk, 1991), producing an increase in the
Pesticides play an important role in pest control, in agriculture, and in activities of catalase, superoxide dismutase, as well as the concentration of
non-agricultural settings. They are applied in high concentrations and malondialdehyde (MDA) in liver and erythrocytes (Akhgari et al., 2003).
their residues cause contamination to the environment, representing risks According to Bujagic et al. (2019); Radovic et al. (2015), there are
to organisms and human health (Ryberg and Gilliom, 2015; Willison et al., high concentrations of malathion found in the sediments of Danube and
2019; Elfikrie et al., 2020; Meftaul et al., 2020). Malathion (diethyl Tisza rivers in Serbia at 5–10 cm depth (2.9 and 69 ng g
1, respectively).
(dimethoxythiophosphorylthio) succinate or S-1,2-bis (ethoxycarbonyl) The previous concentrations are significantly higher than the minimum
ethyl O, O-dimethyl phosphorodithioate) is one of the most widely used levels known (6.7  10
3 ng g
1), which negatively affects benthic or-
organophosphate pesticides worldwide (Singh and Roy, 2017), as an ganisms (Fisher et al., 1993). In surface waters they have found mala-
acaricide and insecticide, it is commonly applied to stored fruit, vegetable thion doses of 0.193 μg⋅L
1 and 25 ng/g, which represents a negative
and grain crops (Climent et al., 2019). When ingested or inhaled, it quickly impact on aquatic ecosystems, especially fish, invertebrates and algae
passes into the bloodstream, interfering with the nervous system by (K€ock-Schulmeyer et al., 2013; Houbraken et al., 2017; Pic o et al., 2018;
inhibiting the enzyme cholinesterase, whose function is to inactivate the Triassi et al., 2019). Additionally, it has been found in some amphibian
acetylcholine neurotransmitter in synapses (Bavcon et al., 2003; Wu et al., tissues at doses of 0.68 ppm and in soils of 13.06 ppm, affecting the
2011; Houbraken et al., 2017). Likewise, malathion has been found in the amphibian's liver metabolomics (Climent et al., 2019). Likewise, at
children's nutritional diet of Japan by 4% (Kawahara et al., 2007). Inter- malathion concentrations of 0.1 and 1.0 mg/L, larval activity decreases
viewing with several farmers, they described negative health symptoms in anuran species (Relyea and Edwards, 2010). In tomato plants specif-
after pesticide application, including vomiting, headache and eye irrita- ically in the fruit, they have found malathion doses higher than 1,000
tion, and skin conditions (Sumon et al., 2016). Furthermore, according to μg/kg in the pericarp, in cabbage of 1000 ng/g and in Chinese cabbage of
Mendoza et al. (2015), exposure to Malathion could cause damage to 330 ppb (Reiler et al., 2015; Wanwimolruk et al., 2015; Pic o et al., 2018),

* Corresponding author.
** Corresponding author.
E-mail addresses: seirantoniosm@ufps.edu.co, salazar663@hotmail.com (S.A. Salazar Mercado), jesusdavidqc@ufps.edu.co (J.D. Quintero Cale~
no).

https://doi.org/10.1016/j.heliyon.2020.e04846
Received 18 April 2020; Received in revised form 27 August 2020; Accepted 1 September 2020
2405-8440/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S.A. Salazar Mercado, J.D. Quintero Cale~
no Heliyon 6 (2020) e04846

which represents a high risk of consuming these foods without being analyzed for the mitotic index and cellular abnormalities. The obtained
washed and processed after harvest, particularly for children. In this data were evaluated using the analysis of variance (ANOVA). Subse-
sense, it is important to educate farmers to use less dangerous pesticides, quently, the averages of each treatment were compared by applying the
in order to guarantee public and ecosystem health (Reiler et al., 2015). HSD multiple range test (Tukey's Honestly Significant Difference) (P 
Searching for bioindicators, plants that can detect toxic substances 0.05). Using the InfoStat program.
due to their high sensitivity to environmental changes have been used
(Salazar and Maldonado, 2019). These have similar characteristics to 3. Results and discussion
mammalian chromosomes and are inexpensive to perform various
toxicity tests (Abdelsalam et al., 2018; Salazar-Mercado et al., 2019). The 3.1. Root length
plants mostly used as bioindicators in research related to cytotoxic and
genotoxic tests are Allium cepa L. (Martins et al., 2016; Haq et al., 2017; Exposing lentil seeds to hydration stimulates cell growth, elongating
de Souza et al., 2017; Silveira et al., 2017; Braga et al., 2018; Verma and their meristematic cells. Furthermore, when the roots are subjected to
Srivastava, 2018; Heikal et al., 2019; García-Medina et al., 2020; Salazar chemical substances, variations in their morphology and coloration occur
and Quintero, 2020), Pisum sativum L. (Salazar-Mercado et al., 2019), (Khanna and Sharma, 2013). The degree of affectation depends on the
Lactuca sativa L. (Andrade-Vieira et al., 2018), Zea mays L. (Reynoso chemical substance and the time of exposure (Salazar-Mercado et al.,
et al., 2015), Triticum aestivum L. (Abdelsalam et al., 2018) and in recent 2019).
studies Lens culinaris Medik (Shahwar et al., 2019; Salazar and Maldo- The results, as shown in Table 1, indicate that at 24, 48 and 72 h the
nado, 2020; Salazar et al., 2020a). highest root growth was achieved at the control treatment and the lowest
Trials conducted by Singh and Roy (2017), who evaluated growth was found at the 30 mgL
1dose, this proves that the root growth
Malathion-induced cytogenetic effects using the A. cepa test. They found inhibition was greater, with the increase of the malathion concentration.
that, at malathion doses of 50, 125, 250, and 375 mgL-1 at different At 24h, the doses of 20, 25 and 30 mgL-1, root growth did not show
exposure periods, it inhibited root growth, reduced the mitotic index, and statistically significant differences, whereas, at 48 and 72 h, the treat-
produced different chromosomal aberrations. In the literature up till ments at 25 and 30 mgL-1 were homogeneous. However, at 24h (0.5, 1,
now, no research has been conducted on the malathion toxic effect on 2.5 mgL-1) and 48h (0.5, 1, 2.5, 5 mgL-1) the doses of malathion did not
L. culinaris cells. As stated above, this study aims to determine the toxic differ from the control treatment. At 72h, only the control treatment was
effect of the malathion pesticide on root growth, cell division and the significantly related to the 0.5 mgL-1 dose.
frequency of chromosomal abnormalities using the L. culinaris test. These results are similar to those reported by Singh and Roy (2017),
where malathion had a negative effect on A. cepa root growth at all
2. Material and methods concentrations for 3 days. For this reason, root inhibition is a parameter
that can be used to measure the toxicity of malathion. This agrees with
2.1. Plant material and treatments studies carried on by Salazar and Maldonado (2019), who submitted
different doses of chlorpyrifos organophosphate pesticide (0, 1, 3, 5, 7, 8,
Malathion was diluted in distilled water at doses of 0.5, 1, 2.5, 5, 10, 10 and 15 mgL-1) in L. culinaris seeds, affecting root growth significantly
15, 20, 25 and 30 mgL-1 and control of distilled water. For L. culinaris seeds at 24, 48 and 72h of exposure. The inhibition of root growth caused by
germination, it was carried out in Petri dishes with cotton and filter paper the malathion pesticide might be due to the disturbance of the Reactive
under controlled environmental conditions (26  2 in the dark). The seeds oxygen species (ROS) in the plant since, according to Mhamdi and Van
were subjected to darkness for three days as described by Salazar and Breusegem (2018), when ROS homeostasis is altered, numerous pro-
Botello (2018); Salazar et al. (2020b); Salazar et al. (2020c) and root cesses are affected, from seed germination to root development, because
growth was measured at 24, 48 and 72 h. Subsequently, the mitotic index ROS interact with hormones (auxin and cytokinin) associated with plant
and the frequency of cellular abnormalities were determined. development.

2.2. Mitotic index and inhibition

3.2. Mitotic index and inhibition

After 72 h, the root tips were cut 4 mm approximately, washed with

In the evaluation of the mitotic index (5000 cells per treatment) after
running water and subjected to toluidine blue metachromatic dye for 30
72 h of exposure to malathion, it shows that the control had a higher MI
min. Subsequently, the roots were taken to the slide, the coverslip was
with statistically significant differences with respect to the different doses
placed and the squash technique was performed, applying uniform
pressure with the fingertip of the thumb to disperse the cells, making
each phase of the cell cycle visible (Salazar and Maldonado, 2019). Table 1. L. culinaris root growth subjected to different doses of Malathion.
Samples were visualized under the Leica DME 500 compound microscope
at 40X and 100X magnification. To determine the mitotic index, 1000 Dose: Malathion (mgL
1) Root length (cm)

cells per repetition (5000 cells per treatment) were analyzed, using the 24h 48 h 72 h
formula Mitotic index (MI): number of dividing cells/number of total T1: Control 2.36  0.23a 4  0.7a 5  0.04a
cells x100. Continuedly, the mitosis inhibition was found according to T2: 0.5 1.86  0.18a,b,c 3.66  0.61a 4.34  0.42a,b
Salazar-Mercado et al. (2019). T3: 1 2.0  0.7a,b 3.64  0.65a 4.04  0.4b
Cellular abnormalities are alterations that occur in the structure and T4: 2.5 1.76  0.11a,b,c 3.22  0.68a,b 3.98  0.04b,c
number of chromosomes and depend on the concentration and type of T5: 5 1.52  0.3 b,c,d
3.02  0.17 a,b,c
3.64  0.46b,c,d
cytotoxic substance (Fatma et al., 2018). Therefore, the following for- T6:10 1.54  0.35 b,c,d
2.54  0.37 b,c
3.14  0.49c,d,e
mula used by Salazar and Quintero (2020) was applied in this research: T7: 15 1.38  0.4b,c,d 2.3  0.44b,c 2.92  0.57d,e
Frequency of chromosomal anomalies (%): Total number of abnormal T8: 20 1.16  0.15d,e 2.14  0.35b,c 2.66  0.46e,f
cells/Total number of cells observed x100. d,e c,d
T9: 25 0.94  0.08 1.58  0.34 2.22  0.34f,g
e d
T10: 30 0.54  0.23 1.04  0.11 1.34  0.39g
2.3. Statistical analysis
The means  SD values with different letter indicate statistically significant
For root development, 25 L. culinaris roots were used per treatment differences, according to Tukey (P  0.05). SD: Standard deviation; cm:
centimeter.
with 5 replications. 1000 cells per repeat (5000 cells per treatment) were

2
S.A. Salazar Mercado, J.D. Quintero Cale~
no Heliyon 6 (2020) e04846

where the mitotic index has been higher in A. Cepa, in relation to the
Table 2. Mitotic index and Percentage of mitosis inhibition of L. culinaris root tip L. culinaris test in equal doses (Salazar et al., 2020a). The mitotic index is
cells, submitted to different doses of Malathion.
a measurement to quantitatively evaluate the cell division of an indi-
Dose: Malathion (mg L
1) Mitotic index (MI: %) Inhibition of mitosis (%) vidual (Lessa and Cariello, 2017). Therefore, if the MI is low compared to
L. culinaris L. culinaris the control treatment, the pesticide is affecting mitosis. According to
T1: Control 18.4  1.5a —— Rosales (2015), organophosphate pesticides cause damage to deoxy-
T2: 0.5 14.2  1.3b 22.8 ribonucleic acid (DNA) at any stage of the cell cycle.
T3: 1 14.4  1.1b 21.7 Regarding mitotic inhibition, the concentration that achieved data
T4: 2.5 14.6  0.7b 20.6
greater than 50% cell cycle inhibition was 30 mgL-1 dose (52.1%:
Table 2), evidencing the mitodepressive activity of malathion. In
T5: 5 13.6  1.1b,c 26.1
contrast, lower doses of mitotic inhibition were found at doses of 0.5, 1,
T6:10 13.2  1.6b,c,d 28.2
and 2.5 mgL-1 (22.8%, 21.7% and 20.6%, respectively). These results
T7: 15 12.8  0.8b,c,d 30.4
differ from those reported by Salazar and Quintero (2020), where 30
T8: 20 11.4  1.5b,d,e 38
mgL-1 of glyphosate in A. cepa, obtained a mitotic inhibition index of
T9: 25 10.6  10.6d,e 42.3
90.8%. Likewise, in L. culinaris roots at 15 mgL-1 of chlorpyrifos, an in-
T10: 30 8.81e 52.1
hibition of 72.41% was found for 72h (Salazar and Maldonado, 2019).
The means  SD values with different letter indicate statistically significant dif-
ferences, according to Tukey (P  0.05). SD: Standard deviation. MI: Mitotic index. 3.2.1. Cellular abnormalities and abnormality rate
As a reference indicator, Figure 1 shows the normal phases of the cell
of the pesticide (18.4%; Table 2). The lowest MI values were found at 20, cycle (interface, prophase, metaphase, anaphase and telophase) and the
25 and 30 mgL-1 concentrations (11.4%, 10.6% and 8.8%, respectively). cellular anomalies found in meristematic cells from L. culinaris roots.
This indicates that the mitotic index decreased depending on the doses of Nine cellular abnormalities were found at all malathion concentrations,
malathion. According to results presented by Singh and Roy (2017), when varying according to each treatment, except in control treatment
subjecting A. cepa to 125 mgL-1 of malathion for 18h, they found a mitotic (Figure 1; Table 3). Micronuclei, metaphase sticky chromosomes, split
index of 8.77%. Likewise, in Vicia faba roots subjected to 320 mgL-1 of chromosomes, nuclear lesions, and irregular anaphase were visualized in
malathion, the mitotic index was 8.99% (Adam et al., 2014). all the doses that contained malathion, with the nuclear lesions being the
In a study, carried out by Srivastava and Singh (2020) in A. cepa root mostly found at 30 mgL-1 dose (116.6  16). Likewise, the anaphase
cells, exposed to different malathion concentrations (50, 130, 260, 390 bridges, binucleated cells, absence of nuclei and telophase bridges
and 520 mgL-1) and exposure time (4, 8 and 18 h). They found that the anomalies were not observed in some treatments (Table 3). In the same
application of malathion affected the MI as the concentration and the way, at 0.5 mgL-1 of malathion, fewer anomalies were found.
treatment periods increased, reducing the growth rate and cell division in These results contrast with the study carried out by Singh and Roy
the A. cepa roots. In the previous study, the 50 mgL-1 dose of malathion (2017) in the A. cepa test, where 50, 125, 250 and 375 mgL-1 of mala-
had an MI of approximately 12%. In contrast, in this research the con- thion doses, produced various chromosomal aberrations (anaphase and
centration of 30 mgL-1, the mitotic index was 8.8%. According to the telophase bridging, multipolarity, chromosomal breakage (sticky and
above, the L. culinari method is more sensitive to malathion than the lagging chromosomes, nuclear lesions and binucleated cells)), but no
A. cepa test. This is corroborated by studies related to other pesticides, micronuclei were found in A. cepa. However, in Vicia faba roots subjected

Figure 1. Cell cycle phases and cellular abnormalities in L. culinaris root cells treated with doses of Malathion. A ¼ Interphase: T1. B¼ Prophase: T1. C ¼ Metaphase:
T1. D ¼ Anaphase: T1. E ¼ Telophase: T1. F ¼ Micronuclei: T10 G ¼ Anaphase bridge: T9. H¼ Sticky chromosomes at metaphase: T8. I ¼ Absence of nucleus: T7. J ¼
Chromosome break: T6. K¼ Binucleate cells: T6. L ¼ Telophase bridge: T10.

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Table 3. Frequency of chromosomal anomalies in L. culinaris treated with different doses of Malathion.

Dose: Malathion (mg L
1) Frequency of Chromosomal Anomalies (Mean  SD)

Mn AB SCM AN CB BC TB NL IA
T1: Control 0a 0a 0a 0a 0a 0a 0a 0a 0a
T2: 0.5 1.2  0.44a 0a 0.4  0.1a 0a 0.6  0.8a 0a 0a 3.6  0.8a 0.2  0.08a
T3: 1 4.0  1.2a,b 0a 1.8  0.8a 0.2  0.1a 3.0  1.3a 0a 0a 7  1.5a 0.2  0.01a
a a a a a a a a
T4: 2.5 4.4  1.5 0 3.8  1.7 0.6  0.8 3.2  0.8 0 0 10  0.9 0.2  0.07a
T5: 5 5.8  0.8a,b 0a 4.2  2.1a 3.4  1.1a 3.6  1.4a 0a 0a 12.2  1.9a 0.4  0.18a
T6:10 13  1.3a,b 0a 4.4  1.4a 5.4  0.8a 5.8  1.5a,b 0.6  0.25a,c 0a 15.8  5.7a 0.4  0.14a
T7: 15 13.6  3.5a,b 0.6  0.1a 4.8  1.7a 8.4  1.1a 8  2.5a,b 1  0.4a 0a 20.2  3.1a 0.6  0.12a
b a a,c b a,c,d a,b b
T8: 20 16.8  2.1 0.6  0.12 5.8  1.1 25.2  3.2 16.6  5.5 1.2  0.43 0.2  0.01 44.8  14.7 0.6  0.1a
T9: 25 37.6  9.8c 2.6  0.9b 11  6.2a,c 35  5.5b 17.4  5.9b 1.2  1.7a,c,d 2.2  1.7b 87.4  18c 0.8  0.2a
T10: 30 47  9.4c 2.8  0.8b 16  7.4a,c 365b 24  7.1 8.0  3.1b,d 2.2  1.1b 116.6  16d 2.6  0.5b

SD: Standard deviation. Mn: Micronuclei. AB: anaphase bridge. SCM: Sticky chromosomes at metaphase. AN: Absence of nucleus. CB: Chromosome break. BC: Binu-
cleate cells. TB: Telophase bridge. NL: Nuclear lesions. IA: Irregular anaphase.
The means  SD values with different letter indicate statistically significant differences, according to Tukey (P  0.05).

to 80, 160 and 320 mgL-1 of malathion, micronuclei were found in all 4. Conclusion
treatments (Adam et al., 2014). According to Doherty et al. (2016), the
micronuclei are fragments of DNA that are separated from the main To conclude, the present investigation determined the toxic effect of
nucleus and have originated from eccentric chromosomes or fragments of malathion on root growth, cell division and formation of cellular ab-
chromatic, can result from alterations of structural type (clastogenic ef- normalities, producing mitodepressive and cytotoxic effects on meriste-
fect) or numerical alterations (aneugenic effect). They are used as a high matic cells at L. culinaris root tip. The presence of changes at the cellular
degree of toxicity parameter (Bhatia and Kumar, 2013). In this investi- level and damage to the chromosomes was observed at 0.5 to 30 mgL-1 of
gation, micronuclei were observed in all doses, being in greater quantity malathion; being the 30 mgL-1 dose the one that produced a greater
in 30 mgL-1 concentration (47  9.4; Table 3). Worrisome data since, it is cellular inhibition and relative abnormality rate. In addition, a high
evident that malathion induces genetic instability in L. culinaris, because frequency of abnormality was found in the micronuclei, which represents
it is a cytotoxic and genotoxic agent. an indicator of a high degree of toxicity at the cellular level. Accordingly,
Table 4 compares the obtained results from the abnormality rate in the use of less toxic and environmentally friendly pesticides is suggested
treatments with malathion in L. culinaris. It is observed that at the con- to farmers. According to the above, it is suggested that farmers use less
centration of 30 mgL-1, a higher rate of relative abnormality (25.5) was toxic and environmentally friendly pesticides since the repeated and
found, indicating that the formation of abnormal cells in L. culinaris roots common use of this molecule can lead to problems in the performance
increases as the dose of malathion is increased. These results also agree and crop yield, thanks to physiological abnormalities that are harmful to
with the observations made by Salazar and Maldonado (2019); Salazar development of the agricultural exploitation.
et al. (2020a), by subjecting L. culinaris to different concentrations of
chlorpyrifos and propanil. Likewise, according to Singh and Roy (2017), Declarations
pesticides alter ROS homeostasis and cause intracellular oxidative stress
(Wu et al., 2017). Consequently, the tubulin polymerization, the mitotic Author contribution statement
spindle, the assembly of the phragmoplast, the dynamics of the nuclear
envelope, the separation and movement of the chromosomes, and the Seir Antonio Salazar Mercado: Conceived and designed the experi-
formation of cell plates are affected, delaying the stages of cell division ments; Performed the experiments; Analyzed and interpreted the data.
(Livanos et al., 2012). Furthermore, according to Singh and Roy (2017), Jesús David Quintero Cale~ no: Contributed reagents, materials, anal-
the toxic and genotoxic activity of malathion is possibly associated with ysis tools or data; Wrote the paper.
an overproduction of pro-oxidant agents and oxidative stress due to the
significant increase in concentrations of malondialdehyde (MDA) caused
by malathion in the plant. These products of lipoperoxidation reactions Funding statement
are also associated with damage to lysosomal membranes and DNA
structure (Cortesía et al., 2015). This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.

Table 4. Relative abnormality rate for each doses of Paraquat.

Competing interest statement
Dose: Malathion (mg L
1) Relative abnormality rate
T1: Control 0 The authors declare no conflict of interest.
T2: 0.5 0.6
T3: 1 1.66
Additional information
T4: 2.5 2.36
T5: 5 2.9
No additional information is available for this paper.
T6:10 4.8
T7: 15 5.56
Acknowledgements
T8: 20 11.1
T9: 25 19.38
To Universidad Francisco de Paula Santander for their valuable
T10: 30 25.5
collaboration.

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S.A. Salazar Mercado, J.D. Quintero Cale~
no Heliyon 6 (2020) e04846

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