An insight into the cytotoxicity, genotoxicity, and

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An insight into the cytotoxicity, genotoxicity, and mutagenicity of smoked cigarette butt leachate by using Allium cepa as test system. Mateus Flores Montalvão1,2.

Environmental Science and Pollution Research https://doi.org/10.1007/s11356-018-3731-2

SHORT RESEARCH AND DISCUSSION ARTICLE

An insight into the cytotoxicity, genotoxicity, and mutagenicity of smoked cigarette butt leachate by using Allium cepa as test system Mateus Flores Montalvão 1,2 & Lorrana Lucas Gomes Sampaio 2 & Huan Henrique Ferreira Gomes 1 & Guilherme Malafaia 1,2,3 Received: 19 August 2018 / Accepted: 9 November 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Smoked cigarette butt (SCB) discharged in the environment became an issue of unknown consequences for plants. Thus, we aim at assessing the impact of water containing SBC leachate on the meristem cells of Allium cepa roots. We defined the following experimental groups: negative control (water), positive control (cyclophosphamide); water with SCB leachate at environmental concentration (1.9 μg/L of nicotine) (EC1× group) and water with SCB leachate concentration 1000 times higher than EC1 (EC1000× group). Mitotic index, total number of abnormal cells, index of abnormal cells per mitotic/phase, relative growth index, and inhibition index were calculated after 48 exposure hours. Root meristems were used to prepare slides in order to investigate chromosomal and nuclear abnormalities. According to our data, plants exposed to SCB leachate presented low relative growth index, high inhibition index, large number of abnormal cells, and high abnormality frequency at metaphase/ anaphase. The exposed A. cepa recorded a wide variety of abnormalities such as diagonal metaphase/anaphase, metaphase/ anaphase presenting chromosome fragments, binucleated cells, displaced nucleus, chromosome bridges, micronuclei, necrotic cells, stick metaphase, chromosome adherence, notched nucleus, among other cell disturbances. The chemicals in the SBC leachate had aneugenic and clastogenic effect on the genetic material of the tested plants, either when they acted individually, synergistically, or additively. Thus, our study is a pioneer in reporting that the mere disposal of cigarette butts in the environment can have cytotoxic, genotoxic, and mutagenic effects on plants. Keywords Cigarette . Mutagenic . Environmental pollution

Introduction Cigarettes are one of the main causes of death worldwide. Their harming effects on human health are well described in the literature (Matt et al. 2011). Cigarette smoke carries several complex chemical components that are toxic to human Responsible editor: Philippe Garrigues * Guilherme Malafaia [email protected] 1

Post-graduation Program in Cerrado Natural Resource Conservation - Biological Research Laboratory, Goiano Federal Institution, Urutaí Campus, Urutaí, GO, Brazil

2

Laboratório de Pesquisas Biológicas, Instituto Federal Goiano, Campus Urutaí, Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, GO, Brazil

3

Post-graduation Program in Chemistry, Federal University of Goiás, Samambaia Campus, Goiânia, GO, Brazil

health (Talhout et al. 2011); consequently, the discharge of smoked cigarette butts (SCBs) in the environment is also highly toxic (Booth et al. 2015). Overall, smoking has harming effects on the environment. According to Moriwaki et al. (2009), Schneider et al. (2012), and Seco Pon and Becherucci (2012), SCB is the most common urban waste worldwide, since it represents 22–46% of the visible garbage in urban areas. According to Novotny et al. (2009), SCBs are taken through surface runoffs into watercourses, reach aquatic ecosystems, and become a relevant environmental issue. Alarming amounts of SCB accumulate in the environment because they take long to degrade (Bonanomi et al., 2015). Therefore, they can severely damage aquatic and terrestrial biota, as previously reported by several authors (Slaughter et al. 2011; Lee and Lee 2015; Osuala et al. 2017; Lawal and Ologundudu 2013; Parker and Rayburn 2017; SuárezRodríguez et al. 2012; Suárez-Rodríguez and Macías Garcia 2014; Bekele 2016; Cardoso et al. 2018; Gill et al. 2018). The

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aforementioned studies are essential to help in better understanding that SCB discharge in the environment has a strong impact on animal biota. However, there are important gaps in the knowledge field about the potential impacts on biota exposed to SCB leachates at environmentally relevant concentrations and about how their constituents affect different organisms. We recently demonstrated that mice chronically exposed to water contaminated with SCB leachate (at environmental concentration) presented deficit in their defensive response to potential predators (cat and snake) (Cardoso et al. 2018). Nevertheless, the action mechanisms of this pollutant in different organisms remain unknown. The cytotoxic potential of these pollutants remains unexplored, although the identification of changes at genetic material level can be very useful for the early detection of systemic harmful effects on individuals. Thus, plant testing systems such as Allium cepa have been used as efficient environmental monitoring models (Leme and Marin-Morales 2009). Despite the significant differences between the metabolic processes of plants and animals, DNA can be used as toxicological target in both systems, since it exists in all living cell forms (Forterre 2002). Thus, DNA damage caused by environmental pollutants in a model system has the potential to produce similar effects on different species. Accordingly, our study presents an insight into the toxicity caused by water contaminated with SCB leachate based on different genetic endpoints. We exposed the species A. cepa to this pollutant in order to evaluate its genotoxic (by analyzing possible chromosomal aberrations), cytotoxic (by evaluating the mitotic index and some nuclear abnormalities), and mutagenic potentials (through the identification of micronucleated cells) on plants. Since cigarettes have a mix of potentially toxic chemical components, we assume that onion bulbs exposed to water contaminated with SCB, even at low concentrations, would present damaged chromosome cells.

Materials and methods Model system and experimental design The Allium cepa model system was used as a bioindicator of cellular damage resulting from the exposure to water contaminated with SCB leachate. Sixteen (16) onions presenting approximate diameters and biomass (5–10 g and 25–30-mm diameter, respectively) were selected in a single local market and distributed in four experimental groups. The negative control group (C) comprised onion bulbs partially immersed in pollutant-free water, whereas the positive control group (CP) comprised onion bulbs exposed to water containing 50 mg/L of cyclophosphamide (Baxter Healthcare S/A, São

Paulo, Brazil), which is well known for its mutagenicity. On the other hand, groups EC1× and EC1000× comprised onion bulbs partially immersed in water contaminated with SCB leachate at predicted environmental concentration in surface water (PECsw) and at concentration 1000 times higher than PECsw, respectively. The onion bulbs were placed in polyethylene containers (32 cm length × 12 cm width × 11 cm height) for 48 h in order to be exposed to the treatments, which were applied under controlled luminosity, temperature (± 20 °C) and humidity (± 45%) conditions. Figure 1 shows the herein adopted general design. The water used in the control or leachate dilution group was treated in a water treatment plant of the Instituto Federal Goiano, following all potability standards required by Brazilian legislation. The definition of PECsw was based on nicotine concentrations previously identified in surface water [1.9 μg/L; Valcárcel et al. 2011], since SCBs are one of the main sources of this compound in urban watercourses (Green et al. 2014). The preparation of the stock solution (20 SCB/L) took into account the approximate amount of nicotine in each SCB (4 mg), according to the Missouri Poison Center (MPC 2014). Thus, the onion bulbs in the EC1× group were exposed to water containing SCB at the concentration 1.9 μg/L nicotine; whereas the onion bulbs in the EC1000× group were exposed to 1900 μg/L of the aforementioned compound.

Cigarette butt leachate preparation and chemical characterization The stock solution was prepared according to procedures described by Cardoso et al. (2018). Briefly, cigarettes (Derby Azul KS, Souza Cruz S/A, Rio de Janeiro, Brazil) were introduced in a smoke-simulating device (Fig. 1) to dismiss the participation of smokers. When the ember of the cigarettes was approximately 1 cm away from the filter, the cigarettes were put out, transferred to a beaker containing 1 L of drinking water and left to rest overnight in an exhaust hood. Subsequently, the resulting solution was filtered and stored at − 20 °C until the dilutions of the treatments were performed. The chemical characterization of SCB leachate was performed via inorganic and organic analyses (through spectrometry), according to procedures described by Guimarães et al. (2016); results were presented in a previous study conducted by our research group (Cardoso et al. 2018).

Microscopic analysis At the end of the exposure period, two slides/root/onion bulbs were formed using root meristems, based on methodological procedures described by Kumari et al. (2011). These procedures consisted in keeping the roots extracted from the onions in 1 M HCl for 6 min, staining them with safranin (40%) (Sigma-Aldrich CAS No. 477-73-6) for 5 min, and in carrying

Environ Sci Pollut Res Fig. 1 (A) Schematic drawing depicting the herein adopted experimental design and (B) realistic condition of contamination of watercourse by cigarette butts

out the analysis (coded and blind) under light microscopy at × 1000 magnification. We initially evaluated the percentage of dividing cells (mitosis or interphase) in each slide: 400 cells/ slide were analyzed, thus totaling 3200 cells per treatment. Next, the frequency of chromosomal abnormalities in 800 dividing cells/treatment was recorded. Cells undergoing division were examined to assess the induction of chromosome and nuclear aberrations at prophase, metaphase, anaphase, and telophase. The following indices were calculated (Eqs. 1–3) [according to Kumari et al. 2011]: TDC  100 TC Tabn Total abnormal cells  100 TDC

Mitotic index ¼

wherein BTDC^ is the total number of dividing cells, BTC^ is the total number of observed cells, BTabn^ is the total number of abnormal cells, and BTabn per phase^ is the total number of abnormal cells observed in each phase (prophase, metaphase, anaphase, or telophase).

Macroscopic analysis

ð1Þ

The length of the roots emerging from the bulbs was measured with a digital caliper (precision ± 0.02 mm) in order to calculate the relative growth (Eq. 4) and inhibition indices (Eq. 5), based on Caetano et al. (2018).

ð2Þ

Relative growth index ¼

ð3Þ

  Lm Inhibition index ¼ 100− Lc

Lm  100 Lc

ð4Þ

Abnormal cells per mitotic=phase ¼

Tabn per phase  100 Tcell per phase

ð5Þ

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wherein BLm^ is the mean root length and BLc^ is the mean control-root length.

Statistical analysis Parametric data were subjected to one-way ANOVA, with Tukey post-test, at 5% probability level, whenever F was statistically significant. Non-parametric data were subjected to the Kruskal-Wallis test, with Dunn’s post-test, at 5% probability level. The correlation analysis was performed based on Spearman’s method. Furthermore, data were analyzed through the chi-square test, using Yates correction—χ2 values higher than, or equal to, 3.84 were set as significant (Centeno 1990). The analyses were performed, and graphs were generated, in the GraphPad Prism software (version 7.0).

Results As previously demonstrated by Cardoso et al. (2018), water contaminated with SCB leachate presented high concentrations of heavy metals such as Pb, Cr, Ni, Ba, Sr, and Ti (Table 1). In addition, the mass spectra evidenced several organic compounds including polyethylene glycol, nicotine, and different monoaromatic and polycyclic aromatic hydrocarbons. The main organic components identified in the leached-SCB stock solution samples are presented below and the details can be seen in Cardoso et al. (2018). Bulbs exposed to water with SCB recorded higher mitotic index (F(3,28) = 4.802; p = 0.008) than the non-exposed ones (Fig. 2A). The control group presented the highest relative growth index (Table 2). Consequently, inhibition indices were higher in the exposed plants than in the non-exposed ones. These changes were followed by a large number of cell abnormalities (H = 20.82; p = 0.0001), which were represented by the sum of chromosomes (Fig. 2B). Thus, we observed that the treatments induced cell changes similar to the ones caused Table 1 Heavy metals identified in drinking water and SCB leachate samples

by cyclophosphamide (PC group), whose clastogenicity and aneugenicity are well known. Plants exposed to the herein adopted treatments presented several abnormalities such as diagonal metaphase (Fig. 3A) and anaphase (Fig. 3B1–2), metaphase with chromosome fragments (Fig. 3C-C1), binucleated nucleus (Fig. 3D), displaced nucleus (Fig. 3E), chromosome bridges (Fig. 3F), micronuclei (Fig. 3G), necrotic cells (Fig. 3H1-2), stick metaphase (Fig. 3I), chromosome adherence (Fig. 3J–K and J1–K1), metaphase anaphase presenting evident chromosome breakage (arrows) (Fig. 3L–M), notched nucleus (Fig. 3N–N1), chromosome fragments at metaphase (Fig. 3O–O1), laggard chromosome at anaphase (Fig. 3P–P1), ghost cells (Fig. 3Q), and metaphase disturbances (Fig. 3R). The highest indices of abnormal cells per mitotic/phase were recorded at anaphase and metaphase (Fig. 4A–B, respectively), the fact that evidenced notorious negative effect on cells (F(3,28) = 10.23; p = 0.0001 and H = 11.71; p = 0.0085, respectively). The PC group recorded the highest abnormality index at prophase (H = 18.18; p = 0.0004) (Fig. 4A), whereas the EC1000× group recorded higher abnormality index at telophase than the control and EC1× groups (H = 13.66; p = 0.0034) (Fig. 4B).

Discussion Water pollution has certainly reached alarming levels in recent years (Pavlidis and Tsihrintzis 2018). The disposal of domestic sewage and industrial effluents, besides the surface runoff of agrochemicals, are the main sources of pollution in river waters. However, we must not neglect that xenobiotics such as the ones found in SBCs can also reach natural environments and, consequently, they constitute an emerging pollutant whose effects on different organisms remain unknown. Thus, the present investigation confirmed the toxic potential of SBC leachate—corroborating our previous study involving animals (Cardoso et al. 2018)—and it was a pioneer in showing that this pollutant causes significant biometric, cytotoxic,

Chemical elements

Drinking water Smoked cigarette (100% or 20 cig/L) Metal concentrations (μg/L)

EC (1×)

EC (1000×)

Pb Ni Cr Ba Sr Ti

ND 13.0 ND ND ND ND

18,000 13,010 0.0006 0.091 0.023 0.0016

18,300 13,240 0.0061 0.092 0.237 0.015

29,000 23,000 0.25 3.81 9.89 0.646

This table was generated based on results recorded in a previous study conducted by our research group (Cardoso et al. 2018), which adopted the same treatments and pollutant used in the present study. Therefore, details about the chemical characterization of pollutants can be seen in the aforementioned study. ND, non-detected; Cig, cigarette; EC, environmental concentration

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Fig. 2 (A) Mitotic index and (B) total abnormal cells of onion bulbs exposed, or not, to water with SCB. Bars indicate mean + standard deviation. Data were subjected to one-way ANOVA (in BA^), Kruskal-Wallis (in BB^), and Tukey and Dunn’s post-tests at 5% probability level, respectively. Different lowercase letters indicate significant differences between groups. C, control group; PC, positive control (cyclophosphamide diluted in water); EC1×, environmental concentration; EC1000×, concentration 1000 times higher than the environmental concentration

genotoxic, and mutagenic damages to plants. We recorded harming changes in plants belonging to groups EC1×, PC, Table 2 Relative growth index, inhibition index, and summary of the chi-square test applied to onion bulbs exposed, or not, to different SCB concentrations in water

Length (mm)

and EC1000×. This outcome makes the matter even more relevant, because low concentrations of the herein investigated pollutant (PECsw) can be as damaging as high concentrations of it. Surprisingly, the mitotic indices of meristematic cells in onion roots exposed to the treatments (Fig. 2A—PC, EC1×, and EC1000× groups) were higher than the indices recorded for non-exposed plants. This index is an indicator of cell proliferation (Gadano et al. 2007), whose decrease or increase is good toxicity biomarkers (Fernandes et al. 2007). As discussed by Leme and Marin-Morales (2009), lower mitotic indices can be explained by the toxic action of pollutants on plant growth and development. On the other hand, increased mitotic indices indicate disordered activation of cell division processes by pollutants, which can lead to the formation of tissue tumors (Leme and Marin-Morales 2009). However, there was no significant correlation between the mitotic levels and the relative plant growth indices in our study (r = 0.2855; R2 = 0.0815; p = 0.4931). Therefore, despite the increased mitotic levels recorded in our study, they did not increase the root growth rates. The increased diameter of roots from plants exposed to the pollutant (non-quantified visible parameter) could explain this outcome, as well as the higher meristematic celldeath frequency recorded for onions exposed to the treatments. This mortality could have been caused by chromosome abnormalities induced by the treatments. Thus, cell losscompensatory mechanisms could have been activated to keep root viability through intense cell division. The significant correlation between the mitotic index and the total number of abnormal cells (r = 0.5806; R2 = 0.3371; p = 0.0005) reinforced the hypothesis that treatments have

Attributes Mean

Indices

Comparisons C vs. PC C vs. EC1× C vs. EC1000× PC vs. EC1× PC vs. EC1000× EC1× vs. EC1000×

C 12.86

PC 11.37

EC1× 10.08

Std. deviation 4.536 5.015 Std. error of mean 0.6352 0.8136 Lower 95% CI of mean 11.58 9.719 Upper 95% CI of mean 14.13 13.02 Coefficient of variation (%) 35.28 44.12 Relative growth index (%) 100.0 88.4 Inhibition index (%) 0.0 11.6 Chi-square test applied to the relative growth index (%) Chi-square; df z value p value 11.76; 1 3.429 0.0006* 22.46; 1 4.739 < 0.0001* 23.46; 1 4.844 < 0.0001* 2.685; 1 1.639 0.1013ns 3.152; 1 1.775 0.0758ns 0.027; 1

0.164

5.276 0.7778 8.509 11.64 52.36 79.1 20.9

EC1000× 10.14 6.627 1.711 6.465 13.81 65.39 78.8 21.2

0.8693ns

C, control group; PC, positive control (cyclophosphamide diluted in water); EC1×, environmental concentration; EC1000×, concentration 1000 times higher than the environmental concentration. *Statistically significant differences; ns: non-significant differences

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Fig. 3 Different chromosome abnormalities observed in the meristematic cells of Allium cepa (2n = 16) exposed, or not, to water with SCB. A: Diagonal metaphase, B1–2: diagonal anaphase, C: metaphase with chromosome fragment (C1: detail, arrow), D: binucleated nucleus, E: displaced nucleus, F: chromosome bridges (arrow), G: micronuclei, H1– 2: necrotic cells (necrotic vacuoles, arrow), I: stickiness in metaphase. J

and K: chromosome adherence (J1 and K1: detail, arrow), L: metaphase and M: anaphase with evident chromosome breakage (arrows), N: notched nucleus (N1: detail, arrow), O: chromosomal fragment in metaphase (O1: detail, arrow), P: laggard chromosome in anaphase (P1: detail, arrow), Q: ghost cells (arrows), R: metaphase disturbances

induced cell proliferation, although it was unbalanced and followed by significant chromosome abnormalities. The

presence of ghost cells (Fig. 3Q) in plants exposed to the treatments is an example that substantiates our assumption.

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Fig. 4 Indices of abnormal cells per mitotic/phase recorded for the prophase, metaphase (A), anaphase, and telophase (B). Bars indicate mean + standard deviation. Data were subjected to Kruskal-Wallis and Dunn’s post-tests, at 5% probability level. Different lowercase letters indicate significant differences between groups. C, control group; PC, positive control (cyclophosphamide diluted in water); EC1×, environmental concentration; EC1000×, concentration 1000 times higher than the environmental concentration

Such cells do not have nuclear and cytoplasmic structures [although their contour remains visible (Khanna and Sharma 2013)], which may have been lost due to the toxic action of the chemical constituents of the SBC leachate on them. Similar results were observed when A. cepa were exposed to different concentrations of refined petroleum products (Obute et al. 2016) and to Inula viscosa extracts (Celik and Aslantürk 2010). Our data also evidenced that chemical components of SBC leachate led to DNA necrosis, in which the genetic material is often randomly cut due to nonspecific nuclease action (Petriccione et al. 2013). The presence of necrotic vacuoles in cells of A. cepa plants exposed to SCB leachate (Fig. 3H) suggested, once again, the direct action of these components on cellular DNA. Therefore, this process may also have happened in cells exposed to the contaminants, similarly to what was observed by Gomes et al. (2015), who used A. cepa to

analyze the cytotoxic and genotoxic potential of the water in Guandu River (Brazil). On the other hand, the highest mitotic index recorded for the root of plants exposed to leached SCB may be associated with the increased expression of genes coding cell proliferation-determining proteins (Rodrigues and Kerbauy 2009), as well as with increased proliferative endocrine stimuli. SBC leachate is a highly complex pollutant; therefore, it is considered a mixture of xenobiotics that can act as phytohormonal disrupters. In this case, it is plausible to suggest that the pollutant may have adversely affected several endogenous hormones that modulate plant growth and development, among them: indole-3-acetic acid (IAA), gibberellins (GA), cytokinins, ethylene (ETH), abscisic acid (ABA), and zeatin (ZT)—one of the major cytokinins. The study by Wang et al. (2015), for example, reinforces our hypothesis by showing that endogenous hormone levels and the interaction between them affected the growth of soybean roots exposed to bisphenol A (BPA). If we take into consideration the singularity of the chemical composition of SCB leachate [both inorganic, when organic—see Cardoso et al. 2018], the action mechanisms leading to the observed changes are also complex and difficult to be understood. However, given the types of chromosomal changes observed in our study, it is plausible to affirm that the pollutant may have synergistically, antagonistically, or additionally acted alone on activated cellular repair mechanisms when the genetic material presented some changes. The literature has extensively reported that unrepaired or erroneously repaired cells can lead to different types of changes such as the chromosomal ones (Iliakis et al. 2004; Asaithamby et al. 2011). The chromosomal losses (laggard, Fig. 3P) observed in the root cells of onions exposed to the pollutant suggested aneugenic effect, in which the negative interference on the mitotic fuse stopped one or more chromosomes from adhering to the fuse fibers during anaphase (Kirsch-Volders et al., 2002). Displaced nucleus (Fig. 3E) and binuclear cells (Fig. 3D) are also associated with incompletely developed microtubules subjected to the control of Ca+2 ions (Vidakovic-Cifrek et al., 2002), whereas chromosome fragments (Fig. 3C) may result from chromosome bridge breaks, as suggested by Fiskesjö (1993). Our study recorded significant frequencies of chromosome breaks at the metaphase and anaphase (Fig. 3L–M) of the root meristem of onions exposed to the pollutant, fact that evidenced clastogenic effect. The formation of micronuclei (Fig. 3G), in its turn, (Fig. 3G) indicated the aneugenic and/ or clastogenic action of the pollutant, as reported by Fenech (2002). On the other hand, the chromosome stickiness (Fig. 3I) indicated a negative effect of the pollutant on processes modulating DNA contraction, condensation, and

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depolymerization—this effect is often irreversible and may lead to cell death. This outcome could explain the presence of ghost cells (Fig. 3Q) in the onion roots exposed to SCB leachate. Finally, although there is no record of previous studies similar to ours, our data meet the ones already recorded for plants exposed to mixtures based on complex pollutants, which present a large diversity of chemical components. Genotoxic effects were also found in A. cepa exposed to wastewater samples (Grover and Kaur 1999) containing industrial (Rank and Nielsen 1993; Chandra et al. 2005; Fatima and Ahmad 2006; Migid et al. 2007), tannery (Gupta et al. 2012; Roy et al. 2015); soap, beverage, paint (Ibeh and Umeham 2018), and pharmaceutical effluents (Ibeh et al. 2018). These studies agree that the action mechanisms of these pollutants can be as complex as their chemical composition. Such issue demands the development of new investigations focused on broadening the knowledge about how these complex wastes, even at low concentrations and in short exposure periods, can affect different organisms.

Conclusion The present study confirmed our initial hypothesis that SBC leachate discharged in water is capable of changing cell division and chromosomal processes in meristematic cells of A. cepa plants, even at low concentrations (PECsw). Thus, we herein presented an insight into the cytotoxicity, genotoxicity, and mutagenicity of this emerging pollutant, besides opening new perspectives to be investigated at cellular and molecular levels. These investigations will certainly be very useful to help in understanding how the simple disposal of cigarette butts in the environment can affect its biota. Acknowledgments The authors are grateful to CNPq for granting the scholarship to the student who developed the study. Moreover, the authors thank Dr. Ivandilson Pessoa Pinto de Menezes for your help in methodological procedures. Funding information This study was financially supported by the Brazilian National Research Council (CNPq) (Brazilian research agency) (Proc. N. 467801/2014-2) and Goiano Federal Institute (Proc. No. 23218.000286/2017-21).

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