Cytotoxic evaluation of glyphosate, using Allium cepa L. as bioindicator

Journal Pre-proofs Cytotoxic evaluation of glyphosate, using Allium cepa L as bioindicator Seir Antonio Salazar Mercado, Jesús David Quintero Caleño PII: DOI: Reference:

S0048-9697(19)34443-2 https://doi.org/10.1016/j.scitotenv.2019.134452 STOTEN 134452

To appear in:

Science of the Total Environment

Received Date: Revised Date: Accepted Date:

1 August 2019 7 September 2019 13 September 2019

Please cite this article as: S.A.S. Mercado, J.D.Q. Caleño, Cytotoxic evaluation of glyphosate, using Allium cepa L as bioindicator, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv. 2019.134452

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Cytotoxic evaluation of glyphosate, using Allium cepa L as bioindicator

Seir Antonio Salazar Mercado a, *, Jesús David Quintero Caleño b, ** a Department

of Biology, Universidad Francisco de Paula Santander. Avenida Gran

Colombia No. 12E-96B Colsag. San José de Cúcuta, Colombia. b Department

of Agricultural Sciences, Universidad Francisco de Paula Santander.

* Corresponding author. E-mail addresses: [email protected], [email protected]. ** E-mail addresses: [email protected]

Abstract Glyphosate is a chemical compound used mainly as a broad spectrum herbicide, it is recognized for its proven effectiveness and easy handling. It represents more than 60% of the world market of non-selective herbicides and is used in both agricultural fields and family gardens. The present study was designed to test the cytogenotoxic potential of glyphosate using the Allium cepa test as toxicity bioindicator. Consequently, bulbs of A. cepa were exposed to different concentrations of glyphosate (5, 10, 15, 25 and 30 mgL-1) and a control (deionized water), for 72 hours; root development was also studied in this period of time. The cytogenotoxic potential of glyphosate was determined by calculating the mitotic index (MI), cellular anomalies (CA) and registering the roots longitudinal growth at 24, 48 and 72h. Regarding root development, a greater growth was observed in the control treatment in the three measurement times. The mitotic phases analysis, determined that the higher the concentration, the lower the mitotic index, in addition the 1

inhibition of the telophase Mitotic Index (TMI) was observed in any of the concentrations. The results indicate that the exposure to glyphosate of A. cepa meristematic cells induces diverse types of chromosomal anomalies, such as micronuclei (MN), chromosome breaking (CB), nuclear notch (Nn), among others. Therefore, in demostrates that glyphosate has a highly cytogenotoxic effect for any of the concentrations used. Key words: celular anomalies, mitosis, chromosome, Allium cepa, cytogenotoxic, DNA.

1. INTRODUCTION

Glyphosate is part of the chemical group of substituted glycines, being classified as an agricultural use herbicide of toxicological class I (Santos et al., 2018). It is part of the organophosphates (Sulukan et al., 2017), which are highly toxic agents, potent binders of acetylcholinesterase, which produces competitiveness in the neuromuscular junction (Greer and Donofrio, 2009). Its mechanism of action belongs to amino acid synthesis inhibitors (phenylalanine, tryptophan and tyrosine), which is systemic and non-selective (Almeida, 2018). Its effect at the metabolic level lies in the inhibition of the enzyme 5-enolpiruvil shikimate-3phosphate synthetase (EPSPS), which catalyzes the reaction between phosphoenolpyruvic acid and 5-phosphoshikimic acid to synthesize a corismic acid precursor, in the pathway of shikimic acid, that takes place in plants, bacteria and fungi (Spósito and Espínola, 2016). Production on glyphosate presents exponential growth since its inception in 1974 and since then it has become the most frequent and intensive herbicide in the world (Almeida et al., 2015; Sosa et al., 2019).

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In 2012, about 127,000 tons of glyphosate were used in the United States and 700,000 tons worldwide (Van et al., 2018). The widespread application of glyphosate to crops has stimulated the spread of tolerant and resistant weeds throughout the world, which in turn has created the need for more frequent applications at higher concentrations (Benbrook, 2016). This increase in its use compromises the health of the human being and the fauna and flora welfare that can lead to significant changes in the biology of several species (Silva et al, 2017; Van et al., 2018). It is important to highlight that, in the long term, environmental pollution can culminate in the population decline of various species of animals, plants and microorganisms (Fonseca, 2007). Therefore, non-lethal concentrations should be monitored, whose silent effects may affect both the morphology, physiology and biochemistry of organic tissues (Silva et al., 2017). Most of the agrotoxics are organic and hydrophobic compounds that tend to be absorbed into suspended particles and dissolved organic matter, accumulating in sediments and aquatic biota (Sucahyo et al., 2008). It is considered that glyphosate decomposes rapidly in the soil, with reported average lives of (DT50) <5 days and DT90 <10 days (Al-Rajab and Schiavon, 2010; Yang et al., 2015a; Bento et al., 2016). However, it was also found that glyphosate persisted in the soil for longer periods of time, under drought conditions and at low temperatures (HeinonenTanski, 1989; Schroll et al., 2006; Bento et al., 2019) or in soils with strong adsorption capacity (Al-Rajab and Schiavon, 2010). Studies have reported on the glyphosate biphasic disintegration behaviour, which means, a rapid initial decomposition rate, followed by a slower one (Eberbach, 1998; Al-Rajab and Schiavon, 2010). Considering that glyphosate can persist in the soil for long periods and the high frequency and application rates in agricultural fields (Benbrook, 2016; Sihtmäe et al., 2013), there is a risk of accumulation in the environment (Van et al., 2018).

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In many Mediterranean areas, high rates of soil loss are exhibited due to the widespread use of glyphosate (Keesstra et al., 2019). Recent findings suggest that glyphosate and its metabolites can also be propagated by wind and water erosion (Silva et al., 2018). The residues that remain in the soil upper layer are transported off-site during the erosive events (Todorovic et al., 2014; Yang et al., 2015a; Yang et al., 2015b). For example, Shipitalo et al. (2008) reported on glyphosate concentrations between 31 and 182 μgL-1 in runoff waters, from Appalachian basins (USA). Studies in different parts of Buenos Aires province, in Argentina, reported the appearance of glyphosate in shallow waters and stream sediments (Peruzzo et al., 2008; Aparicio et al., 2013; Castro et al., 2018). It has also been found in dust within non-agricultural households, suggesting that exposure is not only occupational (Curwin et al., 2005). In addition, according to Camacho and Mejía (2017), spontaneous abortions, dermatological and respiratory diseases were related to glyphosate exposure during aerial fumigation campaigns to eliminate coca plants (Erythroxylum coca) in Colombia (Van et al., 2018). People can be exposed to glyphosate through several routes, such as food, drinking water and in the environment (EPA, 2017). Its zwitterionic nature prevents its inclusion in analytical methods for environmental monitoring. Consequently, despite its extensive use, data on the appearance of glyphosate in the aquatic environment are still scarce (Poiger et al., 2017). In recent years, the carcinogenic potential of glyphosate has been subject to reviewing and debating by multiple authorized and regulatory bodies. In 2015, International Agency for Research on Cancer classified glyphosate as a "probable human carcinogen" (IARC, 2016). Evidence such as the bioaccumulation was observed in rodents (Panzacchi et al., 2018). The indiscriminate use of glyphosate in the urban area (domestic gardens, horticulture and weed control) constitutes a problem for human and animal health (Fernandes et al., 2019; Centner et al., 2019). The water contamination by glyphosate can pose an environmental risk due to its 4

chronic toxicity (Turkmen et al., 2019). In this order of ideas, it is worrisome the fact that Colombian government intention to reuse glyphosate, now with drones, ignoring the IARC classification (Idrovo, 2015). Colombia is once again the only country that has an anti-narcotics strategy that uses aerial fumigation with pesticides (Idrovo, 2019). Therefore, the importance of using other molecules such as ammonium glufosinate, presented by the European Commission as "one of the very few alternatives to glyphosate" (Ravier et al., 2019).

To recognize the effect that chemical pollutants cause on ecosystems, certain plants are used as bioindicators (Ghosh et al., 2017; Biruk et al., 2017; Bortolottoa et al., 2017; Abdelsalam et al., 2018), among the most used plants, A. cepa is found (Pedrazzani et al., 2012; Sommaggio et al., 2018; Liman et al., 2019) due to it has similarity in chromosomal morphology with mammals (Firbas and Amon, 2014; Prajitha and Thoppil, 2016). Among the most used plants is Allium cepa (Fatima and Ahmad, 2005; Kumari et al., 2009; Radić et al., 2010; Laughinghouse et al., 2012; Fernández et al., 2016; Scherer et al., 2019), which represents an important proof for the evaluation of the cytogenotoxic effects of various substances that can affect the genetic material (Ozkara et al., 2015; Garcia et al., 2017; Martins et al., 2016). This test has a direct correlation with the responses obtained in the tests applied in mammals (Moura et al., 2016). In addition, radicular meristems have a high proportion of cells in mitosis, which makes it easier to recognize the incidence of contaminants in cells (Talledo and Escobar, 2018). According to Llontop and Vargas (2014), the Allium cepa species proves to be effective and economical to perform the tests. Consequently, in the following study the cytotoxic activity of glyphosate on A. cepa root meristem is evaluated, using the mitotic index and inhibition, cell abnormalities and chromosomal abnormality rate.

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2. METHODOLOGY 2.1 Assay method Five commercial type glyphosate solutions (360 mgL-1) were prepared, which concentrations were: 5, 10, 15, 25 and 30mg / L (ppm), and a control solution (deionized water). These doses were used taking into account previous studies about DNA damage by reactive oxygen species (ROS) increasing production, and DNA methylation decreasing with the use of 42 mg l-1 glyphosate (Kwiatkowska et al., 2014; Kwiatkowska et al., 2017). Implementing the method described by Causil et al. (2017), with certain modifications. Ten onion bulbs were used in perfect phytosanitary conditions without any trace of pathologies, with white external cataphylls (Disner et al., 2011). The onions used had an average diameter size of 6.30 ± 0.08 cm with an average weight of 78.33 ± 0.87 g as described by Ogeleka et al. (2016). Next, the bulbs were washed in running water and placed in disposable containers (150 mL capacity) sterilized with a 5% chlorine solution for 10 minutes, leaving the root area in contact with the water of each treatment at room temperature (Pereira et al, 2017). Root length was recorded during 72 hours by measuring its growth every 24 hours. Subsequently, at 72 hours, tests for measuring mitosis in the root apices were performed, the mitotic index was calculated for each dose and the number of cell abnormalities was identified for each treatment.

2.2 Microscopic analysis The procedure for the mitosis test is carried out at room temperature and consisted of submerging 3 mm of the root tips in 1N hydrochloric acid for 15 min to break the cell walls

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(Causil et al., 2017). Then, Samples were then transferred to a plate where they were stained with 1 N aceto-orcein for 10 min. Once stained, the roots were placed on slides and covered with a coverslip, then the squash technique was performed to make each of the cellular phases visible (Salazar-Mercado et al., 2019). Subsequently, were observed in a Leica DME 500 optical microscope.

2.3 Mitotic Index calculation The formulas used by Salazar and Maldonado (2019) were applied: general mitotic index (GMI) = number of cells in division / number of total cells; prophase mitotic index (PMI) = number of cells in prophase / number of dividing cells; metaphase mitotic index (MMI) = number of cells in metaphase / number of cells in division; anaphase mitotic index (AMI) = number of cells in anaphase / number of dividing cells; telophase mitotic index (TMI) = number of cells in telophase / number of dividing cells (Salazar and Maldonado, 2019). According to Restrepo et al. (2012), if values lower than those of the control solution were obtained, it is considered that there is inhibition and if higher values are obtained, it would indicate that there is an increase in the division caused by the application of substances.

2.4 Cellular anomalies According to Datta et al. (2018), the cellular anomalies (CA) are any anomaly or irregularity in the number and structure of the chromosomes. The formula used: number of abnormal cells / number of total cells. For each concentration, the methodology proposed in 2019 by Salazar-

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mercado and others was used. For Fatma et al. (2018), the appearance of cellular anomalies is determined by the increase in the concentrations of mutagenic agents. For each concentration the used formula was:

Relative anomaly rate (%) =

Number of anomalous cells 𝑥 100 Number of total observed cells

2.5 Experimental design and statistical analysis In the radicular growth analysis, a factorial design of six treatments and three exposure times (24, 48 and 72h) with ten repetitions was performed, for a total of 60 experimental units. Regarding the mitotic index evaluation and cellular anomalies, the experimental design consisted of a randomized block analysis, with 6 treatments and a single exposure time (72 h). Five replicates of 1000 cells each were evaluated (5000 per treatment). Subsequently, a variance analysis (ANOVA) was performed and the averages were compared using Tukey's HSD (Honest Significant Difference) multiple range test to determine the significant differences at P ≤ 0.05 level (Tukey, 1994). The statistical software Statgraphics Centurion XVI version was used to create and edit the graphs. 3. RESULTS 3.1 Radicular development During 72 hours, the bulbs of A. cepa were exposed to different glyphosate concentrations, taking measurements every 24 hours. It was observed that the greatest longitudinal growth in the 24h period was 3cm in the concentrations of 5 and 10 mg L-1, without significant differences with the control (2.8cm). Likewise, it is observed that the lowest growth found at 24h was 1cm for the 30mg L-1 treatment (Table 1). The measurement done at 48h shows a greater growth for 8

the control treatment (5.2cm) and maintains a high difference with the 30 mg L-1 treatment with 2cm length (Table 1). After 72 hours, the last measurement showed that the control treatment obtained, on average, 7.2cm, with significant differences compared to 2.6 cm of 30 mg L-1 treatment. The Tukey HSD statistical analysis (P≤0.05) allowed the observation of significant differences between the concentrations of 5 and 10 mg L-1 with respect to the 30 mg L-1 treatment at all exposure times.

Table 1. Growth in length of the roots of A. cepa submitted to different concentrations of Glyphosate. Glyphosate concentration: C3H8NO5P (mg L-1) 0 5 10 15 25 30

Root length (cm) 24 hours 2.8±0.8a 3.0±1a 3.0±0.7a 2.2±0.8a,b 1.6±0.54a,b 1.0±1.0b

48 hours 5.2±0.83a 4.0±1.0a,b 3.6±0.54b 3.0±0.7b,c 2.8±0.44b,c 2.0±0.7c

72 hours 7.2±0.83a 5.2±0.83b 5.2±0.83b 4.0±0.7b,c 3.2±0.44c 2.6±0.54c

The means ± SD values with different letter of each column indicate statistically significant differences, according to Tukey HSD test (P≤0.05). SD = Standard deviation.

3.2 Mitotic index In this study, a clear trend could be seen for any of the mitotic indexes analyzed (GMI, PMI, MMI, AMI and TMI). The control treatment had the highest values in all doses (Table 2). The TMI was lower in the trend with 2.4 (Table 2). In general, the control treatment had a higher mitotic activity according to the observed results (Table 2). According to the above, there is a clear influence of the increase in the glyphosate concentration in each phase of cell division in the A. cepa meristematic cells (Figure 1). Likewise, the 30 mgL-1 treatment has lower mitotic 9

rates. It is noteworthy that the use of glyphosate completely inhibited the TMI being 0 for all concentrations tested (Table 2).

Figure 1. Mitosis stages of A. cepa root cells. (A) Prophase. (B) Metaphase (C) Anaphase (D) Telophase.

3.3 Mitosis inhibition The use of 30 mg L-1 of glyphosate there was a 90.8% of mitosis inhibition, this being the highest percentage of the test (Table 3), followed by the concentration of 25mg L-1 with an inhibition percentage of 75.8%. Likewise, the 5 mg L-1 concentration showed the least inhibition with 49.4% (Table 3). The control treatment did not show inhibition of mitosis (Figure 2).

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Table 2. Mitotic indexes of cells from A. cepa root tips, submitted to different concentrations of Glyphosate. (5000 cells per treatment). Glyphosate concentration: C3H8NO5P (mg L-1) 0 5 10 15 25 30

Mitotic index GMI

PMI

MMI

AMI

TMI

17.4±2.9a 8.8±2.1b 7.4±1.6b,c 6.4±3b,c 4.2±1.3c,d 1.6±1.34d

7.4±2a 5.2±0.4a,b 4.6±0.5b,c 2.4±1.6c,d 2.8±0.8b,c,d 0.8±1.3d

3.8±1a 2.4±1.6a,b 1.8±1.3a,b 1.6±0.54b 1.0±0.7b 0.6±0.54b

3.8±1.4a 1.2±0.44b,c 1.2±0.44b,c 2.4±1.9a,b 0.4±0.5b,c 0.2±0.4c

2.4±0.5 0 0 0 0 0

The means ± SD values with different letter of each column indicate statistically significant differences, according to Tukey HSD test (P≤0.05). SD = Standard deviation. GMI= General mitotic index. PMI= Profhase mitotic index. MMI=Metaphase mitotic index. AMI= Anaphase mitotic index. TMI= Telephase mitotic index.

3.4 Chromosomal anomalies In total, nine types of chromosomal anomalies were found (Figure 2, Table 4) with the glyphosate application. In this scenario, the highest frequency of micronuclei (58) was observed with 15 and 30 mg L-1 application. Similarly, the lowest frequency (20) was presented with 5 mg L-1 concentration. Likewise, it was found that the 15 mg L-1 application produced the highest number of bridges in anaphase (AP) with a frequency of 12 and 0 in the 5 mg L-1 concentration, equaling the control treatment compared to this type of anomalies. It was also possible to observe the existence of sticky metaphase (SM), with a higher frequency for 30 mg L-1 concentration (14). SM is the only anomaly presented in the control with a value of 2 (Table 4). An important anomaly that presented the study, was the chromosomes breaking (CB), where the use of 30 mg L-1 showed a frequency of 10. In the case of treatments with 10, 15 and 25 mg L-1 11

concentrations, a frequency of 2 without significant differences with respect to the 5 mg L-1 concentration (Table 4). As a result, chromosomal clustering metaphase (CCM) showed that 5 and 25 mg L-1 treatment produced an average of 2. As well as a frequency of 10 in the 15 mg L-1 treatment.

Figure 2. Abnormalities in dividing cells of A. cepa root tip cells treated with different concentrations of Glyphosate. (A) Micronuclei. (B) Anaphase bridge. (C) Irregular anaphase. (D) Chromosome break (E) Elongated nuclei. (F) Absence of nucleus Table 3. Percentage of mitosis inhibition of A. cepa root tip cells, submitted to different concentrations of Glyphosate. Glyphosate concentration: C3H8NO5P (mg L-1)

(%) Inhibition of mitosis

Contrast (Control) 5 10 15 25 30

---49.4 57.4 63.2 75.8 90.8

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The display of elongated nuclei (EN) is the aberration with the highest values compared to the rest of anomalies, with a frequency of 170 in the 30 mg L-1 treatment and a frequency less than 12 in the case of 15 mg L-1 treatment (Table 4). Next, the appearance of irregular anaphase (IA) was observed, which maintained the trend of the study, where the 30 mg L-1 treatment presented the highest frequency with a value of 6 and a minimum value of 1.6 in the 5 and 10 mg L-1 treatments, only preceded by the control treatment with 0 MA (Table 4). Finally, the study also showed an important aberration, such as nuclear notch (Nn), which highest frequency was 20 in the 15 mg L-1 treatment and 0 for the 5 mg L-1 treatment and control treatment (Table 4). Table 4. Frequency of Chromosomal Anomalies at A. cepa root tip treated with different concentrations of Glyphosate. The data are averages of 5 repetitions (5000 cells per treatment). Glyphosate concentration: C3H8NO5P (mg L-1)

Frequency of Chromosomal Anomalies (Mean ± SD) Mn

AP

SM

CB

CCM

AN

EN

MA

NN

0

0a

0a

2.0±4.4 a,b

0a

0a

0a

0a

0a

0a

5

20±17a

0a

0a

6±8.9a,b

2±4.4a

2.0±4.4a

68±21a,b

1.6±0.9a

0a

10

52±28b

6.0±8.9a,b

8±8.3 a,b,c

2±4.4a,b

6±5.4a,b

4.0±5.4a,b

86±16a,b

1.6±0.5a

11±5.4a,b

15

58±23b

12.0±4.4b

12±4.4 a,b,c

2±4.4a,b

10±0b

6.0±5.4a,b

12±59a,b

4.4±0.9b

20±17b

25

52±19b

2.0±4.4a

9±8.2b,c

2±4.4a,b

2±4.4a

6.0±5.4a,b

136±111a,b

4.4±0.9b

10±6.1a,b

30

58±23b

2.0±4.4a

14±5.4c

10±0b

4±5.4a,b

12.0±4.4b

170±143b

6.0±2.2b

14±5.4a,b

The means ± SD values with different letter of each column indicate statistically significant differences, according to Tukey HSD test (P ≤ 0.05). SD = Standard deviation. Mn= Micronuclei. AP= anaphase bridge. SM= Sticky metaphase. CB= Chromosome break. CCM= Chromosomal clumping at metaphase. AN= Absence of nucleus. EN= Elongated nuclei. IA= Irregular anaphase Nn= Nuclear notch.

3.5 Chromosomal anomalies rate With a 6.7 standard deviation, the highest anomaly rate was obtained with the 30 mg L-1 concentration (Table 5). In addition, a rate of 2.7 and 2.48 is observed for the 15 and 25 mg L-1 concentrations, respectively. In the same way, the control treatment presented a very low rate of 13

0.02. This indicates that a higher concentration of glyphosate increases the rate of anomalies, which leads to an inhibition of the cell cycle in A. cepa roots meristematic zone.

Table 5. Relative abnormality rate for each concentration of Glyphosate. (Mean ± standard deviation)

Glyphosate concentration: C3H8NO5P (mg L-1)

Relative abnormality rate

Control 5 10 15 25 30

0.02±0.015 0.11±2.3 2±3 2.7±4.2 2.48±5.4 3.22±6.7

4. DISCUSSION 4.1 Radicular development By propitiating the hydration of the bulbs, meristematic cells elongation occurs giving origin to A. cepa roots (Camarillo-Ravelo et al., 2015). However, by exposing the roots to toxic chemicals, they will develop traits alterations (Restrepo et al., 2012), which will depend on the toxicity of the substances and the exposure time (Khanna and Sharma, 2013). According to Disner et al. (2011) the radicular growth inhibition test in A. cepa has great value in relation to the glyphosate toxicity test. The radicular growth from the 5 and 10 mg L-1 treatments observed at 24h, slightly higher than the control (Table 1), can be described as a clear example of hormesis (Calabrese and Mattson, 2017), which is defined as a dose-response relationship, there being a stimulatory response at 14

low doses but with an inhibitory response at high doses (Camarillo-Ravelo et al., 2015). On the other hand, the measurements taken at 24, 48 and 72h show a marked trend by greater inhibition of root growth by the 30mg L-1 treatment. Likewise, the control treatment achieved a greater development compared to the other treatments for 48 and 72h measurements. In this sense, what is presented in the different measurement times agrees with what was obtained by Ogeleka et al. (2016), where A. cepa roots length exposed to different glyphosate concentrations (0.625, 1.25, 2.5, 5 and 10 mg L-1) varied between 0.29 ± 0.03 and 4.90 ± 0.12 cm, while the control registered 6.82 ± 0.07 cm on average. Disner et al. (2011) observed that the higher the concentration of glyphosate, the lower the cell division in the roots, since from 10 μl l-1 concentration (6.38cm, P = 0.003), the onions presented differences significant in the root size, compared to the control (> 9.84cm, P = 0.003), checking that even very low concentrations can carry some degree of toxicity to organisms. Similar results were reported by Krüger (2009), who observed inhibition of A. cepa radicular growth for concentrations between 1 to 20 μl l-1, confirming the glyphosate high degree of toxicity. In this order of ideas, it is important to highlight the high sensitivity and low cost (Llontop and Vargas, 2014) and therefore the suitability of the A. cepa test compared to other tests, regarding glyphosate toxicity measurement. For example, the studies conducted by Sposito and Espinola (2016), obtained a maximum effective concentration (EC50) of 37.28 mg / L in Vibrio fischeri and an average lethal concentration (LC50) of 24.58 mg/L in Pseudokirchneriella subcapitata. In synthesis, the reduction in root growth could be due to an alteration in the mitotic cycle duration, as a result of the direct interaction of the A. cepa root meristematic cells with glyphosate (Atoyebi et al., 2015).

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4.2 Mitotic Index The mitotic index is a parameter that allows to quantify the mitotic activity of an organism (Lessa and Carriello, 2017) as well as, estimating the cytotoxicity of a great variety of compounds (Salazar-Mercado et al., 2019). It is also used to know the proportion of cells in the different phases of the cell cycle (Figure 1) and as an indicator of cell proliferation (Ping et al., 2012). In this study, an evident inhibition was observed in the mitosis process, since the obtained values in the GMI with the use of glyphosate proved to be considerably lower than those of the control (Table 2). When comparing the PMI results with the others the indices from the different phases (MMI, AMI and TMI), a greater percentage of the mitotic cell population was observed in prophase, which demonstrates that the majority of the mitotic cell population stops there. This effect is probably due to the fact that glyphosate inhibits the formation or function of the Cyclin B-cdk1 complex, which initiates the genetic material condensation and activates a group of proteins called condensins, responsible for inducing the mitotic spindle assembly, ensuring that the chromosomes join to this and consequently to the processes of genetic material condensation and microtubules polymerization (Aybar and Zabala, 2016). Therefore, cells would not normally transit from prophase to metaphase. Similarly, it is very important to highlight the glyphosate effect in the arresting of the cell cycle, at the anaphase level, being able to observe that its use caused that the counting of the telophase mitotic index (TMI) was 0 for all treatments, except for the control treatment. This could be due to the fact that glyphosate intervenes in the formation or function of the Anaphase / Cyclosome Promoter Complex or APC / C, which is responsible for the destruction of the proteins that keep 16

the sister chromatids together and allows them to separate in the anaphase and move towards opposite poles of the cell (Musacchio and Salmon, 2007; Silva, 2011), allowing telophase to happen. On the other hand, Atoyebi et al. (2015) state that an IM significantly lower than the control indicates a direct alteration in A. cepa DNA. In addition, Prajitha and Topphil (2016) claim that a decrease below 22% in the mitotic index could have a lethal impact on the organism, while a decrease below 50% leads, in general, to sublethal effects, which is called cytotoxic limit value. 4.3 Cellular anomalies and abnormality rate Regarding the results obtained in the mitotic indexes (Table 2) and the percentages of inhibition (Table 3) for each treatment, it was observed that they are directly proportional results, therefore, at higher glyphosate concentrations, the mitotic inhibition grows and the number of cells present in each mitosis phase decreases, to such an extent that the telophase mitotic index is 0. Taking into account the above, we can also observe that the anomalies frequency increases as the glyphosate concentration rises (Table 4). This is directly related to the negative impact on fauna and microorganisms (Van et al., 2018),

In this study we can confirm the high toxicity of glyphosate, by causing nine types of chromosomal anomalies. One of the most important are the micronuclei (Mn). This data are in agreement with the results obtained by Krüger (2009), where a significant increase in the number of micronuclei was observed in roots of A. cepa that were in contact with glyphosate in concentrations between 1 to 20 μl l-1. Regarding this anomaly, Martins et al. (2016) recommend the monitoring of sewage sludge in agricultural areas, due to the high genotoxic and mutagenic activity that the appearance of micronuclei represents. Another characteristic aberration found in 17

this study, are bridges in anaphase (AB), which concentration of 15mg L-1 presented a greater number of this anomaly (12.0 ± 4.4). However, Katabale et al. (2017), found that, by exposing A. cepa roots to 125mg L-1 glyphosate, it produced a lower frequency of bridges in anaphase (8 ± 3.05). We can also compare these results to those obtained by Asita and Mokhobo (2013) where the exposure of A. cepa roots to different agrochemicals produced 0.42% ± 0.61 of cells with Anaphase bridge. There is evidence that several of the alterations presented here, such as sticky metaphase (SM), chromosomal clustering in metaphase (CCM), nucleus absence (NA), anaphase bridge (AB) and nuclear notch (Nn), they are due to changes in chromatin by to the action of a toxic substance, glyphosate in this case, (Quintana et al, 2013). Regarding the appearance of sticky metaphase and chromosomal grouping in metaphase, Silva et al. (2018) characterize them as a failure in the process of chromosomes condensation at some stage of the cell cycle. Finally, it is worth to highlight the appearance of elongated nuclei (EN), an important anomaly due to the large number of cells that presented this condition, with the lowest frequency (68 ± 21) in the 5mg L-1 concentration and the highest in the 30mg L-1 concentration (170 ± 143). These data contrast with the frequency of 0.8 cells per 1000 sampled (0.08%±0.21) found by Asita and Mokhobo (2013) in A. cepa submitted to four types of pesticides.

The anomalies found in this study are consistent with those reported by several authors who performed the test on A. cepa with solutions of different chemical substances with cytotoxic potential (Prajitha and Thoppil, 2016; Causil et al., 2017). Thanks to the incidence of the anomalies found, glyphosate biocidal effect is ratified (Van et al., 2018). Summarizing, these chromosomal changes are an indicator that glyphosate has a direct action on the genetic material and therefore, it can be considered genotoxic (Silva et al., 2018). 18

5. CONCLUSION It is concluded that the use of glyphosate causes a high rate of elongated nuclei (EN), the appearance of a large number of micronuclei from the minimum concentration used. As well as, a considerable number of nuclear clefts and cells with total nucleus absence (NA), among other chromosomal anomalies, capable of generating permanent damage in DNA, causing total inhibition of telophase. In the same way, the suitability of the Allium cepa test is ratified, due to its low cost and practicality as a cytogenotoxicity bioindicator. It is advisable to carry out new investigations with lower concentrations of glyphosate, as well as shorter exposure times. Due to the damage of the found DNA, it is recommended to implement actions to reduce its impact on fauna and flora, since this could compromise the balance of ecosystems.

6. REFERENCES

Abdelsalam, N., Megeed, A., Ali, H., Salem, M., Z., Al-Hayali, M., Elshikh, MS., 2018. Genotoxicity effects of silver nanoparticles on wheat (Triticum aestivum L.) root tip cells. B. Environ. Safe. 155, 76-85. Akinboro, A., Kamaruzaman B., Asmawi, MZ., Sulaiman, SF., Sofiman, OA., 2011. Antioxidants in aqueous extract of Myristica fragrans (Houtt.) suppress mitosis and cyclophosphamide-induced chromosomal aberrations in Allium cepa L. cells. Journal of Zhejiang University SCIENCE B. 12(915), 1862-1783.

19

Almeida, D., Timossi, PC., Lima, SF., Silva, UR., Reis, EF., 2015. Droplets size categories and application volumes in burndown of plant covers. Revista Brasileira de Herbicidas. 14(1), 7382. Almeida, DP. 2018. Volume de aplicação reduzido e concentrações de glyphosate na calda em condições meteorológicas distintas para dessecação de cobertura vegetal em sistema de plantio direto. Tese Doutoral, Universidade estadual paulista - UNESP câmpus de Jaboticabal. Al-Rajab, A., Schiavon, M., 2010. Degradation of 14C-glyphosate and aminomethylphosphonic acid (AMPA) in three agricultural soils. Journal of Environmental Sciences. 22(9), 13741380. doi.org/10.1016/S1001-0742(09)60264-3. Aparicio, VC., De Gerónimo, E., Marino, D., Primost, J., Carriquiriborde, P., Costa, JL., 2013. Environmental fate of glyphosate and aminomethylphosphonic acid in surface waters and soil of

agricultural

basins.

Chemosphere.

93(9),

1866-1873

doi.org/10.1016/j.chemosphere.2013.06.041 Asita, AO., Mokhobo, MM., 2013. Clastogenic and Cytotoxic Effects of Four Pesticides Used to Control Insect Pests of Stored Products on Root Meristems of Allium cepa. Environment and Natural Resources Research. 3(2). doi:10.5539/enrr.v3n2p133. Atoyebi, SM., Oyeyemi, IT., Dauda, BA., Bakare, AA., 2015. Genotoxicity and antigenotoxicity of aqueous extracts of herbal recipes containing Luffa cylindrica (L), Nymphaea lotus (L) and Spondias mombin (L) using the Allium cepa (L) assay. African Journal of Pharmacy and Pharmacology. 9(15), 492-499. doi: 10.5897/AJPP2014. 4219. Aybar, JA, Zavala, F., 2016. Efecto Citotóxico del extracto acuoso del pericarpio de Caesalpinia spinosa “tara” en células meristemáticas de Allium cepa L. var. Arequipeña. Revista Ciencia y Tecnología. 12(2), 185-193.

20

Benbrook, C., 2016. Trends in glyphosate herbicide use in the United States and globally. Environmental Sciences Europe. 28(1), 3. doi.org/10.1186/s12302-016-0070-0. Bento, CP.,

Yang, X.,

Gort, G.,

Mol, HG., Ritsema, CJ., Geissen,

V.,

Xue, S., 2016.

van

Persistence

Dam, R., Zomer, P., of

glyphosate

and

aminomethylphosphonic acid in loess soil under different combinations of temperature, soil moisture and light/darkness.

Science of The Total Environment. 572, 301-31.

doi.org/10.1016/j.scitotenv.2016.07.215. Bento, CPM., van der Hoeven, S., Yang, X., Riksen., M., Mol, H., Ritsema, Geissen, C., 2019. Dynamics of glyphosate and AMPA in the soil surface layer of glyphosate-resistant crop cultivations in the loess Pampas of Argentina. Environmental Pollution. 244, 323-331. doi.org/10.1016/j.envpol.2018.10.046 Biruk, L., Moretton, J., abrizio, A., Weigandt, C., Etcheverry, J., Filippetto, J., Magdaleno, A., 2017. Toxicity and genotoxicity assessment in sediments from the MatanzaRiachuelo river basin (Argentina) under the influence of heavy metals and organic contaminants. Ecotoxicol. Environ. Safe. 135, 302-311. Bortolottoa, T., Silvaa, j., Célio, A., Osowski, K., Geremiasa, R., Angiolettob, E., Pich, C., 2017. Evaluation of toxic and genotoxic potential of a wet gas scrubber effluent obtained from wooden-based biomass furnaces: a case study in the red ceramic industry in southern Brazil. Ecotoxicol. Environ. Safe. 143, 259-265. Calabrese, EJ., Mattson, MP., 2017. How does hormesis impact biology, toxicology, and medicine?. Nature research catalog. 3(13). doi: 10.1038/s41514-017-0013-z Camarillo-Ravelo, D., Barajas-Aceves, M., Rodríguez-vázquez, R., 2015. Evaluación de la fitotoxicidad de jales mineros en cuatro especies empleadas como bioindicadoras de metales pesados. Revista Internacional de Contaminación Ambiental. 31(2), 133-143. 21

Camacho, A., Mejía, D., 2017. The health consequences of aerial spraying illicit crops: the case of Colombia. J. Health Econ. 54, 147-160. Castro, MB., Marino, D., Quiroga, MV., Zagarese, H., 2018. Occurrence and levels of glyphosate and AMPA in shallow lakes from the Pampean and Patagonian regions of Argentina. Chemosphere. 200, 513-522. doi.org/10.1016/j.chemosphere.2018.02.103 Causil, L., Coronado, J., Vega, M., Verbel, L., 2017. Efecto citotóxico del hipoclorito de sodio (NaClO), en células apicales de raíces de cebolla (Allium cepa L.). Revista Colombiana de Ciencieas Hortíolas. 11(1), 97-104. doi.org/10.17584/rcch.2017v11i1.5662. Centner, T., Russell, L., Mays, M., 2019. Viewing evidence of harm accompanying uses of glyphosate-based herbicides under US legal requirements. Science of The Total Environment 648, 609-617. https://doi.org/10.1016/j.scitotenv.2018.08.156 Curwin, BD., Hein, MJ., Sanderson, WT., Nishioka, MG., Reynolds, SJ., Ward, EM., Alavanja, MC., 2005. Pesticide contamination inside farm and nonfarm homes. Journal of Occupational and Environmental Hygiene. 2(7), 357-67. Datta, S., Singh, J., Singh, J., Singh, S., Singh, S., 2018. Assessment of genotoxic effects of pesticide and vermicompost treated soil with Allium cepa test. Sustain. Environ. Res. 28(4), 171-178. doi.org/10.1016/j.serj.2018.01.005. Disner, GR., Rocha, MV., Miranda, GB., 2011. Avaliação da atividade mutagênica do Roundup® em Astyanax altiparanae (Chordata, Actinopterygii). Evidencia: biotecnologia e alimentos. 11(1), 33-42. Eberbach, P., 1998. Applying non‐steady‐state compartmental analysis to investigate the simultaneous degradation of soluble and sorbed glyphosate (N-(phosphonomethyl)glycine) in four soils. Pest Management Science. 52(3), 229-240. doi.org/10.1002/(SICI)10969063(199803)52:3<229::AID-PS684>3.0.CO;2-O. 22

Environmental Protection Agency, 2017. Office of Chemical Safety and Pollution Prevention. Glyphosate. Dietary exposure analysis in support of registration review. In. Washington, DC: United States; p. 1-20. Fatma, F, Verma, S, Kamal, A, Srivastava, A, 2018. Monitoring of morphotoxic, cytotoxic and genotoxic potential of mancozeb using Allium assay. Chemosphere, 195, 864-870. doi.org/10.1016/j.chemosphere.2017.12.052. Fatima, R., Ahmad, M., 2005. Certain antioxidant enzymes of Allium cepa as biomarkers for the detection of toxic heavy metals in wastewater. Science of The Total Environment 346. (1–3), 256-273. Fernandes, G., Aparicio, V., Bastos, M., De Gerónimo, E., Labanowski, J et. al., 2019. Indiscriminate use of glyphosate impregnates river epilithic biofilms in southern Brazil. Science

of

The

Total

Environment.

651,

1377-1387.

https://doi.org/10.1016/j.scitotenv.2018.09.292. Fernández, P., Peropadre, A., Rosal, R., Pérez, J Hazen, M., 2016. Toxicological assessment of third generation (G3) poly (amidoamine) dendrimers using the Allium cepa test. Science of The Total Environment. 563–564, 899-903. https://doi.org/10.1016/j.scitotenv.2015.07.137 Firbas, P., Amon, T., 2014. Chromosome damage studies in the onion plant Allium cepa L. Caryologia. 67, 25-35. Fonseca, MB., 2007. Crescimento e parâmetros toxicológicos em jundiás (Rhamdiaquelen) expostos a uma formulação comercial do herbicida 2,4Diamin. Dissertação de Mestrado. Universidade de Santa Maria/RS. Garcia, C., Souza, R., de Souza, C., Christofoletti, C., Fontanetti, C., 2017. Toxicity of two effluents from agricultural activity: comparing the genotoxicity of sugar cane and orange vinasse. Ecotoxicol. Environ. Safe. 142, 216–221. 23

Greer, DG., Donofrio PD., 2009. CHAPTER 16 - Electrophysiological Evaluations, Editor(s): MICHAEL R. DOBBS, Clinical Neurotoxicology, W.B. Saunders, pp. 201-212, doi.org/10.1016/B978-032305260-3.50022-8. Haq, I., Kumar, S., Raj, A., Lohani, M., Satyanarayana, G., 2017. Genotoxicity assessment of pulp and paper mill effluent before and after bacterial degradation using Allium cepa test. Chemosphere. 169, 642-650. doi.org/10.1016/j.chemosphere.2016.11.101. Ghosh, P., Thakurb, I., Kaushik, A., 2017. Bioassays for toxicological risk assessment of landfill leachate: a review. Ecotoxicol. Environ. Safe. 141, 259-270. Heinonen-Tanski, H., 1989. The effect of temperature and liming on the degradation of glyphosate in two arctic forest Soils. Soil Biology and Biochemistry. 21(2), 313-317. doi.org/10.1016/0038-0717(89)90110-7 Idrovo, AJ., 2015. De la erradicación de cultivos ilícitos a la erradicación del glifosato en Colombia. Revista Universidad Industrial de Santander, Salud. 47(2), 113-114. Idrovo, AJ., 2019. Salud UIS in The Lancet: visibility and quality beyond the impact factor and the

H5.

Revista

Universidad

Industrial

de

Santander,

Salud.

51(1),

5-6.

doi:

10.18273/revsal.v51n1-2019001 INTERNATIONAL AGENCY FOR RESEARCH ON CANCER, 2016. Monographs on the evaluation

of

carcinogenic

risks

to

humans,

Glyphosate.

112.

http://monographs.iarc.fr/ENG/Monographs/ vol112/mono112-10.pdf. Katabale, MK., Esther, NM., Kariuki, D., 2017. Phytochemical screening, Cytotoxic, genotoxic and mutagenic effects of the aqueous extract of Azadirachta indica leaves. International Journal of Herbal Medicine. 5(3), 39-44. Keesstra, SD., Rodrigo-Comino, J., Novara, A., Giménez-Morera, A., Pulido, M, Di Prima, S., Cerdà, A., 2019. Straw mulch as a sustainable solution to decrease runoff and erosion in 24

glyphosate-treated clementine plantations in Eastern Spain. Anassessmentusing rainfall simulation experiments. CATENA. 174, 95-103. doi.org/10.1016/j.catena.2018.11.007 Khanna, N., Sharma, S., 2013. Allium cepa root chromosomal aberration assay: A review. Indian J. Pharm. Biol. 1(3), 105-119. Krüger, RA., 2009. Análise da toxicidade e da genotoxicidade de agrotóxicos utilizados na agricultura utilizando bioensaios com Allium cepa. Dissertação (Mestrado em Qualidade Ambiental) Universidade FEEVALE, Novo Hamburgo, 2009. Kumari, M., Mukherjee, A., Chandrasekaran, N., 2009. Genotoxicity of silver nanoparticles in Allium cepa. Science of The Total Environment. 407, 5243-5246. https://doi.org/10.1016/j.scitotenv.2009.06.024.

Kwiatkowska, M., Huras, B., Bukowska, B., 2014. The effect of metabolites and impurities of glyphosate on human erythrocytes (in vitro). Pestic. Biochem. Phys. 109, 34-43. Kwiatkowska, M., Reszka, E., Woźniak, K., Jabłońska, E., Michałowicz, J., Bukowska, B., 2017. DNA damage and methylation induced by glyphosate in human peripheral blood mononuclear cells (in vitro study). Food Toxicol. 105, 93-98 Laughinghouse, H., Prá, D., Silva-Stenico, M., Rieger, A Frescura, V., et. al., 2012. Biomonitoring genotoxicity and cytotoxicity of Microcystis aeruginosa (Chroococcales, Cyanobacteria) using the Allium cepa test. Science of The Total Environment. 432, 180-188. https://doi.org/10.1016/j.scitotenv.2012.05.093. Lessa, L., Cariello, F., 2017. Adsorção do paracetamol em carvão ativado: regressão da citotóxicidade e mutagênicidade no sistema Allium cepa. HÓRUS. 12(1), 44-54.

25

Llontop, L., Vargas, C., 2014. Efecto citoreparador de Aloe vera L.“sábila” en tejidos embrionarios de Allium cepa L.“cebolla” con daño cromosómico inducido por amoxicilina. Acc. Cietna. 2(2), 1-10. Martins, MN., Souza, VV., Souza, TD., 2016. Genotoxic and mutagenic effects of sewage sludge on higher plants. Ecotoxicol. Environ. Safe 124, 489-496. Martins, M., de Souza, V., da Silva, ST., 2016. Cytotoxic, genotoxic and mutagenic effects of sewage

sludge

on Allium

cepa.

Chemosphere.

148,

481-486

doi.org/10.1016/j.chemosphere.2016.01.071. Moura, AG., Santana, GM., Ferreira, P., Sousa, J., Peron, AP., 2016. Cytotoxicity of cheese and Cheddar cheese food fl avorings on Allium cepa L root meristems. Braz. J. Biol. 76(2), 439443. doi.org/10.1590/1519-6984.20514. Musacchio, A., Salmon, ED., 2007. The spindle-assembly checkpoint in space and time. Nature Reviews Molecular Cell Biology. 8, 379-393. Ogeleka, DF., Okieimen, FE., Ekpudi, FO., Tudararo-Aherobo LE., 2016. Short-term phytotoxicity consequences of a nonselective herbicide glyphosate (Roundup™) on the growth of onions (Allium cepa Linn.). African Journal of Biotechnology. 15(18), 740-744. doi: 10.5897/AJB2014.14355. Ozkara, A., Akyıl, D., Eren, Y., Erdoğmus¸ SF., 2015. Potential cytotoxic effect of Anilofos by using Allium cepa assay. Cytotechnology 67(5), 783-791. doi.org/10.1007/s10616-014-97161. Panzacchi, S., Mandrioli, D., Manservisi, F., Bua, L., Falcioni, L., Spinaci, M., Galeati, G., Dinelli, G., Miglio, R., Mantovani, A., et al., 2018. The Ramazzini institute 13-week study on glyphosate-based herbicides at human-equivalent dose in Sprague Dawley rats: study design

26

and first in-life endpoints evaluation. Environmental Health. 17(1), 52. doi: 10.1186/s12940018-0393-y Pereira, AC., Silva, NC., de Almeida, LM., 2017. Potencial toxicológico de Lafoensia pacari (Lythraceae) usando o sistema teste Allium cepa como bioindicador. IV Congresso de Ensino, Pesquisa e Extensão da UEG. 4. Peruzzo, PJ., Porta, AA., Ronco, AE., 2008. Levels of glyphosate in surface waters, sediments and soils associated with direct sowing soybean cultivation in north pampasic region of Argentina. Environmental Pollution. 156(1), 61-66. doi.org/10.1016/j.envpol.2008.01.015 Ping, KY., Darah, I., Yusuf, UK., Sasidharan, S., 2012. Genotoxicity of Euphorbia hirta on Allium cepa assay. Molecules. 39, 222-225. doi: 10.3390/molecules17077782 Poiger, T., Buerge, IJ., Bächli, A., Müller, MD., Balmer, ME., 2017. Occurrence of the herbicide glyphosate and its metabolite AMPA in surface waters in Switzerland determined with on-line solid phase extraction LC-MS/MS. Environmental Science and Pollution Research. 24(2), 1588–1596. Prajitha, V., Thoppil, J., 2016. Genotoxic and antigenotoxic potential of the aqueous leaf extracts of Amaranthus spinosus Linn. using Allium cepa assay. South Afr. J. Bot. 102, 18-25. http://dx.doi.org/10.1016/j.sajb.2015.06.018. Quintana, RV., Alarcón, JS., Arroyo, SG., Eslava, JC., Waliszewski, S., Fernández, S., Pietrini, RV., 2013. Genotoxicidad de plaguicidas en sistemas vegetales. Rev. Int. Contam. Ambie. 29, 133-157. Radić, S., Stipaničev, D., Vujčić, V., Rajčić, M., Širac, S., et. al., 2010. The evaluation of surface and wastewater genotoxicity using the Allium cepa test. Science of The Total Environment. 408, 1228-1233. https://doi.org/10.1016/j.scitotenv.2009.11.055.

27

Ravier, S., Désert, M., Gille, G., Armengaud, A., Wortham, H., Quivet, E, 2019. Monitoring of Glyphosate, Glufosinate-ammonium, and (Aminomethyl) phosphonic acid in ambient air of Provence-Alpes-Côte-d’Azur Region, France. Atmospheric Environment, 204, 102-109. doi.org/10.1016/j.atmosenv.2019.02.023. Restrepo, R., Reyes, D., Ortiz, M., Ruiz, F., Kouznetsov, V., 2012. Aberraciones cromosomales en bulbos de cebolla Allium cepa inducidas por moléculas híbridas 4-aminoquinolínicas. Universitas Scientiarum. 17(3), 253-261. doi.org/10.11144/javeriana.SC17-3.aceb. Salazar-Mercado, SA., Torres-León., CA., Rojas-Suárez, JP., 2019, Cytotoxic evaluation of sodium hypochlorite, using Pisum sativum L as effective bioindicator. Ecotoxicology and Environmental Safety. 173, 71-76. doi.org/10.1016/j.ecoenv.2019.02.027. Salazar, S., Maldonado, H., (2019). Evaluation of cytotoxic potential of chlorpyrifos using Lens culinaris Med as efficient bioindicator. Ecotoxicology and Environmental Safety. 183. https://doi.org/10.1016/j.ecoenv.2019.109528. Sánchez, JA., Klosterhoff, MC., Romano, LA., Martins, G., 2019. Histological evaluation of vital organs of the livebearer Jenynsia multidentata (Jenyns, 1842) exposed to glyphosate: A comparative

analysis

of

Roundup®formulations.

Chemosphere,

217,

914-924.

doi.org/10.1016/j.chemosphere.2018.11.020. Santos, LJ., de oliveira, PM., Cardoso, CE., 2018. Determinação espectrofotométrica de glifosato em cabelo humano utilizando complexação com Ca2+. Revista Eletrônica TECCEN. 11(1), 54-60. doi.org/10.21727/teccen.v11i1.1155. Schroll, R., Becher, HH., Dörfler, U., Gayler, S., Grundmann, S., Hartmann HP., Ruoss, J., 2006. Quantifying the effect of soil moisture on the aerobic microbial mineralization of selected pesticides in different Soils. Environmental Science & Technology. 40(10), 3305-3312. doi: 10.1021/es052205j. 28

Scherer, M., Sposito, J., Falco, W., Grisolia, A., Andrade L et. al., 2019. Cytotoxic and genotoxic effects of silver nanoparticles on meristematic cells of Allium cepa roots: A close analysis of particle size dependence. Science of The Total Environment. 660, 459-467. https://doi.org/10.1016/j.scitotenv.2018.12.444 Shipitalo, MJ., Malone, RW., Owens, LB., 2008. Impact of glyphosate-tolerant soybean and glufosinate-tolerant corn production on herbicide losses in surface runoff. Journal of Environmental Quality. 37, 401-408. doi: 10.2134/jeq2006.0540 Silva, F., Barp, E., Armiliato, N., 2017. Avaliação da toxicidade celular do glifosato sobre as gônadas de Danio rerio (Cyprinidae). Saúde e meio ambiente: revista interdisciplinar. 6(1), 85-95. doi.org/10.24302/sma.v6i1.1041 Silva, P., Barbosa, J., Nascimento, VA., Faria, J., Reis, R., Bousbaa. H., 2011. Monitoring the fidelity of mitotic chromosome segregation by the spindle assembly checkpoint. Cell Proliferation. 44, 391-400. doi:10.1111/j.1365-2184.2011.00767.x Silva, V., Montanarella, L., Jones, A., Fernandez-Ugalde, O., Mol H., Ritsema CJ., Geissen V., 2018. Distribution of glyphosate and aminomethylphosphonic acid (AMPA) in agricultural topsoils of the European Union. Science of The Total Environment. 621, 1352-1359. Sihtmäe, M., Blinova, I., Künnis-Beres, K., Kanarbik, L., Heinlaan, M., Kahru, A., (2013). Ecotoxicological effects of different glyphosate formulations. Applied Soil Ecology. 72, 215-224. Sosa, B., Fontans-Álvarez, E., Romero, D., da, Fonseca, A., Achkar M., 2019. Analysis of scientific production on glyphosate: An example of politicization of science. Science of The Total Environment. 681, 541-550.

29

Spósito M., Espínola, MJ., 2016. Evaluación in vitro del efecto tóxico de una formulación comercial de glifosato de amonio sobre cinco especies representantes de diferentes hábitats y niveles tróficos. Rev. INNOTEC. 12, 48-53. Sucahyo, D., van Straalen., NM, Krave, A., van Gestel, CA., 2008. Acute toxicity of pesticides to the tropical freshwater shrimp Caridinalaevis. Ecotoxicology and Environmental Safety, 69, 421-427. Sulukan, E., Köktürk, M., Ceylan, H., Beydemir, Ş., Işik, M., Atamanalp, M., Ceyhun, SB., 2017. An approach to clarify the effect mechanism of glyphosate on body malformations during embryonic development of zebrafish (Daino rerio). Chemosphere. 180, 77-85. doi.org/10.1016/j.chemosphere.2017.04.018 Talledo, D., Escobar, C., 2018. El ciclo celular en vegetales. Su estudio, importancia y aplicaciones. Biotempo. 2, 13-31. Todorovic, G., Rampazzo, N., Mentler, A., Blum, WE., Eder, A., Strauss, P., 2014. Influence of soil tillage and erosion on the dispersion of glyphosate and aminomethylphosphonic acid in agricultural soils. International. Agrophysics. 28(1), 93-100. doi: 10.2478/intag-2013-0031. Tukey, JW., 1994. The problem of multiple comparisons. En: H. L. Braun (ed.). The collected works of John W. Tukey. Nueva York: Chapman and Hall. 8(1), 1-300. Turkmen, R., Birdane, YO., Demirel, HH., Kabu, M., Ince S., 2019. Protective effects of resveratrol on biomarkers of oxidative stress, biochemical and histopathological changes induced by sub-chronic oral glyphosate-based herbicide in rats. Toxicology Research. Advance Article. doi: 10.1039/C8TX00287H Van, A., He, M., Shin, K., Mai, V., Jeong, K et. al., 2018. Environmental and health effects of the herbicide glyphosate. Science of The Total Environment. 616-617, 255-268. https://doi.org/10.1016/j.scitotenv.2017.10.309. 30

Yang, X., Wang, F., Bento, CP., Meng, L., VanDam, R., Mol, H., Liu, G., Ritsema, CJ., Geissen, V., 2015a. Decay characteristics and erosion-related transport of glyphosate in Chinese loess soil

under

field

conditions.

Science

of

The

Total

Environment.

530, 87-95

doi.org/10.1016/j.scitotenv.2015.05.082. Yang, X., Wang, F., Bento, CP., Xue, S., Gai, L., van Dam, R., Mol, H., Ritsema, C., Geissen, V., 2015b. Short-term transport of glyphosate with erosion in Chinese loess soil - a flume experiment. Science of the Total Environmental. 512–513, 406-414.

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32

Highlights



The use of glyphosate caused permanent DNA damage, producing nine types of cellular anomalies and telophase inhibition in Allium cepa.



The 30mg L-1 treatment caused an average frequency of 170 elongated nuclei.



The exposition to 15 and 30 mg L-1 glyphosate caused an average frequency of 58 micronuclei.

33