Cytotoxicity and genotoxicity of some agropesticides used in Southern Africa

Pesticides use boosts agricultural yield by reducing crop losses. However, some pesticides are mutagens and active ingredients could produce different effects in different formulations. Wipe-out (WO) (Glyphosate 360 g/l), K-O Gard® (KOG) (Deltamethrin 10 g/l), Cutworm bait (CWB) (Sodium Fluosilicate, 100 g/kg), Snail ban (SB) (Metaldehyde, 30 g/kg and Carbaryl, 20 g/kg) and Coopex (CPX) (Permethrin 250 g/kg) were tested for cytotoxicity and genotoxicity using the onion (Allium cepa) test. Onion seeds were germinated and exposed (mg/l) to below EC50 values of WO (78.1, 156.3, 312.5), KOG (156.3, 312.5, 625.0), CWB (78.1, 156.3, 312.5), SB (156.3, 312.5, 625.0), and CPX (312.5, 625.0, 1250.0) for 24 h. Root tips were cut, fixed, hydrolyzed in 1 N hydrochloric acid, stained, squashed and observed microscopically. Cytotoxicity and genotoxicity induction at each treatment was compared with the negative control using t-test. WO (78.1, 156.3, and 312.5), KOG (312.5, 625.0) and CWB (312.5) were cytotoxic, (P < 0.05). WO (78.1, 312.5), CWB (156.3, 312.5), SB (625.0) and CPX (1250.0) induced genotoxicity (P < 0.05), mostly, sticky chromosomes. The genotoxic pesticides have potential to cause adverse environmental and health effects, because most adverse effects by genotoxins result from genetic damage and genotoxicity of chemicals is likely to result from cell division abnormalities.


INTRODUCTION
Among crops, the actual loss due to agricultural pests (weeds, animal pests, pathogens and viruses) worldwide were estimated at 26 to 30% for sugar beet, barley, soybean, wheat and cotton, and 35, 39 and 40% for maize, potatoes and rice, respectively (Oerke and Dehne, 2004).Mohan and Fields (2002) stated that annual postharvest losses resulting from insect damage and other bio-agents are estimated to be 10 to 40% of world agricultural production.Grain losses caused by insect pests in Africa are quite high and vary from country to country and from region to region.However, annual grain losses of over 50% in cereals (Abraham and Firdissa, 1991) have been reported, although the average stands at 20% (Youdeowi and Service, 1986;Philips and Throne, 2010).In order to cut down losses in agriculture, pesticides have been integrated into modern agricultural process to control weeds, diseases and insect pests that can markedly reduce the amount of harvestable produce and thereby increase outputs and productivity (Aktar et al., 2009).Pimentel et al. (1993) argue that on average, the economic benefits from pesticide use are about four times their direct cost to the users.Without pesticide application, the loss of fruits, vegetables and cereals from pest injury would reach 78, 54 and 32%, respectively (Cai, 2008).
Pesticides are different from other agricultural inputs in that they do not directly boost yields in the way that fertilizers do; instead they reduce crop losses caused by pests (Jha and Regmi, 2009).Though pesticides use reduces crop losses and so increases agricultural output, pesticides are known to produce a wide spectrum of adverse health and environmental effects.According to a report of World Health Organization (WHO) and United Nations Environment Programme (UNEP), worldwide, there are more than 26 million human pesticide poisonings with about 220,000 deaths per year (Richter, 2002).
In a review of the literature on the health effects of pesticides, Mansour (2004) concluded that there is strong scientific evidence that pesticides, as a whole, can induce severe effects to human health ranging from myelotoxicity to cytogenetic damage and carcinogenicity.Other health effects of pesticides include acute and persistent damage in the nervous system (Kamel et al., 2007), lung and respiratory disorders (Hoppin et al., 2008), alterations in the reproductive organs (Hileman, 1994) birth defects (Rojas et al., 2000).Other causes of worry, according to Anwar (1997) are other side effects associated with agricultural chemicals such as the carcinogenic and genotoxic effects, which are considered as being among the most important of the effects.Most of the adverse effects to health, according to Norppa (2004) are the result of the genetic damage induced by genotoxic agents in somatic as well as in germinal cells and any genetic activity of chemicals, it has been suggested, is most likely to result from cell division abnormalities (Parry et al., 1999).Pesticides residues are known to persist in soil (Subbarao, 1999), water (Medina et al., 1999) and in fruits and vegetables (Ahouangninou et al., 2012;Osman et al., 2010) and represent a risk for human health.Genotoxicity and mutagenicity of pesticides for non target organisms and their influence on ecosystems are of worldwide concern (Pimentel et al., 1998).
According to Zhang et al. (2011), worldwide, there are currently about 500 pesticides used widely and in large quantities.In China alone, more than 400 companies are manufacturers of over 300 varieties of original pesticides and 3,000 formulations or commercial names.These pesticides are manufactured as insecticides, fungicides, herbicides, rodenticides and antimicrobials to protect crops.Pesticides of different types are used in agriculture to protect crops and stored foods in order to increase agricultural productivity.In the southern African subregion, commonly used agropesticides include Wipe-out (Glyphosate 360 g/l), K-O Gard® (Deltamethrin 10 g/l), Cutworm bait (Sodium Fluosilicate, 100 g/kg), Snail ban (Metaldehyde, 30 g/kg and Carbaryl, 20 g/kg) and Coopex (Permethrin 250 g/kg).
Data gap exists in the information available on the genotoxicity of some of the active ingredients and/or these formulations of pesticides on A. cepa, because synergistic interaction or potentiation between or among pesticides that are mixed together is a well known phenomenon (Cloyd et al., 2007).For instance, we are not aware of information on the genotoxicity of sodium hexafluorosilicate and metaldehyde on A. cepa.The analysis of chromosomal alterations serves as a mutagenicity test and is one of the few direct methods to measure damages in systems exposed to possible mutagens or carcinogens (Tedesco and Laughinghouse (IV), 2012).Plants are direct recipients of agrotoxins and plant genotoxicity assays are relatively fast, inexpensive and give reliable results (Grant, 1978) and the Allium test is simple and just as reliable as the method where chromosome aberrations were recorded in all types of mitotic cells (Rank and Nielsen, 1997).
This study sought to investigate the cytotoxicity and genotoxicity of different concentrations of Wipe-out, K-O Gard®, Cutworm bait, Snail ban and Coopex, using the anaphase-telophase chromosome aberration assay with root tip meristem cells of onion (A.cepa).This study is a continuation (Asita and MatebesI, 2010) of our efforts to test the pesticides that are sold in garden centres in Maseru, Lesotho, Southern Africa, for cytotoxicity and genotoxicity in view of the known adverse health and environmental effects of pesticides.

Selection of pesticide dosages
A preliminary dose selection experiment was conducted to determine the doses to be used in the actual experiments.A total of 100 onion seeds were spread on filter paper moistened with different concentrations of each pesticide or with water (negative control) for 72 h at room temperature (22 ± 2°C).The effective concentration (EC50) (mg/ml) was the concentration that inhibited the germination of 50% of the seeds or the effective concentration for 50% growth inhibition for relative reduction of root length when onion bulbs were used (Asita and Matabesi, 2012;Yildiz and Arikan, 2008).The effective concentration (EC50) (mg/I) values for the five pesticides were approximately: WO (1250.0),KOG (> 20000), CWB (1500), SB (5000) and CPX (5000).Thus KOG did not inhibit germination even at the limit of solubility.In trial experiments, the EC50s of the test compounds were too toxic therefore not enough cells in division stages could be observed.In the actual genotoxicity experiments therefore, the highest concentration of each pesticides tested was a fraction of each of their EC50 as follows, WO (1/4 EC50), CWB (1/4 EC50), SB (1/8 EC50) and CPX (1/4 EC50).The highest concentration of 6.25 mg/ml for KOG was an arbitrary value determined by solubility consideration.On the basis of the EC50 values, the results of trial experiments and the need for sufficient number of dividing cells on each slide for scoring, the following three concentrations (mg/l) of the pesticides were used in the actual cytotoxicity and genotoxicity experiments; WO (78.1, 156.3, 312.5), KOG (156.3, 312.5, 625.0), CWB (78.1, 156.3, 312.5), SB (156.3, 312.5, 625.0), and CPX (312.5, 625.0, 1250.0).

Genotoxicity assay
The method used was similar to the method of Matsumoto et al. (2006).A. cepa (onion) seeds were spread on water moistened filter paper in a petri dish, at room temperature (22 ± 2°C), until they germinated (about 72 h) and the radicles reached a length of about 2 cm.For each treatment, ten germinated seeds were transferred onto filter paper moistened with the pesticide in a Petridish and kept for 24 h at room temperature.Distilled water was used as a negative control.

Root harvest and slide preparation
After the 24 h exposure, three root tips from three seedlings per treatment were collected at random and assessed.Root tips 1 to 2 cm long were cut from the treated germinated seeds and fixed in alcohol-acetic acid (ethanol: glacial acetic acid in 3:1 ratio) for 24 h in a fridge at 4 to 6°C.The root tips were washed twice with ice cold water for 10 min each and allowed to dry in a watch glass.A solution of 1 N HCl pre-heated to 60°C was added to the root tips for 10 min.The HCl treatment was repeated a second time.Three root tips from each treatment were transferred singly to clean microscope slides and cut 2 mm from the growing tip.The tips were kept and the rest was discarded.Aceto-carmine stain was added to each slide to cover the root tip for about 10 min.A glass cover slip was placed on the root tip on each slide and tapped gently with a pencil eraser to spread the cells evenly into a monolayer to facilitate the scoring process for normal and aberrant cells in the different stages of the cell cycle.The slides were viewed under the light microscope (Olympus CX 21) and the cells were scored under oil immersion at 1000× magnification.The slides were coded and scored blind.The most representative ones for each structural aberration class were photographed using a Zeiss PrimoStar Microscope mounted with a Canon Camera, model, Power Shot A640.

Scoring of slides and data analysis
Cytotoxicity: On each of three slides (n = 3) for each concentration, a total of 2,000 cells, classified into interphase or dividing cells (Prophase, Metaphase, Anaphase or telophase) were scored that is, a total of 6,000 cells each for the control and treatment groups.The mitotic index (MI) was expressed as the number of dividing cells per 1,000 cells scored.The mitotic index of each treatment was compared with that of the negative control group using t-test at a probability level of 0.05.Analysis was performed using the SPSS for windows version 11.0 software.

Genotoxicity:
On each of three slides for each dose, 100 anaphase and telophase cells were examined for chromosome aberration, that is, 300 anaphase and telophase cells per pesticide treatment.The following aberrations, adapted from Parry et al. (1985) were observed and scored: 1. Chromosome fragments (F): Pieces of chromosome broken from whole chromosome as a result of pesticide treatment and lacking centromere; 2. Anaphase or Telophase bridge (AB): Dicentric chromosomes that form a bridge between both poles at anaphase or telophase.Often it indicates paracentric inversions or other possibilities that include breakage and fusion of chromosomes and sister chromatid reunion; 3. Laggard (L): Whole chromosomes that fail to migrate to either pole at anaphase because of damage to the centromere and/or kinetochore; 4. C-Mitosis (C-Mit): Mitotic cells that lack spindle fibres so that the chromosomes lie scattered throughout the cell.The effect is usually produced in cells treated with the spindle poisons, colchicines or colcemid, hence C-Mitosis; 5. Multipolar anaphases and telophases (Multipolar): Anaphase and Asita and Hatane 177 telophase cells with three or more spindle poles instead of the normal two.It is caused by damage to the centrosome; 6. Sticky chromosomes (S): Sticky chromosomes fail to condense completely so that at metaphase, the chromosomes are still long like prophase chromosomes and remain entangled with each other.
In extreme cases, chromatin masses, undistinguishable as chromosome are seen as clumps.The cells lack spindle fibres.The percentage of anaphase and telophase cells with aberrations in each treatment group of three slides at each concentration of pesticide was compared with that of the negative control group using t-test.

Cytotoxicity of the pesticides
The results of the effects on the mitotic index, of the treatment of onion root tip meristem cells with different concentrations of the pesticides are presented in Table 1.WO (78.1,156.3,312.5 mg/l), KOG (312.5, 625.0 mg/l) and CWB (312.5 mg/l) had mitotic indices significantly lower than that of the control (P < 0.05) and were adjudged cytotoxic.None of the three treatment concentrations of SB and CPX that were tested induced significantly depressed mitotic index when compared to the control and were accordingly adjudged not cytotoxic.

Genotoxicity of the pesticides
Photographs of the most representative pictures of normal mitotic cells and cells containing the different types of chromosome aberrations that were observed and scored are presented in Figure 1

DISCUSSION
The results of this study have shown that WO, KOG and CWB significantly depressed the mitotic index (MI) in treated root tip meristem cells of A. cepa, when compared to the control at one or more concentrations (P < 0.05).SB and CPX were not cytotoxic.Rank and Nielsen (1997) showed that when the EC 50 value was chosen as the highest concentration for the genotoxicity test, the mitotic index was never below 50% of the control.With the exception of KOG that the correct EC 50 could not be determined even at the limit of its solubility, the highest concentrations of each pesticide that was tested was below the EC 50 .
In the present study, the mitotic indices of meristem cells of A. cepa were thus reduced to below 50% of the control value when they were treated with concentrations of WO, KOG and CWB that were below their respective EC 50 s.In each case, a lower concentration of the pesticide was required to achieve a reduction of the MI of treated cells to below 50% of control than to achieve the EC 50 .The MI was thus a more sensitive end point than the EC 50 which was the concentration of pesticide that inhibited the germination of 50% of seeds after 72 h treatment at room temperature.In trial experiments when the EC 50 s of the pesticides were used as the highest concentration in a genotoxicity experiment, the MI was so depressed that not enough cells in division stages were available for observation and scoring.
The results of the present study showed that when the EC 50 value was chosen as the highest concentration for the genotoxicity test, the mitotic index reduced to below 50% of the control, which was in contrast to the observations of Rank and Nielsen (1997).The difference in the results could be because while Rank and Nielsen tested pure compounds (N-methyl-N-nitrosourea, maleichydrazide, sodiumazide and ethylmethanesulfonate), the pesticides tested in the present study were mixtures, and one aspect of pesticide mixtures is the opportunity for complex interactions including synergism or antagonism (Cloyd et al., 2007).No concentration of WO tested was cytotoxic.
Another formulation that contained glyphosate called Roundup UltraMax (450 g/l glyphosate) was however shown to reduce MI at 100, 250 and 500 mg/L in the A. cepa test (Cavuşoğlu et al., 2011) and in tests using Vicia faba (El-Tayeb and Zaki, 2009).The results of the present study thus contradicted those of the studies cited above which could be due to the fact that the highest concentration of the WO tested was 312.5 mg/l which was ¼ of the EC 50 and lower than the top concentration tested in the studies cited above.Another reason for the difference in the results of the present study and those of previous glyphosate in the formulation used in the present study was 360 g/l, lower than the 450 g/l glyphosate in Roundup UltraMax.The two highest concentrations of KOG (312.5, 625.0 mg/l) tested in this study significantly reduced the mitotic index (P < 0.05) and were cytotoxic.Reduction of the mitotic index  following treatment of A. cepa roots with deltamethrin, the active ingredient in the KOG, has been demonstrated previously (Chauhan et al., 1986;Saxena et al., 2009).
Only the highest concentration of CWB significantly reduced the mitotic index (P < 0.05) and was cytotoxic.We are not aware of any study in which Sodium hexafluorosilicate, the active ingredient in CWB, was tested for the effect on mitotic index in a plant system.None of the three concentrations of Snail ban (SB) significantly reduced the mitotic index.Therefore, none of the concentrations of Snail ban tested was cytotoxic.None of the three concentrations of coopex (CPX) that was tested in the present study reduced the mitotic index.The present study provides information on the reduction of mitotic index in a plant genotoxicity test system following treatment with pesticide formulation containing permethrin.Mitotic index is considered a parameter that allows estimates to be made of the frequency of cellular division (Marcano et al., 2004) and inhibition of mitotic activities is often used for tracing cytotoxic substances (Linnainmaa et al., 1978;Smaka-Kincl et al., 1996).A depression of the mitotic index has been recorded by many investigators as a result of treatment with pesticides (Panda and Sahu, 1985;Amer and Farah, 1974).
Treatment of the root-tip meristem cells of A. cepa with WO, CWB, SB or CPX caused cytological abnormalities at one or two concentrations of the pesticide screened in this work.The sticky chromosomes, multipolar and C-mitosis aberrations together constituted between 96 to 100% of the aberrations in all cases.Wipe-out induced genotoxicity at 78.1 and 312.5 mg/l in A. cepa root tip cells.Treatment of A. cepa root meristem cells with another pesticide formulation, Roundup UltraMax that contained 450 g/l glyphosate, induced chromosomal aberrations at 100, 250 and 500 mg/L in the A. cepa test (Cavusoglu et al., 2011).
Roundup treatment also induced chromosome aberrations in Vicia faba (El-Tayeb and Zaki, 2009).However results of mutagenicity and genotoxicity assays of glyphosate itself have been been negative (Weed Science Society of America (WSSA), 1994).No concentration of K-O Gard® tested in the present study induced genotoxicity in A. cepa root tip cells.KOG was not toxic in preliminary toxicity tests even at the limit of solubility.However, treat-ment of garlic cloves/onion bulbs (Saxena et al., 2009) or root meristems of A. cepa (Chauhan et al., 1986;Bhanu et al., 2011) with pure deltamethrin induced mitotic and chromosomal aberrations.The studies of Chauhan et al. (1986) employed deltamethrin concentrations of between 0.05 to 2 ppm that is, 0.05 to 2 mg/l.Only the top two doses (156.3 and 312.5 mg/l) of CWB induced genotoxic effects in the present study with A. cepa root tip cells.However the pure compound, sodium hexafluorosilicate was negative in the Salmonella/microsome test, micronucleus test on mouse bone marrow and sex-linked recessive lethal mutations in Drosophila (Gocke et al., 1981;IARC, 1987a).The induction of genotoxicity by a pesticide formulation containing sodium hexafluorosilicate in the present plant assay system is therefore informative.
Snail ban induced mitotic and chromosomal aberrations at the highest concentration (625.0 mg/l) tested in the present study with A. cepa root tip cells.No mutagenic effect was observed in tests of 98% pure metaldehyde in several strains of Salmonella typhimurium, with or without metabolic activation (IPCS, 1996).According to the EPA ( 2006), the mutagenicity data for metaldehyde are deficient and new data are required, but examination of the old data suggests that metaldehyde is not a mutagen.For carbaryl however, a joint meeting of the Food and Agriculture Organization (FAO) panel of experts on pesticide residues in food and the environment and the WHO core assessment group in 1996 noted that, carbaryl has been evaluated for mutagenicity in a number of tests in vitro and vivo and been found to induce disturbances in the spindle fibre mechanism in plant and mammalian cells in vitro and chromosomal damage in human, rat and hamster cells and in plants treated in vitro with high doses of carbaryl (FAO and WHO, 1996).
Coopex induced mitotic and chromosomal aberrations at the highest concentration of 1250.0 mg/l tested in A. cepa root tip cells.The result of the present study with a plant assay system agreed with another study with an insect assay system where it was found that exposure to Ambush (a permethrin-containing insecticide) during larval development increased sex-linked lethal mutations in fruit flies (Kale et al., 1995).
According to All et al. (1977), Comins (1986) and Cloyd et al. (2007), one aspect of pesticide mixtures is the opportunity for complex interactions including synergism or antagonism.One well known example of synergism or potentiation (enhanced efficacy) is glyphosate, which alone rarely caused genetic damage in laboratory tests and Roundup, a glyphosate product, which demonstrated mutagenicity (Rank et al., 1993).It is therefore not unlikely that some of the differences in the results of the present study compared with results of some of the studies cited above have been caused by synergistic and/or antagonistic interactions in the pesticides mixtures used in the different studies.
Stickiness has been shown to be as a result of DNA condensation (Österberg et al., 1984) and entanglement of inter-chromosomal chromatin fibers which lead to subchromatid connections between chromosomes (Patil and Bhat, 1992).The results of the present study suggested that treatment of A. cepa root tip meristem cells with WO, CWB, SB and CPX induced DNA condensation, chromosome coiling and entanglement of interchromosomal chromatin fibers.Levan (1938) described colchicine mitosis (c-metaphase or c-anaphase) as an inactivation of the spindle followed by a random scattering of the condensed chromosomes in the cell.According to Yildiz and Arikan (2008), a large number of laggard chromosomes and c-anaphases indicate a test compound acted as a potent spindle inhibitor.The induction of vagrant chromosomes according to Elghamery et al. (2003), leads to the separation of unequal number of chromosomes in the daughter nuclei and subsequently formation of daughter cells with unequal sized or irregularly shaped nuclei at interphase.
In the present study, only the treatment with WO induced C-mitosis in the onion root tip meristem cells.The induction of anaphase/telophase bridges has been attributed to chromosome breaks, stickiness and breakage and reunion of the broken ends (Parry et al., 1985;Badr et al., 1992).The presence of the anaphase/ telophase bridges suggested a clastogenic effect of CPX.

Conclusion
The exposure of A. cepa root-tip meristem cells to concentrations below their respective EC 50 s of WO (78.1,156.3,312.5 mg/l); KOG (312.5, 625.0 mg/l) and CWB (312.5 mg/l) resulted in a significant reduction (P < 0.05) in the mitotic index compared with the negative control, which was indicative of mitodepressive effect of the pesticides.The exposure of A. cepa root-tip meristem cells to concentrations of WO (78.1, 312.5 mg/l); CWB (156.3, 312.5 mg/l); SB (625.0) and CPX (1250.0mg/l) caused significant increase (P < 0.05) in cytological abnormalities at one or two concentrations of the pesticides.The following types of chromosome aberrations were recorded in anaphase-telophase cells: S (65% average) > F (5% average) > C-mit (3% average) = multipolar > A.B (1% average) = L.The sticky chromosomes, multipolar and C-mitosis aberrations together constituted between 85 to 100% of the aberrations in each case.The most prevalent type of aberration was the sticky chromosome type which was induced by WO, CWB, SB and CPX.Because most adverse health effects by genotoxic agents are the result of genetic damage and any genetic activity of chemicals is most likely to result from cell division abnormalities, the genotoxic pesticides have potential to cause adverse environmental and health effects.
Photographs of the most representative pictures of normal mitotic cells and cells containing the different types of chromosome aberrations that were observed and scored are presented in Figure1.Six main types of chromosome aberrations were recorded in anaphasetelophase cells: F = fragment; AB = Anaphase or Telophase bridge; L = Laggard; C-Mit = C-Mitosis; S = Sticky chromosomes.The result of the determination of genotoxic effects of the pesticides on onion root tip meristem cells are presented in Table2.WO (78.1, 312.5 mg/l), CWB (156.3, 312.5 mg/l), SB (625.0 mg/l), and CPX (1250.0mg/l) induced genotoxicity (P < 0.05).No concentration of KOG induced genotoxicity.WO induced L, C-Mit, Multipolar and S types.CWB induced F, Multipolar and the S types.SB induced the S types only.CPX induced multipolar and the S type of aberrations.Sticky chromosomes were induced by all the pesticides that induced genotoxicity.This type of aberration was more prevalent and contributed upwards of 60% of the aberrations induction in each case.In Table3are presented the averages of prevalence of the six types of aberrations.The order was; S (65% average) > F (5% average) > C-mit (3% average) = multipolar > AB (1% TC = Test Compound; SD = Standard deviation; WO = Wipe-out; KOG = K-O.Gard; CWB = Cutworm Bait; SB = Snail Ban; CPX = COOPEX; MI = Mitotic Index; Interph = Interphase; Proph = Prophase; Metaph = Metaphase; Anaph = Anaphase; Teloph = Telophase; ‡ = Significant difference from negative control at P < 0.05 in the t-test, n = 3. average) = L.The sticky chromosomes, C-mitosis and multipolar aberrations together constituted between 85 to 100% of the aberrations induced by the individual pesticides.

Table 2
Sticky chromosomes were induced by all the pesticides that induced genotoxicity.This type of aberration was more prevalent and contributed upwards of 60% of the aberrations induction in each case.In Table3are presented the averages of prevalence of the six types of aberrations.The order was; S (65% average) > F (5% average) > C-mit (3% average) = multipolar > AB (1%

Table 1 .
The Cytotoxic effects of pesticides treatments on the different cell cycle stages of Allium cepa.

Table 2 .
Chromosome aberrations induced in root tip cells of Allium cepa following treatment with pesticides.

Table 3 .
Comparative induction of different chromosome aberration types in root tip cells of Allium cepa treated with different pesticides.