Cytogenetic Effects of Two Synthetic Pesticides on Mitotic Chromosome on Root Tip Cells of Allium Cepa

Cytogenetic Effects of Two Synthetic Pesticides on Mitotic Chromosome on Root Tip Cells of Allium cepa

Mona S. Al-Ahmadi*

Department of Biology, Science College of Girls, Dammam University, Dammam, Saudi Arabia

Received June 18, 2012; accepted October 16, 2012

Abstract In this study, two agricultural synthetic insecticides were examined for their genotoxic and cytotoxic effects on root tip cells of Allium cepa, namely, Emar non-systemic Insecticide, which is used to eliminate worms on rodents, and Confidor Systemic insecticide that targets sucking insects.Two concentrations (0.2 and 0.1 ml/L) of Emar and one concentration (0.15 ml/L) of Confidor were used for four different time periods (8, 12, 16 and 24 h); tested dosages were assessed for genotoxic and cytotoxic effects to mitotic chromosomes of Allium cepa root tip cells. The results indicate that both insecticides were genotoxic, as they decrease the mitotic index and increase chromosomal aberration. The observed effects were significant after treatment with 0.1 ml/L and 0.2 ml/L of Emare; also, treament with 0.15 ml/L of Confidor was significant at p>0.05 and cytotoxic, especially after treatment for a long time (16, 24 h). The types of chromosome aberration observed were stickiness, distributed, C-metaphase, C-anaphase, lagging chromosome, binucleate cell and bridge.

Key words Cytogenetic, Synthetic pesticides, Mitotic chromosome, Allium cepa root tip.

The use of pesticides to control plant diseases and pests has been steadily increasing at an an- nual rate of about 14% since the mid 1950s; their application remains the most effective means for protecting plants from pests, especially crop yields; the most widely used pesticides are insecticides to control diseases of the foliage and of other above ground parts of plants (Agrios 1988). The us- ages of pesticides have increased many folds and have dominated the field of disease control with little consideration for their harmful side effects on plants, animals and humans (Ajay and Sarbhoy 1987). The mutagenic and carcinogenic actions of herbicides, insecticides, and fungicides on exper- imental animals is well known and several studies have shown that chronic exposure to low levels of pesticides can cause mutations and/or carcinogenicity (IARC 1990, 1991, Yu 2005, Bull et al. 2006). The genotoxicity of pesticides has been demonstrated in many plant systems and several re- ports have shown that many of the chemicals used in pest control affect cell division, induce chromosomal aberration and decrease cell division. In this study, two insecticides are used as an agricultural chemical to control plants pests, Emar non-systemic Insecticide to eliminate worms and rodents (the active ingredient is Emamectine Benzoate) and Confidor Systemic insecticide which targets sucking insects (the active ingredient is Imidacloprid). The Allium cepa assay is an efficient test for chemical screening for genotoxicity of environmental contaminants and has been widely used to study the genotoxicity of many pesticides reveal- ing that these compounds can induce chromosomal aberrations in root meristems of Allium cepa (Ma et al. 1994, Fernandes et al. 2007)

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.78.3 4 M. S. Al-Ahmadi Cytologia 78(1)

Materials and methods

Allium cepa bulbs obtained from local markets were used for testing geotoxicity and cytotox- ity of the two insecticides used. The loss outer scales and old roots were scraped to expose the root primordia to two concentrations, (0.2 ml/L), (0.1 ml/L) of Emar and one concentration (0.15 ml/L) of Confidor 8, 12, 16, 24 h. The concentration of each substance was freshly prepared in distilled water. The bulbs were germinated until the roots reach 2–3 cm long then were detached, fixed in freshly prepared 3:1 (v/v) alcohol–glacial acetic acid for 24 h. For Cytological preparations, root tips of Allium cepa were hy- drolyzed in 1 N HCL 60 ml for 8 min, roots tips were then washed with distilled water several times and staind with 1% acetocarmin, five temporary slides were prepared using the squash tech- nique, two root tips on each slide were examined for the effects of the Emar and Confidor on mitotic index (MI) . The same slides were analysed for the types and frequencies of chromosomal abnormalities produced by the examined insectcides.

Scoring of slides and data analysis The slides were viewed under a light microscope (Phenix P H 50 DB047 VU) using the 40X objective lens immersion. The most representative examples for each structural aberration were photographed using a Phenix Micro Image analyzer Software 2008 En V2, 2.

Mitotic index On one slide for each treatment, a total of 2,000 cells, were scored. The mitotic index (MI) was expressed as the number of dividing cells per total cells scored.

Cytotoxicity The mitotic index of the treated cells at each dose of each insecticide was compared with that of the negative control group.

Genotoxicity test Chromosomal aberration per dose of each insecticide was examined; the percentage of cells with aberrations of each dose for each insecticide were compared with that of the negative control using the SPSS 16.0 for Windows statistical package. Two-way analysis of variance was the statistical method used for determining the signiﬁcance of difference at p = 0.05.

Results and discussion

Effect on mitotic index (MI) and aberration frequency (AF) Table 1 shows the result of tested dosages of Emar and Confidor for different time periods. Both insecticides were observed to affect the MI and AF. The increase of dosages combined with the increase of treatment time result in decreasing (MI) and it was significant after treatment with 0.1 ml/L and 0.2 ml/L of Emare, also treament with 0.15 ml/L of Confidor was significant at p>0.05. The treatment causes increasing the (AF) compared with the control (root tip treated with distilled water only) and was significant after treatment with 0.1 ml/L of Emare and 0.2 ml/L at p>0.05 also treament with 0.15 ml/L of Confidor (Bu et al. 2011).

Type of chromosome abnormalities Table 2 show the types of chromsomal aberration observed after treatement with different concentrations for different time periods. 2013 Cytogenetic Effects of Two Synthetic Pesticides on Mitotic Chromosome on Root Tip cells of Allium cepa 5

Table 1. Total number of examind cells, mitotic index and aberration frequancy after treatment with different concentration of the tested insecticids for different period of time

Type of Total Total number Name of Test Total cells Mitotic index Aberration treatment and number of of abnormal pesticide concentration observed % frequancy duration (h) divided cell cell

8 Distilled water 2215 219 5 9.9 0.02

12 Distilled water 2107 204 6 9.7 0.03 Control 16 Distilled water 2391 265 4 11.1 0.02

24 Distilled water 2123 244 1 11.5 0.004

8 0.1 2061 193 27 9.4 0.14

12 0.1 2010 199 28 9.9 0.14

16 0.1 2102 190 35 9 0.18

24 0.1 2063 184 22 8.9 0.12 Emar 8 0.2 2081 175 32 8.4 0.18

12 0.2 2079 151 40 7.3 0.27

16 0.2 2121 267 61 12.6 0.23

24 0.2 2252 159 30 7.06 0.19

8 0.15 2096 140 19 6.68 0.14

12 0.15 2043 144 25 7.05 0.17 Conﬁdor 16 0.15 2054 139 30 6.77 0.22

24 0.15 2134 145 18 6.79 0.12

The treatment causes several types of chromosomal aberration compared with control, though the aberration freuency of chromosomal aberration decreases after treatment with 0.1 ml/L and 0.2 ml/L for 24 h; this may be due to the lower number of divided cells after treatment with Emar insecticide. The chromosomal aberrations observed were: stickineses which is a physiological effect involving alternation in the chromosomal proteins (Signa and Vanakuma 1994), and is also a result of entanglement on interchromosomal chromatin fibers, and leads to subchromatid connec- tions between chromosomes (Mehmet et al. 1994); disturbed chromosomes, C-metaphase, C- anaphase due to the inhibition of spindle formation because of the cytotoxic substances that are in- hibiting mitosis by affecting the microtubule configuration (Armbruster et al. 1991, Kaymak and Pinargoc 2009) in addition, lagging chromosomes were observed in high frequency after treatment with Confidor which indicates the ability of Confidor to inhibit spindles (Baeshen et al. 1999). Other types of chromosomal aberration were anaphase bridges at low frequency compared with control and chromsomal bridges formed because of the fusion of stiky ends of separating chroma- tids (Signa and Vanakuma 1994). Although star anaphase was also observed, this can also be a result of the failure of the spindle apparatus to organize and function in a normal way (Ozlem et al. 2008). Binucleated cells reveal the inhibition of cell wall formation between cells (El-Abidin et al. 1993). 6 M. S. Al-Ahmadi Cytologia 78(1) 24 2.07 2.07 0.69 1.38 2.07 0.69 0.69 1.38 0.69 0.69 11.73 16 3.6 5.04 7.91 0.72 0.72 17,9 Confidor 0.15 ml/L 12 2.77 2.77 6.94 0.69 0.69 3.47 17.3 8 0.04 0.71 0.71 4.29 2.14 0.71 0.71 13.6 24 0.02 0.04 0.01 0.02 0.05 0.03 0.03 20 16 0.03 0.06 0.003 0.01 0.007 0.01 0.004 0.08 0.03 0.03 0.68 23 0.2 ml/L 12 0.05 0.1 0.02 0.02 0.07 0.01 26.17 8 0.05 0.006 0.02 0.006 0.03 0.005 0.03 18 Emar 24 0.02 0.03 0.005 0.005 0.01 0.005 0.04 11.5 16 0.005 0.05 0.005 0.01 0.04 0.02 0.005 0.02 0.02 0.005 0.005 0.01 18.4 0.1 ml/L 12 0.01 0.05 0.01 0.04 0.005 0.03 0.005 0.005 14.1 8 after treatment with different concentration of the tested insecticids for period time 0.05 0.04 0.005 0.04 0.005 0.005 14 24 Allium cepa 0.004 0.4 16 0.01 1.6 Control 12 0.01 0.02 0.01 3 Distilled water 8 0.009 0.009 0.005 2.3

(h) Pesticide and duration Concentration Sticky Distirb Lagging C-Metaphase S-Metaphase Sticky Distirb Lagging Colch Bridg Polar S-Metaphase Lagging C-Anaphase Distirb Lagging Colch Table 2. Type of chromosomal aberration on root tip cells Metaphase Anaphase Telophase Nucli AF% 2013 Cytogenetic Effects of Two Synthetic Pesticides on Mitotic Chromosome on Root Tip cells of Allium cepa 7

Figs. 1–10. Type of chromosomal aberration. 1 and 4. Bridge at anaphase. 2. Binucheated cell. 3 and 10. C-Metaphase. 5 and 6. Lagging chromosome at anaphase. 7. Disturbed metaphase. 8. Disturbed anaphase. 9. Star metaphase.

Conclusion

Emare and Conﬁdor are synthetic insecticides and in this study the tested dosages indicate genotoxic ability to affect the root tips of Allium cepa, inducing several types of chromosomal aberration (stickiness, disturbance, C-metaphase, C-anaphase, lagging chromosome, binucleated cell) because the effect of the two insecticides on the spindles and coiling of chromosomes. The results did not indicate, any cytotoxic ability, since cells were in telophase stage except for those tested with high dosages for 24 h; here there was a decrease in the level of cell division due to the effect on cell division. 8 M. S. Al-Ahmadi Cytologia 78(1)

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