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Genotoxicity Assessment of Water Samples from the Sungai Dua River in Penang, Malaysia, Using the Allium cepa


Akeem Akinboro*, 1Kamaruzaman bin Mohammed, 1Selestin a/p Rathnasamy and 1Vijay Raj s/o

1 2

School of Biological Sciences, Universiti Sains Malaysia, 11800 USM Pulau Pinang, Malaysia Department of Pure and Applied Biology, Ladoke Akintola University of Technology, P. M. B. 4000,

Ogbomoso, Nigeria *Corresponding author: Abstract: Unwanted side effects from a polluted water body may not be limited to the flora and fauna, they may also be transferred to the organisms along the food chain. Four water samples collected immediately and five days after rainfall from two locations inside polluted Sungai Dua River (SGD) were tested for toxicity using the Allium cepa assay. The samples were analysed for metal content and were both macroscopically and microscopically evaluated. The water samples contained more Na+ and Ca2+ than the control tap water, and they showed root growth and mitotic inhibitions in A. cepa. However, the inhibitory effects were not dose-dependent. No chromosomal aberration was induced at 100.00% (undiluted water sample). These results suggest the water samples from SGD had weak mitodepressive and genotoxic effects on the A. cepa cells. Keywords: Mitosis, Water Samples, Pollution, Metals, Chromosome Aberration

INTRODUCTION Water is an indispensable natural product that finds its use in virtually all aspects of human life. Thus, there is a great need for ensuring that water used by humans does not contain hazardous substances. The pollution of water bodies is a global problem because of the danger that polluted waters may contain mutagenic and carcinogenic substances that may cause or promote the occurrence of human diseases such as cancer, atherosclerosis, cardiovascular disease and premature aging (Radić et al. 2010). Several tests have used microorganisms, diatoms, plant and mammalian cells, the presence and distribution of aquatic organisms, and chemical analysis to ascertain the quality of water sources (Smaka-Kincl et al. 1996; Wan & Mashhor 2002; Ohe et al. 2003; Zegura et al. 2009). The plant assay Allium cepa L. (2n = 16) has been used extensively to evaluate the cytotoxicity and genotoxicity of water samples (Rank & Nielsen 1998; Monarca et al. 2003; Monarca et al. 2005; Fatima & Ahmad 2006; Radić et al. 2010). This assay is low cost, is easy to use, and produces similar results to animal tests because of similarity in their genetic compositions, hence same response to mutagens. The presence of metacentric chromosomes in A. cepa cells allows easier and better microscopic assessment.


Water sample A2 (WSA2) was collected at location A five days after rainfall on 3 January 2010. who established sources and confirmed the occurrence of pollution in the Pinang river basin. 1996. We hope that our findings will reveal the extent of pollution in this river. there is no known information on the adverse effects from polluted SGD waters on cellular organisms. The sampling site was divided into two locations: A and B. Malaysia. Location A is the part of the river that receives domestic wastewaters from the houses in the area. The samples were labelled as follows. cell division. 2 . cepa. SDG’s course extends to the Hamna area of Gelugor. Tørsløv et al. therefore. Sampling Technique The water samples were collected immediately and five days after rainfall from our chosen locations A and B using a plastic pail tied to a rope. MATERIALS AND METHODS Study Area and Sampling Sites SGD is found in the town of Sungai Dua (Latitude 5. Webber et al. Klöpffer 1996. such as nitrosamines. 1997). Many hazardous organic chemicals have also been detected in wastewaters. polychlorinated dibenzop-dioxins and furans (PCDD/F). Water sample A1 (WSA1) was collected at location A immediately after rainfall on 28 December 2009. The samples were kept separately inside thoroughly washed 5 l plastic bottles. and some of these. Water sample B1 (WSB1) collected at location B immediately after rainfall on 28 December 2009. At each sampling time. Malaysia. and chromosome morphology in A.Sungai Dua in Bahasa Malaysia means two rivers. The Hamna area of Gelugor is one of the residential locations where the SGD extends and where the water samples were collected for this study. 1). An evaluation of water samples for toxicity on cell division and chromosomes in a eukaryotic organism like A. with full knowledge that the produce is being sold to market for consumption. This study.333333°) in eastern Penang Island. The regular pollution of the river through wastewater discharge and refuse by residents poses dangers not only to the fauna and flora in it but also to higher animals. The Sungai Dua River (SGD) is found in the town of the same name in Penang. and polychromatic hydrocarbons (PHA) are known to induce damage to DNA (Connor et al. and location B is the place where farmers along the river take water to irrigate their farmlands (Fig. Rogers 1996. including humans along the food chain. The gravity of the hazards caused by a polluted river increases when heavy metals are the major contaminants. polychlorinated biphenyls (PCB). Apart from the reports of Wan and Mashhor (2002). This is because the river has as its primary producers the plants being watered by this polluted water. the samples were quickly transferred to the laboratory and stored at 4°C for metal analysis and toxicity screenings. seeks to assess the toxic effects of water samples from SGD on root growth.45° Longitude 100. 1992. cepa (onion) will be a useful for predicting likely side effects on humans from agricultural production in farmlands watered by the polluted SGD. Water sample B2 (WSB2) was collected at location B five days after rainfall on 3 January 2010.

A2. The negative control was set up with tap water only. (2n = 16). Allium cepa Test Procurement and preparation of onions Allium cepa L. 50% and 100% (undiluted) concentrations. were analysed for the presence and concentration of sodium (Na+). lead (Pb2+). The onions were sun dried for a week. calcium (Ca2+). The outer scales were carefully removed. The metal standards were prepared to known concentrations (Table 1). Cat No 1001 110) that had been rinsed with distilled water. cadmium (Cd2+). The already fixed root tips were hydrolysed in 1N HCL at 60°C for 5 minutes.25%. Penang. Microscopic evaluation The root tips from the remaining three onions were cut and fixed in ethanol/acetic acid (3:1) inside universal bottles and kept at 4°C for 24 h before use. A2. Thereafter. 1.5%. Excess stains were removed. commonly called onions. and the cover slip’s edges were sealed as suggested by Grant (1982). This method necessitated analysis of the distilled water (DW) for the presence of the above metals to remove any possible influence on the results of the analysed metals in the water samples and controls. the concentrations of the five metals in water samples A1. but with the renewal of the previous water sample after 24 h. Three root tips were squashed on each slide and stained with aceto-orcine for 10 minutes. Qualitative Circles 110 mm Ø. together with controls tap water 1 (TW1) and 2 (TW2). Ten equally sized onions per concentration were suspended on the water sample inside 50 ml beakers and kept in the dark for 48 h. Macroscopic evaluation Each water sample was diluted with tap water to obtain 6. (2009). the root lengths of at least 20 roots from each of 7 onions per concentration were measured using a ruler. The water samples and controls were filtered using Whatman® filter paper (No. and those attacked by fungi were discarded at the beginning of the experiment. The hydrolysed root tips were washed several times with distilled water. 25%. without tampering with the primordial root ring. A total of 5000 cells from 5 slides per concentration were observed (at 1000 x magnification) for different mitotic stages and chromosomal aberrations using a Nikon Eclipse (E400) light microscope. Graphs of the concentrations against the absorbance of each of the standards for the metals were plotted. Malaysia. and zinc (Zn2+) using an atomic absorption spectrometer (AAS) (PerkinElmer A Analyst 100). The absorbance of the standards. B1. water samples and controls was taken in triplicates. The effective concentration at 80% root growth of the control was determined as opposed 50% root growth of the control reported by Akinboro and Bakare (2007) and Yildiz et al.Metal Analysis Water samples A1. and B2. and kept inside plastic bottles that were pre-cleansed with concentrated nitric acid and distilled water. labelled. After 48 h. B1. TW1 and TW2 were interpolated from their respective graphs. were purchased at the Jusco shopping complex. 12. 3 . B2.

The same amount of Ca2+ was detected in WSA2 and B1.0 for Windows.05. which were then subjected to Duncan multiple comparison and Dunetts tests in a one-way ANOVA. However.00% and 100. the inhibition of root growth caused by B1 at 50. Microscopic Evaluation The cytotoxicity and genotoxicity of A1. Zn2+.00% was significantly different from the control (p≤0. Bakare et al.00%) was reduced between the two sampling times. the same were recorded for Ca2+ in A1 and TW2. Significant differences were set at p≤0. 2000). Pb2+. The amount of sodium and calcium ions in the four water samples was higher than that of TW1 and TW2.04 ppm) concentrations of Na+ were recorded for A2 and TW1. but not in a dose-dependent manner (Table 3). RESULTS Metal Analysis Table 2 shows the concentrations of Na+. A2. There was 0. there was promotion of growth above the controls. The three heavy metals were not detected by the AAS in the other samples. The inhibition of root growth by the undiluted water samples (100.0%) > A2 (5. but they were not significantly different.The mitotic index (MI) and the frequency of chromosomal aberrations (CA) was calculated (Fiskesjo 1997. Ca2+. All the water samples reduced the growth of the onion’s roots. The order of toxicity to the root growth. amounting to lower inhibitions. Higher but insignificantly different (from 4 .00 ppm of Zn2+and Cd2+ in A1 and TW2. was B1 (4. and Cd2+ in the water samples. cepa were compared. cell division and chromosomes of A. At 6. based on the values of EC20. The highest (13.50% and 25.00% concentrations of A1 and B2. 12. respectively.05).2%) > B2 (50. using SPSS version 15. with higher inhibitions from A1 and B1.50 ppm) and lowest (4. A2 and B2 induced more root growth. MI = Number of dividing cells Total number of cells counted X 100 Frequency of CA (%) = Number of aberrant cells Total number of cells counted X 100 Statistical Analysis Quantitative data were summarised as means ± standard deviation and percentages.25%. The effects of the water samples and controls on the root growth.2%).0%) > A1 (95. Macroscopic Evaluation The effects of the water samples on the roots suggest levels of toxicity in terms of growth inhibition. B1 and B2 were determined based on their impacts on cell division and chromosome behaviour in the onions.

25.05) MI values were obtained at 6. which could not be obtained due to the poor inhibitory effects of the water samples on the root growth of A. B1 and B2. cepa due to higher concentrations of Na+ compared to the tap water control (Table 2). which consequently leads to the loss of turgor pressure. 2009. The results of macroscopic and microscopic evaluations usually support each other (Akinboro & Bakare 2007). Furthermore. cepa has been observed (Arambasić et al. 25. Carruyo et al. 1995.00% and 50. Samardakiewick & Woźny 2005. WSA1.00% for A1. cytotoxicity.25% (A2). The MI has been used to measure the level of toxic effects that any tested substances have on cell division and chromosomes (Yildiz et al. hence. 2008). B1 and B2 could also be responsible for their poor root growth inhibition.00% (B1). The induction of higher MI 5 .00% and 100. sticky chromosomes. However.00% for A1. 2010). and 6. the lack of root growth inhibition at the lower concentrations of WSA1 and B2 and the non-dose-related inhibition of B1 and A2 suggests that the water samples had weak toxicity. In fact. Lower and significantly different (p≤0. Cd2+ and Zn2+ in WSA1. Because sodium chloride (NaCl) is commonly used domestically. 2). Therefore. rather than EC50. This conclusion is corroborated by the EC20 value recorded for WSA1. thus decreasing cell growth and division (Nilsen & Orcutt 1996). A2 and B2 may have inhibited root growth in A. 2009. cepa. The mechanism of NaCl root growth inhibition may be osmotic stress or salt toxicity. Yildiz et al. the inhibition of root growth indicates the reduction of cell division and. wastewaters from the houses around the SGD may contain Na+. 2008. the tested water samples and controls induced different types of chromosomal aberrations. A2. such as disturbed spindles. 2009). A2. chromosome lag. our metals analysis results established that the WSA2 collected five days after rainfall from the location where the wastewater discharge comes from the residential buildings contained higher amounts of Na+ than the WSA1 collected immediately after rainfall from the same location. However. anaphase bridges and chromosome fragmentation (Fig.00% (B2) as well as 100.control) MI values were recorded at all concentrations except 100. B1 and B2 (Table 4). thus increasing the levels of ions in the water samples. Carruyo et al. (2004) reported the inhibitory effects of NaCl on the root growth of Chrysanthemum morifolium Ramat. red raspberry (Neocleous & Vasilakakis 2007) and four vegetables (Jamil et al. but they were significantly different from the controls at 25. 2006). DISCUSSION The cytotoxic and genotoxic screening of polluted water bodies and other substances using the Allium cepa test has is easy and fast way of elucidating the effects of pollutants and/or contaminants on cell division and chromosomes (Rank & Nielsen 1998. The mitotic activity in the test organism was suppressed at various concentrations of the water samples. and B2. which also implies that the SGD does not contain highly toxic pollutants. Teerarak et al. B1.25% (B2). B1. Na+ from refuse dumped into the SDG is also possible.00% (A1). It is worth noting that the induction of lower MI values by these water samples was not concentration dependent (Table 4).00% for A2 and 50. The implication of some heavy metals in the root growth reduction of A. Radić et al. In the present investigation. The absence of Pb2+. These aberrations were not also dose related. Hossain et al.

29(2): 497–503. Journal of Environmental Biology 21: 263–271.00% of A2 and 50. which contained a high level of calcium (9. Mosuro A A and Osibanjo O. A2. Zinc). (1995). This difference could also be partly due to the low level of calcium ions in B2. The greater observed suppression of mitosis by WSB2 than A1. Lead. (2007). Shahbudin at the Ecology Laboratory of the School of Biological Sciences.00% of A1. Sabrija B and Gordana S. These results are consistent with the assertion that the water in this river did not contain highly toxic substances. However. It is therefore recommended that the authorities responsible for protecting the environment provide proper and adequate places for people to dump their refuse and that they educate the people on the dangers of disposing wastes into flowing rivers.values by WSA1. B1 and B2 at lower concentrations. as the same mitodepressive effect was not seen with A1. as well as the non-concentration-dependent A2 (Table 4).). B1 and B2.70 ppm). phenol and sodium on Allium cepa L. Arambašić M B. B1 and B2. It should be remembered that lead and other heavy metals that have been reported to inhibit root growth and cause mitodepresssion were not found in WSA1.: Comparative investigations and the practical applications. and Daphnia magna St.. Effects of simulated leachate on chromosomes and mitosis in roots of Allium cepa (L.00%. aberrations significantly different from the controls were induced by the water samples at 25. Conclusion and Recommendations The assessments of water samples from the two locations in SGD suggest that the water body did not contain highly toxic pollutants that might have found their way in through the indiscriminate disposal of refuse and wastewater discharge. Nevertheless. Universiti Sains Malaysia. REFERENCES Akinboro A and Bakare A A. supports the macroscopic evaluation results. Wat. It is interesting that there was concordance between the first two results and that of the CA caused by the water samples. (2000). B1 and A2 could be the result of synergistic reactions between Na+. The CAs were not concentration-dependent. for assisting us in determining the metal content of the water samples. This result could also be interpreted as the weak genotoxic action of the water samples because of the unexpected induction of lesser and not significantly different CAs at 100. Acute toxicity of heavy metals (Copper. Journal of Ethnopharmacology 112: 470–475. Ca2+ and other suspended organic matter in the water body. Bakare A A. ACKNOWLEDGEMENT We are thankful to Mr. Res. 6 . Cytotoxic and genotoxic effects of aqueous extracts of five medicinal plants on Allium cepa Linn. our findings do not support the continuation of the pollution of the SGD because of the health implications of these unhygienic habits on the people who consume agricultural produce from the farmlands being watered by this polluted water. Lepidium sativum L.

Effect of NaCl stress on red raspberry (Rubus idaeus L ‘Autumn Bliss’). (1996). 125: 276–285. Draženka. Environmental and Molecular Mutagenesis 41: 353–359. 112: 282–289. Cent.). (2008). Physiology of plant under stress. Boca Raton. Sources. (2010). 308–333. Siniša Š and Branka P K. Claudia Z. Mutation Research 99: 273–291. 185: 3–26. behaviuor and fate of organic contaminants during sewage treatment and in sewage sludges. (2003). (1996). Grant W F. (2005). Mutat. In W Wang. White P A and DeMarini D M. (2003). NaCl stress-its chromotoxic effects and antioxidant behavior in roots of Chrysanthemum morifolium Ramat. Yusmary F. Zani C. New York: CRC Lewis Publisher. Rogers H R. Mutation Research 609: 81–91. Fiskesjo G. PB92–153931. Sci. (1982). Environmental hazard assessment of chemicals and products: Part V. evaluation of cytologic parameters. S. (2006).Carruyo I. Mandal A K A. Plant Sci. (1996). Biol. Total Environ. Jamil M. 7: 273– 282. Bioavailability to plants of sludge-borne toxic organics.. Jung K Y. Ashraf M. Donatella F. Rank J and Nielsen M H. New York: John Wiley & Sons Inc. Valerija V. Silvia C and Bianca G. Effect of salt (NaCl) stress on germination and early seedling growth of four vegetables species. Agric. Plants for environmental studies. J W Gorsuch and J S Hughes (eds. (1998). Res. 166: 215–220. Hossain Z. Science of the Total Environment 408: 1228–1233. Correlation of toxicity with lead content in root tip cells (Allium cepa L. Chromosome aberrations assays in allium report of the USEPA gene tox program. J. Shukla R and Datta S K. Marco R. 7 . Rizzoni M. Genotoxicity of industrial wastewaters obtained from two different pollution sources in northern India: A comparison of three bioassays. Feretti D and Zerbini I. Anthropogenic chemicals in sewage sludge. Letty M. Chemosphere 3(6): 1067–1081. Xiomara M and Zaida T. Res. Connor O G A. Genet Toxicol Environ Mutagen 534: 101–112. (2006). Genotoxicity testing of wastewater sludge using the Allium cepa anaphase-telophase chromosome aberration assay. Chaney R L and Ryan J A. The evaluation of surface and wastewater genotoxicity using the Allium cepa test. Ohe T. Nilsen E T and Orcutt D M. Eur. Lee D B. (1992). Cincinnati. Mutation Research 418: 113–119.). (2007). Gustavino B. Lee S C and Rha E S. OH: USEPA. Radić S. (1997). Trace Elem. Marija M R. Hortic. Alberti A. Monarca S. Mutagenic characteristics of river waters flowing through large metropolitan areas in North America. Genotoxicity of surface water treated with different disinfectants using in situ plant tests. Monarca S. Environmental and Molecular Mutagenesis 46: 96– 103. Allium test for screening chemicals. Fatima R A and Masood A. Klöpffer W. (2004). Genotoxicity of drinking water disinfectants in plant Bioassays. Sci. Neocleous D and Vasilakakis M.

0 8. Ibrahim H C. Combination of in vitro bioassays for the determination of cytotoxic and genotoxic potential of wastewater. Rasmussen J O and Kristensen P. (2005).0 0. Sompop T and Chamroon L. non-analytical methods. (2002). Total Environ. (1997). Černoša A and Filipič M. Monitoring and prioritisation of organic contaminants in sewage treatment and in sewage sludges using specific chemical analysis and predictive. Table 1: Concentrations (ppm) of standards for the analysed metals.0 1. Wan M W O and Mashhor M. Malaysia. Aquatic pollution assessment based on attached diatom communities in the Pinang River Basin. (2009). Tørsløv J. Mutation Research 368: 171–179.6 8 . environmental risk assessment and recommendations for quality criteria.0 1. Kisana B.0 0. Scientia Horticulture 121: 228–232. Use of waste products in agriculture. Contamination level.0 3. Denmark: Danish Environmental Protection Agency. (1996). Heath E. Determination of genotoxic effects of copper sulphate and cobalt chloride in Allium cepa root cells by chromosome aberration and comet assays.0 4. Environmental Project No 366. Samsøe-Petersen L. Metal Sodium (Na+) Calcium (Ca2+) Lead (Pb2+) Zinc (Zn2+) Cadmium (Ca2+) 2.0 0.9 1. Watts C D. cell division. (2009).2 8. Muhsin K. Davs R D and Scoffin R. Hydrobiologia 487: 229–241. Lovka M and Toman M J. The evaluation of waste.8 6.0 4. surface water and drinking water samples. Žegura B. (1996). Yildiz M.0 2. The impact of sodium chloride on root growth. Boxall A B A.4 Concentration (ppm) 4.6 0. (2009). Sci. Chemosphere 75: 934–938.2 1.Samardakiewicz S and Woźny A.3 0. Smaka-Kincl V. and interphase silver-stained nucleolar organizer region (AgNORs) in root tip cells of Allium cepa L. Chemosphere 75: 1453–1460. Fatih A F and Hakan T. 185: 27–44.0 2. Stegnar P. Cell division in Lemna minor roots treated with lead. Webber M D. Teerarak M.0 6. Rogers H R. Aquatic Botany 83: 289–295. surface and ground water quality using the Allium cepa test procedure.

70 9.65 6. non detectable.Table 2: Concentrations (ppm.00 ND ND ND ND ND ND ND ND ND ND 0.00 ND ND ND ND Na+ Ca2+ Zn2+ Pb2+ Cd2+ Note: ND.50 10.75 7.75 ND ND 0.04 4.69 6.13 9.13 7. Metal Water sample TW1 TW2 A1 A2 B1 B2 TW1 TW2 A1 A2 B1 B2 TW1 TW2 A1 A2 B1 B2 TW1 TW2 A1 A2 B1 B2 TW1 TW2 A1 A2 B1 B2 Concentration (ppm.) of metals detected in the water samples and controls.82 12. 9 .24 9.) 4.50 13.

00 0.41 79.84 87.50 25.08 85.00 0.75 1.52 53.00 129.00 1.22 4. (%) AV.00 0.70 1.00 50.00 100. concentration.52 123.00 69.83 ± 0.71 1.25 4.76* 100.58 ± 0..48 46. * Values are significantly different from control (p≤0.83 ± 0..58 0.01* 1.20 ± 0.92 1.42 95.81 113.84 50.14 18.98 3. SD.65 1.00 74.65 79. ± SD root length (cm) A2 Root growth (%) Root growth inhibitio n (%) 0.61 1. AV.00 3.16 12.68 ± 0. ± SD root length (cm) B2 Root growth (%) Root growth inhibitio n (%) 0. 10 .25 1.86 81.60 3.00 18.28 1.46 ± 0.49 ± 0.37 ± 0.00 0.21 ± 0.78 95. TW..Table 3: Effects of the water samples and control on root growth of A.92 Notes: Conc.37 1.05).42 81.00 30. average.38 29.85 2.56 ± 0.10 ± 0.08 AV.58 18.00) 6.65 98.16 49.58 4.25 12. cepa.56 ± 1.59 20.06 81.00 20.25 2.33 AV..08 ± 0.05 Root growth inhibitio n (%) 0.67 1.49 ± 0.49 2. ± SD root length (cm) B1 Root growth (%) Root growth inhibitio n (%) 0.76 ± 1.03 2.97 ± 0.90 ± 1.25 AV.92 14.52 1.64 2.54 100.00 25.62 70.42 107.61 100.15 ± 0.95 3. ± SD root length (cm) A1 Root growth (%) TW (0.49 ± 0.74 ± 0. standard deviation.00 130.74 ± 0.38 ± 0.49 ± 0. Conc.15 ± 0.31 3.39 100.49 1. tap water.

.28 ± 0.10 0..40 1.21 3.22 MI ± SD 2.32 ± 0.24 ± 0.14 0.22 ± 0.14 0.09 MI ± SD 3.40 ± 0.23 1.62 2.19 ± 0.14 ± 0.21 3.36 ± 0.40 ± 0.50* A1 CA ± SD 0.08 ± 0. * Values are significantly different from control (p≤0.37 ± 0.44 ± 0.74 ± 0.20 ± 0.30 2.84 ± 0.70 2.16 ± 0.22 ± 0.98 ± 0. Conc.00 ± 0.20 ± 0. cepa cells.00 50.56 1.48 3.10 MI ± SD 3.11 ± 0.00 2. 11 .88 ± 0.72 1.19 ± 0.05 0.07 0.00 100.Table 4: MI and frequency of CA induced by the water samples and control in A.40 ± 0.23* 1.15 2.18 ± 0.24* B1 CA ± SD 0.36 ± 0.08 ± 0.12* 0.46 ± 0.75 2. CA.40 ± 0.34 ± 0.22 ± 0.05 0.68 ± 0.50 ± 0.26* 0.25 A2 CA ± SD 0.52 2.56 3.19* 0. chromosomal aberration.26 ± 0.17 0.22 ± 0.32 ± 0.08 0.14 ± 0.10 0.50 25.24* 0.09 0.65 ± 0.04 0.25 12.43* 1.34 ± 0.54 ± 0.10 0.24 ± 0.06 ± 0. (%) MI ± SD TW 6.10 0.15 0.37* B2 CA ± SD 0.14 0.84 ± 0.62 ± 0.36 2.50* 2.04 Notes: MI.05).10 0.56 1.45 2.92 ± 0.26 ± 0. mitotic index.60 ± 0.11 ± 0.

Figure 1: 12 .

c b a d e g f h i Figure 2: 13 .