Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(1): 55-63, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1067 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Anup Kumar Sarkar, Ranita Saha, Rupak Halder (2022) Chromosomes damage by sewage water studies in the Allium cepa L. and Zea mays L.. Caryologia 75(1): 55-63. doi: 10.36253/ caryologia-1067 Received: August 29, 2020 Accepted: March 20, 2022 Published: July 6, 2022 Copyright: © 2022 Anup Kumar Sarkar, Ranita Saha, Rupak Halder. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID AKS: 0000-0002-6777-740X Chromosomes damage by sewage water studies in the Allium cepa L. and Zea mays L. Anup Kumar Sarkar*, Ranita Saha, Rupak Halder Department of Botany, West Bengal State University, Berunanpukuria, Malikapur, North 24 Parganas, Barasat, Kolkata–700126, West Bengal, India *Corresponding author. E-mail: ak94sarkar@yahoo.com Abstract. The effect of sewage water sample of the three locations Khardah (22.7003° N, 88.3753° E), Titagarh (22.7383° N, 88.3737° E), Madhyamgram (22.6924° N, 88.4653° E), and Control (distilled H2O) in the district of North 24 Parganas (22.6168° N, 88.4029° E), West Bengal, India on the damage of chromosomes in the onion (Alli- um cepa L.) and maize plant (Zea mays L.) were investigated by employing mitotic chromosomal aberration assay. Physiochemical analysis of sewage water samples showed the pH is 5.10-5.30 in nature. Few heavy elements: Fe, Mn and Zn in the sam- ple from Khardah (22.7003° N, 88.3753° E) sewage water exceeded the Indian Stand- ard 10500:2012 and WHO’s (2006) permissible limits. Whereas Cl, Cu, Pb, Cr, and Cd are more or less within limit of the standard condition. The obtained data exhibited a decline in reproductive capacity of cells and the occurrence of deviation from the normal mitotic cell division. The mitotic index (MI) decreased significantly (p < 0.05) in both the cases and is given as Control (57.03 %) > Madhyamgram (41.70 %) > Tita- garh (33.85 %) > Khardah (31.57 %) in Allium cepa L. and Control (49.33 %) > Tita- garh (21.45 %) > Madhyamgram (26.47 %) > Khardah (24.05 %) in Zea mays L. The chromosomal aberrations (CAs): Karyorrhexis, Karyolysis, Fragments, Lagging chro- mosome, Anaphase bridges are present in significant amount in the crops treated with sewage water sample than the one with control condition. Heavy metals act as pollut- ants in the sewage water sample which has cytotoxic effect on cells, threat to water eco- system and human health. Keywords: sewage water, mitotic index, indian standard, heavy metal, cytotoxic, eco- system. INTRODUCTION Rapid industrialization in the last four decades has resulted in the mush- rooming of production units even in the vicinity of semiurban and rural areas of the country. Several hazardous chemical industries discharge their untreated effluents into the atmosphere. Water and soil along with the eco- environmental profile of the area are adversely disturbed. The endemic expo- sure to pollutants causes toxicity, morbidity, early mortality, genetic and cytogenetic damage and various other pathological symptoms in the exposed human and plant populations. 56 Anup Kumar Sarkar, Ranita Saha, Rupak Halder This study was undertaken to evaluate the cyto- toxic effects of effluents in sewage water of the three locations Khardah (22.7003° N, 88.3753° E), Titagarh (22.7383° N, 88.3737° E), and Madhyamgram (22.6924° N, 88.4653° E) of North 24 Parganas (22.6168° N, 88.4029° E), West Bengal, India. For more than forty years officially accepted “Allium test” is used widely for assessment of the environmental water pollution (Fiskesjo 1985, 1997; Ivanova et al. 2002, 2005; Rank 2003). Various investigators (Al-Sabti 1989; Smaka- Kink l et al. 1996; Rank and Nielsen, 1998; Moraes and Jordao, 2001) advocate different plant test systems which are useful for studying cy totoxicity of heav y metals. Currently, the physio-chemical and cyto-tox- icological evaluation of sewage water discharges from the three locations (T1, T2, and T3) by different ways has not been documented, thereby no information on their hazardous effect on agricultural field and the ecosystem is recorded. With this background, the pre- sent work was undertaken to investigate chromosomal damage (cytotoxic) impact of sewage effluents collected from three different locations on root tip meristematic cells of Allium cepa L. and Zea mays L. with special reference to analysis of physio-chemical parameters of the liquid waste. MATERIALS AND METHODS Bluish and blackish sewage water was collected from the three main drains of T1 = Khardah (22.7003° N, 88.3753° E), T2 = Titagarh (22.7383° N, 88.3737° E), and T3 = Madhyamgram (22.6924° N, 88.4653° E), of North 24 Parganas (22.6168° N, 88.4029° E), West Ben- gal, India at the depth of six inches from three random points within the drain of each location. The sewage water samples were filtered four times by muslin cloth and then stored in a clean plastic jar for chemicals anal- ysis and setting experiment along with distilled water as a control (T4) on two species namely, Allium cepa L. and Zea mays L. Physicochemical parameters were analyzed from the three locations’ (T1, T2, and T3) sewage water sam- ples (filtered four times) for a standard physicochemi- cal property (chloride) according to IS:3025 (Part 32): 1988, RA 2003. The eight heavy metals, i.e., Copper (Cu), Chromium (Cr), Nickel (Ni), Iron (Fe), Zink (Zn), Cadmium (Cd), Lead (Pb), Manganese (Mn) were deter- mined in mg/l, following the methods described in APHA 22nd edition 3125B and WHO-2006 limits (Olo- runfemi et al. 2014). Chromosome preparation was made from the treated root tips of both the species Chromosome preparation was performed in both the species following the protocol adapted by Sharma and Sharma (1980). The root tips of treated and control sets of both species were fixed in Carnoy’s fluid-I for overnight followed by treatment with 45% acetic acid for 10 minutes at room temperature. The resultant root sam- ples were stained for 45 minutes with a mixture of 2% Aceto-Orcein:1N HCl (9:1) and warmed lightly at 60°C. The meristematic tip portion (~ 1mm size) of onion and maize roots were cut and placed on a clean grease free slide in a drop of acetic acid (45%) and squashed, later temporarily sealed with paraffin wax. Slides were pre- pared from five randomly drawn root tips from each treatment of both the species. Five random microscopic fields from each slide were scored under Olympus with the Prog-Res Capture Pro 2.1 photo system. The mitotic indices were calculated for all the treated materials of each treatment. Statistical Analysis The experiment was organized according to a rand- omized complete design (RCD) with three replications. A two-way ANOVA was performed for test of signifi- cance at p<0.05, employing F-test. Data were expressed as mean ± standard error (SEM) (Gomez and Gomez 1984). The mean mitotic index of each treatment was compared with those corresponding to control employ- ing “t” test for significant difference, if any. RESULTS Heavy metals and chloride determination in the sewage water samples The heavy metal and chloride analysis of the sew- age water sample of the three locations have been shown in the Table 1. The sewage water collected from differ- ent locations were acidic in nature on pH scale: 5.30 (T1), 5.15 (T2) and 5.10 (T3) during the middle of February, 2017.The sewage water sample from the three locations attained a higher range of iron concentration i.e., 0.34- 0.52 mg/l as compared to the limit of 0.001-0.30 mg/l. The contents of copper, chromium, cadmium and lead were found less than the permissible limit (0.001 mg/l) in the sample of T1, T2, and T3 treatments, except cop- per (0.006 mg/l) in T3 treatment within limit compared with National (APHA 22nd edition 3125 B and IS: 10500: 57Chromosomes damage by sewage water studies in the Allium cepa L. and Zea mays L. 2012) and WHO’s (2006) standards. Th e result revealed that the concentration of manganese (0.321 mg/l) con- tent is higher in the sample of Khardah (22.7003° N, 88.3753° E) location while it was found in the range of limit (0.001-0.03 mg/l) in the sewage water sample of Titagarh (22.7383° N, 88.3737° E), and Madhyamgram (22.6924° N, 88.4653° E) respectively. General toxicity-root growth inhibition and deformity of Allium cepa L. & Zea mays L. test Th ere was a signifi cant (p< 0.05) root growth inhi- bition of the two species in the wastewater samples of three locations compared with distilled water (Figure 1). Root length in distilled water was higher than that in wastewater samples for both of them (Figure 2). Sew- age water trials were compared with control treatment (distilled water). Th e mitotic index (MI) signifyingly decreased along with an increase in chromosomal aber- rations (CAs) of the root tips meristematic cells of onion and maize were found (Table 2, Figure 5). Table 1. Contents of Heavy metals, Chloride and pH in the experimental sewage water samples (T1, T2 and T3). Sl. No. Diff erent parameters in the water samples of the three locations Limit Requirements As per IS 10500: 2012. Maximum Result of the three locations or treatments (T1, T2, & T3) WHO (2006) Limit T1=Khardah (22.70030 N, 88.37530 E) T2=Titagarh (22.73830 N, 88.37370 E) T3=Madhyamgram (22.69240 N, 88.46530 E) 1 Copper (Cu)mg/l 0.001 Max:1.5 < 0.001 < 0.001 0.006 - 2 Chromium (Cr)mg/l 0.001 Max: 0.05 < 0.001 < 0.001 < 0.001 0.05 3 Nickel (Ni) mg/l 0.001 Max: 0.02 0.006 < 0.001 0.002 0.02 4 Iron (Fe) mg/l 0.001 Max: 0.30 0.340 0.501 0.520 - 5 Zink (Zn)mg/l 0.001 Max: 15.0 0.450 < 0.001 0.002 0.01 6 Cadmium (Cd)mg/l 0.001 Max: 0.003 < 0.001 < 0.001 <0.001 0.003 7 Lead (Pb) mg/l 0.001 Max: 0.01 < 0.001 < 0.001 < 0.001 0.01 8 Manganese (Mn) mg/l 0.001 Max: 0.30 0.321 0.275 0.101 - 9 Chloride (Cl)(mg/l) N/A Max: 1000 89.19 79.55 269.98 - 10 pH - - 5.30 5.15 5.10 6.5-9.5 11 Colour - - Bluish Blackish Blackish - Contents of heavy metals & chloride present in the three experimental fi elds (T1, T2, & T3) done by efrac (Edward Food Research & Analy- sis Centre Limited, Subash Nagar, P.O. Nilgunj Bazar, Barasat, Kolkata-700121, India. Email: efraclab@cfrac.org, Ph. No.91-3371122800. Figure 1. Eff ect of Sewage water and control samples (T1, T2, T3, and T4) on Germination % and Disinhibition root length % in Alli- um cepa L. and Zea mays L. Figure 2. Percentage root growth of Allium cepa L. and Zea mays L. roots exposed to the test Sewage water and control samples (T1, T2, T3 and T4). 58 Anup Kumar Sarkar, Ranita Saha, Rupak Halder Allium cepa L. test with sewage water samples Allium cepa L. meristematic cells in the root tips after 72 hours exposure to the different sewage water treatments exhibited various chromosomal aberra- tions (CAs) in comparison to distilled water (control) that included karyorrhexis, karyolysis, fragmenta- tion, laggard, and anaphase bridge (Figure 3). T1 has significantly decreased anaphase bridge (5.60 ± 1.27), laggard chromosome (5.60 ± 1.27), mitotic index (31.57%). T2 has extensively decreased fragmentation (2.00 ± 0.79) only. T3 has significantly decreased kar- yorrhexis (22.00 ± 1.76), karyolysis (19.80 ± 7.09) and aberration frequency (8.09%). The activities of differ- ent types of abnormal cells and aberration frequency % were seen to have a higher value in all samples (T1, T2, and T3) as compared to control (T4) in the onion (Table 2). Zea mays L. test with sewage water samples Chromosomal aberrations (CAs) induced in Zea mays L. root tips meristematic cells after 72 hours exposure to different waste water treatments (T1, T2, and T3) in comparison with distilled water (T4) were summarized in Figure 4. The treatment T1 has significantly decreased only mitotic index (24.05%). T2 has considerably decreased laggard chromosome (0.60 ± 0.61) and anaphase bridge (0.60 ± 0.61). T3 has drastically decreased karyorrhexis (53.60 ±16.32), fragmentation (2.80 ± 1.22) and aberration frequen- cy (10.13%). No effect on karyolysis was found in T1, T2 and T3 samples. The activities of different types of abnormal cells and aberration frequency % was observed to have higher values in all sewage water samples (T1, T2, and T3) as compared to control (T4) in the maize (Table 2). Different types of abnormal cells The aberration shown at level of nucleus is of two types: (i) Karyorrhexis and Karylysis (ii) Chromo- somes. Karyorrhexis It is the manner of destructive fragmentation of the nucleus of dying cells where the chromatin is irregularly distributed throughout the cytoplasm (Figure 3A, E, I, and Figure 4A, D, F). Ta bl e 2. F re qu en ci es o f d iff er en t t yp es o f c el ls a fte r tr ea tm en t w ith d iff er en t w at er s am pl es ( T 1, T 2, T 3 a nd T 4) M ea n ± SE & A N O VA in b ot h sp ec ie s. Species L oc at io ns To ta l C el ls D iv id in g C el ls C el ls o f di ff er en t s ta ge s A bn or m al c el ls M ito tic In de x (% ) A be rr at io n Fr eq ue nc y (% ) Pr op ha se M et ap ha se A na ph as e Te lo ph as e K ar yo rr he xi s K ar yo ly si s Fr ag m en te d L ag ga rd A na ph as e br id ge Allium cepa L. K ha rd ah 60 8. 80 ± 13 1. 53 19 2. 20 ± 49 .4 6 17 3. 20 ± 50 .2 6 7. 40 ± 1. 69 8. 00 ± 1. 76 3. 60 ± 1. 86 29 .2 0± 5 .4 1 30 .4 0± 5 .9 7 3. 40 ± 0. 99 5. 60 ± 1. 27 5. 60 ± 1. 27 31 .5 7 11 .2 7 T ita ga rh 58 2. 60 ± 46 .4 4 19 7. 20 ± 57 .7 1 17 4. 80 ± 59 .9 0 6. 80 ± 2. 14 12 .0 0± 3 .0 5 3. 60 ± 1. 69 65 .4 0± 1 8. 11 29 .0 0± 9 .5 7 2. 00 ± 0. 79 8. 60 ± 2. 17 8. 60 ± 2. 17 33 .8 5 18 .0 2 M ad hy am gr am 62 0. 20 ± 90 .7 0 25 8. 60 ± 44 .1 7 23 8. 40 ± 39 .2 3 10 .6 0± 3 .2 0 6. 80 ± 2. 88 2. 80 ± 1. 83 22 .0 0± 1 .7 6 19 .8 0± 7 .0 9 2. 60 ± 1. 69 5. 80 ± 2. 77 5. 80 ± 2. 77 41 .7 0 8. 09 D is til le d w at er 6 78 .2 0± 6 1. 68 38 6. 80 ± 3 3. 06 35 7. 00 ± 2 7. 86 17 .6 0 ± 8. 92 7. 6± 1 .8 6 4. 60 ± 2. 68 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 57 .0 3 0. 00 A N O V A N S ** ** ** ** N S ** Zea mays L. K ha rd ah 59 0. 40 ± 11 4. 33 14 2. 00 ± 34 .6 3 13 0. 80 ± 33 .3 3 9. 60 ± 2. 56 1. 40 ± 1. 69 0. 40 ± 0. 61 90 .8 0± 2 4. 85 0. 00 ± 0. 00 6. 40 ± 2. 31 0. 80 ± 0. 50 0. 80 ± 0. 50 24 .0 5 16 .6 0 T ita ga rh 75 7. 20 ± 40 .0 5 21 4. 60 ± 66 .9 9 13 2. 80 ± 29 .8 2 9. 80 ± 4. 81 1. 80 ± 1. 45 0. 80 ± 0. 93 15 6. 40 ±6 8. 61 0. 00 ± 0. 00 5. 40 ± 2. 90 0. 60 ± 0 .6 1 0. 60 ± 0. 61 28 .3 4 21 .4 5 M ad hy am gr am 56 4. 60 ±1 42 .0 5 14 9. 40 ± 53 .4 6 13 7. 20 ± 51 .7 4 7. 00 ± 2. 84 1. 80 ± 0. 93 3. 40 ± 1. 69 53 .6 0± 1 6. 32 0. 00 ± 0. 00 2. 80 ± 1. 22 0. 80 ± 0. 50 0. 80 ± 0. 50 26 .4 6 10 .1 3 D is til le d w at er 6 54 .0 0± 3 9. 87 32 2. 6 ± 64 .7 3 28 7. 40 ± 5 3. 67 29 .8 0± 1 3. 47 4. 00 ± 2. 08 1. 40 ± 2. 44 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 0± 0 0 49 .3 3 0. 00 A N O V A * ** ** ** * * ** A na ly si s of v ar ia nc e (A N O VA ): Si gn ifi ca nt e ffe ct s ar e in di ca te d by a s * = p < 0. 05 ( 5% ); ** = p < 0 .0 1 (1 % ) an d w hi le n on -s ig ni fic an t e ffe ct s ar e in di ca te d by N S. 59Chromosomes damage by sewage water studies in the Allium cepa L. and Zea mays L. Karyolysis Enzymatic dissolution leads to complete suspension of the chromatin in a dying cell. After karyolysis the whole cells will be stained uniformly (Figure 3B, F, J). Chromosome laggard Particular concentration of few turbagens which has affinity for thiol groups that induce various types of spindle disturbances at all stages of mitosis division in most of cells were confined to one or more number of chromosomes. The movement of chromosomes deviated from the main mass and were often seen to be lost. Such aberrant chromosomes have been called “laggards” (Fig- ure 3K). It may be present in the location of spindle area or outside of it (Figure 3K). Anaphase bridge Chromatin bridge is a mitotic event that forms when telomeres of sister chromatids combine together and fail to completely segregate into their respective daughter cells. This event mostly occurs during the anaphase stage that is why called anaphase bridge (Figure 3C, G, K and Figure 4C, E, G). Chromosome fragmentation Chromosome fragmentation results are indicators of a clastogenic action from the numerous breaks in the chromosome arms where there is loss of integrity of the chromosome. Disintegration can range from partial to total breakup of the chromosome (Figure 3D, H, L and Figure 4B). DISCUSSION The research survey works (Ma 1999; Fatima and Ahmed 2005, 2006b) on industrial eff luent samples that were taken from different parts of the city of Ali- garh and Ghaziabad, UP in India. It may be used as a bio indicator for aquatic atmosphere. In our experi- ment different elements and microorganism present in the wastewater samples of the three locations might have induced cytological effects on the roots of both species i.e., Allium cepa L. and Zea mays L. It may have direct or indirect risk on their life due to irriga-Ta bl e 2. F re qu en ci es o f d iff er en t t yp es o f c el ls a fte r tr ea tm en t w ith d iff er en t w at er s am pl es ( T 1, T 2, T 3 a nd T 4) M ea n ± SE & A N O VA in b ot h sp ec ie s. Species L oc at io ns To ta l C el ls D iv id in g C el ls C el ls o f di ff er en t s ta ge s A bn or m al c el ls M ito tic In de x (% ) A be rr at io n Fr eq ue nc y (% ) Pr op ha se M et ap ha se A na ph as e Te lo ph as e K ar yo rr he xi s K ar yo ly si s Fr ag m en te d L ag ga rd A na ph as e br id ge Allium cepa L. K ha rd ah 60 8. 80 ± 13 1. 53 19 2. 20 ± 49 .4 6 17 3. 20 ± 50 .2 6 7. 40 ± 1. 69 8. 00 ± 1. 76 3. 60 ± 1. 86 29 .2 0± 5 .4 1 30 .4 0± 5 .9 7 3. 40 ± 0. 99 5. 60 ± 1. 27 5. 60 ± 1. 27 31 .5 7 11 .2 7 T ita ga rh 58 2. 60 ± 46 .4 4 19 7. 20 ± 57 .7 1 17 4. 80 ± 59 .9 0 6. 80 ± 2. 14 12 .0 0± 3 .0 5 3. 60 ± 1. 69 65 .4 0± 1 8. 11 29 .0 0± 9 .5 7 2. 00 ± 0. 79 8. 60 ± 2. 17 8. 60 ± 2. 17 33 .8 5 18 .0 2 M ad hy am gr am 62 0. 20 ± 90 .7 0 25 8. 60 ± 44 .1 7 23 8. 40 ± 39 .2 3 10 .6 0± 3 .2 0 6. 80 ± 2. 88 2. 80 ± 1. 83 22 .0 0± 1 .7 6 19 .8 0± 7 .0 9 2. 60 ± 1. 69 5. 80 ± 2. 77 5. 80 ± 2. 77 41 .7 0 8. 09 D is til le d w at er 6 78 .2 0± 6 1. 68 38 6. 80 ± 3 3. 06 35 7. 00 ± 2 7. 86 17 .6 0 ± 8. 92 7. 6± 1 .8 6 4. 60 ± 2. 68 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 57 .0 3 0. 00 A N O V A N S ** ** ** ** N S ** Zea mays L. K ha rd ah 59 0. 40 ± 11 4. 33 14 2. 00 ± 34 .6 3 13 0. 80 ± 33 .3 3 9. 60 ± 2. 56 1. 40 ± 1. 69 0. 40 ± 0. 61 90 .8 0± 2 4. 85 0. 00 ± 0. 00 6. 40 ± 2. 31 0. 80 ± 0. 50 0. 80 ± 0. 50 24 .0 5 16 .6 0 T ita ga rh 75 7. 20 ± 40 .0 5 21 4. 60 ± 66 .9 9 13 2. 80 ± 29 .8 2 9. 80 ± 4. 81 1. 80 ± 1. 45 0. 80 ± 0. 93 15 6. 40 ±6 8. 61 0. 00 ± 0. 00 5. 40 ± 2. 90 0. 60 ± 0 .6 1 0. 60 ± 0. 61 28 .3 4 21 .4 5 M ad hy am gr am 56 4. 60 ±1 42 .0 5 14 9. 40 ± 53 .4 6 13 7. 20 ± 51 .7 4 7. 00 ± 2. 84 1. 80 ± 0. 93 3. 40 ± 1. 69 53 .6 0± 1 6. 32 0. 00 ± 0. 00 2. 80 ± 1. 22 0. 80 ± 0. 50 0. 80 ± 0. 50 26 .4 6 10 .1 3 D is til le d w at er 6 54 .0 0± 3 9. 87 32 2. 6 ± 64 .7 3 28 7. 40 ± 5 3. 67 29 .8 0± 1 3. 47 4. 00 ± 2. 08 1. 40 ± 2. 44 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 00 ± 0. 00 0. 0± 0 0 49 .3 3 0. 00 A N O V A * ** ** ** * * ** A na ly si s of v ar ia nc e (A N O VA ): Si gn ifi ca nt e ffe ct s ar e in di ca te d by a s * = p < 0. 05 ( 5% ); ** = p < 0 .0 1 (1 % ) an d w hi le n on -s ig ni fic an t e ffe ct s ar e in di ca te d by N S. B CA D H LKJI E F G Figure 3. Photomicrographs of cytological aberration in Allium cepa L. (2n=16) root tip cells treated with different sewage water samples. (A) Karyorrhexis (B) Karyolysis (C) Anaphage bridge and (D) Fragmented, respectively in Khardah water; E. Karyorrhexis, F. Karyolysis, G. Anaphage bridge and H. Fragmented, respectively in Titagarh water; I. Karyorrhexis, J. Karyolysis, K. Laggard, Anaphase bridges and L. Fragmented in Madhyamgram water. 60 Anup Kumar Sarkar, Ranita Saha, Rupak Halder tion with sewage wastewater to the food plants (Iqbal et al. 2016; Chary et al. 2008). Trace elements accumula- tion in the food chain may harm different organism in the ecosystem along with humans. Several studies have shown that presence of various metal in the Industrial waste water can cause the various nature of chromo- somal aberrations like lagging chromosome, fragmented chromosome, anaphase bridge and binucleated cells etc. in the meristematic root tip cells of Allium cepa L. and plants with such abnormalities which may induce alter- ations in the genetic constitution not only the future progenies but correspondingly have triggered further complication in mankind when consumed as nourish- ment materials (Sabeen et al. 2020). Surveys on sludge samples from thirty-four cities in the USA have reports that there were no effects of chromosomal aberration A B C D E GF Figure 4. Photomicrographs of cytological aberration in Zea mays L. (2n=20) root tip cells treated with different sewage water samples. A. Karyorrhexis, B. Fragmented, C. Anaphage bridge, in Khardah water; D. Karyorrhexis and E. Anaphage bridges in Titagarh water; F. Kary- orrhexis, and G. Anaphase bridge in Madhayagram water. 61Chromosomes damage by sewage water studies in the Allium cepa L. and Zea mays L. due to the treatment of effl uents (Babish et al. 1983). Conversely, there are also studies, revealing cytologi- cal eff ect of extracts from wastewater sludges collected from various American cities on the test of Salmonella typhimurium sample (Mumma et al. 1988; Brown et al. 1991; Blevins and Brennan 1990). In 1998, White and Rasmussen demonstrated that in the large areas of metropolis cities, wastewaters of diff erent municipali- ties are a multifaceted combination of effl uent resourc- es from domestic and industrial sewage, containing a widespread series of heavy or light constituents from a source of diff erent varieties. Siddiqui et al. 2011 had strongly recommended that seed germination of diff er- ent species such as Brassica oleracea var. capitata, Pen- nisetum glaucum and Cucumis sativus are remarkable living beings for heav y metal toxicological monitor- ing of industrial effl uents and XAD concentrated river water. Furthermore, it was reported that significant quantities of diff erent types chromosomal abnormalities including fragmentation, bridges and stickiness were found by Allium cepa test. Cytogenetic eff ect of the car- bon black factory industrial effl uents in Allium sativum root meristem cells not only retarded germination per- centage and radical growth but also induced chromo- somal aberrations: karyolysis, fragmentation, laggards (Ray and Saha 1992). In the absence of telomeres, chro- mosomes turned out to be adhesive in nature which may join the end part of other fragmented chromo- somes in the root tip of meristem cells of Allium cepa in presence of alprazolam chemical compound (Nefi c et al. 2013). Th e presence of breaking fragments, laggards, chromosome bridges and stickiness with other abnor- malities are viewed as mitotic irregularities are due to an-eugenic agents (Zang and Yang 1994; Silveira et al. 2017; Haq et al. 2017). Grant (1982) told that chromo- somes stickiness probably occurred due to degradation or de-polymerization of DNA segment of the chromo- some. It was also reported that the sticking of chro- mosomes resulted from DNA compression and adhe- siveness of inter-chromosome fi bers (Schneiderman et al.1971). One of the abnormalities, which is stickiness and it shows high toxic substances are present along with irreversibility while acentric fragments that appear in anaphase stages are the result of chromatids or chro- mosome interruptions, representing interference with DNA. Bridges in Anaphase stage are the outcome of the disruptions and joining of chromatids or chromosomes (Turkoglu 2007). It is also described that anaphase bridges occur as an output of adhesiveness of chromo- somes, unequal process of translocation or inversion in the segments of chromosome (Gomurgen 2005). Stud- ies by Nagajyoti et al. (2010) and Fashola et al. (2016) indicates that among the heavy metal cadmium (Cd) is known to be carcinogenic and mutagenic in biologi- cal system. As per the investigation reported by Adhi- kari (2019) indicated that lead (Pb) one of heavy metal act as a robust mutagenic mediator on Lathyrus sativus. Nickel with magnesium can be the cause for chroma- tin condensation of the cells (Lee et al. 1995). Whereas it also stated that the combination of Nickel and Chro- mium aff ected the cell division of mitotic spindle lead- ing to chromosomal aberration in the root’s tips of Allium cepa (Anderson 1985). Th e trace amount of few metals such as Mn, Fe, Zn and Cr combined together or individually has caused the observed cytogenotoxic eff ects and reported to induce aberrations in the larvae of Newt (Godet et al, 1993). It also reported that heavy metals induced the toxicity and mutagenicity on Zea mays L. (Vojtechova and Leblova, 1991). Th e Mitotic index (MI) inhibition has been accredited to the eff ect of diff erent environmental substances on DNA and syn- thesis of protein of the living organism (Chauhan et al. 1998). Nefi c et al. 2013 revealed that the occurrence of high concentration of heavyweight metals in the earth sample triggered the downward movement of the Mitot- ic index of the meristematic root tips cells of Allium cepa L. Several heavy metals inhibit the cell division along with reduction of MI in the cortex of the meris- tematic root tips of Zea mays L. (Kozhevnikova 2009). In this study, the Allium cepa L. and Zea mays L. roots anaphase-telophase assay at diff erent stages of cells established that all the three wastewater samples had approximately same levels of toxicity. In the Titagarh (22.7383° N, 88.3737° E) samples, the aberration frequen- cy percentage was however higher in both species than Figure 5. Eff ect of sewage water and control samples (T1, T2, T3, andT4) on Chromosomal aberration frequency % in Allium cepa L. and Zea mays L. 62 Anup Kumar Sarkar, Ranita Saha, Rupak Halder the other two samples of wastewater. Even the mitotic index percentage in all three wastewater location sam- ples were half of the control sample (distilled water). CONCLUSION The study indicates that the heav y metals present in the wastewater samples in Khardah (T1); Titagarh (T2); and Madhyamgram (T3) in the district of North 24 Parganas, West Bengal, induced chromosomal aber- rations: Kar yorrhexis, Kar yolysis, Fragmented, Lag- gard and Anaphase bridge, reduced the Mitotic index and morphological structure also such as germina- tion % and root length inhibitions % in Allium cepa L. and Zea mays L. It may be concluded that presence of heav y metals such as Ni, Ld, Mn, Fe, Cd leads to decrease cell reproduction and increase in the chro- mosome mutation frequency, posing a great potential threat to water ecosystem and human health as well. 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