Middle East Journal of Applied Sciences ISSN 2077-4613

Volume : 05 | Issue : 05 | Oct.-Dec. | 2015 Pages: 112-124

Corresponding Author: Hamdy H. M. Abdel-Rahman, Genetic Engineering Division, Genetic and Cytology Department, National Research Centre, 33 El bohoth St. P. Box 12622, Dokki, Giza, Egypt.

Tel: +0201226333300; E-mail: hamdyna2010@yahoo.com

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Cytogenetical, Biochemical and Chemical Analytical Studies for Assessment of the Water Quality of Nile Water Using Three Bioassays 1Hamdy H.M. Abdel-Rahman, 2Hala M. Abdel Migid, 1Samy A. Attia and 1Aida A. Rizkalla 1Genetic Engineering Division, Genetic and Cytology Department, National Research Centre, 33 El Bohoth St. P. Box 12622, Dokki, Giza, Egypt. 2Botany Dept., Faculty of Science, Mansoura University, Egypt. 2Biotechnology Dept., Faculty of Science, Taif University, KSA.

ABSTRACT The quality of surface Nile water in Mansoura city was subjected to evaluation for its safety for crops irrigation with three plant biotests. The results revealed that water samples taken from the Nile reduced mitotic activity of the three plants (Allium cepa, Vicia faba var. Giza 3 and V. faba var. Giza Blanca), increased chromosomal aberrations and induced disturbances in mitotic phases. SDS-PAGE (Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis) protein revealed different responses by appearance and disappearance of bands as well as differences in nucleic acid quantities. Water samples were chemically analyzed for different elements, heavy metals and other parameters, many of them were found in unsafe limits for plants, animals and human life. Key words: Chromosomal aberrations, Mitotic index, River Nile, Plant bioassay, SDS-PAGE protein banding,

Water quality.

1. Introduction

Water is essential to the life on earth and dominates chemical composition of all biota. All animals and humans are more than 50% water by weight. Water is an excellent solvent capable of dissolving a wide range of substances. Pollution considered a critical danger to our environment that the increasing discharge of chemicals into the environment has affected natural ecosystems balance (Mattar et al., 2014). Nowadays drinking water shortage becomes a serious problem, and many factors involved in this situation; the water bodies' pollution occurs accidentally all over the world, there is lack of control and management policies regarding the basic sanitation and appropriate effluents treatment. And so the understanding on the reasonable water use, efficient monitoring and treatment policies, establishing strict aims for water quality control, are increasingly critical, to ensure the survival of all forms of life (Barberio, 2013). Egypt facing many-water related challenges, first of all limitation of rainfall water make the country dependent essentially on water of River Nile (Agrama and El-Sayed, 2013). The Nile water is of high quality as the river reaches Cairo. Deterioration in water quality occurs when river Nile divided into Rosetta and Damietta branches due to the discharge of municipal, industrial and agricultural drainage besides flows are decreasing (World Bank Country Environmental Analysis, 1992-2002). In Egypt population continuously growing makes a great pressure on water resources resulted in water pollution, (AWF (African Water Facility), 2007). In recent years, there has been growing concern about the toxic effects of chemical substances in the aquatic environment (Hikal et al., 2015). River Nile pollution is a serious danger to our environment in the country. This consequently attracts awareness of many official agencies and researchers to health of living organisms (Leme and Marin-Morales, 2009). Environmental pollution constitutes a great health hazard to human and animals. Pollution has adverse effects on land, water and its biotic and abiotic components (Al-Dulaimi et al., et al, 2012). Damages due to chemical agents for livings are including genotoxic and mutagenic effects. These effects have shown to be worrying; due to its ability to induce genetic alternation that can lead to several health problems and also affect future generations, since these change can be heritable (Ribeiro, 2003). Water quality testing determines if water is safe for drinking or irrigation. Many monitoring programs must be carried out to ensure the quality of surface waters (Bobeldijk et al., 2001). Many substances are found naturally in water but man activities can increase amounts to where it is not possible to use the water safely. Sources include fertilizer, sand and salt from roads, erosion from fields, mining and industrial wastes. Metals

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like mercury, lead, cadmium, zinc, iron and copper are also inorganic pollutants which can create serious health problems even if they found in very low concentrations. Both Allium cepa and Vicia faba are considered popular edible crops in Egypt for human and animal. Hence two plants have been recommended to be used in the present study. Due to these advantages over conventional analytical techniques, several biotests are already being employed (Brüggemann, 1995). The present research aims to evaluate the surface water quality of River Nile in Mansoura city. Investigating the water quality using Canadian Council of Ministers of the Environment Water Quality Index (CCME-WQI) (2001) based on Egyptian guidelines of law 48/1982 (2013).

2. Materials and Methods 2.1. Sampling: Nile water samples were regularly (monthly for one year) collected from the River Nile at Mansoura city, El-Dakahlia province. The samples were kept in the dark until reaching the laboratory, for chemical analysis and biotests investigations. Tap water was used as control. Onion bulbs (Allium cepa) and two varieties of broad bean (Vicia faba, var. Giza 3 (G3) and var. Giza Blanca (GB)), were obtained from Agriculture Research Center, Legume and Onion, Research Sections, Giza, Egypt. 2.2. Chemical analysis for water sample: As soon the water samples reach the laboratory, they were filtered (except those which used for estimation dissolved oxygen and biological oxygen demand). Then the following chemical parameters were measured. Water salinity (%) and electric conductivity (moh l-1) were measured directly using YSI model 33 S.C.T. Total dissolved salts (mg l-1) were measured using CORNING (Cole-Parmer model Check-mate 90- London- UK). The pH values determined using pH meter Model 5995. Chlorides as described in APHA (1985). Dissolved oxygen (DO) was determined using Wood (1975). Biological Oxygen Demand (BOD) described by APHA (1989). Dissolved organic nitrogen was estimated by Golterman et al., (1978). Total dissolved phosphorus (T.D.P) (mg l-1) was determined according to APHA, (1985). Sulfate was determined as described by APHA, (1989). Total hardness (Ca++ and Mg+) was determined according to Diehl et al., (1950) and APHA (1985). The cations (Na+, K+ and Ca++) were determined by using a flame photometer while, Mg++ was estimated by UNICAM SP 90A SERIES 2 atomic absorption spectrophotometer and heavy metals (Cu, Fe, Zn, Pb and Co) were analyzed using atomic absorption spectro-photometer type PERKIN ELMER, 2380 (Chandler, Arizona, USA). 2.3. Genotoxicity biotests: I- Allium test: The test was carried out as described by Rank and Nielsen (1993)]. Bulbs were submerged in treatment water, which changed daily. Roots were grown to length of 1-5 cm be ready for cytological and biochemical analysis. II- Vicia test: The seeds of V. faba were soaked in distilled water over night and then germinated in soil (2 sand: 1clay). The roots were let to grow to a length of 1-5 cm for cytological and biochemical analysis. 2.3.1. Cytological analysis: On the 5th day of treatment, root tips of both A. cepa and V. faba, were chosen randomly from each treated tests, excised and fixed in ethanol: acetic acid (3:1). After fixation roots were hydrolyzed in 1N HCL at 60 °C for 3 min. and then transferred into the Feulgen stain. The following parameters were determined: Mitotic index (MI), Phase index (PI), Relative Division Rate (RDR) and chromosomal aberrations (CA). RDR calculated by the formula according to Hoda et al. (1991). "RDR % = % of dividing cells in treated sample-% of dividing cells in control sample x 100" 100 - % of dividing cells in control plants 2.4. Field experiments: The selected seeds of the two V. faba varieties were surface sterilized in 1% HgCl2 solution. Pots were filled with a mixture of clay and sand at a ratio of 2: 1 (v/v). The presoaked seeds were planted in pots. The planted pots were separated into 2 groups, one group irrigated with Nile water and the other irrigated with tap water as control. After maturation, F1 harvest seeds (May) were used for biochemical analysis.

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2.5. Biochemical analysis: I-Nucleic acids analysis: DNA was estimated by diphenylamine (DPA) color reaction described by Burton 21. RNA was quantitatively determined according to Dische (1962) II- Estimation of total protein: Protein content was determined spectrophotometrically by Bradford (1976). III- SDS gel electrophoresis: SDS-PAGE technique based on Laemmli (1970). The data obtained from the scanning process of gel were analyzed using total lab version 1.11 and labimaging version 2.7 programs. 2.6. Statistical analysis of data: SPSS program (Nie et al., 1975) was used. Least Significant Difference (L.S.D.) was used to determine significance (p=0.05) in the data compared to the control.

3. Results and Discussion 3.1. Chemical analysis: Chemical analysis of the studied water samples and the guidelines of parameters used in the Water Quality Index (WQI) calculations are represented in tables (1&2). These guidelines are according to Egyptian Ministry of Water Resources and irrigation “law 48/1982”, for protection of the Nile River and Waterways from Pollution (i.e. decree No. 49 in the amended executive regulations of the law by Minister Decision No. 92/2013). According to this WOI chlorides and sulphate were found in unsafe limits or near this limits but other parameters recorded safe limits. Meanwhile, heavy metals analysis cobalt, cadmium, and zinc recorded unsafe values (Table 2). Table 1: Chemical analysis of elements and other parameters in water samples by mg l-1 or % (E.C. by µmhos/cm), (Permissible limits according to Egyptian Ministry of Water Resources and irrigation.

Parameter Control Nile sample Permissible Limit Chlorides 141.48 319.14 250 Total nitrogen 15.4 17.8 2 Total alkalinity 114 139 200 DO (Dissolved Oxygen) 10.36 12.19 6 Sulphate 37.58 147.64 200 Total hardness (Ca++ and Mg+) 73.44 85.68 300 Total Phosphorus 0.08 0.14 2 Calcium 5.1 4.67 75 ‰ Salinity 0 0 0 pH 7.6 8.05 6.5-8.5 E.C. (Electric Conductivity) 447 429 3 BOD (Biological Oxygen Demand) 5 17 6 COD (Chemical Oxygen Demand) 10 15 10 Sodium 9.5 97.52 100 Potassium 8.6 14.4 10

Table 2: Chemical analysis of heavy metals in water samples by mg l-1, (Permissible limits according to Egyptian Ministry of Water

Resources and irrigation. Heavy metal Control Nile Permissible Limit Cobalt 0.002 0.14 0.05 Iron 0.028 0.15 5.00 Cupper 0.00 0.00 0.20 Lead 0.09 0.47 5.00 Cadmium 0.00 0.09 0.01 Zinc 0.006 9.70 5.00 Total 0.045 10.55 1.00

Generally chemical analysis of the studied water samples revealed that, the concentrations of some estimated elements and parameters exceed the safety limits or near the maximum approved values. In the present study chlorides were found in unsafe limits in Nile sample. Different liquid pollutants, industrial discharges and domestic wastes are reported to be the main pollutants which cause increase in chloride content (Aziz, Q., 1996) as well as agriculture fertilizers (Golterman, 1999). Chloride was increased proportionally with pollution (Saad and Abbas, 1985). High concentration of chloride content is known as a cause of toxicity to plants. Dissolved oxygen is widely accepted as an efficient and sensitive indicator for the water quality

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condition (Leester, 1975). Increasing organic wastes may leads to the deoxygenation of water (Juttner et al., 1996). Chemical analysis of water samples revealed that, DO values found in safe limit. Sang and Li, (2004) found that, there is a correlation between the genotoxicity in Vicia faba and the chemical measurement (COD) of leachate. Freshwater must have a pH from 5.0 to 8.5. An acid level of less than 5.0 tells that mine drainage or acid manufacturing wastes may have polluted the water. Industrial alkaline wastes are indicated when pH is 8.5- 9.0. A neutral pH 7.0 is considered the best for human utilization (Pratt, 1972). It is known that, total phosphorous is normally present with high quantity in polluted water (Shaaban-Dessouki, et al., 1994) and if exceed 0.1 mg/L it’s a sign that agricultural wastes has polluted the body of water in this study total phosphorus recorded 0.14 mg/L . Nitrogen (Nitrate) is essential for plant growth and it is naturally found in water at low level. For safe drinking water, the nitrate should not exceed than 10 mg/l. Nitrates come mainly from polluting water with fertilizers and industrial wastes. Concentration of nitrogen in water sample found to exceed the safe limits for use. Toxicity of heavy metals could be also assessed by growth inhibition, which has been shown for a wide range of plant species (Kozhevnikova et al., 2009). When penetrating into the cells, heavy metals interact with some functional groups of proteins. This results in protein conformation changes and inactivation of numerous enzymes (Ivanov et al., 2003). Zinc is essential for plant growth but in very small quantity. Lead is known as inhibitory of plant growth, root elongation, seed germination, seedling development and cell division (Wierzbicka, 1995). In the present study lead concentration in Nile water exceeding the safety limits, its toxicity is due to mimics many aspects of the metabolic behavior of calcium and inhibits many enzyme systems (Mengel and Kirkby, 1982). Cadmium is one of the most toxic environmental pollutants (Unyayar et al., 2006). These findings are confirmed by the results obtained in this study were cadmium recorded in unsafe concentration in tested water sample. Cobalt is a common metal contaminant in soils (Woodard, T.L.,). Cobalt exceeds approved and save limits in this study. These results are in agreement with many authors findings (Abd-El Magid and Soliman, 2003; Mahmood, 2006). 3.2. Cytological analysis: 3.2.1. Germination of A. cepa Root: The morphological data of onions treated with Nile water samples recorded shorter root growth than control (Table 3 and Plate 1). The shorter root growth could be due to the toxicity of heavy metals (as cobalt, cadmium and zinc) which are exceeding the approved limits in Nile water samples. Root growth is determined by cell division and elongation (Evseeva et al., 2005). There are only few reports about heavy metals effects on these two processes (Seregin and Kozhevnikova, 2006). However, it is not known until now how metal growth-inhibiting action is related to the specificity of their translocation and partition and what are the reasons for different action of different metals.

Plate 1: Root length of Allium cepa root after treated with different treatments after 5 days. Table 3: Root lengths of Allium cepa through 3 days of treatment.

Treatments

Root length (mm) 24 H. 48 H. 72 h.

Control 12.5 35 75 Nile water 11 28 57.5

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3.2.2. Mitotic Index: The recorded mitotic indices of V. faba Var. G3 and A. cepa decreased significantly after treated with Nile water, where the decrease in V. faba Var. GB V. was not significant compared to control. Mitotic index is considered as parameter that allows estimating the frequency of cellular division and used as an indicator of cell proliferation biomarkers which measures the proportion of cells in the mitotic phase of the cell cycle. Hence, the decrease in the mitotic index of A. cepa meristematic cells could be interpreted as cellular death (Ping et al., 2012) It is an acceptable measure of cytotoxicity for all living organism. Mitotic inhibition has been attributed to a number of factors (Deysson, 1968). Among these is the blocking of mitotic cycle during interphase. The significant reduction in mitotic index by cytotoxic substances and polluted water in the present study may be due to the effect on microtubule configuration or may be due to the blocking of the mitotic cycle during interphase stage (Smaka-Kinel et al., 1996), inhibition of DNA synthesis (Chand, and Roy, 1981) which could be due to blocking of G1, by suppressing DNA synthesis (Schneiderman et al., 1971). Blocking in G2 prevents the cell from entering mitosis (Van’t Hoff, 1968) and inhibits nuclear protein synthesis in the cell cycle (Kim and Bendixen, 1987) which leads to inhibit the formation of various metabolic events necessary for mitosis (Rost and Morrison, 1984) leading to low ATP content, which is essential for progress of mitosis (Amin, 2002). Table 4: Percentage of Mitotic Index (M.I.), Relative Division Rate (RDR) and phase index in the three plants after treatment (Mean ± S.D., *=significant at p=0.05).

Plant Treatment % M.I. RDR % Phase Index

Prophase Metaphase Anaphase Telophase V. faba

G.3. Control 20.2±0.76 - 21.38±2.89 34.64±2.74 25.88±5.41 20.82±3.86

Nile Water 17.4±1.08* -3.80 18.52±7.91 36.64±5.05 23.16±4.89 20.14±3.73 V. faba

G.B. Control 15.7±1.03 - 19.02±5.72 34.52±6.62 23.56±6.77 23.46±4.78

Nile Water 14.1±0.96 -1.34 22.16±4.88 31.9±5.02 20.38±3.97 25.5±5.3

A. cepa Control 17.6±1.02 - 17.1±1.02 34.8±5.17 27.7±5.0 20.42±2.07

Nile Water 14.3±1.75* -2.72 21.98±5.44* 31.94±4.69 25.36±4.12 22.44±2.4

In the present study the followings heavy metals zinc, cadmium and cobalt are found in unsafe limits for plants, which could exert the inhibition in mitotic indices. Zinc containing compounds exerts toxic action resulting in mutations that persist in the F2 generation (El-Ghamery et al., 2003). It is found that, zinc had an inhibitory effect on cell division even at non-lethal concentration. It caused a decrease in the mitotic index and produced a number of mitotic abnormalities in cells of Nigella sativa and Triticum. The inhibitory effect of zinc on cell division could be attributed to the action of zinc on the spindle fibers. Data of chemical analysis in the present study showed a high concentration of zinc in Nile sample. Consequently, inhibition in cell division increase in abnormalities percentage may be in part led from zinc effect. Cadmium affecting cytogenetically the various organisms, inhibition in lengths of primary roots, decreases the mitotic index and increase chromosomal abnormalities recorded in rice seedling (Abdel-Azeem, and El-Nahas, 1996), also cadmium reduces total proteins and RNA. Reddy and Venugobal (Reddy and Venugobal, 1991) reported that treatments of some crops with cadmium induced a reduction in protein content. Cobalt may affect rate of cell division by retardation the process of karyokinesis and cytokinesis, also cobalt decreased mitotic index and repressed the passage of interphase cells into mitosis (Palit et al., 1994). In beans, an irregular arrangement of nuclear chromatin with the appearance of micronuclei and small granules were observed (Komczynski et al., 1963) and caused despiralization of prophase chromosomes in meristematic cells (Bobak, 1974). Cobalt compounds have been shown to affect mitotic spindle in A. cepa causing c-mitosis and formation of chromosomal aberrations as micronuclei, chromatin bridges, fragmentation binucleate cell and stickiness (Palit et al., 1994). High concentrations of cobalt slow down RNA synthesis and then protein content decrease, generally cobalt decrease total nucleic acids. Lead was found to cause reduction in mitotic index, DNA, RNA synthesis and reduce protein content (Wierzbicka, 1999). Rucinska et al. (2004) studied the effect of lead on lupine roots, and reported that, only limited data is available on the effect of lead on plant genetic material. It was previously revealed that it causes a definite effect on the growth of seedlings and mitotic index (Rucińska et al., 1999) The various plant genotoxic tests demonstrated that lead strongly induce DNA damage in a concentration-dependent manner (Kovalchuk et al., 2001). 3.2.3. RDR (Relative Division Rate): Table (4) recorded negative values in case of all three test plants for relative division rate (RDR). Increase in negative value of RDR was directly proportional to the severity of the mitotic inhibition (Hossain et al., 2003). Thabet, (1986) stated that mitotic index was represented in the form of RDR. The RDR at each plant and treatment was calculated and in all cases the values were negative indicating inhibition of mitotic division. 3.2.4. Phase Index: Disturbance in phase index were found (Table 4). The results indicate that water pollutants may induce an imbalance in the frequency of mitotic phases in root meristems of both plants. In general, the frequencies of

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prophase and/or metaphase stages were increased on the expense of other mitotic stages in both plants; this indicates that studied water affect the relative duration of each stage as compared to control. The accumulations of these stages indicate that the substances in water samples influence the sequence of mitotic division by blocking the process at the end of prophase. It may also be due to the interaction with the spindle which can cause an arrest of the cell division at metaphase. Compared to control, there was slight significant increase in the frequency of metaphase cells in roots of V. faba plants irrigated by mixed water treatment while larger increase was observed in plants irrigated by agricultural drainage water treatments. Also, frequency of anaphase cells was significantly increased in plants treated with agricultural drainage water, while plants treated with mixed water treatment produced a significant decrease in the frequency of anaphase cells (Egito et al., 2007). 3.2.5. Chromosomal Aberrations: Total chromosomal interphase abnormalities (Table 5) in faba Var. G3 and A. cepa increased significantly after treatment with Nile water and V. faba Var. GB increased non-significantly. While in case of total chromosomal aberrations in mitotic phases (Table 6) the highest abnormality percent was recorded in V. faba var. G3 after Nile water treatment (51.3±4.56*) compared to control (16.34±5.56). Table 5: Different types of chromosomal aberrations in interphase of Vicia faba and Allium cepa. (Mean ± S.D., *= significant at p=0.05).

Treatment

% interphase abnormalities % Total Abnormalities

Micronucleus Bi/multinucleated cell

V. faba G.3.

V. faba G.B.

A. cepa V.

faba G.3

V. faba G.B.

A. cepa V. faba G.3. V. faba G.B. A. cepa

Control 1.2 2.2 0.4 0.2 1.0 0.8 0.88±0.34 1.9±0.87 0.72±0.26 Nile Water 6.4* 2.8 1.2 1.8* 1.6 1.2 4.96±1.73* 2.56±0.93 1.4±0.63*

Table 6: Chromosomal aberrations in mitotic phases of Allium cepa and Vicia faba after treatment. (Mean±S.D., * significant at p=0.05).

(Mic = micronucleus, Mis = misorientation, Dis = disturbed, Lag = laggard chromosome).

TREATMENT % CHROMOSOMAL ABERRATIONS

Prophase Metaphase Anaphase Telophase %Total Aberrations Stickiness Mic stickiness Mis Dis Bridge Lag Breaks Mic Bridge

Allium cepa

Control 1.0 0 0.8 1.4 0.8 0.8 0 0.4 0 0.6 16.52±7.54 Nile 1.81 0 1.2 1.6 1.0 1.6 0.2 2.4* 0.2 0.8 38.02±4.33*

Vicia faba G3

Control 0.2 0.2 0.4 1.8 0.4 0 0.2 1.4 0.2 0.4 16.34±5.56

Nile 2.6* 2.8* 1.0 1.8 0.8 0.8 0.6 1.8 1.4* 1.2 51.3±4.56*

Vicia faba GB

Control 1.2 0.2 1.2 1.2 0 0.2 0.6 0 0.2 1.2 25.66± 5.91

Nile 1.6 0.6 1.2 1.6 0.8 0.8 0.2 0 0.8 0.8 41.2± 12.4*

Generally, chromosomal aberrations may be attributed to the exceeding values of toxic substances found in the studied water samples, hence this proof their cytotoxicity and mutagenicity especially if they were used either in irrigation or drinking Nile water. Stickiness which is the most frequent aberration showed in this study may be due to defective functioning of one or two types of specific nonhistone proteins involving chromosome organization (Turkoglu, 2007). It may be also occurring through immediate reactions with DNA during its inhibition periods, causing DNA-DNA or DNA-protein cross linking. Sticky chromosomes indicate a highly toxic, irreversible effect probably leading to cell death (Fiskesjö, 1988). Based on our data we can strongly suggest that pollutants affect DNA and consequently affecting telomeres which save chromatids and chromosomes from being stick together- so, chromosomes aggregate together in a sticky mass as previously reported by Darlington and Mcleich, (1951) who suggested that stickiness might be due to degradation or depolymerization of chromosomal DNA. The production of chromosomal abnormalities by chemical compounds is regarded as a reliable evidence of the genotoxicity of these water pollutants (Grant, 1982). Higher percentage of different chromosomal aberrations due to using polluted water was observed by Mattar et al., (2014) The toxic effects of the studied water samples, on the mitotic activity paralleled their effects on nucleus and chromosomal morphology. The treatments caused an increase in the percentage of the total abnormalities in the meristematic cells of V. faba and A. cepa roots. In the present study, different types of the observed aberrations in mitotic cells possibly a result of the effect of genotoxic compounds found in the studied water samples. Such toxic compounds (domestic chemicals, detergents, chlorophenols, heavy metals etc.) caused severe changes in chromatin, spindle apparatus and centromeres, leading to impairment of chromosome arrangement onto metaphase plate, anomalous spindle orientation, abnormal chromosome movement and c-mitosis. Abnormal direction of chromosomes can attribute to altered quality and quantity of kinetochore heterochromatin (Jannifer et al., 1988).

V.

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Chromosomal aberrations arise because of lesions in both DNA and chromosomal spindle protein causing genetic damage. The suppression of mitotic behavior was often used for tracing cytotoxicity. This suppression has been reported to be associated with high percentage of chromosomal aberrations in particular, c-mitosis, sticky and abnormal chromosome orientation. It is clear from data of cytological analysis, that, the inhibition in mitotic activity was accompanied with high percentage of chromosomal aberrations. Chromosomal abnormalities were scored in all stages of mitosis, where chromosome stickiness was the most common type of abnormalities. Other types of abnormalities were also observed, such as micronucleus, bridges, chromosomal fragments and disturbed phases. Clastogenic pollutants are which stimulate break in genetic material and aneugenic ones are those cause chromosome disjunction during division. Micronuclei and bridges are examples of results clastogenic, while c-mitosis and stickiness, aneugenic. In the present study bridges appeared in a significant frequency. Chromosomal breakage which recorded in a significant in case of effect of Nile water on A. cepa has been regarded to involve an action on DNA (Grant, 1978). Micronucleus arises from anomalous disjunction of chromosomes resulting in formation of acentric fragments, digenetic chromosomes and chromatin bridges (Grover et al., 1999) also, may be the result of lagging chromosome or a chromosome segment produced during a preceding division]. In this study micronucleus almost disappeared in A. cepa test, and found in both V. faba varieties, more pronounced in var. G3. in significant increase. An increase in the frequency of micronucleated cells is considered a result of chromosomal and genomic damage caused by clastogens or spindle poisons (Miller et al., 1997). Stickiness and micronucleus were the most frequent chromosomal aberrations were recorded (Plates 2-3).

Plate 2: Types of chromosomal aberrations was recorded in Allium cepa root meristematic cells after treatment

with Nile water, (a) stickiness, (b) fragment, (c) micronucleus, (d) bridge, (e) stickiness with micronucleus and (f) vagrant.

Plate 3: Types of chromosomal aberrations was recorded in Vicia faba root meristematic cells after treated with

Nile water, (a-c) micronucleus, (d) stickiness, (e) c-mitosis, (f) laggard chromosome, (g) fragment and (k) bridge.

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3.3. Biochemical analysis: Total nucleic acids and total protein are recorded in tables (7-8). Data revealed that total protein increased excessively in case of A. cepa with little change in case of both V. faba varieties. While total nucleic acids in case of V. faba Var. GB both DNA and RNA increased after treatment and decreased in V. faba Var. G3 and A. cepa. New bands were appeared and other bands were disappeared in protein banding pattern (Plate 4).

Results of biochemical analysis showed that the treatments of A. cepa and V. faba with Nile water induced changes in both protein and nucleic acids contents. Also, changes in protein banding pattern were occurred. The data of total nucleic acids revealed that, the amounts of both nucleic acids (DNA & RNA) in A. cepa decreased after treatment. However, in case of V. faba total DNA decreased and total RNA increased. Inhibition of cell division was found to be a cause in decrease in both DNA and RNA contents (Badr, and Ghareeb, 1987). Reduction in mitotic activity could be due to blocking at G1, preventing DNA synthesis or blocking at G2 after DNA synthesis (Zaki and EL-Din, 1985) giving an inhibition effect on DNA and RNA synthesis. Chand and Roy suggested that inhibition of DNA and RNA may occur due to reduction in oxidative phosphorylation resulting in low ATP levels. Ghotbi Kohan and Morgan, (2007) have revealed the direct relation between increase of pollution and the level of stress protein. They explained that stress proteins have high sensitivity to changes in the environment. Table 7: Total nucleic acids (DNA & RNA) of Vicia faba seeds and Allium cepa by µg/gm following treatment.

Treatment

Total Nucleic acids BY µg/gm V. faba G. B. V. faba G. 3. Allium cepa

DNA RNA DNA RNA DNA RNA Control 4.04 3865.74 2.05 8836.8 0.192 0.423

Nile 4.16 4571.75 1.21 7864.58 0.143 0.261 Table 8: Total protein of both varieties of Vicia faba and Allium cepa treatment by µg/gm.

Treatment Total protein µg/gm

V. faba G. B. V. faba G. 3. A. cepa Control 23.37 23.37 2.14

Nile 23.27 23 6.23

var. G.3. var. G.B.

Nile Control Marker Control Nile.

Plate 4: SDS-PAGE of Vicia faba seed protein banding pattern. . In this study the reduction in mitotic activity in A. cepa and V. faba was associated with a reduction in the amounts of DNA. Similar results were reported by Badr and Ghareeb (1987) who found that the reduction in mitotic activity was accompanied with the inhibition of DNA synthesis in mitotic cycle. Other authors suggested that inhibition of cell division may cause an inhibitory effect on DNA and RNA synthesis (Yoshida, Y., 1983). The common effects which observed by environmental agents is chemical change of the DNA leads to disturb essential processes, like DNA duplication and transcription, gene regulation and cell division, and leading cells to pathologic processes and/or cell death. Heavy metals are especially toxic due to their capability to combine with proteins and stop DNA replication (Kar et al., 1992). The pattern of protein synthesis during the cell cycle is characterized for each individual. It was observed in this study that an increase in RNA content occurred in both varieties of V. faba while it decreased in

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A. cepa cells. Changes in ribosome content could be due to changes in the rates of synthesis of the three major classes of cytoplasmic RNA. Changes in ribosome content, synthesis or activation or inhibition of elongation factor, t-RNA or some other activators or inhibitors may lead to changes in RNA content (Stanner and Becker, 1971). Consequently, quantitative changes in protein content were observed in this study, which could be also attributed to the changes in rates of either DNA or RNA synthesis. Total protein content of both varieties of V. faba slightly decreased with treatment, but showed increase after treatment in case of A. cepa. Mutagenic chemicals interact with DNA causing changes in its structure. This may result in the loss, addition or replacement of bases, thus altering their sequence in the DNA and affecting the fidelity of the genetic message. Amino acid changes within a protein, due to mutational events, can result in altered protein migration rates when the proteins are compared on polyacrylamide gels (Hussein and Salam, 1985). Also these changes could be traced back to the induction of cytological abnormalities. The induction of bridges, breaks, laggards, and micronuclei lead to loss of some of the genetic material. Therefore some electrophoretic bands disappeared due to the deletion of their corresponding genes (Gamal et al., 1988). Bands disappearance could be also explained on the basis of mutational events at the regulatory genes that suppress transcription, while the appearance of new bands could be explained on the basis of mutational events at the regulatory system of an unexpressed gene(s) that activate it (Abdelsalam et al., 1993)]. The occurrence of additional bands in PAGE profile may be the result of the synthesis of new protein controlled by the mutation (activation) of structural genes (Hassan, H.Z., 1996). Primary DNA lesions (as oxidation of bases, formation of DNA-adducts, base-dimerization, or cross-links) are resulted from genotoxic factors, theses lesions are either repaired or otherwise induce irreversible changes of the DNA or lead cell death. Mutations occur on both, the gene level (gene mutations) and on the chromosomal level (structural and numerical aberrations) (Majer, et al., 2005). Data of present study revealed that polluted water displays a strong mutagenic potentiality as indicated by the observed changes in DNA, RNA content and protein banding pattern. Therefore, keeping high attention to river Nile water pollution sources and getting rid any of these pollutants to be safe for irrigation in economic way to eliminate pollution. Besides keeping attention to rive Nile water sources through elimination of all sorts of pollution to be used safety by humans. The effect of chemicals on the process of chromatin synthesis is another factor which may lead to inhibition in mitotic activity (Topaktas and Rencuzogullari, 1996). The inhibition in cell division also has been attributed to the inhibition in protein synthesis. Such mitodepressive effect has been attributed to the inhibition in synthesis of certain types of nuclear proteins essential in the mitotic cycle. Mitotic inhibition has also been attributed to the inhibiting effect of chemicals on the formation of various metabolites which are necessary for normal sequence of mitosis. Moreover, the transition points from G1 to S and from G2 to mitosis are carbohydrate dependent, and that cells starved of either carbohydrates or phosphate failed to make this transition (Van’t Hoff, 1985a,b) Evaluation of water quality is a complicated process undertaking several parameters capable of causing various stresses on water quality generally. To assess water quality from numerous samples, each containing concentrations for many parameters is difficult (Almeida et al., 2007). Water quality can be compromised by the presence of toxic chemicals, infectious agents and radiological hazards (World Health Organization, 2012a). More than 10% of people worldwide consume foods irrigated by wastewater that can contain chemicals or disease-causing organisms. 1.1 billion People practiced open defecation in 2010 (World Health Organization, 2012b). Pollutants enter aquatic systems via numerous pathways, including effluent discharge, industrial, urban and agricultural run-off, as well as airborne deposition (Younis and Nafea, 2015). In Egypt, the Nile River is the most important source of water for man and animal drinking or plant irrigation. Nile River water quality was the object of a great number of studies. Meanwhile, efforts are devoted to keep the water of Nile River always good for irrigation, by avoiding the pollution which comes from industries or wastewater. On the other hand domestic sewage and municipal wastewater, agricultural and industrial effluents all contribute significantly to the water pollution problem in Nile water. Many water quality indices have been formulated world wide which can easily judge out the overall water quality in a specific area (Bharti and Katyal, 2011). As US National Sanitation Foundation Water Quality Index (NSFWQI) (Sharifi, 1990) and Canadian Council of Ministers of the Environment Water Quality Index (CCMEWQI) (Lumb et al., 2002), these indices are based on the comparison of the water quality parameters to regulatory standards and give a single value to the water quality of a source (Khan et al., 2003). The Nile water treatment using different tested plants induced mitotic changes in root tips. These changes vary from the reduction of mitotic index, changes in phase index also induction of percentage and types of chromosomal aberrations. As well as it stimulated changes in quantity of nucleic acids and protein banding pattern. The analysis of Nile water samples showed that Nile water may be polluted and could not be acceptable for using in drinking or irrigation (Egyptian Ministry of Water Resources and irrigation,2013; Pratt, 1972; National Academy of Sciences and Engineering, 1972; World Health Organization (WHO),1984; FAO,1985) guidelines for classification of irrigation water. More efforts must be paid to avoid the Nile pollution.

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