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Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba



A.W. Amin, F.K. Sherif, H. El-Atar and H. Ez-Eldin
 
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ABSTRACT

Field experiments were conducted to evaluate the effect of using sewage sludge as organic fertilizer on different cytological and yield parameters of Vicia faba. Sewage sludge treatment rates were 0, 10, 20, 30 and 40 T/F and applied to the soil in three successive additions during 1999-2001. Different rates of residual and repeated application of sewage sludge increased heavy metals concentrations in the soil before sowing and after harvesting of Vicia faba. Sludge treatments did not affect some yield parameters, but mature plant height and number of tillers/plant were increased or decreased at different treatments of sludge. The seed index and fresh and dry weights of shoots and roots were increased. The number of seeds/pot recorded the highest value by 10 T/F of the one residual addition, while the lowest value was obtained at 20 T/F two additions. Also, sludge treatments increased mature plant height, seed index, seed density, the number of nodules/plant and fresh and dry weights of nodules except at the highest treatments of two and three additions. Generally, it decreased nodules efficiency percentage. The root tolerance index was increased by sludge treatments. The sludge treatments increased the mean mitotic index and those of three cumulative additions were higher than that of one residual addition treatment. Sludge treatments included a number of abnormalities in all mitotic phases and non dividing cells. The percentage of abnormal cells was increased by three cumulative additions treatments than the one residual addition.

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A.W. Amin, F.K. Sherif, H. El-Atar and H. Ez-Eldin, 2009. Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba. Research Journal of Environmental Toxicology, 3: 60-75.

DOI: 10.3923/rjet.2009.60.75

URL: https://scialert.net/abstract/?doi=rjet.2009.60.75
 

INTRODUCTION

Wastewater sludge produced by sewage treatment plants are represented as a good source of macro- and microelements and generally contain a high quality of organic matter (Bierman and Rosen, 1994; Logan and Harrison, 1995). Their application in agriculture translates into improved soil fertility. Sludge also maintains soil structure, soil water holding ability, soil cation exchange capacity and soil biology activity (Lindsay and Logan, 1998). Although, the recycling of sludge is an attractive alternative, its use often brings about certain risks to the environment caused by accumulation of heavy metals and organic compounds and potential contamination from the pathogenic organisms (Alloway and Steinnes, 1999).

Heavy metals toxicity may affect growth, morphology and biochemical activities of microorganisms (Ibekwe et al., 1996). Variations in effective Rhizobium population size were apparent in relation to sludge application rate and type, heavy metal concentrations and soil pH (Obbard, 2001).

Repeated applications of heavy metal-contaminated sewage sludge can result in an accumulation in toxic metals in the soil which causes injuries to soil microorganisms (McGrath et al., 2000).

Mench et al. (1993) reported that the highly metal-polluted sludge reduced the yield of leaves of maize. However, Khalil et al. (2000) and Mater (2000) found that the addition of different organic biosolid increased significantly the dry matter yield of plants.

Plants have been the material of choice to study the cytotoxic and mutagenic effects of metals and can provide a good system for studies related to environmental monitoring (Fiskesjo, 1988). Because of the similarity of effects and target materials in all organisms, the effects of metals on plants can be usefully applied to other systems as well (Minissi et al., 1998; Rank and Nielsen, 1998; Zhang and Xiao, 1998; Amin, 2001; Steinkellner et al., 2002; Ivanova et al., 2005), found that the frequencies of chromosomal aberrations increased significantly in plants exposed to different concentrations of heavy metals.

In order to increase accuracy and reduce environmental risks, an evaluation of the bioavailability of metals to the plants and the heavy metal content in soil solution and ground water is necessary. Therefore, assessment of the degree of solubility in water is important. The goal of this study is to compare the efficiency of different rates of sludge as residual or cumulative treatments on the chemical nature of soil, the genome of Vicia faba (as a short plant bioassay to be excellent for mutagenicity studies) and some of its yield parameters (e.g., plant height, pod length, seeds pod-1, seed index, seed density, nodules number and activity and fresh and dry weight).

Plant bioassay have many advantages among them are being less expensive, similar in chromosome morphology and response to mutagens to mammals and suitability to in situ experiments.

MATERIALS AND METHODS

Physical and Chemical Properties of Sludge
This investigation was carried out at the field station of the Faculty of Agriculture, Alexandria University at Abis area during the period (1999-2001). Composted sewage sludge was collected from Alexandria General Organization of Sanitary Drainage (AGOSD) of Alexandria city in February, 1999. The chemical composition of sludge and physical and chemical characteristics of the soil were determined according to Page et al. (1982) and are presented in Amin and Sherif (2001).

On March 1999 (before the experimental study started), sewage sludge was amended to the soil at the rates of 0, 10, 20, 30 and 40 T/F and was planted with Zea mays. After harvesting, the plot was divided into two subplots; one received a second sludge addition at the previous rates while the other was left without further addition to compare the effect of the residual and accumulation treatments. Vicia faba Giza 461 was sown in the two subplots (following Zea mays) on November, 1999. On July, 2000 another study using Zea mays was grown following Vicia faba Giza 461 on the same soil without any additions. In the following year (2000-2001) a third experiment was conducted by using Vicia faba on the same soil but the subplot which have the second sludge addition received a third sludge addition at the same rate.

Soils Sampling and Analysis
Surface soil samples were collected from each plot, before cultivation and after harvesting at soil depth 20 cm. The collected samples were air-dried, ground, passed through 2 mm sieve and stored in polyethylene bags for analyses. Heavy metals analysis of AB-DTPA (Ammonium Bicarbonate-diethylene triamine-pentacetic acid) extracts were made for heavy metals analysis using Atomic Absorption Spectrophotometer (Parken Elmer model 3300).

Plant Analysis
Thirty, 45 and 75 days of sowing and mature plant samples of Vicia faba were collected . Also, mature seeds from each plot were sampled, washed with distilled water, oven dried at 70°C, then stored in paper bags for analysis. Leaves and mature seeds from each treatment were homogenized and wet digested using concentrated sulfuric acid and hydrogen peroxide (FAO, 1980) and analyzed for extractable heavy metals.

Chlorophyll Estimation
Seventh leaf stage samples were collected, washed with tap water, then with distilled water and dried between paper towels. Known weight (about 0.1 g fresh weight) of leaves were immersed in 10 mL N, N-dimethyl formamide (DMF) and kept overnight at 4°C. After incubation, Chlorophyll a and b was determined in the extract according to Inskeep and Bloom (1985). Absorption spectra of the extracts were measured at 645 and 647 nm using spectrophotometer (uk) using the following formula:

Chlorophyll a = 10.4 E645-E647
Chlorophyll b = 19.6 E647-E645

Morphological Measurements
After 4 weeks of planting, seedling height and number of tillers per plant were determined. At maturity ten characters were recorded: plant height, seed index (100 seeds weight), number of seeds pod-1, number of sterile and fertile seeds, seeds pod-1, seed color, hilum color and nodules number and activity. The nodule activity (efficiency) was measured according to the density of red colored of the nodules; dark red is highly efficiency, light red is low and colorless in not active. Seed density was calculated by dividing the weight of 10 seeds by its volume (Kharkwal and Chaudhary, 1997).

The rate of root growth day-1 was determined by sowing five replica each of fifteen homogenous seeds in the different treated soil samples in pots. Root tolerance index was calculated after 2 and 3 days of planting (root growth increase expressed as a percentage of the control, Wierzbicka, 1999).

Cytological Studies
Homogenous seeds of Vicia faba were germinated in sewage sludge treatments after soaking and incubation. Three replica root meristems were stained with the conventional Feulgen technique (Sharma and DPhil, 1980); squashed and permanent slides were examined to determine the mitotic index and mitotic distribution, in addition to the rate and types of abnormal dividing (ADCs) and non dividing cells (ANDCs).

Statistical Analysis
Statistical analysis for data were evaluated by applying the analysis of variance and least significant differences test, using COSTAT program.

RESULTS

Effect of Sludge Treatments on Heavy Metals Content in Soil
Using one residual addition, soil heavy metals increased significantly with increasing sludge application rates before cultivation (21 months from 1st addition) (Table 1 ). After harvesting (25 months from 1st addition), the heavy metals content decreased but still higher than the control. In the meantime, cumulative effect of three additions showed that extractable Zn, Cd and Pb increased significantly before cultivation (20 months from 1st addition+12 months from 2nd addition+8 days from 3rd addition) and after harvesting (25 months from 1st addition +17 months from 2nd addition +5 months from 3rd addition).

Table 1: Heavy metal concentrations in the sludge amended soil before Vicia faba sowing and after harvesting using one addition and three cumulative additions
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range test

Table 2: Heavy metals concentrations in leaves and seeds of Vicia faba plants using one addition and three cumulative additions
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range test, -: Missed data, nd: Not detected

Effect of Sludge Treatments on Heavy Metals Content in Plant
One residual addition significantly reduced the concentration of Cu in leaves in all sludge treatments (Table 2). However, the three cumulative additions reduced Cu content in seeds.

Table 3: Effect of different rates of sludge treatments on yield components of Vicia faba
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range test

Table 4: M2 seed characters of Vicia faba grown on soil amended with sludge
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range test

Effect of Sludge Addition on Plant Yield and Nodule Formation
One Residual Addition and Two Cumulative Additions Effect
Some sludge residual treatments increased the mature plant height. In the meantime, the number of tillers per plant for the two additions treatments was significantly higher than that of the residual treatment (mean treatments were 8.75 and 7.81, respectively). The number of seeds per pod recorded the highest value by 10 T/F of the one residual addition, while the lowest value recorded by 20 T/F of the two cumulative additions. On the contrary of that, seed index increased significantly. Generally, increasing of sludge rates increased significantly the fresh and dry weights of shoot and root (Table 3).

One residual addition treatments showed low sterility percentage compared to the two cumulative additions (Table 4). Seeds with white hilum were recorded in three treatments only. M2 Seed coat color was found to be uniform green or red and blotched black, beige or red instead of brown after sludge treatments (Table 4). Mottled, green and red colors were also found in 17 and 52% of seeds after residual and cumulative treatments, respectively.

The number of mature nodules plant-1 exhibited significant increase in all residual treatments except at 10T/F (Table 5). Nevertheless, the lowest treatment of two cumulative additions recorded the highest significant increase. In the same time, while the fresh and dry weights of nodules plant-1 increased significantly by all sludge treatments, the levels of nodules efficiency percentage decreased (Table 5).

Table 5: Effect of different rates of sludge treatments on nodules of Vicia faba
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range test

Table 6: Effect of sludge treatments on mature plant productivity of Vicia faba
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter are non-significantly different at 5% level according to Duncan´s multiple range test

Table 7: Effect of different rates of sludge treatments on root and shoot fresh and dry weights of Vicia faba
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range test. FW: Fresh weight, DW: Dry weight

Cumulative Effect of Three Additions
The different yield parameters increased significantly by all sludge treatments (Table 6, 7). This was accompanied by the decrease of the fresh weight of shoots after 75 days.

The number of nodules per plant was significantly increased in most sludge treatments (Table 8). However, the mean percent of efficiency (high and low) was higher at one residual addition than at repeated applications of sewage sludge.

Effect of Sewage Sludge on Chlorophyll Mutation and Photosynthetic Pigment
Table 9 indicated that chlorophyll b content increased significantly by 10 and 40 T/F one residual addition treatments and 20 and 30 T/F three cumulative additions treatments.

Effect of Sewage Sludge on Cell Division and Chromosomes
Root Growth, Mitotic Activity, Phase Distribution and Cell Aberrations
Root tolerance index (Fig. 1 ) increased by sludge treatments and by increasing the addition rates, which indicated that stimulation of root growth occurred.

The effect of sludge residual treatments and three cumulative additions on mitosis and chromosomes is presented in Table 10. An increase in mitotic activity (MI) was found in all treatments. The increase in cell division by sludge treatment was accompanied by an increase in the frequency of metaphase. As indicated from Table 10, metaphase-anaphase transition was increased generally by sludge treatments.

Table 8: Effect of different rates of sludge treatments on nodules number, efficiency [low (+), high (++)] and fresh (FW) and dry weight (DW) of Vicia faba
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range test. nd: Not detected

Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Fig. 1: Root tolerance index of root growth after two and three days germinated seeds grown on sludge amended soil

Table 9: Chlorophyll a and b in Vicia faba
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range test

Table 10: Root growth, mitotic activity, phase distribution and abnormal dividing (ADCs) and non dividing cells (ANDCs) of Vicia faba primary roots grown on sludge amended soil
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range, M/P: Metaphase Prophase ratio, A: Anaphase, T: Telophase, M: Metaphase, MI: Mitotic index, ADCs: Abnormal dividing cells, ANDCs: Abnormal nondividing cells

Aberration Spectrum
The percentage of abnormal dividing cells was increased by the three cumulative additions treatments (Table 11, Fig. 2a-f). Chromosome stickiness was the most dominant abnormality apparent in all phases.

Drastic changes in spindle apparatus and centromeres leading to impairment of chromosome alignment (scattered chromosomes) on metaphase plate, abnormal spindle orientation, abnormal chromosome movement and c-mitosis also occurred (Table 11, Fig. 2).

Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Fig. 2: Non- dividing and dividing cells of Vicia faba root meristems affected with different rates of sewage sludge. (a) Interphase with large micronucleus, (b) Binucleate with two small micronuclei, © Sticky metaphase, (d) Fragmented metaphase (e) Sticky multigroups metaphase and (f) Telophase with bridge and with sticky and ring laggards

Table 11: Effect of different rates of sewage sludge treatments on the chromosome behavior of root meristems of Vicia faba plants
Image for - Effect of Residual and Accumulative Sewage Sludge on Heavy Metals Bioaccumulation: Gene Action and Some Yield Parameters of Vicia faba
Mean values within a column followed by the same letter(s) are non-significantly different at 5% level according to Duncan´s multiple range test. Abn: Abnormal, DC: Dividing cells, NDC: Non dividing cells, Chrom. Mov: Chromosome movement, Pi: Prophase with micronucleus, Ori: Orientation

Multigroups in metaphase and anaphase-telophase cells and abnormal group orientation represented other forms of spindle disturbance were also scored and recorded. Micronuclei were found to increase by additive sludge treatments (Table 11, Fig. 2).

Generally, chromosome fragments are frequently increased after sludge treatments. In the present study, a considerable number of micronuclei were also recorded in all treatments.

Abnormal interphase cells increased by sludge treatments and gave the highest values by the cumulative treatments (Table 11). The highest percentage of multinucleate and binucleate cells was recorded for the 40 T/F of one residual treatment. Dissolution of the chromatin material was also observed in some cells. Most of the aberrant dividing cells contained one type of aberrations. However, 1.2 aberrations per cell was found after 20 and 30 T/F (Table 11).

DISCUSSION

Effect of Sludge Treatments on Heavy Metals Content in Soil and Plant Parts
Soil heavy metals increased significantly with increasing sludge application rates before cultivation. However, it decreased after harvesting but was still higher than the control. These results were in agreement with McBride et al. (2000), Sherif et al. (2000), Granto et al. (2004) and Ashworth and Alloway (2004).

The concentration of Cu, Pb and Zn in leaves in all one residual addition sludge treatments were significantly reduced, this is similar to that reported by Tiffany et al. (2000), Antoniadis and Alloway (2002) and Martinez et al. (2003). However, the three cumulative additions reduced Cu content only in the seeds. This may be due to its accumulation in roots or its chelation by the high organic matter content of soil (Shorrocks, 1984; Amin et al., 2007; Bose et al., 2008). Also, Kim et al. (2007) stated that significant Cu loss in soybean seeds was related with the degree of Cu mobilization.

Effect of Sludge Addition on Plant Yield and Nodule Formation
Seed yield is a complex trait and quantitatively inherited with low heritable value (Saravanan et al., 2000; El-Shakhess and Sommer, 2003). So, high yield ability must be associated with its components; number of pods/plant, plant height, length of fruiting structure and number of tillers plant-1 (Zhang and Yishow, 2002).

The number of tillers plant-1, increased significantly in the two additions treatments than that of the one residual treatment. While, seeds per pod and seed index increased in one residual addition than that of two addition treatment. Generally, increasing of sludge rates increased significantly the fresh and dry weights of shoot and root. This may be due to increasing the concentration of macronutrients (N and P) of plants (Khalil et al., 2000; Meyer et al., 2004).

Seeds with white hilum were recorded in three treatments only. Higgins et al. (1988) and Bould and Crofton (1987) found that hilum color of most recent varieties is controlled by two alleles (N and n) with the black allele being completely dominant to white. Also, M2 seed coat color was found to be green, red and blotched black (mottled), beige or red instead of brown after residual and cumulative sludge treatments. Ricciardi et al. (1985) reported that the mottled seed coat color is dominant over the uniform one.

Therefore, it can be concluded that the sludge treatments may mutate the N allele to n revealing that the genotype of black hilum was either homozygous for N (NN) or heterozygous (Nn). Also, all two cumulative additions and 20 T/F of one residual addition were more effective on gene mutations of seed coat color.

Toxicity threshold of sewage sludge application on plants and soil microorganisms was estimated herein by counting the number and efficiency of root nodules. The inhibition of nodulation may be due to several sludge additions. This is disagree with Iqbal et al. (2007), who stated that soil with residual sludge provide better nodule formation of L. leucocephala. In the same time, while the fresh and dry weighs of nodules/plant increased significantly by all sludge treatments, the levels of nodules efficiency percentage decreased. This can be interpreted as the plant got its all nitrogen requirement from sludge (Chaudrie et al., 2000) and in the meantime the heavy metals content of sludge caused toxicity to soil microorganisms. This would lead to the decrease of nodule efficiency percentage as well as affecting host signal transduction genes decreasing host-microsymbiont interaction (Werner, 1992; Ibekwe et al., 1996; Siddique and Loss, 1999). Accordingly, nodulation of plants can be used as sensitive bioindicator towards toxic chemicals in soil by nodule genes (nod genes, Werner, 1992).

The significant increase in different yield parameters by all sludge treatments of three additions may indicate that the crop fulfilled its requirements of N, P and essential nutrients from sewage sludge (Korcak and Fanning, 1985). However, the decrease of the fresh weight of shoots after 75 days may be due to the increase of soil salinity (induced by cumulative sludge addition) leading to the decrease of yield as reported by Sherif et al. (2001). Anyhow, Kirkham (1976) attributed the drop of yields at high rates of sludge additions to the production of phytotoxic substances during sludge decomposition.

Effect of Sewage Sludge on Chlorophyll Mutation and Photosynthetic Pigments
Many gene mutations are known to affect the main pigments associated with photosynthesis which form chlorophyll a and b protein complexes (King, 1991). Chlorophyll b content and chlorophyll a/b ratios increased significantly by one residual addition and three cumulative additions. The increase in total chlorophyll content of the leaves could be attributed to the increase in nitrogen content of the sludge amended soil. Similar results were obtained by Amin and Sherif (2001). However, mutations also occurred in chlorophyll b genes (chlorina f2; King, 1991) more than those of chlorophyll a giving high a/b ratio and revealing that the conversion of chlorophyll a to chlorophyll b was affected (Westhoff and Kloppstech, 1998).

Effect of Sewage Sludge on Cell Division and Chromosomes
Parameters such as root growth, frequency of mitosis and abnormal cell divisions were analyzed to estimate the cytotoxicity, genotoxicity and mutagenecity of environmental mutants.

Stimulation of root growth (root tolerance index) occurred by sludge treatments and by increasing the addition rates, which indicated that this stimulation may affect certain metabolic processes (Vygas et al., 1985) leading to the increase of endogenous growth regulators (Grover and Tejpaul, 1981; El-Anatably et al., 1994). Since root tolerance index is a way to asses the phytotoxicity (Wierzbicka, 1999), herein, sewage sludge treatments enhanced root tolerance especially after the repeated applications. An increase in mitotic activity (MI) was also found in all treatments, which proved that the addition of sludge induced an increase in both cell division and cell elongation. However, herein, there is no relation between high MI and root elongation. This means that sludge treatments disturb the growth hormones responsible for cell cycle and cell elongation (Chen, 2001; Mouchel et al., 2004).

Reversible protein phosphorylation is an important regulatory mechanism in the control of cell cycle progression. Central to this regulation are the Cyclin-Dependent Kinases (CDK) and their activating proteins-cyclins. The Mitotic Cyclin-Dependent Kinase (MCDK) complex consists of a Cdc2 catalytic subunit and a cyclin B regulatory subunit. A critical event in the regulation of Cdc2/cyclin B involves its dephosphorylation by Cdc25C (Xiaoqi Liu and Erikson, 2002). The increase in cell division by sludge treatments was accompanied by an increase in the frequency of metaphases, in the present study, might be due to the activation of spindle checkpoint genes through cyclin B leading to a general increase in M/P ratio (Tsukaya and Beemster, 2006). In the meantime, the increase of rate of biosynthesis of nucleic acids and Mitosis Promoting Factors (MPF) would lead also to the acceleration of the rate of cell division (Kornberg and Baker, 1992). Recently it has been shown that when the tension associated with proper attachment is absent the kinetochore becomes phosphorylated and anaphase is delayed. It has been proposed that the kinetochore protein dephosphorylation caused by tension is the all-clear signal to the checkpoint. The involvement of Cyclin B/cdc2 in the events surrounding this mitotic checkpoint have yet to be documented, but the fact that Cyclin B/cdc2 is degraded at the metaphase-anaphase transition, suggests that MPF is the direct target of this checkpoint (Xiaoqi Liu and Erikson, 2002).

Herein, metaphase-anaphase transition was increased generally by sludge treatments, probably due to stimulation of the synthesis of Anaphase Promoting Complex (APC) which is a check point protein required to reduce B-cyclin levels as cells pass through anaphase and telophase regulates the transition, during mitosis (Yu et al., 2000). Moreover, the APC/C is required to coordinate chromosome separation at the metaphase-to-anaphase transition in a process that requires Cdk activity (Harper et al., 2002).

In other word, Abnormal spindle (Asp) is affected by Microtubule-Associated Protein (MAP), found at the poles of mitotic spindles. Abnormal spindle mutants exhibit a mitotic metaphase checkpoint arrest with abnormal poles; that reflects a requirement for Asp for the integrity of microtubule organizing centers (MTOCs). The absence of a strong spindle integrity checkpoint enables Asp mutant cells to proceed through anaphase and telophase (Mendes-Bonato1 et al., 2006). However, the central spindle region is not correctly organized and cells frequently fail to complete cytokinesis.

The increase in the percentage of abnormal dividing cells by the three cumulative additions treatments could be due to the cumulative effect of genotoxic compounds found in sewage sludge, high heavy metals content, household detergents and chlorophenols (Ivanova et al., 2005). Chromosome stickiness which were recorded in appreciable percentage in this study and increased by all sludge treatments is probably caused through immediate reactions with DNA causing DNA-DNA or DNA-protein cross linking (El-Kodary et al., 1990; El-Ghamery et al., 2000; Kovaleva, 2008). consequently, sticky chromosome bridges were also recorded.

Spindle disturbances caused by drastic changes in spindle apparatus and centromeres leading to impairment of chromosome alignment (scattered chromosomes) on metaphase plate, abnormal spindle orientation (and split spindle and then Multigroups), abnormal chromosome movement (laggards and then micronuclei) and c-mitosis also occurred. The mechanistic background to spindle disturbances with compounds might be partially based on the partitioning (sharing) of some household chemicals and metals into hydrophobic compounds of the cell (Onfelt, 1987). In addition, the presence of Ca+2 (El-Bagouri, 1999) as well as chlorophenols (UNESCO, 1996) in sewage water and consequently sewage sludge, could be the cause of the occurrence of c-mitosis. Dipankar and Crothers (1986) suggested that Ca+2 neutralizes the negative charges on chromatin fibrils, which would increase chromosome repulsion and chromatin condensation causing c-mitosis. Also, scattered chromosomes, herein, might be a consequence of the increase of Ca+2 concentration of the soil (Amin and Sherif, 2001) and afterwards, at the cellular level during cell division affecting glutathione and ATP contents causing the disruption of spindle mechanism (Onfelt, 1987). Heavy metals like Pb was found to be associated with the spindle protein tubulin (Johnson, 1998) leading to inhibition of polymerization and/or microtubule formation (Aardema et al., 1998) disrupting chromosome movement (Oshimura and Barrett, 1986). Moreover, Pb could block the combination of spindle microtubules with the associated proteins essential for the sliding function of microtubules during anaphase and thus disrupt the movement of the chromosomes (Oshimura and Barrett, 1986). However, in addition to all previous causes for spindle abnormalities, the possibility of mutation of one or more of the genes responsible for the assembly and function of microtubules (Onfelt, 1986) could not be neglected. The abnormal orientation of some chromosomes was found to be due to altered quantity and quality of kinetochore heterochromatin (Graves and Zelesco,1988) or mutation of one or more of the genes responsible for pole determination (Rajendra and Bates, 1981). Micronuclei are true mutagenic indicators of the exposure to genotoxic agent (Minissi et al., 1998; Grover and Kaur, 1999; Kovaleva, 2008) were found

herein to increase by additive sludge treatments. The increase of multinucleate and binucleate interphase cells by sludge treatments is an indication of the inhibition of cell wall formation at the end of mitosis.

In conclusion, this study suggests that sewage sludge is good source of nitrogen content and macro- and micronutrients leading to high plant productivity. This was reflected as increased mean mitotic index. However, it also contains heavy metals and its application lead to harmful accumulation in soil and plants but no phytotoxic effects were detected. However, chromosome aberrations and gene mutations occurred and were reflected as: (A) the disturbance of balance between chlorophyll type a and b, (B) affecting nodulation genes and © occurrence of gene mutations of genes that regulate the seed hilum and coat color. Taking into consideration, the long-term applications of sewage sludge would carry a risk of progressive heavy metal toxic levels. This might be considered as a yellow but rather a red light of warning to the hazardous effect of using sewage sludge as a recycle of wastes.

Thus regular sludge, soil and plant analysis are needed to check for low levels of sewage sludge-borne metals used as soil amendments. Further studies should be encouraged towards more investigations on the different methods for secondary treatments of sewage sludge reducing hazardous compounds (e.g., heavy metals and house hold detergents) to benefit from high nitrogen content and micro- and macro nutrients found in sewage sludge. This type of study might be useful to urge decision makers to design guide lines to the producers and users of sewage sludge in agriculture.

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