HEAVY METAL TOXICITY: EFFECT ON PLANT GROWTH AND METAL UPTAKE BY WHEAT, AND ON FREE LIVING AZOTOBACTER

RANA ATHAR1 and MASOOD AHMAD2 1 Institute of Agriculture, Aligarh Muslim University, Aligarh (U.P.) 202002, India; 2 Department of
Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh (U.P.) 202002, India ( author for correspondence)

(Received 4 July 2000; accepted 1 August 2001)

Abstract. A pot study was conducted to investigate the toxic effects of certain heavy metals on the plant growth and grain yield of wheat (Triticum aestivum L.). The results revealed that heavy metals brought about signicant reductions in both parameters, Cd being the most toxic metal followed by Cu, Ni, Zn, Pb and Cr. Moreover, the presence of Cd in the soil resulted in the maximum inhibition (84.9%) in the number of free living Azotobacter chroococcum cells over the control. The phytotoxicity was apparently due to the susceptibility of the free living Azotobacter chroococcum cells to the toxic doses of heavy metals. Protein content decreased from 19.071.4% in metal exposed plants at metal concentrations equivalent to those found in polluted soil. Metal uptake by grains was directly related to the applied heavy metal with greater concentrations of metals found in cases where metals were added separately rather than in combinations. The toxic effects on the plant growth, nitrogen content in plant parts, and protein content in grains, exerted by two metals in combination were not additive, but rather only as severe as for the most toxic metal alone. Keywords: heavy metals, heavy metal uptake, nitrogen xation, phytotoxicity, wheat

1. Introduction Industrial wastes are a major source of soil pollution and originate from mining industries, chemical industries, metal processing industries and others. These wastes include a variety of chemicals like heavy metals, phenolics etc. (Mueller et al., 1989; Van Assche and Clijsters, 1990). Use of industrial efuent and sewage sludge on agricultural land has become a common practice in India as a result of which these toxic metals can be transferred and concentrated into plant tissues from the soil. These metals have damaging effects on the plants themselves and may become a health hazard to man and animals. Above certain concentrations and over a narrow range, the heavy metals turn into toxins (Babich and Stotzky, 1980; Babich et al., 1982). Moreover, these metals adversely affect natural microbial populations leading to disruption of vital ecological processes (Sterritt and Lester, 1980; Nriagu and Nieboer, 1988; Brynhildsen and Rosswall, 1997). Currently, microorganisms are being used as potential bioindicators for the assessment of chemical risk to the ecosystem (Bitton and Dutka, 1986). The effects of heavy metals on the growth of plants and microorganisms have been investigated
Water, Air, and Soil Pollution 138: 165180, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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TABLE I Physiochemical properties and heavy metal concentration of the test soil used in this study Texture Type pH Cation exchange capacity (CEC) (cmol kg1 ) Water holding capacity Organic matter Organic carbon Available nitrogen (kg ha1 ) Anion exchange capacity (AEC) (cmol kg1 ) Lead (Pb)a Zinc (Zn) Copper(Cu) Nickel (Ni) Cadmium (Cd) Chromium (Cr)
a All metal concentrations are in mg kg1 . b Standard deviation. c N.D. = Not detected.

Sandy clay loam Alluvial 7.7 11.7 40.6 0.62 0.36 170 5.1 30.02.0b 96.02.0 18.62.1 12.92.5 N.D.c 15.02.0

by several workers (Skujins et al., 1986; Coppola et al., 1988; Lorenz et al., 1992; Baccouch et al., 1998). Abiotic stresses like heavy metal stress, air pollutants stress etc negatively affect processes associated with biomass production and grain yield in almost all major eld grown crops (Agarwal et al., 1999). Every metal and plant interact in a specic way, which depends on several factors such as type of soil, growth conditions and the presence of other ions. The objective of this study was to examine the toxic effect of heavy metals on a free living nitrogen xer viz. Azotobacter sp. as well as on the growth of wheat plants to gain an insight on the loss of agricultural productivity of a very important cereal crop.

2. Materials and Methods The soil in which the experiments have been conducted was a sandy clay loam and had received no exogenous input of metals. The physiochemical properties and the heavy metal concentrations of the soil are given in Table I. The soil was sieved (<2 mm) and homogenized and the test heavy metals were added as the solutions of their chloride salts. The amounts of heavy metals added were equivalent to normal, half and double the concentrations found in the polluted soil in

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TABLE II Amounts of heavy metals added to the soil at various dose levels Heavy metal Concentration of heavy metals (mg kg1 ) at 0.5 Pb Zn Cu Ni Cd Cr 97.09 2558.7 367.7 150.9 6.2 37.23 1 194.18 (100.6)a 5117.4 (1158.0) 735.5 (107.2) 301.9 (112.6) 12.4 (5.0) 74.46 (20.1) 2 388.36 10234.8 1471.0 603.8 24.8 148.92

a The values in parentheses indicate the concentrations

of bio-available forms of heavy metals.

Aligarh city receiving industrial efuents of lock manufacturing and plating industries for over a decade and are given in Table II (R. Athar and M. Ahmad, personal communication). 2.1. P OT EXPERIMENTS A pot experiment was conducted with wheat, Triticum aestivum L. var. PDW 154, as test crop. Three and a half kilograms of soil were taken in each pot for the different treatments and three replicates were taken for each treatment. An extra set of pots which contained no added heavy metals were also taken which served as a control. The heavy metals Zn, Pb, Ni, Cd, Cr and Cu singly and in combinations were added once as chloride salts in solution to the soil before sowing. Sufcient water was added to bring the soil to 50% of its water holding capacity. The soil was preincubated for 2 weeks before sowing, and was also fertilized with 120:60:50 kg ha1 of nitrogen, phosphorus and potassium (NPK). Wheat seeds obtained from the Division of Genetics, the Indian Agricultural Research Institute (IARI), New Delhi were surface sterilized in 0.1% mercuric chloride solution and washed with six changes of sterile distilled water. The carrier based strain inoculant of Azotobacter chroococcum (1.5 1010 cells g1 ) obtained from the Indian Agricultural Research Institute (IARI), New Delhi was used to treat the seeds of wheat before sowing at

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the rate of 1 g per 5 g of seeds approx. (Vincent, 1970). Four seeds of wheat were sown in each pot. Seedlings were thinned to two plants per pot, and plants were watered as and when required. The pots were randomised on alternate weeks to minimize any positional effects. 2.2. B IOMASS PRODUCTION The test plants for biomass production were harvested after 90 days of germination. Roots and shoots were dried at 80 C for 18 hr and then weighed separately. The grade of growth inhibition (GGI) was evaluated by the comparison of dry matter production of metal treated and control plant tissues (Purves, 1985). 2.3. G RAIN YIELD Grain yield was also recorded at the harvest of the crop. 2.4. E STIMATION OF AVAILABLE SOIL NITROGEN Available soil nitrogen of the treated and control soil was estimated by the Kjeldahl method using alkaline permanganate (Subbiah and Asija, 1956; Ghosh et al., 1983) after about four weeks of germination of seeds. The procedure involves distilling the soil with alkaline potassium permanganate solution and determination of the ammonia liberated which serves as an index of the available nitrogen status. 2.5. Azotobacter COUNT IN METAL TREATED AND UNTREATED SOIL The soil dilution and plate count method of Timonin (1940) was used for counting the Azotobacter population in the rhizosphere region of wheat plants after about 90 days of sowing of seeds. The wheat plants were carefully uprooted and brought to the laboratory under aseptic conditions. The roots were removed and were transferred along with adhering soil particles into a ask having sterilized distilled water. After thorough shaking of the ask the roots were removed and the rhizosphere soil sample was serially diluted and a 0.1 mL aliquot from the nal dilution was poured in sterilized petri plates containing 2025 mL of nitrogen free Jensens agar medium and was spread with a sterilized glass spreader. The plates were incubated at 282 C for 34 days, and the resulting colonies were identied and scored. 2.6. E STIMATION OF NITROGEN IN PLANT PARTS At harvest, the shoots and roots were dried at 80 C for 18 hr, weighed and ground to pass through a 2 mm pore size stainless steel sieve and the nitrogen in roots and shoots was determined by the Kjeldahl method (Bremner, 1965).

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2.7. E STIMATION OF PROTEIN CONTENT IN GRAINS At harvest, grains were dried, weighed and ground. The protein content was then determined by the Kjeldahl method (Bremner, 1965). 2.8. H EAVY METAL UPTAKE BY GRAINS The heavy metal concentrations of the most toxic (Cd) and the least toxic (Cr) metals found in this study in the grains of wheat were determined in hot concentrated HNO3 digests of the ground grain samples. Heavy metals were determined by Atomic Absorption Spectrophotometer (AAS) model Unichem FP 1900 series. 2.9. S TATISTICAL ANALYSIS The results were analysed statistically by analysis of variance and critical difference (CD) at 5% level according to standard procedures (Chaddha, 1990). 3. Results Toxicity of heavy metals on the growth of wheat is presented in Table III. These data indicate that the heavy metals were toxic to the growth of wheat plants. Shoots of plants had noticeable and gradual stunted growth. These symptoms were more obvious in treatments containing Cd alone and a combination of all the heavy metals (Table III). The reduction in dry weight of wheat plants as a result of treatment with heavy metals was minimum with Cr and Pb. Phytotoxic effect of heavy metals was in the following order: Cd > Cu > Ni > Zn > Pb > Cr The higher the concentration of heavy metal in the soil, the greater was the toxic effect on the plant. The results in Table III show that the effects of combinations of two metals were not additive, rather the effects were only as severe as the most toxic metal alone. The lowest reduction in the grain yield was recorded with Cr at all the test doses and the highest was recorded in the plants having been treated with all the test metals (Table III). Decrease in the grain yield was less than 40% with Cr as against 83.9% by Cd at 2 concentration. Perusal of the data in Table III clearly indicates that though Cr appeared to be the least toxic metal, it also led to substantial losses (>40%) in the dry matter at 2 concentration. Results in Table III show that the dry weights of shoot and root of wheat plant, respectively, were reduced 63.4 and 70.5% by Cd; 58.5 and 55.8% by Cu; 51.2 and 46.1% by Ni: 26.3 and 29.1% by Pb; 31.7 and 39.7% by Zn; 17.0 and 13.8% by Cr at 0.5 concentration. Zn appears to be less toxic than Cu and Ni despite the relatively higher amount used in the study (Table II).

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TABLE III

Dry matter and grain yield of wheat plant exposed to various concentrations of heavy metals added either separately or in combination Heavy metal treatment Dry shoot weight (g/pot) at 0.5 1 2 Dry root weight (g/pot) at 0.5 1 2 Grain weight (g/pot) at 0.5 1 2

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3.02 2.80 1.70 3.40 2.00 1.50 1.50 2.60 1.74 1.05 0.80 Sig.a 0.78 0.71 0.69 0.65 0.65 0.60 1.40

2.50 2.10 1.30 2.70 1.70 1.10 1.10 2.00 1.40 0.84 0.75 4.10 Sig.

1.80 1.73 1.00 2.21 1.50 0.90 0.86 1.75 1.01 0.63 0.43 Sig.

2.41 2.05 1.50 2.93 1.83 1.00 1.05 2.05 1.23 0.83 0.40 Sig.

1.81 1.71 1.10 2.05 1.60 0.80 0.85 1.45 0.91 0.45 0.31 3.40 Sig.

1.30 0.91 0.68 1.78 0.82 0.63 0.49 1.08 0.85 0.35 0.19 Sig.

4.02 3.64 2.28 4.71 2.83 1.87 1.78 2.71 2.05 1.06 0.70 Sig.

3.75 3.16 1.54 4.09 2.34 1.30 0.94 2.30 1.74 0.77 0.36 5.30 Sig. 1.40

3.03 2.51 1.05 3.53 2.05 0.85 0.55 1.77 1.16 0.40 0.29 Sig. 0.72

Pb Zn Cu Cr Ni Cd Ni+Cd Ni+Cr Cr+Cd Ni+Cr+Cd Ni+Cr+Cd+Cu+Zn+Pb Control Statistical analysis (F test) CDb at 5%

Values are mean of three replicates. a Sig. = Signicantly different over control. b CD = Critical difference.

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TABLE IV Available soil nitrogen (mg kg1 ) in the rhizosphere soil of wheat under different metal treatments Heavy metal treatments Available soil nitrogen at various concentrations of heavy metal treatment 0.5 Pb Zn Cu Cr Ni Cd Cr+Cd Ni+Cr Ni+Cd Ni+Cr+Cd Ni+Cr+Cd+Cu+Pb+Zn Control Statistical analysis (F test) 95.05.0a 94.45.1 91.63.6 96.04.0 92.21.6 88.66.3 86.53.7 90.21.3 85.41.7 84.32.0 82.32.5 N.S.b 1 92.62.1 90.21.3 88.76.2 94.22.0 89.35.3 83.43.0 82.11.0 88.46.2 81.12.1 80.02.0 79.62.5 114.06.0 N.S. 2 89.35.3 91.02.6 86.23.7 92.54.3 86.53.2 80.73.0 81.02.0 80.12.2 79.11.8 77.03.0 72.81.0 N.S.

Values are mean of three replicates. a Standard deviation. b N.S. = Not signicant.

Results in Table IV show that the reduction in nitrogen content in the soil with metal treatments at all the three concentrations was found to be statistically insignicant. There was hundred percent inhibition of Azotobacter population with the treatment having the combination of six metals at all the test doses, though some cells were present in the soil treated with the combination of three metals i.e. Ni + Cr + Cd. (Table V). A marginal toxicity towards the Azotobacter population was observed with Cr while the highest bactericidal effect was obtained in case of Cd. Even at concentrations equal to half the usual concentrations detected in contaminated soil (i.e. 0.5 ), the metals Cd, Cu and Ni signicantly inhibited the Azotobacter population. Although Cr did not exhibit the microbiocidal behaviour at the levels it was maximally present in the polluted soil, it could bring about pronounced effect in the Azotobacter count (TAC) at 2 concentration (Table V). The order of toxicity of various heavy metals was as under: Cd > Cu > Ni > Zn > Pb > Cr

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TABLE V Total number of Azotobacter chroococcum cells in the rhizosphere of wheat expressed in terms of standard deviation and mean log values Heavy metal treatment TAC ( 102 CFU g1 ) at the metal concentrations of 0.5 Pb Zn Ni Cu Cd Cr Ni+Cr Cr+Cd Ni+Cd Ni+Cr+Cd Ni+Cr+Cd+Zn+Pb+Cu Control Statistical analysis (F test) CDd at 5% 40.96.6a 39.08.8 36.43.8 26.75.3 21.35.9 44.05.3 35.06.9 20.43.3 11.11.8 8.52.6 N.D.b Sig.c 21.4 1 28.43.8 22.23.9 19.92.8 16.32.0 15.23.3 35.63.7 19.46.8 11.04.1 8.22.1 5.52.0 N.D. 61.07.6 Sig. 18.7 2 20.75.2 15.33.5 13.84.7 12.31.6 9.22.4 28.43.3 11.31.7 7.03.1 3.81.7 2.80.9 N.D. Sig. 16.4 TAC (log10 CFU g1 ) at the metal concentrations of 0.5 3.54 3.53 3.40 3.30 3.26 3.56 3.50 3.20 3.00 2.70 N.D. Sig. 0.29 1 3.40 3.30 3.20 3.16 3.13 3.43 3.20 3.00 2.83 2.66 N.D. 3.70 Sig. 0.23 2 3.16 3.16 3.10 3.04 2.90 3.36 3.00 2.76 2.50 2.36 N.D. Sig. 0.23

Values are mean of three replicates. a Standard deviation. b N.D. = Not detected. c Sig. = Signicantly different over control. d CD = Critical difference.

The nitrogen content in shoots and roots decreased in metal treated plants. Data in Table VI indicates that there was a signicant reduction in nitrogen (%) in shoots and roots of wheat plants compared with the control. In both the shoots and roots, the percent of nitrogen varied inversely with the amounts of metals added, Cd and Cu causing the greatest effect. The percent of nitrogen in shoot and root was reduced by 68.6 and 79.4% in the presence of Cd at 2 concentration. Data in Table VII indicates that heavy metal treatment under different concentrations resulted in decreased protein content in grains with Cr recording the highest protein content in grains. Protein content was signicantly lower in metal treated grains compared with the control. The metal (Cd and Cr) uptake pattern by wheat grains is given in Table VIII. It was observed that as the concentration of Cd and Cr in the grains increased, the yield was reduced and the metal accumulation in grains was also found to be directly related to that applied to the soil. The concentrations of individual metals

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TABLE VI Percent nitrogen content in shoots and roots of wheat plant as inuenced by different heavy metal treatments Heavy metal treatment % N in shoot at various concentrations of metal treatments 0.5 Pb Zn Ni Cu Cd Cr Ni+Cr Cr+Cd Ni+Cd Ni+Cr+Cd Ni+Cr+Cd+Pb+Zn+Cu Control Statistical analysis (F test) CDb at 5% 1.84 1.52 1.42 1.34 1.22 2.00 2.00 1.33 1.40 1.00 0.72 Sig.a 0.85 1 1.64 1.32 1.20 1.01 0.90 1.86 1.70 1.00 1.20 1.10 0.53 2.30 Sig. 0.63 2 1.42 1.20 0.94 0.90 0.72 1.70 1.51 0.92 0.88 0.70 0.33 Sig. 0.50 % N in root at various concentrations of metal treatments 0.5 1.50 1.30 0.97 0.90 0.86 1.60 1.10 0.96 0.44 0.42 0.28 Sig. 0.50 1 1.10 0.66 0.53 0.62 0.53 1.40 0.91 0.73 0.18 0.35 0.20 1.70 Sig. 0.37 2 0.62 0.50 0.46 0.40 0.35 1.00 0.82 0.60 0.15 0.16 0.11 Sig. 0.29

Values are mean of three replicates. a Sig. = Signicantly different over control. b CD = Critical difference.

in grains were usually found to be greater for metals added separately than in combinations of all metals in the pot.

4. Discussion Treatment of wheat plant with heavy metals resulted in decreased dry matter and grain yield, reduced nitrogen content in plant tissues and lowered protein content in grains. The data in Table III indicates that heavy metals exerted an adverse effect on the growth and yield of wheat plants substantiating the reported phytotoxicity of these metal ions (Woolhouse, 1983). The effects on plants of environmental stresses are determined by the responses of the individual cells in which the integrity of structure and function is affected (Ciamporova and Mistrik, 1993). In the present investigation, Cr was found to be the least but signicantly phytotoxic metal as

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TABLE VII Protein content in wheat grains as inuenced by different heavy metal treatments Heavy metal treatments Pb Zn Ni Cu Cd Cr Ni+Cr Cr+Cd Ni+Cd Ni+Cr+Cd Ni+Cr+Cd+Zn+Pb+Cu Control Statistical analysis (F test) CDb at 5% Protein (%) in wheat grains at 0.5 1 2 18.0 16.2 14.5 13.0 10.7 19.0 15.6 10.0 9.3 9.5 7.2 Sig.a 8.3 15.6 12.5 11.5 11.2 8.1 17.0 12.5 8.7 7.2 8.0 6.0 21.0 Sig. 7.4 13.1 10.0 8.0 9.5 6.2 15.0 10.0 7.0 5.6 6.2 4.0 Sig. 6.5

Values are mean of three replicates. a Sig. = Signicantly different over control. b CD = Critical difference.

compared to other metals added separately in the soil. Low levels of phytotoxicity of Cr(III) have been attributed to its insolubility under most soil conditions (James and Bartlet, 1984), and it did not affect the plant growth unless the concentrations were very large (Smith et al., 1992). Previous investigations indicated that Cr3+ added in sand culture of wheat under glasshouse conditions brought about signicant reduction in biomass, chlorophyll and activities of catalase and peroxidase while it enhanced the acid phosphatase and ribonuclease activities (Sharma and Sharma, 1996). Cd at all levels tested was found to be the most toxic metal for the free living nitrogen xer i.e. Azotobacter chroococcum as well as for the wheat crop and caused the most severe reduction in the dry weight of shoot, root and grain yield followed in order by Cu, Ni and Zn. Previous studies have also demonstrated a relatively higher phytotoxicity of Cd and Cu than that of Zn (Kalyanaraman and Sivagurunathan, 1993). In general, the reduction in the dry weight of roots was more severe than the dry weight of shoots following treatment with heavy metals added separately or in combination (Table III). This is supported by the ndings of Karataglis et al. (1991) who reported that the inuence of relatively higher amounts of Cu, Zn, Pb, Ni, Cr and Cd in wheat cv. Vergina resulted in depressed shoot growth but the most evident

TABLE VIII Cd and Cr contents (mg kg1 ) in wheat grains as inuenced by metal amendments added singly or in combination Heavy metal treatment 0.5 0.220.03a 0.110.01 0.420.07 0.300.08 N.D.b 0.700.17 0.510.18 0.5 0.930.12 0.720.40 Cd Cr Ni+Cr+Cd+Zn+Pb+Cu Control Cd concentration at 1 2 Cr concentration at 1 2 3.700.20 0.640.14 0.110.1 10.00.1 9.63.0
HEAVY METAL TOXICITY

Values are mean of three replicates. a Standard deviation. b N.S. = Not detected.

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symptoms were on roots. Baccouch et al. (1998) showed that the accumulation of carbohydrates in maize shoots treated with Ni might, at least in part be the cause of root growth inhibition. Amendment of soil with the heavy metals at concentrations higher than the normal levels resulted in a striking decrease of root and shoot biomass expressed in terms of dry weight (Table III). It has earlier been reported that increasing Cu supply resulted in decreased root biomass indicating the alterations of physiology and metabolism of test plants (Ouzounidou et al., 1995). Biomass loss (fresh weight) under metal treatment has also been reported by many workers (Lolkema et al., 1984; Verkleij and Prast, 1989). When two heavy metals were added in combinations, instead of an additive effect on the phytotoxicity, the effect was only as severe as for the most toxic metal alone. This might be due to the antagonistic effect of the two metals. Cd is reported to antagonize the inhibitory effect of Zn on the total amount of mineralised carbon (Bewley and Stotzky, 1983). The use of non-symbiotic nitrogen xer, Azotobacter sp. as a bioinoculant is known to benet a wide variety of crops, due to its properties like nitrogen xation, secretion of growth promoting substances, vitamins, anti-fungal metabolites and phosphate solubilization (Mishustin and Shilkinova, 1971; Brown, 1972; Martinez Toledo et al., 1988). Nitrogen xing capacity of biological nitrogen xing organisms (BNFs) has been found to be sensitive to small concentrations of heavy metals added experimentally or in connection with mining (Letunova et al., 1985; Skujins et al., 1986). Table V shows that there was a signicant depletion in the number of Azotobacter cells with complete absence of Azotobacter population in the rhizospheric soil containing all the test metals. This is due to their high sensitivity to heavy metals (Maliszewska et al., 1985). Azotobacter sp. were found to be sensitive to the heavy metals present in the sludge following its application to soil (Martensson and Torstensson, 1996). Previous studies have also shown that as the pH was increased from acidic (e.g. pH 5) to alkaline (pH 8 and 9) value, the toxicity of Cd to bacteria and fungi was increased, suggesting that Cd OH+ , which was formed at these alkaline pH levels, was more toxic than was divalent Cd2+ (Babich and Stotzky, 1977a, b). A signicant decrease in the grain protein content was observed with heavy metal treatment in this study (Table VII). This is in accordance with the ndings of Salgare and Acharekar (1992) who reported that growth performance, as well as pigment, carbohydrate and protein content showed a decreasing trend with increase in the level of industrial pollution. Decreased levels of protein content in heavy metal exposed tissues have been reported by many workers (Gupta, 1986; Satyakala and Jamil, 1997). Relatively strong afnities of heavy metal ions for side chain ligands of protein indicate that enzyme and other functional proteins are one of the primary targets of metal toxicity (Hampp et al., 1976).

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Our results on the metal uptake by wheat grains are given in Table VIII. The uptake of metals in the grains was greater at least in the case of 1 concentration when the metals were added separately than in combination. This is in accordance with the ndings of Smilde (1981) who demonstrated that the total amounts of metals in plant tissues were higher for metals added separately than for combined metals. It is an established fact that the soils and plants under waste water irrigation from various industries contained higher concentrations of heavy metals than those irrigated with tubewell water (Bansal, 1998). Moreover, the heavy metals deposited in soil were bound preferentially to interaggregate soil material, and accumulation preferentially occurred in parts of the soil where plant roots were concentrated and in the forms easily accessible for plants (Wilcke et al., 1998). Cd is of particular concern to the human health as it is concentrated by many cereal and vegetables (leafy and roots) as well as fruits (Wagner, 1993; Cieslinski et al., 1996) which can lead to unexpected human intoxication when it is consumed (Jarup et al., 1998). Albering et al. (1999) showed that the legal standard for Cd as endorsed by commodities act was exceeded in wheat crops grown in soil contaminated with heavy metals and the main exposure pathways for the general population was through the consumption of food crops grown in these soils. The different heavy metals used in this study were found to vary in their phytotoxic effects with Cd being the most toxic and Cr the least toxic. Most of the desirable soil microbiological activities of Azotobacter and nodule forming bacteria of legumes are adversely affected as the acidity increases. Therefore, the alkaline pH of the test soil in the present system presumably make it easier to monitor the toxicity of heavy metals alone. We can conclude that soils contaminated by heavy metals bring about a marked depletion of non-symbiotic nitrogen xers and interfering with nitrogen uptake mechanism in plants which probably leads to substantial losses in dry matter and grain yield of wheat plant.

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