Effect of heavy metal (Zn) on different growth attributes of Zea mays L. in Punjab, Pakistan: A case study

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1 BIOLOGIA (PAKISTAN) June, 2015, 61 (1), PKISSN (Print) ISSN X (On-Line) Effect of heavy metal (Zn) on different growth attributes of Zea mays L. in Punjab, Pakistan: A case study ABRAR HUSSAIN 1, FAHIM ARSHAD 2, NABILA ABBAS 3, *ZAFAR IQBAL KHAN 4, MUHAMMAD MANSHA 1, KAFEEL AHMAD 4, ZILE HUMA 1, IRFAN MUSTAFA 4 & ALIREZA SEIDAVI 5 1 Department of Botany, University of Education, Township Campus, Lahore. 2 Department of Botany, University of Education, Okara Campus, Lahore. 3 Department of Chemistry, University of Education, Township Campus, Lahore. 4 Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan. 5 Department of Animal Science, Rasht Branch, Islamic Azad University, Rasht, Iran. ABSTRACT Present scenario is concerned with the toxic effects of Zinc Sulphate on different growth parameters viz.; germination, growth of the seedling, root shoot length, fresh and dry weights of root and shoot, zinc uptake through roots and shoots and total protein contents of Zea mays L. At all Zinc concentrations, the percentage germination potential, root shoot length, root shoot fresh and dry weights and total protein contents of Zea mays were reduced. The reduction in the total protein contents in roots was prevalent with the increased amount of Zinc in the media. Zinc uptake is more in roots as compared to the shoots. The growth inhibition was attributable to metal s contamination that resulted in disruption of various biochemical and physiological functions. Key Words: Growth Attributes, Zinc Uptake, Zea mays L. INTRODUCTION Heavy metals like Pd, Zn, Cu, and Cd were found to be more or less toxic to plants and microorganism (Kachout et al., 2009; Nies, 1999). The anthropogenic release of heavy metals in the prevailing atmosphere is toxic to the soil surface, natural biological systems, human beings, animals as well as plants (Baccio et al., 2005). Plants provide the main foundation of food chains through which trace metals can be concentrated in human beings (Alfani et al., 1996). Traffic and industrial establishments are the major urban sources of pollutants that are added from various sources (Yilmaz et al., 2006). Soil and water pollution mainly occurred due to industrial wastes including various chemicals such as various phenolic compounds, and heavy metals (Mueller et al., 1989; van Assche & Clijsters, 1990; Iqbal et al., 2001). The agricultural areas in various underdeveloped countries are being commonly practiced by the use of sewage water and industrial effluents which in turn result in their accumulation in the plant tissues from the contaminated soils (Doncheva et al., 1996). Among transition metals, Zinc forms an essential micronutrient in different biochemical as well as physiological processes (Baccio et al., 2005) but its increased concentration may become toxic to biological life. Zinc constitutes the major component of trace metals in agroecosystems (Madhoolika & Rajesh, 2006). In underdeveloped countries, soil contamination in the agricultural fields occurs due to the over usage of sewage sludge and that will be ultimately transferred to human beings through food chain (Mozumder et al., 2008). Zinc (Zn) is the mineral/micronutrient that plants need in very trace amounts (Marschner, 1986; Pahlsson, 1989). Like other heavy metals (Doncheva et al., 1998), excessive accumulation of Zinc in plant tissues causes alteration in vital growth processes. Toxic level of Zinc for different varieties of crops has varied from 64 grams per liter for sorghum to 2000 grams per liter for cotton (Ohki, 1984). The restricted zinc uptake in plants plays a key role in protection against the damaging effect of its excessive amount (Baccio et al., 2005). This protection may results from zinc binding to the walls of root cells or due to an intracellular mechanism (Mathys, 1977). The main purpose of the present investigation was to analyze the accumulation of Zinc in Zea mays L. for assessing the toxic effects of heavy metal on various growth attributes like germination of seed, fresh and dry weights of root and shoot, and root - shoot length, that provide tolerance mechanisms of the Zea mays L. As a major cereal crop in the world, along with rice and wheat (Stephen et al., 2006), the potential ability of uptake of the metal, its presence and effect of metals on the growth of Zea mays was sought out. *Corresponding author: zikhan11@gmail.com

2 158 A. HUSSAIN ET AL BIOLOGIA (PAKISTAN) MATERIALS AND METHODS Certified seeds of Zea mays L. taken from Punjab Seed Corporation, Pakistan were initially surface sterilized through the addition Hydrogen Per Oxide solution followed by thoroughly washing in deionized water before sowing. Further methodology followed Hussain et al. (2013). Various concentrations like 0, 1, 25, 50, 100, 200, 500 mm of Zinc metal were prepared. Earthen pots were used for sowing seeds. Before filling with soil, pots were washed under running tap-water followed by several times decantation in distilled water. Soil was added in the earthen pots followed by propagating ten sterilized seeds in every pot. During sowing, 10 ml. of varied concentrations of Zinc Sulphate were added in all pots. For the germination of seeds, earthen vessels were put into darkness for 3 days at ordinary temperature and pressure. After 72 hours, when germination was ensured, the pots were placed in luminous conditions. After 14 days, seedlings were uprooted. For each experiment, three replicates were made and mean values of all replicates for each experiment were obtained for the compilation of whole data viz., germination of seeds, fresh and dry weight of root and shoots, total protein content and root-shoot length to check the Zinc uptake by the plant. Statistical data analysis was carried out following the research of Iqbal & Rafique (1987). Total Protein Kjeldahl s method was applied for the estimation of total protein contents (Egli, 2008). Ashing of Plant Material For observing the presence of metal content in the shoots as well as roots, the second replicate of fully grown seedlings were taken, followed by the treatments of various concentrations of Zinc. Dry ashing method (Rieth et al., 1974) was employed to estimate the Zinc content. Shoot and root samples were initially dried in Memmert GmbH Oven Model No. D Schwach and measurements were noted. The samples of root and shoot were placed in porcelain crucible and heated lightly for 5 to 7 hours at a temperature of o C in muffle furnace up till whole samples were changed to white ash. The black residues often appeared were thoroughly soaked in concentrated Nitric acid (HNO 3 ) and heated further for two hours for their removal till white ash became apparent. 05 ml of 6N HCl were added to dissolve the ash following heating the sample for the removal of any other residues. Filtrate of the material was obtained from the acid resistant filter paper. The samples were decanted many times with deionized water for the elimination of acidic residues. 100 ml of volume of the material were formed in distilled water. Zinc contents were determined using Atomic Absorption Spectrophotometer (Hitachi, Model ) equipped with a Zinc hollow cathode lamp activated at nm. The calculations of Zinc contents were made by comparing the normal treatments with same reagent that was the addition of 5 m1 of 6N HC1 for each sample. RESULTS Effect on Percentage Germination Germination of seeds was reduced at 1.0 to 500mM concentration of Zinc content. 10% decrease at 1.0 mm and 100% at 500 mm was observed on germination percentage of seeds thus showing significant decrease in relation to control (Table I). Effect on Seedling Growth The deleterious effects were observed on the seedlings growth following Zinc treatment. At all Zinc concentrations, the shoot length was decreased and even at 1.0 mm treatment, the reduction on shoot growth (12.07%) was observed, however 100% decrease at 500 mm was prevalent. In roots, a marked decrease in length was observed comparable to shoots (Table I). A sharp decrease of 93.23% at 200 mm was prevalent in relation to control. At 0.1mM, the root length was decreased (11.5%) while at 500 mm percentage decrease in root length over control was 100%. Effect on Dry Weight of Root and Shoot The data on dry weight of shoot and root was in general similar to the shoot-root length. The dry weight decreased for both shoots and roots. The decrease in shoot length and shoot dry weight was small comparable with roots. The shoot dry weight was reduced to 0% at 1.0 mm concentration comparable to 17.78% of root in control. At 200 mm concentration, the relative decrease was 35.19% and 68.89% for shoot and root, respectively (Table II). Effect on Fresh Weight of Root and Shoot The fresh weight decreased for both shoots and roots. The shoot fresh weight was reduced to 21.99% comparable to 9.1% of root in control at 1.0 mm concentration. At 200mM concentration, marked decrease in fresh weight was recorded to be 88.66% and 65.91% for shoot and root respectively.

3 VOL. 60 (2) EFFECT OF HEAVY METAL (ZN) ON GROWTH OF ZEA MAYS 159 Effect on Total Protein Contents A varied effect on total protein content of shoots and roots was observed following Zinc Zn (mm) treatments (Table III). At mM concentration a marked increase in total protein content was exhibited by the shoots as determined through Duncan s Multiple Range Test of composite means. Table I: Effect of ZnSO 4 on Percentage Germination of Seed and Root and Shoot Length Zea mays L. in 14 days old seedlings Germination (%) Shoots (Mean %age) Root (Mean %age) b a ± a ± c b ± b ± d c ± c ± e d ± d ± f ± e ± e ± f ± f ± f ± /- = % Increase or decrease over control. = Standard Error of the Mean. a, b, c, d, e, f = Values having different alphabets are significantly different from each other at 5% level of significance (Duncan s Multiple Range Test of composite means). Zn (mm) Table II: Effect of ZnSO 4 on Percentage of Fresh and Dry Weight of Root and Shoot of Zea mays L. in 14 days old seedlings Mean Shoot Fresh Weight ±S.E. Mean Shoot dry Weight ±S.E. Mean Root Fresh Weight ±S.E Mean Root Dry Weight a ± a ± a ± a ± b ± a ± b ± b ± c ± b ± c ± c ± d ± d ± d ± c ± e ± e ± e ± d ± f ± c ± f ± e ± ± /- = % Increase or decrease over control. = Standard Error of the Mean. a, b, c, d, e, f = Values having different alphabets are significantly different from each other at 5% level of significance (Duncan s Multiple Range Test of composite means). ±S.E

4 160 A. HUSSAIN ET AL BIOLOGIA (PAKISTAN) Table III: Effect of Zinc Sulphate on Total Protein Content and uptake of Zinc by Root and Shoot in 14 days old seedlings of Zea mays L. Zn (mm) Protein content of Shoot Protein content of Root Absorption Absorption a ± a ± b ± b ± c ± c ± d ± d ± e ± d ± f ± e ± /- = % Increase or decrease over control. = Standard Error of the Mean. a, b, c, d, e, f = Values having different alphabets are significantly different from each other at 5% level of significance (Duncan s Multiple Range Test of composite means). DISCUSSION Zea mays L. provides the most important and one of the essential cultivated cereal crops across the world (Stephen et al., 2006). Therefore research work is being undertaken to study the potential ability of uptake of metals, their occurrence and impact of metals on the growth of Zea mays. The present work deals with the effect of Zinc Sulphate on percentage germination of Zea mays. Zn uptake by shoots and roots and total protein contents of Zea mays has also been investigated. Data revealed the suppression of germination of seeds at all Zinc treatments (Iqbal et al., 1991). A sharp decrease in the crop productivity was recorded in the soils contaminated with Zinc content thus creating a serious problem for agriculture (Johnson & Eaton, 1980). The fresh and dry weight of shoots and roots and root shoot length also exhibited reduction at all Zinc concentrations. Excessive accumulation of Zinc in plant tissues altered the vital growth processes (Doncheva et al., 1996) thus producing negative effects on growth. Dahiya et al. (1987) also described a marked decrease in the dry weight yield of Zea mays. Zinc content affects on soil properties, soil types and species of plants. At lower concentration of Zinc, total protein content of shoots was increased following the decrease at higher concentrations. In roots, total protein content was reduced as the Zinc concentration in the media was increased. Such growth retardation due to heavy metals was accompanied by physiological as well as biochemical processes viz.; rate of photosynthesis, chlorophyll synthesis, protein contents and respiration rate are slowed down. Zn Uptake was recorded more in roots as compared to the shoots (Iqbal et al., 1991). Maximum increase in uptake of zinc was observed at 200 mm concentration. Beyond that limit, roots and shoots showed decrease in the uptake of Zinc content. Such growth retardation was attributable to the loss of various biochemical cum physiological processes in Zea mays. Acknowledgements Authors pay special thanks to Dr. Shahid

5 VOL. 60 (2) EFFECT OF HEAVY METAL (ZN) ON GROWTH OF ZEA MAYS 161 Mehmood, Director, Applied Chemistry Research Centre, Lahore, Pakistan for his guidance and helpful discussion in the compilation of present research work. REFERENCES Alfani, A., Baldantoni, D., Bartoli, G., & Santo, A., Temporal and spatial variation in C, N, S and trace element contents in the leaves of Querecus ilex with the urban area of Naples, Italy. Pl. Nutr., 17: Anonymous, Official Methods of Association of official Analytical Chemists. Association of Official Analytical Chemists, Web site: folder/official-methods-of-analysis-vol.-i.- 15th-Ed.-Association-of-Official-Analytical- Chemists-Arlington-va.-pdf.php. Anonymous, Agricultural statistics of Pakistan ( ), Ministry of Food, Agriculture and Livestock, Government of Pakistan, Islamabad. Baccio, D., Kopriva, S., Sebastiani, L., & Rennenberg, H., Does glutathione metabolism have a role in the defiance of poplar against zinc excess? New Phytol., 16: Dahiya, S. S., Antil, G. S., & Karwasara, S. P. S., Effect of farmyard manure and cadmium on dry matter yield and nutrient uptake by maize. J. Ind. Soc. Soil. Sci., 35(3): Doncheva, S., Nikolov, B., & Ogneva, V., Effect of copper excess on the morphology of the nucleus in maize root meristem cells. Physiol. Plan, 96: Doncheva, S., Copper-induced alterations in structure and proliferation of root meristem cells. J. Plant Physiol., 153: Egli, H. (2008). Kjeldahl Method. BUCHI Labortechnik AG, CH-9230 Flawil, Switzerland. pp: 58. Hussain, A., Abbas, N., Arshad, F., Akram, M., Khan, Z. I., Ahmad, K., Mansha, M. & Mirzaei, F., Effects of diverse doses of Lead (Pb) on different growth attributes of Zea mays L. Agri. Sci., 4(5): Iqbal, J., & Rafique, N., Toxic effects of Ba and C l2 on germination early seedling growth, soluble proteins and acid phosphates in Zea mays L. Pak. J. Bot., 19: 1-8. Iqbal, M. Z., Shafiq, M., & Kausar, A. S Toxic effects of lead and cadmium individually and in combination on germination and growth of Leucaena lucocephala. Pak. J. Bot., 33: Iqbal, J., Hussain, A., Salah-ul-Din, & Salariya, A. M., Cadmium uptake and distribution in young pea seedlings and its effect on growth. Acta Sci., 1(1): Johnson, M. S. & Eaton, J. W., Environmental contamination thljm98rough residual trace metal dispersal from a derelict lead-zinc mine. J. Environ. Qual., 9: Kachout, S. S., Leclerc, J. C., Mansoura, A. B., Rejeb, M. N., & Ouerghi, Z., Effects of heavy metals on growth and bioaccumulation of the annual halophytes Atriplex hortensis and Altahea rosea. J. App. Sci. Res., 8: Madhoolika, A., & Rajesh, K. S., Single and combined effects of Cd and Zn on carrots; Uptake and Bioaccumulation. J. Pl. Nutr., 29: Marschner, H., Mineral Nutrition of Higher Plants. (2 nd Ed.). Academic Press, New York. 674 pp. Mathys, W., The role of malate and mustard oil glucosides in the evolution of Zincresistance in herbage plants. Physiol. Pl., 40: Mozumder, S. I., Khan, M. R., & Islam, A., Kinetics and mechanism of Cr (III) adsorption on tea- leaves waste. Asia- Pacific. J. Chem. Eng., 3(4): Mueller, J. G., Chapman, P. J., & Pritchard, P. H., Creosote Contaminated Sites. Environ. Sci. Technol., 23: Nies, D. H., Microbial heavy metal resistance. App. Microbiol. & Biotechnol., 51: Ohki, K., Zinc nutrition related to critical deficiency and toxicity levels for Sorghum. Agron. J., 76:

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