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Phytotoxicity and remediation of heavy metals by Alfalfa (Medicago sativa) in soil-vermicompost media.

Exponential increase in the use of heavy metals heavy metals, metallic compounds, such as aluminum, arsenic, cadmium, lead, mercury, and nickel. Exposure to these metals has been linked to immune, kidney, and neurotic disorders.
 in industrial processes and products, human exposure to heavy metals has risen dramatically in the last 50 years. Inorganic pollutants occur as natural elements in the earth's crust or atmosphere and human activities such as mining, industry, agriculture and military activities promote their release into the environment, leading to toxicity (Fargasova, 1994). The term heavy metals refers to metals and metalloids having densities greater than 5 g cm' and is usually associated with pollution and toxicity although some of these elements (essential metals) are required by organisms at low concentrations (Adriano, 2001). Heavy metals may enter the human body through food, water, air, or absorption through the skin (Life Extention, 2003). Heavy metal toxicity and the danger of their bioaccumulation in the food chain represent one of the major environmental and health problems of our modern society. High levels of metals in soil can be phytotoxic phytotoxic /phy·to·tox·ic/ (fi´to-tok?sik)
1. pertaining to phytotoxin.

2. poisonous to plants.

1. Poisonous to plants.

. Heavy metals interferes with several metabolic processes, causing toxicity to the plants as exhibited by reduced seed germination germination, in a seed, process by which the plant embryo within the seed resumes growth after a period of dormancy and the seedling emerges. The length of dormancy varies; the seed of some plants (e.g. , root and shoots growth and phytomass, chlorosis chlo·ro·sis
A form of chronic anemia, primarily of young women, characterized by a greenish-yellow discoloration of the skin and usually associated with deficiency in iron and protein. Also called chloremia.
, photosynthetic impairing, stunting and finally plant death (Gardea-Torresdey et al., 2004, Roy et al., 2005). Plant roots participate primarily in the heavy metal cation cation (kăt'ī`ən), atom or group of atoms carrying a positive charge. The charge results because there are more protons than electrons in the cation.  uptake (Lasat 2002). The toxic effect of heavy metal activity is connected to their extremely high concentration in cells. This concentration cause disturbances in cell membrane Cell membrane

The membrane that surrounds the cytoplasm of a cell; it is also called the plasma membrane or, in a more general sense, a unit membrane. This is a very thin, semifluid, sheetlike structure made of four continuous monolayers of molecules.
 functioning in the photosynthetic and mitochondrial mitochondrial

pertaining to mitochondria.

mitochondrial RNAs
a unique set of tRNAs, mRNAs, rRNAs, transcribed from mitochondrial DNA by a mitochondrial-specific RNA polymerase, that account for about 4% of the total cell RNA that
 electron transport electron transport
The successive passage of electrons from one cytochrome or flavoprotein to another by a series of oxidation-reduction reactions during the aerobic production of ATP, with the electrons originating from an oxidizable substrate and
 and in the inactivation inactivation /in·ac·ti·va·tion/ (in-ak?ti-va´shun) the destruction of biological activity, as of a virus, by the action of heat or other agent.  of many enzymes active in the basic cell metabolism regulation, which as the result leads to diminishing energy balance and disturbances in cell mineral nutrition (Gondek and Filipek-Mazur, 2003).

The soil has been traditionally the site for disposal for most of the heavy metal wastes which needs to be treated. Unlike organic compounds, metals cannot be degraded (Salt et al., 1995) and their cleanup requires their immobilization Immobilization Definition

Immobilization refers to the process of holding a joint or bone in place with a splint, cast, or brace. This is done to prevent an injured area from moving while it heals.
 and toxicity reduction or removal. Currently, conventional remediation methods of heavy metal contaminated soils include electrokinetical treatment, chemical oxidation or reduction, leaching, solidification, vitrification vit·ri·fi·ca·tion
The process of using heat and fusion to convert dental porcelain to a glassy substance.

, excavation and off-site treatment. But a majority of these technologies are costly to implement and cause further disturbance to the already damaged environment (Bio-Wise, 2003). Furthermore, bare soil is more susceptible to wind erosion and spreading of contamination by airborne dust. In an attempt to overcome the aforementioned problems associated with more traditional remediation techniques, scientists and engineers have been investigating the ability of live plants and inactivated biomaterials as remediation alternatives (Peralta et al., 2001). The techniques that involve the use of living organisms include bioremediation bi·o·re·me·di·a·tion  
The use of biological agents, such as bacteria or plants, to remove or neutralize contaminants, as in polluted soil or water.
, phytoextraction, phytovolatilization, phytostabilization, rhizofiltration and phytoremediation phy·to·re·me·di·a·tion  
The use of plants and trees to remove or neutralize contaminants, as in polluted soil or water.


See under bioremediation.
 (Yang et al., 2005). Phytoremediation using trees provides a potential opportunity to extract or stabilize metals. Phytoextraction (uptake) involves the use of high yielding plants that readily transport targeted metals from soil to vegetation, allowing removal of metals by harvesting the plants, without damaging the soil or requiring its disposal to landfill. The process takes longer, but meanwhile allows greening of the land and harvested plants can be used as bioenergy crops. Some plants have developed the ability to remove ions selectively from the soil to regulate the uptake and distribution of metals in their tissues. Most metal uptake occurs in the root system, usually via absorption, where many mechanisms are available to prevent toxic effects due to the high concentration of metals in the soil and water (EPA EPA eicosapentaenoic acid.

eicosapentaenoic acid

EPA, See acid, eicosapentaenoic.

, 1996). Phytoremediation can be used to remove not only metals (e.g. Ag, Cd, Co, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Zn) but also radionuclides (e.g. ([sup.90]) Sr, ([sup.137]) Cs, ([sup.239]) Pu, ([sup.234]) U, ([sup.238]) U) and certain organic compounds (Andrade and Mahler., 2002).

Plants have shown the capacity to withstand relatively high concentration of contaminants without toxic effects. A wide range of plants can be useful in cleaning up toxins. Some of the species now being studied--or already in use--are mustards, alfalfa alfalfa (ălfăl`fə) or lucern (lsûn`), perennial leguminous plant (Medicago sativa , vines, bamboo, cord grass and sunflowers. Some trees, including willows and poplars, also make good phytoremediators. The plant material may be used for non-food purposes; alternatively, it can be ashed followed by recycling of the metals or as disposal in a landfill (Angel and Linacre, 2005). Alfalfa has a number of characteristics that make it a prime candidate for mitigation of environmental contamination problems. This includes deep rooted (commonly 9-16 feet), an active rhizosphere rhi·zo·sphere  
The soil zone that surrounds and is influenced by the roots of plants.


The soil zone that surrounds and is influenced by the roots of plants.
 and its ability to absorb water, nitrates and other heavy metals (Putam, 2001). In the present study we examine the potential of alfalfa plant for phytoremediation (uptake) of heavy metals. Medicago sativa (alfalfa) is a good source of plant tissues, because it has been found to tolerate heavy metals and grow well in contaminated soils (Baligar et al., 1993). Gardea-Torresdey et al. (2000) have shown that alfalfa is a potential source of biomaterials for the removal and recovery of heavy metal ions.

Phytoremediation is essentially an agronomic a·gron·o·my  
Application of the various soil and plant sciences to soil management and crop production; scientific agriculture.

 approach and its success depends ultimately on agronomic practices applied at the site (Chaney et al., 1999). Biological processes such as composting followed by vermicomposting to convert vegetable waste (as valuable nutrient source) in agriculturally useful organic fertilizer would be of great benefit. The composting followed by vermicomposting of vegetable waste with earthworm earthworm, terrestrial, cylindrical segmented worm of the class Oligochaeta. There are 2,200 earthworm species, found all over the world except in arid and arctic regions and ranging in size from 1 in. (2.5 cm) to the 11-ft (330-cm) giant worms of the tropics.  (Eisenia foetida) develops in to a natural fertilizer (Maharashtra Nature Park Bulletin, 2003). The vermicompost contain high nutrient value, increases fertility of soil and maintains soil health (Suthar et al., 2005). Application of compost and vermicompost in contaminated soil improves soil fertility and physical properties as well as helps in successful approach to phytoremediation which has been demonstrated by Zheljazkov and Warman (2004). It also enhances quality of growing plants and increased biomass which could suggest that more metal can be taken up from the contaminated growth media and the tolerance to the metal toxicity is improved (Tang et al., 2003). The use of vermicompost developed from vegetable waste by vermiculture biotechnology with soil would provide natural environment for phytoremediation (Elcock and Martens, 1995).

In the work presented we addressed the application of vermicompost developed from vegetable waste in soil contaminated with heavy metals for phytoremediation studies. The objectives of this study is to determine the effects of heavy metals on seed germination, plant growth, biomass and examine their uptake by alfalfa (Medicago sativa) in soil- vermicompost media.

Materials and mehtods

Soil Sampling, Processing and Characterization

Soil was collected from a depth of about 0-15 cm along the banks of Surya River, Palghar (located 100 km away from Mumbai). Stones and plant tissues were carefully removed from the soil prior to drying process under laboratory condition. The soil was screened through 2 mm stainless steel stainless steel: see steel.
stainless steel

Any of a family of alloy steels usually containing 10–30% chromium. The presence of chromium, together with low carbon content, gives remarkable resistance to corrosion and heat.
 sieve and stored in a plastic bag at room temperature until use. Concentrations of Pb, Zn, Cu, Ni and Cd were measured by atomic absorption spectrophotometer spectrophotometer, instrument for measuring and comparing the intensities of common spectral lines in the spectra of two different sources of light. See photometry; spectroscope; spectrum.  (APHA, 1998). The physicochemical parameters were measured by standard methods(Table 1).

Soil texture was determined by the Bouyoucos hydrometer hydrometer (hīdrŏm`ətər), device used to determine directly the specific gravity of a liquid. It usually consists of a thin glass tube closed at both ends, with one end enlarged into a bulb that contains fine lead shot or mercury to  method. The moisture content of soil was calculated by the weight difference before and after drying at 105 [degrees]C to a constant weight. The pH and electrical conductivity (EC) were measured after 20 min of vigorous mixed samples at 1: 2.5:: Solid: deionized water ratio using digital meters [Elico, Model LI-120] with a combination pH electrode and a 1-cm platinum conductivity cell respectively. Total nitrogen and total phosphorus were determined according to the standard methods of the American Public Health Association (1998). Cation exchange capacity In soil science, cation exchange capacity (CEC) is the capacity of a soil for ion exchange of positively charged ions between the soil and the soil solution. A positively-charged ion, which has fewer electrons than protons, is known as a cation due to its attraction to cathodes.  was determined after extraction with ammonium acetate at pH 7.0 and the organic carbon was determined by using Walkley-Black method (Jackson, 1973).

Vermicomposting Development

The vermicompost was produced from vegetable waste (cabbage, french bean French bean

see phaseolus.
, cauliflower cauliflower (kô`lĭflou'ər, käl`ĭ–), variety of cabbage, with an edible head of condensed flowers and flower stems. Broccoli is the horticultural variety (botrytis); both were cultivated in Roman times. , lady finger, spinach, carrot and raddish) collected from the vegetable market. In the process dry leaves, coconut fibers and fresh grasses having high lignin lignin (lĭg`nĭn), a highly polymerized and complex chemical compound especially common in woody plants. The cellulose walls of the wood become impregnated with lignin, a process called lignification, which greatly increases the strength and  content were taken with the vegetable waste in appropriate quantity. About '/2 Kg of exotic varieties of earthworms (Eisenia foetida) was spread on bedding materials. Everyday, 200 to 300 gm of vegetable waste collected from market was supplied for a period of two and half months as a source of food for the earthworms. The physicochemical parameters were measured during vermicomposting as described in soil analysis. After two and half month, vermicompost was collected, air dried, sieved (2-mm) and a portion of it was taken for nutrient analysis in order to prove its potency as biofertilizer. The nutrients in dried sample of vermicompost was digested with concentrated nitric acid nitric acid, chemical compound, HNO3, colorless, highly corrosive, poisonous liquid that gives off choking red or yellow fumes in moist air. It is miscible with water in all proportions.  and 30% hydrogen peroxide hydrogen peroxide, chemical compound, H2O2, a colorless, syrupy liquid that is a strong oxidizing agent and, in water solution, a weak acid. It is miscible with cold water and is soluble in alcohol and ether.  and then determined by an atomic absorption spectrophotometer [AAS, Perkin Elmer] (APHA, 1998).

Greenhouse Experiments

Green house pot culture experiments were conducted to study the effect of heavy metals viz Cd, Ni, Cu, Pb and Zn on seed germination, root growth, shoot growth and phytoremediation (uptake) by Alfalfa. The ratio 3:1 of alluvial soil and vermicompost were used as media. Nutrient and trace elements Trace elements
A group of elements that are present in the human body in very small amounts but are nonetheless important to good health. They include chromium, copper, cobalt, iodine, iron, selenium, and zinc. Trace elements are also called micronutrients.
 concentrations in the vermicompost are presented in table 2. This soil-vermicompost media was then amended with the heavy metals: Cd as Cd ([NO.sub.3]), 4[H.sub.2]O; Cu as CUS CUS Course
CUS Centro Universitario Sportivo (Italian: Universtity Sport Center)
CUS See You Soon (chat)
CUS Concordia University System
CUS Confederación de Unificación Sindical
[O.sub.4]. 5[H.sub.2]O; Ni as Ni([NO.sub.3]).sub.2]; Pb as Pb ([NO.sub.3].sub.2], and Zn as Zn ([NO.sub.3]).sub.2]. 6[H.sub.2.O]. The concentrations of each heavy metal used in this study were 0, 5, 10, 20, 40 and 50 ppm. Alfalfa seeds were obtained from seed supplier Ratanshi Agro-Hortitech (Byculla, Mumbai). Seeds were immersed in 3% (v/v) of formaldehyde for 5 minutes and washed with distilled water several times to avoid fungal contamination. The soil- vermicompost media contaminated/amended with the heavy metal was used as potting media. To determine the effect of heavy metals; 10 seeds of uniform size were placed in every Petri dish pe·tri dish
A shallow circular dish with a loose-fitting cover, used to culture bacteria or other microorganisms.

Petri dish

a shallow, circular, glass or disposable plastic dish used to grow bacteria on solid media such as agar.
 (the lids were left on until 3 days after the germination) for study of seed germination. The Petri dish were kept in the dark and observed for germination. The seeds were considered germinated with the emergence of radicles. Small plastic pots/glasses were used for studying shoot and root growth. Ten plants were grown in 2 Kg capacity plastic pots for phytoremediation study. Soil-moisture content was adjusted regularly by weight to about 60% of water-holding capacity with deionized water.

To prevent loss of nutrients and trace elements out of the pots, plastic trays were placed under each pot and the leachets collected were put back in the respective pots. Each treatment of plant consisted of three replicate for statistical purpose. The seeds were set under 12/12 hrs light/dark cycle and temperatures of 30[degrees]C during the day and 27[degrees]C during the night. The average relative humidity relative humidity
The ratio of the amount of water vapor in the air at a specific temperature to the maximum amount that the air could hold at that temperature, expressed as a percentage.
 was recorded to be 75%. The seedlings were harvested after two weeks; germination rate and shoot/root length were recorded. For the phytoremediation study plants were harvested after 10 weeks. The plants were then separated in to roots and shoots Roots & Shoots is a program of The Jane Goodall Institute. It was started by Dr. Jane Goodall in 1991 with 16 high school age kids from Tanzania. Since then it has grown to more than 8,000 groups in 96 countries. . The plant samples were washed with distilled water and dried in an oven at 70[degrees]C for 3 days and the dry weight of biomass was determined, after which these samples were stored in the brown paper bags. The samples were considered for analysis of metal content digested with concentrated nitric acid and 30% hydrogen peroxide and then the heavy metal content was determined by an atomic absorption spectrophotometer [AAS, Perkin Elmer] (APHA, 1998).

Statistical Analysis

Each treatment for % seed germination, plant root/ shoot growth and uptake consisted of three replicate for statistical purpose. The data presented for each treatment in this study is represented as mean of samples with standard deviation (X [+ or -] S.D.) calculated by standard statistical methods (Mahajan Mahajan is an Indian surname, found among the Vaishya castes (business communities). In India surname Mahajan is used by two communities: - one residing in North of India(mainly on the Amritsar to Jammu belt) and another belonging to North Maharashtra. , 1997).


The present research study has been carried out in glass house in pot culture experiments to study the uptake of heavy metals at various concentrations in soil -vermicompost media. The alfalfa plant was used for uptake of heavy metals. The soil and developed vermicompost were analyzed for physicochemical characteristic. The soil was amended with heavy metals (Pb, Zn, Cu, Ni and Cd) for phytoremediation. The results are presented below:

Soil Analysis

After selecting the probable site, soil was collected and subjected to extensive analysis of various physicochemical parameters, which influence root establishment in soil (Table 1). Soil, texture, which was found to be sandy loam, had profound effect upon the properties of soil including its water supplying power, rate of water intake, aeration aeration /aer·a·tion/ (ar-a´shun)
1. the exchange of carbon dioxide for oxygen by the blood in the lungs.

2. the charging of a liquid with air or gas.

, fertility and ease of tillage. The pH was 7.2, which lies within the recommended value for proper growth and efficient uptake of nutrients and compounds from soil. The percentage of organic matter and nitrogen were found to be 0.80 and 0.05 respectively. Macronutrients including metals were also present in substantial amount. Further to augment the existing native state of soil, it was spiked with vermicompost, which gives all the nutrients conducive to plant growth for phytoremediation studies. There was no history of heavy metal (Cd, Ni and Ph) contamination found in the soil collected.

Vermicomposting Analysis

The vermicompost developed was characterized and found to have high concentration of nutrients such as Ca, Zn, Cu, Mg, Fe and Mn (Table 2). This vermicompost developed by the vermiculture biotechnology was then used as a natural fertilizer for phytoremediation studies of heavy metals.

Effect of Heavy Metals on Seed Germination

The present research demonstrated a concentration dependent inhibition of the seed germination with regards to Alfalfa species (Table 3). The results of this study indicated that Cd, Ni and Pb at 5 ppm levels had very low toxic effects on seed germination while Copper at the same doses increased seed germination. At the 10 and 20 ppm concentrations of Cd, Ni and Pb reduced the seed germination while 5 and 10 ppm doses of Cu promoted seed germination. The seed germination inhibited at 40 and 50 ppm levels as compared to the control for all the four metals i.e. Cd, Ni, Pb and Cu. Delayed germination was also observed in all cases at higher i.e 40 and 50 ppm concentrations. However, in the same study Zinc (Zn) being the only metal which did not reduce the seed germination. The resulting rank order of toxicity for metals on seed germination was Cd > Cu > Ni > Pb >Zn. However, seed germination increased at all Zn concentrations.

Effect of Heavy Metals on Root growth

Increase in the heavy metal concentration in the soil- vermicompost media caused root length decrease with stunt growth of roots (Table 3). The dose of 5ppm of Cd, Ni, Pb, Cu and Zn promoted the root growth of the plants as compared to the root growth of the control plants. The heavy metals Cu, Ni, Zn and Pb at 10 ppm level further increased the root growth over the control root size. However at the same dose Cd reduced the root size on comparison with the control root elongation. Cd, Ni, Cu and Pb demonstrated a concentration dependant inhibition of root growth at 20, 40 and 50 ppm dosages. All the Zn concentrations increased the root length than the control root length alfalfa plants. Root toxicity symptom included browning, reduced number of roots hair and growth. In comparison to the control treatment without heavy metals (Cd, Pb, Cu and Ni), Plant roots were healthy and normal. The color of the roots receiving higher heavy metals treatment (40 and 50 ppm) except Zn, changed gradually over time from creamy white color to dark brown, an indication of intense suberification. Plants treated at lower concentrations were not significantly affected by the metals. All Zinc, concentrations showed increase in root growth. Lateral roots were observed in almost all treated samples of Zn, Cd, Cu, Pb and Ni demonstrated concentration dependant inhibition of root growth at higher ppms.

Effect of Heavy Metals on Shoot Growth

The effects of heavy metals on the shoot growth are different from their effects on root growth (Table 3). The shoot length was found slightly reduced than the control alfalfa plants at the 5 ppm Cd dose. On the otherhand, the 5 ppm dose of Cu, Pb, Ni and Zn increased the shoot lengths as compared to the control treatment. These results indicate that low concentrations of Cd, Cu, Ni and Pb have micronutrient-like effects on the sorghum sorghum, tall, coarse annual (Sorghum vulgare) of the family Gramineae (grass family), somewhat similar in appearance to corn (but having the grain in a panicle rather than an ear) and used for much the same purposes.  plants and all the plants appeared to be healthy. The heavy metals Cd, Ni and Pb at 10 and 20 ppm levels reduced the shoot growth; however, Cu at the same dose increased the shoot size. When the concentration of these above said four metals was increased to 40 and 50 ppm dose, the shoot size of the plants found a concentration dependant inhibition of shoot growth as compared to the control plants. All plants grown in the media contaminated with Zn showed increase in the shoot elongation than the plants grown in media without Zn contamination.

Effect of Heavy Metals on Plant Biomass

The biomass results after 10 weeks of experiments indicated that the mean plant biomass of alfalfa showed increasing tendency as the concentrations increased from 5 to 10 to 20 ppm for Cd, Cu and Ni (Table 4). Biomass decreased gradually as the concentration of Cd, Cu and Ni in the soil- vermicompost media increased to 40 and 50 ppm. The biomass yield affected by the higher ppm levels of Cd caused reduction in the plant biomass. Lead showed low effect on plant biomass. There was positive effect seen in all Zn concentrations and increase in biomass yield as compared to control ones.

Heavy Metal Uptake by Plant Tissue

The heavy metals concentration in the plant is affected by two factors that is the metal content supplied in the soil-vermicompost media and the plant tissue as well as by the interaction between these factors. The mean uptake of metals Cd, Ni, Pb, Cu and Zn by alfalfa increased as the concentrations of these metals in the soil vermicomposting media increased (Table 5). In plant, shoot and root were observed to have a characteristic uptake capacity for different metals. The heavy metals were untaken by the alfalfa plants in the following order: Zn> Cu>Cd>Ni>Pb.


Vermicomposting Analysis

Several researchers have demonstrated that earthworm castings (vermicompost) have excellent aeration, porosity, structure, drainage and moisture-holding capacity. The vermicompost is a rich source of beneficial microorganisms and nutrients (Paul, 2000) and is used as a soil conditioner or fertilizer (Hattenschwile and Gaser, 2005). Increase in crop yield, soil nutrients status and nutrients uptake was reported due to application of vermicompost (Singh and Sharma, 2003). Peng et al. (2005) and Yang et al. (2005) have reported that application of compost or manure can increase the bioavailability and in-plant mobility of copper. Significantly more copper was found in grains and straw of oat treated with vermicompost as compared with the application of mineral fertilizers. Experimental work has shown that earthworm activity enhances tree seedling growth associated with enhanced soil organic matter, improved nutrition (including [N0.sup.-.sub.3], [NH.sup.+.sub.4] and [Ca.sup.+.sub.2]) and increased mycorrhizal colonization (Welke and Parkinson, 2003). Yield of a tropical leguminous le·gu·mi·nous  
1. Of, belonging to, or characteristic of the family Leguminosae, which includes peas, beans, clover, alfalfa, and other plants.

2. Resembling a legume.
 woody shrub, Leucaena leucocephala Leucaena leucocephala

tree in the legume family Mimosaceae; contains the toxic amino acid mimosine; causes hair loss, goiter, infertility and weight loss. Called also L. glauca, lead tree. The disease is called also ekoa, jumbey, koa haole, lamtoro.
, in amended Pb-Zn mine tailings has been found to be increased by 10 to 30% in the presence of burrowing earthworms (Pheretima spp.) (Ma et al., 2003). The earthworms increased available forms of N and P in soil, increased metal bioavailability and raised metal uptake into plants by 16 to 53%. Some evidence indicates that earthworms increase metal bioavailability in relatively low-level metal-contaminated soils with higher organic matter contents. This agrees with results of experiments in which the addition of exogenous humic acid to soil has been shown to increase plant-available metals (Halim et al., 2003).

Effect of Heavy Metals on Seed Germination

The research demonstrated concentration dependent inhibition of the seed germination. Early growth period such as germination is more sensitive to heavy metal pollution. Generally the germination started after 24 hours of sowing and was more enhanced during early hours (before 72 hours after showing). Peralta et al. (2001) found that 20 and 40 ppm of Cu, Cd and Ni inhibited ability of seeds of Medicago sativa to germinate and grow in the contaminated medium, whereas Zn did not reduce the seed germination. Compared to the control, at and above 10 ppm Cr (VI) concentration, significant inhibitory effect on seedling growth of tested rice cultivars were detected by Xiong (1998). The experiment conducted by Peralta-Videa et al. (2004) showed that the susceptibility of living alfalfa plants to Cd, Cu and Zn was correlated to the age of the plants. He also reported that after four days germination, Cr, Cd, Ni, except Zn, had lethal effects on the alfalfa seedlings. The toxic effects of copper in plant cells appear to be largely attributable to competitive inhibition of essential ion pathways by copper and to the redox redox (rē`dŏks): see oxidation and reduction.  production of reactive oxygen species. Jonak et al. (2004) mentioned that an excess of copper ions activated four different mitogen-activated protein kinase Mitogen-activated protein (MAP) kinases (EC are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate various cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis.  (MAPK MAPK Mitogen-Activated Protein Kinase
MAPK Map Kinase
) pathways in alfalfa seedlings: SIMK, MMK MMK

In currencies, this is the abbreviation for the Myanmar Kyat.

The currency market, also known as the Foreign Exchange market, is the largest financial market in the world, with a daily average volume of over US $1 trillion.
2, MMK3 and SAMK. The same pathways appear to respond to cadmium, but with a greater time delay than with copper. They suggested that this may indicate the MAPK response is mediated by reactive oxygen species.

Effect of Heavy Metals on Root growth

Increase in the heavy metal concentration in the soil- vermicompost media caused root length decrease with stunt growth of roots. One of the explanations for roots to be more responsive to toxic metals in environment might be that roots were the specialized absorptive organs so that they were affected earlier and subjected to accumulation of more heavy metals than any of the other organs This could also be the main reason that root length was usually used as a measure for determining heavy metal- tolerant ability of plant (Xiong 1998). According to Chaignon and Hinsinger (2003), higher concentrations of Copper can inhibit root growth before shoot growth and can accumulate in the roots without any significant increase in its content of the aerial parts. Heavy metals are found to be more toxic for root growth because they accumulate on root and retard cell division and cell elongation. Gyawali and Lekhak, (2006) mentioned that the order of metal toxicity to new root primordia in Salix viminalis is Cd>Cr>Pb, whereas root length was more affected by Cr than by other heavy metals studied.

Effect of Heavy Metals on Shoot Growth

The results indicate that low concentrations of Cd, Cu, Ni and Pb have micronutrient-like effects on the alfalfa plants. Ormrod et al. (1986) investigated that Nickel caused stunted and deformed growth of shoot with symptoms of chlorosis. Statistically significant differences (P<0.05) between the shoot length of the control treatment plants and the length of plants of alfalfa grown in the presence of the heavy metal mixture was reported by Peralta- Videa et al. (2006). When Cr was added at 2, 10 and 25 ppm to nutrient solutions in sand cultures in oats (Avena sativa Avena sativa,
n See oats.
 L.), Gyawali and Lekhak, (2006) noted that the 11%, 22% and 41% reduction in plant height, respectively, over control. Generally, it was seen that degrees of inhibition of shoot and root growth started from 10 ppm concentration. In this respect Peralta- Videa et al. (2004) reported that that Cd affected young plants more than old plants of P. coccineus. and Cd applied to the younger plants caused a stronger reduction in growth parameters such as leaf area and fresh weight accumulation and reduce shoot growth by reducing the chlorophyll content and the activity of photosystem Photosystems (ancient Greek: phos = light and systema = assembly) are protein complexes involved in photosynthesis. They are found in the thylakoid membranes of plants, algae and cyanobacteria (in plants and algae these are located in the chloroplasts), or in the  I.

Effect of Heavy Metals on Plant Biomass

The biomass yield affected by the higher concentrations of metals and caused reduction in the plant biomass. Higher doses of heavy metal can affect physiology, reduced plant growth and dry biomass yield (Grifferty and Barrington, 2000). Nwosu, et al. (1995) mentioned that the mean plant biomass decreased in both lettuce and radish, as the concentration of Cd and Pb in soil increased. Grifferty and Barrington (2000) showed that the increased Zn concentration from 25 to 50 mg/kg had a significantly positive effect on dry biomass yield. Higher levels of organic matter (882.30g kg-') and nutrients content in the compost had beneficial influence on soil chemical and biochemical properties and plant growth, thus increasing biomass yields (Yang et al., 2005). The plant biomass may be incinerated either to reduce volume, recover energy, disposed offusing appropriate techniques or recycled to recover valuable metals (Angel and Linacre, 2005). The alfalfa produced greater biomass which result in a higher concentrations uptake of metals was reported by Pivetz, (2001). The phytotoxicity This article or section is in need of attention from an expert on the subject.
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 of cadmium on growth and dry matter production of a number of cultivated plants have been determined by Gondek and Filipek-Mazur (2003). Corn, alfalfa and sorghum were found to be effective due to their fast growth rate and large amount of biomass produced.

Heavy Metal Uptake by Plant Tissue

The mean uptake of metals Cd, Ni, Pb, Cu and Zn by alfalfa increased as the concentrations of these metals in the soil- vermicomposting media increased. Laboratory experiments have determined that alfalfa possesses the ability to bind various heavy metal ions. Alfalfa shoot biomass has demonstrated the ability to bind an appreciable amount of copper, nickel, cadmium, chromium, lead and zinc from aqueous solutions (Tiemann et al., 1998). In addition, alfalfa biomass is also able to reduce the oxidation state of other metals such as Cr and An (GardeaTorresdey et al., 2000). Increased in lead uptake by alfalfa (Medicago sativa) using EDTA EDTA: see chelating agents.  and a plant growth promoter was reported by Lopez et al. (2005). The roots preferentially explored metals in the contaminated area. The large surface area of roots and their intensive penetration of soil, may reduce leaching, runoff and erosion via stabilization of soil, offer advantages for phytoremediation. Most crop species tend to accumulate Cd at the highest concentrations in the root tissue, followed by leaves, then by seeds or storage organs. Several studies have demonstrated that the metal concentration in the plant tissue is a function of the heavy metals content in the growing environment (Cui et al., 2004; Grifferty and Barrington, 2000).

Plants use photosynthetic energy to extract ions from the soil and concentrate them in their biomass, according to nutritional requirements nutritional requirements,
n the food and liquids necessary for normal physiologic function.
 (Kramer and Chardonnens, 2001). The essential elements (Cu and Zn) are required in low concentrations and hence are known as trace elements or micronutrients, whereas nonessential non·es·sen·tial
Being a substance required for normal functioning but not needed in the diet because the body can synthesize it.
 elements (Cd, Ni and Ph) are phytotoxic (Gerard et al., 2000). Zn is relatively mobile in soils and is the most abundant metal in root and shoot of contaminated plants as it is in soils. This metal is necessary as a minor nutrient and it is known that plants have special zinc transporters to absorb this metal (Zhu et al., 1999). However, an excessive accumulation of this element in living tissues leads to toxicity symptoms. The phytoavailable Lead is usually very low due to its strong association with organic matter, Fe-Mn oxides, clays and precipitation as carbonates, hydroxides and phosphates (Shen Shen, in the Bible, place, perhaps close to Bethel, near which Samuel set up the stone Ebenezer.  et al., 2002). An ultrastructural study using transmission electron microscopy revealed the retention of unchelated Pb mainly in cell wall of roots, particularly around intercellular spaces (W enger et al., 2003).

Cadmium also is considered to be mobile in soils but is present in much smaller concentrations than Zn (Zhu et al., 1999). Moreover, many studies have demonstrated that Cd taken up by plants accumulates at higher concentrations in the roots than in the leaves (Boominathan and Doran, 2003). Alloway (1995) mentioned that Alyssum alyssum (əlĭs`əm), any species of the genus Alyssum of the family Cruciferae (mustard family), annual and perennial herbs native to the Mediterranean area. A few species, notably the perennial golden tuft (A.  species which are naturally adapted to serpentine soils can accumulate over 2% Ni. The uptake by some plants has been confirmed for Cd (up to 0.2% Cd in shoot dry biomass), Ni (up to 3.8% Ni in shoot dry biomass) and Zn (up to 4% Zn in shoot dry biomass) by Kramer and Chardonnens, (2001). The application of peat and manure in contaminated soil increased Cu, Zn and Ni accumulation by wheat (Schmidt, 2003).

Organic matter in soil could effectively increase the activity of metals in soil and improve metal mobility and distribution in soil. The application of natural fertilizer (compost and vermicompost) in soils has helped in increase in metal mobility through the formation of soluble metal-organic complexes (Yang et al., 2005). In addition, exudation exudation /ex·u·da·tion/ (eks?u-da´shun)
1. the escape of fluid, cells, and cellular debris from blood vessels and their deposition in or on the tissues, usually as the result of inflammation.

2. an exudate.
 of organic compounds by plant roots, such as organic acids, influence ion solubility and uptake (Klassen et al., 2000) through their effects on microbial microbial

pertaining to or emanating from a microbe.

microbial digestion
the breakdown of organic material, especially feedstuffs, by microbial organisms.
 activity, rhizosphere physical properties and root-growth dynamics (Yang et al., 2005). The higher concentrations uptake of heavy metals (Cu, Zn, Fe, Al and Mn) by alfalfa (Medicago sativa) was reported by Rehab et al. (2002).


This research work deals with phytoremediation of heavy metals by Alfalfa in the soil- vermicompost media. Medicago sativa (alfalfa) is a good source of plant tissues, because it has been found to tolerate heavy metals and grow well in contaminated soils. The phytoremediation techniques for the heavy metal management proves to be very effective as its cost is approximately one tenth that of conventional soil cleansing procedures and in some cases, the plant material can be further utilized to recoup the cost of the operation or even turn a profit. Another advantage of phytoremediation is that it leaves the soil fertile and has less adverse environmental effects as compared to conventional procedures. The low-doses of heavy metals applied stimulated the root and shoot elongation of Alfalfa plants. At higher concentrations i.e. 40 and 50 ppm of Cd, Cu, Ni and Pb reduced the ability to germinate. However the plants were able to germinate and grow efficiently at any Zn concentration evaluated in this study. The study shows that heavy metals were efficiently uptaken at all concentrations using vermicompost media and the uptake was increased along the increasing concentrations in soil. Alfalfa is a very fast-growing, deep rooted with a high biomass producing plant and maybe used for energy production and metal-enriched biomass can be harvested using standard agricultural methods and smelted to recover the metals. The present technology will help to remediate the higher concentrations of metals by the application of vermicompost as a natural fertilizer in soil. This technology will be applicable at the site to remediate the heavy metals. Thus, an increase in the plant resistance to heavy metal toxicity (Zn, Pb and Cd) seems possible by means of addition of nutrient (vermicompost) supply.


The authors are grateful to the University of Mumbai Most of the colleges in the city of Mumbai (Bombay) and the districts of Thane, Raigad, Ratnagiri and Sindhudurg are affiliated to the University of Mumbai. The University of Mumbai offers Bachelors, Masters and Doctoral degrees to students.  for providing financial assistance to C.D. Jadia.

Chhotu D. Jadia and M.H. Fulekar, Phytotoxicity and Remediation of Heavy Metals by Alfalfa (Medicago sativa) in Soil-vermicompost Media, Adv. in Nat. Appl. Sci., 2(3): 141-151, 2008


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Corresponding Author: M.H. Fulekar, Environmental Biotechnology Laboratory, Department of Life Sciences, University of Mumbai, Santacruz (E), Mumbai -400 098, Mumbai, India. E.mail:

Chhotu D. Jadia and M.H. Fulekar

Environmental Biotechnology Laboratory, Department of Life Sciences, University of Mumbai, Santacruz (E), Mumbai -400 098, Mumbai, India.
Table 1: Physicochemical properties of experimental Soil. ([dagger])

Soil parameters                    Values       S.D.

Clay %                             25.9         [+ or -] 1.8
Silt %                             21.7         [+ or -] 2.5
Sand %                             50.4         [+ or -] 2.8
pH                                 7.2          [+ or -] 0.1
Organic matter %                   0.80         [+ or -] 0.045
Nitrogen %                         0.05         [+ or -] 0.02
C EC * c mol/ 100 gm soil          11.27        [+ or -] 0.76
EC ([double dagger]) d[S.sup.-1]   1.1          [+ or -] 0.1
Potassium mg/kg                    22.73        [+ or -] 2.63
WHC ** %                           62           [+ or -] 4.0
Moisture Content %                 34           [+ or -] 1.8

Heavy metal ppm
Cu                                 3.6          [+ or -] 0.5
Cd                                 ND
Ni                                 ND
Ph                                 ND
Zn                                 12           [+ or -] 1.5

([dagger]) Values are averages of three replicates [+ or -] S.D.

* Cation exchange capacity

([double dagger]) Electrical conductivity.

** Water Holding Capacity

Table 2: Chemical and Nutrient Status of Vermicompost. ([dagger])

Parameters                            Values   S.D.

pH                                    6.8      [+ or -] 0.173
EC ([double dagger]) dS [m.sup.-1]    10.55    [+ or -] 0.01
Total C %                             13.5     [+ or -] 0.7
Total N %                             1.33     [+ or -] 0.015
Available P %                         0.47     [+ or -] 0.09
Sodium mg /100gm                      354.68   [+ or -] 9.44
Magnesium mg/100gm                    832.48   [+ or -] 22.48
Iron mg/100gm                         746.26   [+ or -] 23.39
Zinc mg/100gm                         16.19    [+ or -] 0.55
Manganese mg/100gm                    53.86    [+ or -] 2.84
Copper mg/100gm                       5.16     [+ or -] 0.36

([dagger]) Values are averages of three replicates [+ or -] S.D.

([double dagger]) Electrical conductivity.

Table 3: Seed germination. root and shoot length of alfalfa
(Medicago sativa). after two weeks of exposure to heavy metals.

        Dose     Germination
Metal   (ppm)      rate (%)     Root length (cm)    Shoot length (cm)

Cd        0     80 [+ or -] 4   4.9 [+ or -] 0.8    5.5 [+ or -] 0.6
          5     77 [+ or -] 5   5.5 [+ or -] 0.8    5.3 [+ or -] 0.6
         10     70 [+ or -] 4   4.5 [+ or -] 0.9    4.7 [+ or -] 0.5
         20     65 [+ or -] 5   3.9 [+ or -] 0.7    3.8 [+ or -] 0.4
         40     57 [+ or -] 7   3.4 [+ or -] 0.3    4.1 [+ or -] 0.5
         50     49 [+ or -] 7   2.9 [+ or -] 0.4    3.6 [+ or -] 0.5

Cu        5     83 [+ or -] 7   5.1 [+ or -] 0.55   5.7 [+ or -] 0.38
         10     85 [+ or -] 6   5.6 [+ or -] 0.48   6.7 [+ or -] 0.77
         20     72 [+ or -] 7   6.5 [+ or -] 0.34   7.8 [+ or -] 0.89
         40     60 [+ or -] 4   4.1 [+ or -] 0.54   4.7 [+ or -] 0.44
         50     58 [+ or -] 6   3.8 [+ or -] 0.82     4 [+ or -] 0.79

Ni        5     79 [+ or -] 7   5.2 [+ or -] 0.52   5.7 [+ or -] 0.76
         10     75 [+ or -] 4   5.4 [+ or -] 0.56   5.2 [+ or -] 0.9
         20     70 [+ or -] 8   4.8 [+ or -] 0.64     5 [+ or -] 0.8
         40     64 [+ or -] 5   4.5 [+ or -] 0.55   4.7 [+ or -] 0.54
         50     60 [+ or -] 7   4.1 [+ or -] 0.4    4.5 [+ or -] 0.5

Ph        5     79 [+ or -] 8     5 [+ or -] 0.4    5.9 [+ or -] 0.97
         10     77 [+ or -] 5   5.3 [+ or -] 0.55   5.3 [+ or -] 0.27
         20     74 [+ or -] 6   4.7 [+ or -] 0.59     5 [+ or -] 0.64
         40     66 [+ or -] 5   4.3 [+ or -] 0.25   4.9 [+ or -] 0.64
         50     63 [+ or -] 3     4 [+ or -] 0.38   4.7 [+ or -] 0.58

Zn        5     81 [+ or -] 5   5.1 [+ or -] 0.73   5.6 [+ or -] 0.48
         10     83 [+ or -] 6   5.4 [+ or -] 0.27   6.8 [+ or -] 0.82
         20     88 [+ or -] 5   6.1 [+ or -] 0.28   7.2 [+ or -] 0.68
         40     91 [+ or -] 5   6.8 [+ or -] 0.54   7.7 [+ or -] 0.78
         50     95 [+ or -] 3   7.5 [+ or -] 0.67   8.2 [+ or -] 0.55

Table 4: Biomass of alfalfa after 10 weeks of growth in heavy metals
enriched soil-vermicompost media. ([dagger])

Metal   (ppm)    Root Dry Weight (g)    Shoot Dry Weight (g)

Cd        0     0.530 [+ or -] 0.051    0.789 [+ or -] 0.097
          5     0.545 [+ or -] 0.035    0.802 [+ or -] 0.089
         10     0.578 [+ or -] 0.039    0.862 [+ or -] 0.029
         20     0.421 [+ or -] 0.070    0.605 [+ or -] 0.03
         40     0.340 [+ or -] 0.035    0.478 [+ or -] 0.046
         50     0.285 [+ or -] 0.027    0.361 [+ or -] 0.021

Cu        5     0.571 [+ or -] 0.029    0.816 [+ or -] 0.045
         10     0.672 [+ or -] 0.02      1.05 [+ or -] 0.05
         20     0.500 [+ or -] 0.029     0.74 [+ or -] 0.05
         40     0.455 [+ or -] 0.027    0.621 [+ or -] 0.028
         50     0.352 [+ or -] 0.017    0.487 [+ or -] 0.036

Ni        5      0.58 [+ or -] 0.012    0.802 [+ or -] 0.039
         10      0.68 [+ or -] 0.029    0.892 [+ or -] 0.031
         20     0.516 [+ or -] 0.008     0.77 [+ or -] 0.06
         40     0.489 [+ or -] 0.032    0.675 [+ or -] 0.036
         50     0.417 [+ or -] 0.043    0.562 [+ or -] 0.041

Pb        5     0.535 [+ or -] 0.033    0.795 [+ or -] 0.054
         10     0.503 [+ or -] 0.015    0.776 [+ or -] 0.045
         20     0.583 [+ or -] 0.032    0.805 [+ or -] 0.036
         40     0.623 [+ or -] 0.028    0.835 [+ or -] 0.054
         50     0.515 [+ or -] 0.026    0.700 [+ or -] 0.041

Zn        5      0.58 [+ or -] 0.019    0.862 [+ or -] 0.049
         10     0.625 [+ or -] 0.035    0.987 [+ or -] 0.048
         20     0.705 [+ or -] 0.014     0.95 [+ or -] 0.031
         40     0.892 [+ or -] 0.051     1.17 [+ or -] 0.19
         50     0.962 [+ or -] 0.047     1.32 [+ or -] 0.19

([dagger]) Values are averages of three replicates [+ or -] S.D.

Table 5: Metal concentration in roots and shoots and uptake
(compared to control treatment). ([dagger])

                               Metal Dose (ppm)
Metal   (ppm)            Roots                  shoots

Cd        5       0.732 [+ or -] 0.05     0.152 [+ or -] 0.014
          10      0.817 [+ or -] 0.033    0.198 [+ or -] 0.014
          20      2.032 [+ or -] 0.093    0.652 [+ or -] 0.031
          40      3.816 [+ or -] 0.116    1.557 [+ or -] 0.056
          50      5.928 [+ or -] 0.136    2.715 [+ or -] 0.075

Cu        5       1.302 [+ or -] 0.087    2.710 [+ or -] 0.17
          10      1.850 [+ or -] 0.19     3.620 [+ or -] 0.022
          20      2.380 [+ or -] 0.19     5.460 [+ or -] 0.25
          40      3.690 [+ or -] 0.18     7.800 [+ or -] 0.2
          50      5.380 [+ or -] 0.12     9.750 [+ or -] 0.24

Ni        5       1.108 [+ or -] 0.012    0.473 [+ or -] 0.012
          10      1.595 [+ or -] 0.094    0.708 [+ or -] 0.007
          20      3.625 [+ or -] 0.109    0.834 [+ or -] 0.015
          40      5.216 [+ or -] 0.095    1.102 [+ or -] 0.093
          50      5.905 [+ or -] 0.034    1.575 [+ or -] 0.074

Pb        5       0.417 [+ or -] 0.033    0.098 [+ or -] 0.019
          10      0.789 [+ or -] 0.027    0.175 [+ or-] 0.023
          20      1.780 [+ or -] 0.082    0.334 [+ or -] 0.028
          40      2.701 [+ or -] 0.08     0.452 [+ or -] 0.026
          50      3.890 [+ or -] 0.18     0.614 [+ or -] 0.078

Zn        5       1.500 [+ or -] 0.03     3.500 [+ or -] 0.05
          10      2.170 [+ or -] 0.09     4.010 [+ or -] 0.17
          20      2.950 [+ or -] 0.15     8.230 [+ or -] 0.08
          40      5.610 [+ or -] 0.12    10.760 [+ or -] 0.15
          50      7.120 [+ or -] 0.11     11.37 [+ or -] 0.17

([dagger]) Values are averages of three replicates [+ or -] S.D.
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Title Annotation:Original Article
Author:Jadia, Chhotu D.; Fulekar, M.H.
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9INDI
Date:Sep 1, 2008
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