Amaranthus dubius is a nutritious leafy vegetable widely distributed in Africa, Asia and South America. The rapid growth and great biomass makes them one of the highest yielding leafy crops which may be valuable for phytoremediation. The potential of A. dubius for the hyperaccumulation and distribution of chromium (Cr), mercury (Hg), arsenic (As), lead (Pb), copper (Cu) and nickel (Ni) in the different plant organs was investigated under controlled conditions in a tunnel house using the bioconcentration factor and translocation factor. The metal accumulated was investigated using Inductively Coupled Plasma Mass Spectroscopy (ICPMS). Following exposure to 25 ppm, 75 ppm and 100 ppm of Cr, Hg, As, Pb, Cu and Ni respectively for four days, A. dubius accumulated Cr, Hg and Pb at the low concentration (25 ppm) and translocated it to the shoots, but at higher concentrations (75 ppm and 100 ppm) the metal was only stored in the roots. In the case of As accumulation at all concentrations, the metal was translocated to the shoots, Cu and Ni indicated that the metal was stored in the roots mainly. This results of study showed that A. dubius has limited potential for the bioaccumulation of Cr, Hg, Pb, Cu and Ni, but is capable of hyperaccumulating As.
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Keywords: Phytoremediation, Amaranthus dubius, bioconcentration factor, translocation factor, Cr, Hg, Pb, As, Cu, Ni
Concern has been expressed with regard to the accumulation of toxic heavy metals such as chromium (Cr), mercury (Hg), lead (Pb) and arsenic (As) and their impact on both human health and the environment (Gardea-Torresdey et al., 2004; Basta et al., 2001; Kamal et al., 2004). These metals are environmental pollutants from industrial and agricultural activities, sewage sludge leaching and mining waste (Gratao et al., 2005). Fertilizers from sewage sludge, mining waste and paper mills all contribute to the continuous deposition of heavy metals into soils. Another point of concern is the effect of leaching on these contaminated sites which in turn contaminate water tables (Gratao et al., 2005). Living organisms require a trace amount of some heavy metals which include copper (Cu), iron (Fe), nickel (Ni) and zinc (Zn) and are often referred to as essential elements (Odjegba and Fasidi, 2004; Bigaliev, Boguspaev and Znanburshin, 2003).
These environmental pollutants are usually removed by physicochemical methods, however for the remediation of large areas where the pollutants occur at low concentrations use is made of bacteria, algae or plants (Rulkens, Tichy and Grotenhuis, 1998). With current trends moving towards greener technologies, focus is shifting to phytoremediation, where plants are used to take up metals or pollutants from the environment or transform them into harmless compounds (Berti and Cunningham, 2000; Salt et al., 1994). Phytoremediation presents a cheap, noninvasive, and safe alternative to conventional cleanup techniques and can be accomplished by phytoextraction, phytodegradation, phytostabilization, phytovolatilization and rhizofiltration (Glick, 2003). In general, phytoextraction and phytovolatization are considered as the main options for the removal of heavy metals and other elemental compounds, whereas phytodegradation and phytostabilisation are applied mostly to organic contaminants (Meagher, 2000; Guerinot and Salt, 2001).
The capacity of plants to concentrate metals has usually been considered a detrimental trait since some plants are directly or indirectly responsible for a proportion of the dietary uptake of toxic heavy metals by humans (Chaney et al., 1997; Cunningham, Berti and Huang, 1995). The dietary intake of heavy metals through consumption of contaminated crop plants can have long-term effects on human health (Ow, 1996).
Numerous plant species have been identified for bioremediation with certain plant species, known as hyper accumulators, being targeted, as they are able to accumulate potentially phytotoxic elements in their shoots at concentrations 50-500 times higher than average plants (Gisbert et al., 2008). These plants are of great interest both scientific, for understanding the biological mechanisms of hyperaccumulation, and economic, as plants that can be utilized for phytoremediation (Raskin, Smith and Salt, 1997; Salt et al., 1995). Currently, there are approximately 400 known hyper accumulators (Salt and Kramer, 2000), however most are not appropriate for phytoextraction as result of their slow growth and small size. For plants to be classified as hyperaccumulators they need to develop extended and abundant root biomass and maintain a low translocation of metals from roots to shoots (Mendez and Maier, 2008). There have been studies conducted by several researchers who have screened fast-growing, high-biomass accumulating plants, including agronomic crops, for their ability to tolerate, translocate and accumulate metals in their shoots (Kumar et al., 1995; Salt et al., 1995; Blaylock et al., 1997; Huang, Berti and Cunningham, 1997; Vamerali, Bandiera and Mosca, 2009). Many metal-tolerant plant species, particularly grasses, escape toxicity through an exclusion mechanism and are therefore better suited for phytostabilization than phytoextraction (Baker, 1981).
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The high bioconcentration factor, which is the ability of the plant to extract metals from the soil and the efficient root to-shoot transport system endowed with enhanced metal tolerance provide hyper accumulators with a high potential detoxification capacity (Yoon, Cao and Zhou, 2006). The choice of plant species to be used is an important decision in phytoremediation strategies, especially phytostabilisation (Rizzi et al., 2004).
In some places amaranths are considered to be cosmopolitan weeds in that they are capable of growing anywhere. The common names for amaranths are African spinach, Indian spinach, amaranth, bush greens, Chinese spinach and green leaf to mention a few (Hutchings, 1996). Research on the genus Amaranthus include Amaranthus hybridus (Jonnalagadda and Nenzou, 1997), which was used to determine the effect of coal mine contamination on the uptake and distribution of lead, cadmium, mercury, nickel, manganese and iron; Amaranthus tricolor and Amaranthus retroflexus (Bigaliev, Boguspaev and Znanburshin, 2003) which were used for the uptake of cadmium, mercury, zinc and copper; and Amaranthus spinosus (Prasad and de Oliveira Freitas, 2003) that was used for the accumulation of cadmium, zinc and iron.
For the purpose of this study Amaranthus dubius (marog or wild spinach) which is a popular nutritious leafy vegetable crop, rich in proteins, vitamins and minerals (Odhav et al., 2007), and consumed in Africa, Asia and South America was chosen to evaluate its potential as a hyperaccumulator. Their quick growth and great biomass makes them some of the highest yielding leafy crops which would be beneficial as a primary food source, thus preventing starvation and malnutrition in the third world countries. (Grubben, 1976) mentioned that because of their high yield, ability to grow in hot weather conditions, high nutritive value and their pleasant taste and the fact that they grow all year; makes the Amaranth a popular vegetable.
Materials and Methods
Soil and plant culture
Wild source seeds of A. dubius were obtained from a cultivated area. Flower heads were collected, air dried and the seeds removed and stored in glass bottles in a refrigerator. The seeds were germinated for one week in potting soil at a pH of 7-8, a moisture content controlled at 14%, and with 140 ppm potassium, 77 ppm of phosphorous, 1850 ppm of calcium, 520 ppm magnesium, and 77 ppm sodium. Following this, specimens were put into seedling trays in friable loamy soil. Six week old seedlings were transplanted into the pots, with 12 plants of uniform length (± 30 cm) placed a uniform distance apart in each pot. The pots were kept in a tunnel house at an ambient temperature. Salts of Cr, Hg, As, Pb, Cu and Ni [potassium dichromate (K2Cr2O7), mercuric chloride (HgCl2), arsenic trioxide (As2O3), lead nitrate (Pb(NO3)2), cupric nitrate[Cu(NO3)2.3H2O] and nickel sulphate (NiSO4.6H2O)]were added to the potting soil as described by (Gardea-Torresdey et al., 2004; Odjegba and Fasidi, 2004; Ahalya, Kanamadi and Ramachandra, 2005). Final concentrations were 25ppm, 75ppm and 100ppm for each metal under investigation. A separate pot with untreated soil was used to serve as a control. The pots containing the plants were placed in drip trays so as to prevent any leachate from being lost (Giordani, Cecchi and Zanchi, 2005; Kos, GrÄ‡man and Leštan, 2003).
The plants were watered daily with 500 ml of water per pot and all collected leachate was returned to the respective experimental pot. Three plants from each treatment were harvested after four days without damaging the roots and rinsed in distilled water to remove dust and soil mineral particles. The remaining plants were observed at four day intervals over a 16 day period for any phenotypical changes as a result of metal exposure. Plant samples were then separated into roots, stems and leaves and dried at 60°C in a convection oven for 48 h. Samples were milled to a fine powder using a Waring Commercial Laboratory Blender, placed in aluminum covered 500 mL Schott bottles and stored. The heavy metals were extracted using a microwave digestion procedure described by (Zunk and Planck, 1990). A mls 1200 mega - high performance microwave digestion unit [Milestone Microwave Laboratory Systems - supplier (Italy)] was used. Samples were weighed out in the digestion vessel and HNO3 and H2O2 added (Roy et al., 2005). The metals were analyzed using ICPMS according to the procedure outlined by (Zayed et al., 1998) and (Wang et al., 1999). Results obtained from ICPMS Analysis were mg/L and converted to parts per million (ppm).
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To evaluate the potential of A. dubius for phytoextraction, the translocation factor (TF) was calculated. This ratio is an indication of the ability of the plant to translocate metals from the roots to the aerial parts of the plant (Marchiol et al., 2004) . It is represented by the ratio:
Metal concentration in aerial parts
Metal concentration in roots
Metals that are accumulated by plants and largely stored in the roots of plants are indicated by TF values < 1, with values greater indicating translocation to the aerial part of the plant.
The Bioconcentration Factor (BCF) of metals was used to determine the quantity of heavy metal absorbed by the plant from the soil. This is an index of the ability of the plant to accumulate a particular metal with respect to its concentration in the soil (Ghosh and Singh, 2005) and is calculated using the formula:
Metal concentration in tissue of whole plant
Initial concentration of metal in substrate i.e. soil
The higher the BCF value the more suitable is the plant for phytoextraction (Blaylock et al., 1997) BCF Values > 2 were regarded as high values.
The uptake of Cr by A. dubius ranged from 16 ppm to 310 ppm, with the highest concentration of Cr being accumulated in the root system (Fig 1). In the samples exposed to Cr the highest concentration of Cr was accumulated by the plants grown in the soil containing 75 ppm. The plants exposed to Hg showed no visible phenotypical changes, with the samples exposed to each of the different Hg concentrations showing a uniform growth rate. The uptake of Hg ranged from 3 ppm to 666 ppm with the root system accumulating the highest concentration of Hg. In the soil containing As the pattern was similar to that of the plants grown in the soil containing Cr. The plant samples in the soil containing 25 ppm of As showed no phenotypical, changes however the samples exposed to 75 and 100 ppm showed both chlorosis and severe wilt. The concentration of As accumulated ranged from 4 ppm to 200 ppm.
The accumulation of As was different in comparison to the other modes of accumulation thus far with the lowest accumulation in the roots (Fig 1). The plant samples exposed to Pb showed chlorosis among all three concentrations (25, 75 and 100 ppm) with a uniform growth rate between the 25 ppm and 75 ppm Pb concentrations, while the 100 ppm showed a slightly slower growth rate. Leaves in this treatment were larger in comparison to plants exposed to other metals. The concentration of Pb accumulated ranged from 2 ppm to 138 ppm with the highest concentration accumulated by the roots (Fig 1). In the plants grown in the soil containing Cu there were no visible phenotypical changes between the three concentrations, however the plant exposed to 25 ppm showed a slower rate of growth in comparison to those grown in 75 ppm and 100 ppm. The concentration of Cu accumulated ranged from 20 ppm to 172 ppm with the highest concentration being accumulated by the roots (Fig 1). The plants grown in the soil containing Ni showed no phenotypical changes apart from the leaves appearing darker green in colour when compared to the plants exposed to the other metals. Concentrations of Ni accumulated ranged from 8 ppm to 205 ppm with highest concentration being stored in the roots. There was a difference in the growth rates between the different concentrations with the plants exposed to 100 ppm being the highest followed by 25 ppm and 75 ppm.
The Bioconcentration Factor (BCF) indicates the ability of the plant to absorb metals from soil. In this study BCF values > 2 were regarded as high (Table 1). The Translocation Factor (TF) of each metal under investigation (Cr, Hg, As, Pb, Cu and Ni) and each of the 3 different concentrations (25 ppm, 75 ppm and 100 ppm) for each metal is recorded in Table 1. From the TF results the closer to 0, the higher the concentration of the metal stored in the roots instead of being translocated to the shoots/aerial parts of the plant.
From the results, A. dubius accumulates Cr, Hg and Pb at 25 ppm and translocates it to the shoots, but at higher concentrations (75 ppm and 100 ppm) the metal is stored in the roots. In the case of As accumulation at all the concentrations the metal is translocated to the shoots. The Cu and Ni results indicate that the metal is stored mainly in the roots.
A. dubius clearly demonstrates that it can tolerate difficult soil conditions furthermore plant samples collected had well-developed rooting structures at lower concentrations (25 ppm and 75 ppm). The impact of metal contamination in the physiology of plants depends on the metal speciation, which is responsible for its mobilization, subsequent uptake and resultant toxicity in the plant system. All metals were found in the roots of A. dubius lead to an increase in root length corresponding with an increase in metal concentration for Pb, Cu and Ni. However, for Hg there was a decrease in root length with an increase in metal concentration and for Cr there was no visible relationship between root length and concentration. In the study it was found that the soil treated with Cr at 25 ppm showed a higher rate of growth in comparison to the 75 ppm and 100 ppm concentrations with exposure to higher concentrations also showing signs of chlorosis and wilting. The uptake of Cr ranged from 17 ppm to 308 ppm, with the highest concentration of Cr being accumulated in the root system (Tiwari et al., 2009). Soil treated with 100 ppm of Cr(VI) indicate that at this concentration there is toxicity to the plant, or interference to absorption by other metals present in the soil. Cr is not translocated to the aerial parts of the plant however it is bioaccumulated from the soil to the roots. This indicates that A. dubius can accumulate and convert Cr (VI) to Cr (III) which is the less toxic form.
Plants exposed to Hg showed no visible phenotypical changes. The uptake of Hg ranged from 3 ppm to 667 ppm with the root system accumulating the highest concentration of Hg. Bioaccumulation of Hg can occur at high Hg soil concentrations as shown in the field studies. In the in vitro study, soil containing 100 ppm Hg, approximately 1160 ppm Hg was accumulated by the plant. Bioaccumulation of Hg from the soil to the roots occurred at all soil concentrations. However, the translocation index for Hg was greater than one indicating that although Hg is phytoremediated, it is not sequestered to the leaves and thus phytovolatisation does not occur.
The plant samples grown in the soil containing 25 ppm of As showed no phenotypical changes, however the samples exposed to 75 and 100 ppm showed both chlorosis and severe wilt. The concentration of As accumulated, ranged from 4 ppm to 201 ppm. The accumulation of As was different in comparison to the other modes of accumulation thus far, with the lowest rate of accumulation in the roots. Maximum bioaccumulation of As (447 ppm) were observed in the soil containing 100 ppm As. At this high soil concentration, arsenic was bioaccumulated from the soil to the roots as well as translocated from the roots to the aerial parts of the plant.
The plant samples exposed to Pb showed chlorosis among all three concentrations (25, 75 and 100 ppm) with a uniform growth rate between the 25 ppm and 75 ppm Pb concentrations, while the 100 ppm showed a slightly slower growth rate. The ability of the plants to move Pb to the aerial parts of the plant is limited, and only the plants exposed to 25 ppm showed a TF > 1 (Table 1). Thlaspi rotundifolium and T. caerulescens show similar results (Reeves and Brooks, 1983). Leaves were however larger in comparison to the plants exposed to other metals. The concentration of Pb accumulated ranged from 2 ppm to 138 ppm, with the highest concentration accumulated by the roots.
In the plants grown in the soil containing Cu there were no visible phenotypical changes between the three concentrations. However the plant exposed to 25 ppm showed a slower rate of growth in comparison to those grown in 75 ppm and 100ppm. The concentration of Cu accumulated ranged from 20 ppm to 172 ppm, with the highest concentration being accumulated by the roots. It is well known that elements such as Cu, Mo, Ni, Cr, and Zn, among others, are essential trace metals for plant growth in low concentrations (Taiz and Zeiger, 1998). Nevertheless, beyond certain threshold concentrations, these same elements become toxic for most plant species (Blaylock and Huang, 2000).
The plants grown in the soil containing Ni showed no phenotypical changes apart from the leaves appearing darker in colour when compared to the plants exposed to the other metals. Concentrations of Ni accumulated ranged from 8 ppm to 205 ppm, with highest concentration being stored in the roots. Bioaccumulation of Pb and Ni showed the highest accumulation in in vitro studies with soil containing 75 ppm and 100 ppm respectively. Both metals showed similar results with translocation of the metals from the soil to the roots of the plant at low concentrations in soil.
The translocation Factor (TF) of each metal under investigation (Cr, Hg, As, Pb, Cu and Ni) and each of the 3 different concentrations (25 ppm, 75 ppm and 100 ppm) for each metal show that A. dubius accumulates Cr, Hg and Pb at 25 ppm and translocates it to the shoots, but at higher concentrations (75 ppm and 100 ppm) the metal is only stored in the roots. In the case of As accumulation at all the concentrations, the metal is translocated to the shoots. The Cu and Ni results indicate that the metal is stored in the roots mainly.
This study shows that A. dubius can be defined as a hyperaccumulator of As since it has the ability to extract As and can tolerate high levels of this metal. A. dubius is a cosmopolitan plant with a rapid growth rate, yields a high biomass and is easy to harvest and the metal is sequestered from the soil and transported to the aerial parts.