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A pot culture experiment was conducted in the green house of Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore, India to study the potential chromium (Cr) phytoaccumulatory capabilities of 4 promising agroforestry trees species viz., Albizia amara, Casuarina equisetifolia, Tectona grandis, Leucaena luecocephala. Possibility of enhancement of Cr uptake by chemical (citric acid) and biological {vesicular arbuscular mycorrhizal fungi (VAM)} amendments was also tried. Both biologically stable speciation of Cr (trivalent and hexavalent) were used. Cr(VI) was more toxic to the tree growth in terms of CD increments in all the tree species than Cr(III). In general roots accumulated more Cr than shoots in all the trees species. There was more than 10 fold increase in root Cr content in comparison with shoot Cr content in all the trees at all concentration of Cr and all sources of Cr. Citric acid significantly increased the Cr content in the tissues of roots in all the species under both speciation of Cr. The highest increase in Cr content brought by 20mM citric acid addition was in the case of A. amara. Unlike citric acid, VAM treatment did not bring about a significant increase in the Cr contents of the entire tree species studied although there was a slight increase in the Cr content of all the trees. Results suggests that Albizia amara is a potential Cr accumulator with citric acid as soil amendment. This tree can be taken up for future long term on site trials in Cr contaminated lands to establish its usefulness as Cr phytoaccumulator.

Key words: chromium speciation, phytoremediation, agroforestry, organic acids, VAM

  • Present address: National Research Centre for Agroforestry, Jhansi, Uttar Pradesh, INDIA


Chromium (Cr) is the chief heavy metal contaminant found in the tannery effluent. Cr used by the leather industry to tan hides is not taken up completely by leather and relatively large amounts escapes into the effluent. Due to chrome leather tanning processes, large quantities of Cr compounds are discharged through liquid, solid, and gaseous wastes into the environment and can have significant adverse biological and ecological effects. Several reports have shown that the values for Cr in tannery effluent are considerably higher than the safe limits prescribed by International standards (Khan, 2001). Soil and water ecosystems have been contaminated to an overwhelming extent in the vicinity of leather industry and this has rendered arable land unproductive and underproductive. Cr is a toxic element to higher vascular plants and is detrimental to its growth, development and reproduction (Cervantes et al., 2001). The physiological impact of Cr contamination in soil and water is dependant on the speciation viz., Cr(III) and Cr(VI). Speciation of Cr in the soil differentially affects the mobilisation of the metal, subsequent uptake and resultant toxicity in the plant system. Cleaning up of the Cr contaminated sites is a challenging task. Phytoremediation by trees is an emerging technology that can be considered for remediation of contaminated sites because of its cost effectiveness, aesthetic advantages, and long term applicability. Phytoremediation is well suited for use at very large field sites where other methods of remediation are not cost effective or practicable; at sites with low concentrations of contaminants, where treatment is required over long periods of time. Phytoextraction refers to the use of metal accumulating plants that translocate and concentrate metals from the soil in roots and above ground shoots or leaves (Cunningham and Ow, 1996). Tree species in association with mycorrhizae have shown promising prospects for phytoremediation of Cr contaminated lands in and around tannery industrial areas (Khan, 2001). Organic acids have been used to enhance extraction of immobile metals from soils due its ability to complex with metals and increase its availability (Wu et al., 2003). Very little work has been done to integrate multipurpose trees into phytoremediation efforts. There is a distinct lack of literature on Cr phytoaccumulatory potential of trees in general and multipurpose trees in particular. The present study was thus taken up to understand the Cr uptake pattern in potential agroforestry tree species under biological (VAM) and chemical amendment (citric acid) conditions.


Tree culture and treatments

Three month old seedlings of potential agroforestry tree species Albizia amara, Casuarina equisetifolia, Tectona grandis, Leucaena luecocephala tree species of equal height and collar diameter was used in the study. Pot mixture was prepared by mixing thoroughly two parts of soil and one part each of well-decomposed farmyard manure and sand and filled in pots with 10 Kg of soil. Fertilizer was added in the soil at the following rate: Urea - 100 mg of N Kg-1, DAP- 50 mg of P Kg-1, KCl - 50 mg of K Kg-1. The treatments consisted of Control , Cr (III) as Cr2 (SO4)3.2H2O @ 250mg kg-1 of soil ,Cr (VI) as K2Cr2O7 @ 100mg kg-1 of soil, Cr (III) as Cr2 (SO4)3.2H2O @ 250mg kg-1 of soil + VAM, Cr (VI) as K2Cr2O7 @ 100mg kg-1 of soil +VAM, Cr (III) as Cr2 (SO4)3.2H2O @ 250mg kg-1 of soil + 20mM citric acid, Cr (VI) as K2Cr2O7 @ 100mg kg-1 of soil + 20mM citric acid. The soil containing added chromium treatments were thoroughly mixed with chromium salts before filling into the pots. The inoculum of Glomus mossae was applied at 2000 spores per pot as a band at the bottom third of the container, then covered with soil and the roots of the tree seedlings were allowed to grow into the band and colonize.

The soil properties of the pot culture experiment are given in Table 1. The Cr status of the soil at midway stage (6 months) is given in table 2. Normal plant protection measures were adopted through out the seedling growth period. Replications were seven and the design was CRD. The experiment was repeated two times. Plant samples were drawn one year after the initiation of the experiment from each treatment for recording growth and chromium uptake pattern.

Chromium content and growth

Growth in terms of collar diameter (CD) was recorded using a measuring tape and expressed in cm. Measurement of chromium content (mg g-1) was made on individual seedling roots and shoots. Plants were harvested and roots washed with running tap water. Roots and shoots were separated and oven dried for three days at 80oC. Samples were then ground into fine powder using a grinding mill. The conditions used for digestion were modified from Cary and Olsen (1975). Five millilitres of concentrated HNO3 was added to 0.25g of dried sample in a 50ml digestion tube and allowed to stand overnight at room temperature. The digestion tubes were placed in a heating block for one hour at 150oC, tubes were then removed allowed to cool and 2ml of 30 per cent H2O2 was added.The tube contents were mixed by swirling, and then heated for 2 more hours at 150oC. After cooling the solution was diluted to 50 mL total volume and the upper clear portion was used for chromium estimation. During dilution, NH4Cl was added at 2 per cent and CaCl2 was added at 0.5 per cent to each sample and standard to control interference caused by iron (Fe) and phosphorus (P) respectively during spectrophotometer analysis. Digested samples were analysed for Cr in atomic absorption spectrometer (Varion Spectra AA-220) with air- acetylene flame at 358nm with 0.2mm spectral slit width.

The accumulation factor (ACF) of the tree species was estimated according to (Baker et al., 1994).

Seven replication were taken for every estimation and the experiment was repeated twice. The data was analysed statistically using a general linear model (Wilkinson et al., 1996) for analysis of variance in completely randomised design. Critical difference (CD) was used to compare treatments for significance at 0.05 probability level.


Among the tree species highest CD increase was seen in C. equisetifolia, this was followed by L. leucocephala and A.amara (Table3). The least growth was observed in T. grandis. Among the treatments Cr(VI) affected the CD increments significantly in comparison to control in all the tree species (1.38 cm in control and 1.65cm in Cr(VI)). A similar trend was seen in all the other tree species. The decrease in CD caused by Cr(III) was not significantly different from Cr(VI), although both were significantly different from control. The general pattern of Cr accumulation (Table 4) was highest in A. amara followed by T. grandis, C. equisetifolia and the least accumulation was seen in L. leucocephala. Among the Cr treatments the Cr(III) treated trees accumulated significantly more Cr in tissue in comparison to Cr (VI). Cr(III) treated trees exhibited a Cr content of 476, 524, 532 and 623 in the roots of L. leucocephala, C. equisetifolia, T . grandis and A. amara respectively. In general roots accumulated more Cr than shoots in all the trees species. There was more than 10 fold increase in root Cr content in comparison with shoot Cr content in all the trees at all concentration of Cr and all sources of Cr. Citric acid significantly increased the Cr content in the tissues of roots in all the species under both speciation of Cr. The highest increase in Cr content brought by 20mM citric acid addition was in the case of A. amara followed by C. equisetifolia, T. grandis and the least was seen in L. leucocephala in the same treatments in roots of the respective tree species. Unlike citric acid, VAM treatment did not bring about a significant increase in the Cr contents of the entire tree species studied although there was a slight increase in the Cr content of all the trees. Although the total content of Cr was more in Cr(III) treated trees the ACF values were more in Cr(VI) treated trees irrespective of the tree species studied. L. leucocephala had ACF of 1.88 in Cr(III) treatment as against 3.79 in Cr(VI) treatment. VAM and citric increased the ACF values in both the Cr speciation in all the trees species. The increase brought citric acid was higher than VAM. The highest accumulation factor was observed in A.amara followed by T. grandis, C. equisetifolia and the least ACF was seen in L. leucocephala irrespective of the speciation of Cr added to the soil.


Soil culture experiments are more precise in providing results closer to the natural effect of Cr in view of the fact that the bioavailability of Cr is greatly affected by the soil properties. Potential toxicity of Cr speciation added to soils may be affected by the interactions between oxidation-reduction and organic complexation. Plants cannot usually access the total pool of a metal present in the growth substrate. Instead, the fraction of the metal, which plants can absorb, is known as the available or bio available fraction. Metals present in a soil can be divided into a number of fractions including; the soluble metal in the soil solution, metal-precipitates, metal absorbed to clays, hydrous oxides and organic matter, and metals within the matrix of soil minerals (Reichmann, 2002). The deleterious effect of Cr on the growth of all the tree species was evident from the reduction in collar diameter of the seedlings. The reduction in collar diameter could have been due to Cr induced toxicity to the outer cells of the tree barks affecting the secondary thickening processes of the trees. The possibility of reduced root growth and resultant impaired water and nutrient status of the trees could have caused decrease in collar diameter increments. The results obtained are in confirmation with Pulford et al., (2001). The possibility of modification of the Cr form after it is supplied in either Cr(III) or Cr(VI) cannot be ruled out the experiment. This can occur because of action of root exudates with the capacity to reduced or oxidise either form of Cr or by the action of organic complexes or soil micro flora or fauna with the capacity to participate in the redox reactions of Cr in soil The reason of the high accumulation in roots of the trees could be because Cr is immobilised in the vacuoles of the root cells to render it non-toxic, which may be a natural toxicity response of the plant. Since both Cr(VI) and Cr(III) must cross the endodermis via symplast, the Cr(VI) in cells is probably readily reduced to Cr(III) and the Cr(III) is retained in the root cortex cells Zayed et al.(1998). Another important reason for the lack of transport of Cr from roots to shoots could be because the plants lack any specific mechanism of transport of Cr, as it is a toxic and nonessential element in itself. The accumulation factor for both Cr speciation derived in the present study in tree species revealed the differing capacity of the four tree species studied. The results are in confirmation with the reports of Khan (2001). This could be because the uptake pattern of Cr from soil depends on the tree species and that within the tree species the concentration largely differs between different parts of the trees (Pulford et al., 2001). In general it is possible that the restriction of Cr to roots is more in non-accumulator species. The increase although not significant brought about by VAM could have been due to the ability of VAM to mobilize Cr in soil. There was a pronounced enhancement of Cr uptake in tree species treated with citric acid. Similar results have been reported by Shahandeh and Hossner (2000) and Srivastava et al. (1999). The reason for this could be that the organic acids modified soil properties, increased the labile fraction of Cr and mobilised Cr for easy uptake by roots (Wu et al., 2003). The poor transfer of Cr from root to shoot means that the prospects for using trees as phytoremediators on chromium-contaminated sites are poor, but it should be noted that Cr is very poorly translocated in all the higher vascular plants (Zayed et al.,1998). Hence the use of these trees as potential phytoremediators can only be proven over very long period of time Our results suggests that A.amara is a potential Cr accumulator with citric acid as soil amendment. Although fodder is one of the economic component of this tree in an agroforestry system, the tree if only used for firewood and timber and not for fodder would ensure no reentry of the Cr accumulated by the tree into biological systems. This tree can be taken up for future long term on site trials in Cr contaminated lands to establish its usefulness as Cr phytoaccumulator.


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