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Chemosphere 64 (2006) 161–173

Chemical fractionation and heavy metal accumulation in the plantof Sesamum indicum (L.) var. T55 grown on soil amended

with tannery sludge: Selection of single extractants

Amit K. Gupta, Sarita Sinha *

Ecotoxicology and Bioremediation Group, Environmental Science Division, National Botanical Research Institute, Rana Pratap Marg,

Lucknow 226 001, India

Received 29 June 2005; received in revised form 4 October 2005; accepted 11 October 2005Available online 5 December 2005

Abstract

A pot experiment was carried out to study the single and sequential extractions of metals in different tannery sludge amendment andthe potential of the plant of Sesamum indicum L. var. T55 (sesame) for the removal of metals from tannery waste contaminated site. Themetal extraction efficiency obtained with each extractants was slightly different and follow the order; EDTA > DTPA > NH4NO3 >NaNO3 > CaCl2. The correlation analysis between extractable metals in the different amendments of sludge and metal accumulationin the plant (lower and upper parts) showed better correlation for most of the tested metals with EDTA extraction. In this study, asequential extraction technique was applied on different amendments of tannery sludge. The results showed that Mn, Zn, Cr and Cdwere mostly associated with Fe–Mn oxide fraction in most of the amendments, K and Ni was found in residual (RES) fraction, Feand Cu was bound with organic matter (OM) and RES fractions and Na was associated with carbonate (CAR) fraction. The metal accu-mulation after 60 d of growth of the plant was found in the order of K > Na > Fe > Zn > Cr > Mn > Cu > Pb > Ni > Cd and its trans-location was found less in upper part. The accumulation of toxic metals (Cr, Ni and Cd) in the plants was found to increase with increasein sludge ratio, in contrast, the accumulation of Pb decreased. In view of growth parameters and metal accumulation in the plant, it wasobserved that lower amendments (25%) of tannery sludge were found suitable for the phytoremediation of most of the studied metals.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Tannery sludge; Metals; EDTA; DTPA; CaCl2; NH4NO3; NaNO3; Sesamum indicum

1. Introduction

It is impossible to visualize a soil without trace levels ofheavy metals. Further, anthropogenic activities have con-centrated some of these elements in certain areas up to dan-gerous levels for living organism. Activities such as mining,sludge disposal and agriculture have polluted extensiveareas throughout the world. There is an increasing interestin the agricultural application of sewage sludge obtainedfrom wastewater treatment plants due to the possibility ofrecycling valuable components: organic matter, N, P and

0045-6535/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.chemosphere.2005.10.016

* Corresponding author. Tel.: +91 522 205831 35x221; fax: +91 522205839/205836.

E-mail address: sinha_sarita@rediffmail.com (S. Sinha).

other plant nutrients (Wong et al., 2001). The characteriza-tion of sewage sludge metals is an important requirementprior to sludge disposal or application to farmland becausethere is a risk of toxic element accumulation in the soil(Obrador et al., 1997; Alvarez et al., 2002). Although, sew-age sludge has been shown to increase crop production(Hernandez et al., 1991; Reddy et al., 1998), however, itmay contain certain trace elements at injurious level toplants and the food-chain. It is well known that elementssuch as Cu, Mo, Ni, Zn, Mn and Fe, among others areessential for the plant growth in low concentrations (Reevesand Baker, 2000). Nevertheless, beyond certain thresholdconcentrations, these elements become toxic for most ofthe plant species (Blaylock and Huang, 2000; Monniet al., 2000). The environmental hazards of trace element

162 A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173

pollution depend on geochemical and biochemical proper-ties of a given element and are related to several processes.

Several methods have been developed in an attempt topredict phytoavailability of metals, which have largely beenapplied for agricultural plants and soil. In this context,phytoavailability has often been defined through a one-stepsoil extracting procedures. The amount of heavy metalsextracted by such methods gives an idea of the size of apool that might be depleted by a plant during the growthperiod; however, the extent of extracting methods dependson the soil tested.

The determination of total heavy metal content of soilsamples is not sufficient to evaluate the possible mobilityand, consequently, the bioavailability of toxic metals to liv-ing organism. The application of sequential extraction pro-cedures allows the determination of the chemical ‘‘forms’’,in which the elements appear to be associated in the sam-ple. It can help to assess how strongly they retained in soiland how easily they may be released into soil solution. Dif-ferent sequential extraction techniques, such as the five stepprocedure of Tessier et al. (1979) are commonly applied toevaluate both the actual and potential mobility of metals inthe environment (Chlopecka et al., 1996; Sanchez et al.,1999; Kaasalainen and Yli-Halla, 2003). Indeed, theseheavy metal pools may be selectively affected by plantuptake and change in their proportions may give an ideaon the mechanism responsible for heavy metal uptake incomparison with chemical pool in the soil.

In the context of phytoextraction, it is necessary to iden-tify hyperaccumulators plants and more specifically thepotential of the plants to mobilize or deplete metals fromcontaminated soil. This allows assessment of the potentialrisk or benefits of using plants to remediate heavy metal-contaminated soils as well as long-term efficiency of thetechnique. It has been reported that some of the plant spe-cies are capable of tolerating high concentrations of heavymetals which opened new possibilities for the remediationof contaminated soils (Salt et al., 1995; Ebbs and Kochian,1997; Blaylock and Huang, 2000; Singh et al., 2004a,b).Shahandeh and Hossner (2000) also compared the accumu-lation of Cr in thirty-six plant species and found thatIndian mustard and sunflower accumulated more Cr thanother agricultural plant species from the soil. Thus, theoil bearing plants have shown potential for the deconta-mination of the soil.

In view of above, the proposed work was aimed to studythe single and sequential speciation of metals in differentamendment of tannery sludge and the potential of the oilbearing plant, Sesamum indicum L. var. T55 (sesame) forthe removal of metals from tannery waste contaminated site.

2. Materials and methods

2.1. Experimental setup

The uncontaminated garden sub soil collected fromNational Botanical Research Institute, Lucknow (Uttar

Pradesh, India) was used to make the various amendmentsfor experimental studies. Dried tannery sludge cakes werecollected from the sludge beds of UASB Wastewater Treat-ment Plant, Jajmau, Kanpur (Uttar Pradesh, India), inlarge plastic bags and brought to the field laboratory.The tannery sludge (TS) and soil were air dried, finely pow-dered and sieved to 2 mm mesh size before use. The variousamendments of tannery sludge (10%, 25%, 35%, 50% and100%) were prepared using garden soil, which served ascontrol (C). The seeds of S. indicum L. var. T55 (sesame)were sterilized with 0.1% mercuric chloride for 5 min toavoid fungal contamination, washed with distilled waterfor three times. The soaked seeds were evenly sown in pots(12 in. in diameter), which were filled with different amend-ments of tannery sludge along with one set of control, eachin three replicates. Ten seeds were sown in each pot to adepth of 0.5 cm. The pots were watered daily till seed ger-mination. When the seedlings have developed 5 or 6 leaves,they were thinned out to retain 6 uniform ones per pot andallowed to grow. Pots were placed in the field laboratory atan average diurnal temperature of 25–45 �C. The waterlevel was made up as and when required using tap water.The plants were harvested after 60 d after sowing. All theplants were free from any disease in whole of the experi-ment duration.

2.2. Metal accumulation

Plants were uprooted from the pots with the help of finejet of water causing minimum damage to the roots, washedthoroughly with distilled water, and blotted dry. Differentplant parts were separated manually, cut in small piecesand oven dried (80 �C). The oven-dried samples wereground and digested in HNO3 (70%) using MicrowaveDigestion System MDS 2000 and metal contents were esti-mated using atomic absorption spectrophotometer (GBCAvanta R).

2.3. Physico-chemical analysis of different amendments

Physico-chemical analyses were carried out in triplicateon ground dry samples of soil and tannery sludge and theirdifferent amendments before the growth of the plants of S.

indicum. The pH of the different amendments was mea-sured in 1:2.5 soil water suspension using Orion pH meter(Model 420); electrical conductivity (EC) was measuredusing Orion Conductivity meter (Model 150). Organic car-bon (OC) and organic matter (OM), cation exchangescapacity (CEC) are estimated by the method of Kalraand Maynard (1991).

2.4. Single extraction of different amendments

DTPA extractable fraction was obtained by mechanicalshaking of sample (4 g) with 40 ml of 0.5 M DTPA, 0.01 MCaCl2, 0.1 M TEA (triethanolamine) buffered at pH 7.3 for2 h (Lindsay and Norvell, 1978). For CaCl2 extraction, 5 g

A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173 163

soil with 50 ml of 0.01 mol l�1 CaCl2 solution was mechan-ically shaken for 2 h (Houba et al., 1997). For EDTAextraction, 5 g soil with 25 ml of 0.05 M EDTA solutionwas mechanically shaken for 1 h (Lakanen and Ervio,1971). In case of NH4NO3 extraction (Symeonides andMcRae, 1977), 10 g of soil was added in 50 ml of 1 MNH4NO3 and shaken for 2 h at room temperature. ForNaNO3 extraction, 10 g soil with 25 ml of 0.1 M NaNO3

solution was mechanically shaken for 2 h (Houba et al.,1997). All the analyses were carried out in triplicates.GBC Avanta R atomic absorption spectrophotometer(AAS) was used for the estimation of metals.

2.5. Sequential extraction

Metals in different amendments of tannery sludge andgarden soil were fractionated by the sequential extractionprocedure of Tessier et al. (1979) in triplicates. The chem-ical reagent, extraction conditions and corresponding frac-tion are listed in Table A.

2.6. Quality control and quality assurance

The standard reference material of metals (E-Merck,Germany) was used for the calibration and quality assur-ance for each analytical batch. Analytical data quality ofmetals was ensured through repeated analysis (n = 6) ofEPA quality control samples (Lot TMA 989) for metals(Cd, Cr, Cu, Pb) in water and the results were found tobe within ±3.57% of certified values. The reference solutionof multi-elements and single element provided by NationalPhysical Laboratory (NPL), New Delhi, India was alsoused for calibration of analytical equipment and validationof test methods and their certified and observed values aregiven.

Sr.no.

Code no.(elements)

Certifiedvalues

Observedvalues

1 BND 1101.02Zn 99.69 ± 0.94 101.34 ± 0.87Fe 100.12 ± 0.78 99.32 ± 0.44Cu 99.95 ± 0.84 101.43 ± 0.51

2 BND 102.03 (Pb) 2.01 ± 0.02 2.05 ± 0.033 BND 402.02 (Cr) 2.00 ± 0.02 1.98 ± 0.034 BND 1001.02 (Ni) 1.00 ± 0.02 0.99 ± 0.01

Table ASequential extraction procedure and the corresponding fraction

Steps Fractions Extraction procedures

1 Exchangeable (EXC) 1 g of soil sample, 8 ml 1 mol l�1

2 Bound to carbonate (CAR) 8 ml 1 mol l�1 CH3COONa, adjus3 Bound to Fe–Mn oxides (Fe–Mn) 20 ml 0.04 mol l�1 NH2OH ÆHCl i4 Bound to organic matter (OM) 3 ml 0.02 mol l�1 HNO3, 30% H2

CH3COONH4 in 20% (v/v) HNO5 Residual (RES) 3 ml HNO3 + HClO4 + HF under

For plants, recoveries of metals from the plant tissueswere found to be more than 98.1% as determined by digest-ing three samples each from an untreated plant with knownamount of metals. The blanks were run in triplicate tocheck the precision of the method with each set of samples.

2.7. Statistical analysis

The experiment was performed in completely random-ized block design involving six amendments of tannerysludge with soil with three replicates. Student�s t-test (onetailed) and correlation coefficients analysis was performed.Pearson�s correlation coefficients, a measure of the linearassociation of two variables, were determined for plantmetal accumulations versus different extractants and differ-ent physico-chemical parameters of different amendment.Cluster analysis were applied on experimental data stan-dardized through z-scale transformation in order to avoidmisclassification due to wide difference in data dimension-ality and allowed the assessment of homogenous groups forthe impact of the tannery sludge amendments observed inthis study.

3. Results and discussion

3.1. Physico-chemical analysis

The analysis of the results of physico-chemical proper-ties (Table 1) of different amendments of tannery sludgeand control soil before growth of the plant showed signif-icant increase in pH from 6.85 to 7.73 with an increase insludge amendment ratio from 10% to 100% TS. The valueof EC decreased with an increase in sludge amendmentratio up to 35% TS followed by increase in higher amend-ments. The level of CEC, OC and OM was found toincrease with increase in sludge amendments in comparisonto control.

The observed increase in soil pH with increase in tan-nery sludge amendment was attributed to the �alkalising�effect of tannery sludge. Organic matter and pH are mostimportant factors that control the availability of heavymetals in the soil (Karaca, 2004). Raising the soil organicmatter can increase the soil CEC, a factor that may affectboth soluble and exchangeable metal levels (Yoo andJames, 2002). However, in the current study, the effectsof tannery sludge on soil pH seemed to be the one of the

MgCl2, pH 7, shake 1 h, room temperatureted pH to 5.0 with CH3COOH, shake 5 h, room temperaturen 25% CH3COOH, pH 2.0, water bath, 96 �C, 6 h, occasional shakingO2 (adjusted to pH 2.0), water bath, 85 �C, 5 h, 3.2 mol l�1

3, shake 30 minhigh pressure, 170 �C

Table 1Physico-chemical analysis of different amendments of tannery sludge before the growth of S. indicum

Parameters Amendments

C 10% TS 25% TS 35% TS 50% TS 100% TS

pH (1:2.5) 6.69 ± 0.0 6.85 ± 0.01 7.08 ± 0.0 7.12 ± 0.0 7.38 ± 0.01a 7.73 ± 0.01a

EC (lS cm�1) 461.0 ± 0.0 716.25 ± 0.5b 617.0 ± 0.0 589.0 ± 0.0 615.5 ± 5.19b 3830 ± 10b

CEC Cmol (p+) kg�1 32.20 ± 2.60 42.22 ± 3.01 50.32 ± 1.21c 53.49 ± 4.89d 46.65 ± 6.72 174.77 ± 11.91c

OC (%) 0.42 ± 0.08 0.57 ± 0.04 0.81 ± 0.01d 0.86 ± 0.07d 1.15 ± 0.01c 5.87 ± 0.03b

OM (%) 0.72 ± 0.02 0.99 ± 0.0c 1.39 ± 0.02b 1.48 ± 0.03b 1.97 ± 0.02b 10.13 ± 0.06b

All the values are mean of three replicates ± SD. Students t-test (one tailed as compared to control).a p < 0.05.b p < 0.02.c p < 0.01.d p < 0.001.

164 A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173

promising factor, which affects availability and uptake ofmetals. Thus, pH effects were greater than that of metalcomplex formation by soluble OM.

3.2. Single extraction

The analysis of the results (Table 2) revealed that per-centage of EDTA extractable Fe, Mn and K decreased withan increase in sludge ratio from 10% to 100% TS in con-trast, the level of Cr, Cu and Cd increased. The level ofsodium was found almost equal in all the amendmentsexcept 100% TS.

The levels of all the metals (Table 3) extracted withDTPA were found significantly high in tannery sludge thancontrol soil except Mn. In control soil, the level of Cr andNi were not detected in all the amendments except 100%TS.

In case of CaCl2 (Table 4) extraction, the levels of Fe,Mn and Pb were found to decrease with increase in the tan-nery sludge ratio. The level of Cd decreased up to 50% TSfollowed by increase. Zinc and Ni was not detected in allthe amendments, whereas, Pb, Cr and Cd were not detectedin control soil. Maximum level of Na and K was recordedin 100% TS and lowest in 10% TS.

Table 2EDTA extractable metals (mg kg�1 dw) in different amendments of tannery sl

Metals Amendments

C 10% TS 25% TS

Na 1006.98 ± 7.74 1008.37 ± 24.20 1008.59 ± 16.95K 9.63 ± 0.00 25.69 ± 2.62a 23.10 ± 1.54a

Fe 48.00 ± 2.06 69.02 ± 2.52a 62.0 ± 2.84c

Zn 11.10 ± 0.47 14.25 ± 0.07a 26.11 ± 1.58a

Mn 13.91 ± 0.28 59.23 ± 6.45a 47.86 ± 1.73b

Cu 5.79 ± 0.22 9.17 ± 0.76d 10.83 ± 0.23a

Cr 0.15 ± 0.06 6.20 ± 0.16b 12.47 ± 0.68a

Pb 2.00 ± 0.18 10.75 ± 2.14c 9.10 ± 0.22b

Ni ND 2.18 ± 0.05b 1.81 ± 0.01Cd 0.15 ± 0.06 1.29 ± 0.02a 2.34 ± 0.09b

All the values are mean of three replicates ± SD, ND, not detected. Studentsa p < 0.05.b p < 0.02.c p < 0.01.d p < 0.001.

In NH4NO3 extraction (Table 5), the percentage of Fe,Cu and Pb were found to increase with increase in sludgeamendments i.e., 1.20–1.86 and 0.29–2.38 mg kg�1 dw forFe and Cu, respectively, except Pb. The level of Cr andCd were found to decrease with increase in sludge amend-ment up to 35% TS and not detected in control soil. Zincand Ni was not detected in all amendments except 100%TS.

In case of NaNO3 (Table 6) extraction, the level of Cu,Pb, Cd and Cr were found to decrease with increase insludge amendments up to 35% TS followed by increase,in contrast, Fe and K was found to increase with increasein sludge amendment ratio. The level of Mn, Zn and Nidecreased with increase in sludge amendments ratio.

Among all the extractants (Tables 2–6), EDTA extrac-tion showed better extractability of all the tested metalsexcept K, it was found maximum with CaCl2. Overall anal-ysis of single extraction procedures showed the extractionefficiency was as follows EDTA > DTPA > NH4NO3 >NaNO3 > CaCl2. These extractions provide different infor-mation while predicting the mobility of the metals in theplant.

Several reports are available (Hammer and Keller, 2002;Sahuquillo et al., 2003) where EDTA was expected to

udge and control soil before the growth of S. indicum

35% TS 50% TS 100% TS

1008 ± 31.81 1011.64 ± 4.92 897.97 ± 9.30a

22.19 ± 1.91a 15.64 ± 3.54 5.13 ± 0.03b

49.75 ± 5.00d 33.30 ± 2.70a 21.48 ± 0.3118.94 ± 0.21a 20.76 ± 0.45a 38.58 ± 0.48b

46.68 ± 1.44b 37.58 ± 2.45a 11.81 ± 0.25a

10.98 ± 0.02b 11.57 ± 0.04b 20.78 ± 0.04b

12.64 ± 0.42b 10.77 ± 0.41b 11.59 ± 5.218.95 ± 0.05b 8.26 ± 0.05b 13.43 ± 0.24b

1.63 ± 0.01 1.28 ± 0.01 1.75 ± 0.012.52 ± 0.04b 3.34 ± 0.05b 14.85 ± 0.80b

t-test (one tailed as compared to control).

Table 3DTPA extractable metals (mg kg�1 dw) in different amendments of tannery sludge and control soil before the growth of S. indicum

Metals Amendments

C 10% TS 25% TS 35% TS 50% TS 100% TS

Na 189.71 ± 5.73 256.86 ± 4.6a 217.53 ± 4.34b 202.94 ± 11.65 255.54 ± 4.38a 641.39 ± 17.07c

K 33.42 ± 2.16 50.93 ± 4.15b 34.30 ± 6.52 31.21 ± 4.5 38.00 ± 2.83 104.92 ± 6.2a

Fe 10.24 ± 0.21 12.59 ± 0.30a 13.66 ± 0.20a 14.00 ± 0.46a 14.89 ± 0.70a 94.05 ± 4.66a

Zn 6.23 ± 0.65 7.15 ± 1.22 13.65 ± 0.36a 7.47 ± 0.51c 10.21 ± 0.28c 55.06 ± 1.48c

Mn 17.99 ± 1.01 15.06 ± 0.34b 13.33 ± 0.19c 12.95 ± 1.06b 11.89 ± 0.68c 8.61 ± 0.40a

Cu 1.79 ± 0.22 2.68 ± 0.09b 3.47 ± 0.04a 3.24 ± 0.15c 4.11 ± 0.10a 25.10 ± 0.57c

Cr ND ND ND ND ND 3.55 ± 0.72Pb 1.07 ± 0.10 2.73 ± 0.31d 3.36 ± 0.08a 3.50 ± 0.10a 3.87 ± 0.20a 19.66 ± 0.51c

Ni ND ND ND ND ND 4.63 ± 0.17Cd ND 1.21 ± 0.10a 1.64 ± 0.02c 1.71 ± 0.02c 2.14 ± 0.04c 23.24 ± 2.06a

All the values are mean of three replicates ± SD. ND, not detected. Students t-test (one tailed as compared to control).a p < 0.05.b p < 0.02.c p < 0.01.d p < 0.001.

Table 4CaCl2 extractable metals (mg kg�1dw) in different amendments of tannery sludge and control soil (v/v) before the growth of S. indicum

Metals Amendments

C 10% TS 25% TS 35% TS 50% TS 100% TS

Na 185.92 ± 9.50 179.28 ± 0.31 193.97 ± 0.06 172.68 ± 3.90 229.99 ± 1.03a 537.89 ± 3.81b

K 97.66 ± 12.29 52.12 ± 8.75c 62.79 ± 4.59c 63.32 ± 3.11c 65.30 ± 1.84c 116.71 ± 1.31Fe 7.37 ± 0.01 1.96 ± 0.21b 0.20 ± 0.04b 0.58 ± 0.05b 0.02 ± 0.00 NDZn ND ND ND ND ND NDMn 5.32 ± 0.009 1.90 ± 0.02b 1.73 ± 0.03b 1.66 ± 0.09b 0.77 ± 0.06b 0.26 ± 0.08b

Cu 0.23 ± 0.003 0.36 ± 0.01 0.32 ± 0.02a 0.28 ± 008 1.01 ± 0.02b 0.95 ± 0.02b

Cr ND ND ND 0.55 ± 0.03b 2.16 ± 0.04b 5.35 ± 0.09b

Pb ND ND ND 0.84 ± 0.01 0.76 ± 0.03 0.71 ± 0.03d

Ni ND ND ND ND ND NDCd ND 0.90 ± 0.03d 0.54 ± 0.01 0.32 ± 0.01 0.23 ± 0.01 1.33 ± 0.01

All the values are mean of three replicates ± SD. ND, not detected. Students t-test (one tailed as compared to control).a p < 0.05.b p < 0.02.c p < 0.01.d p < 0.001.

A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173 165

extract more heavy metal than DTPA and NaNO3 irre-spective of the soil properties because of its low pH. Fur-ther EDTA is assumed to extract both carbonate andorganically bound fractions of heavy metals in soils,whereas, DTPA buffered with triethanolamine is consid-ered suitable for calcareous soils.

Karaca (2004) reported the effect of organic waste appli-cation on the extractability of Cd, Cu, Ni and Zn in theclay loam soil (typic xerofluvent) which is depended onpH, OM content of the organic wastes, the metals studiedand the time after its application. They give special atten-tion on the effects of OM and pH on the extractability ofmetals and observed an increase in DTPA-extractable Znand Cd by the addition of organic waste. Mandal andHazra (1997) observed that organic matter applicationand low pH arrested a decreasing trend in Zn adsorption,however, extractability of Zn increased with increase inthe levels of soil organic matter. In the present study, thelevel of extractable Zn and Cd increased with increase in

sludge amendments in both (DTPA, EDTA) the extract-ants which may be due to increase in OM content. Theaddition of tannery sludge also increased the extractabilityof Ni. Arnesen and Singh (1999) also reported similar effectof peat on the DTPA-extractable Ni. During et al. (2003)found that the level of EDTA-extractable heavy metalswas not consistent but NH4NO3 extractable concentrationswere lower in the no tillage (NT) area.

3.3. Sequential extraction

The heavy metals in sludge amended soil were catego-rized in the five fractions: exchangeable (EXC), carbonate(CAR), Fe–Mn oxide fraction (Fe–Mn), organic bound(OM) and residual fraction (RES). In control soil and100% TS, maximum level of Fe, Cu, K, Pb and Ni wasfound in RES fraction except Cu and Pb which was foundin OM fraction in 100% TS. Manganese, Zn and Cd wasmostly associated with OM fraction in 100% TS, whereas,

Table 5Metals (mg kg�1 dw) extracted from NH4NO3 from different amendments of tannery sludge and control soil before the growth of S. indicum

Metals Amendments

C 10% TS 25% TS 35% TS 50% TS 100% TS

Na 86.10 ± 0.38 96.92 ± 1.61a 113.58 ± 0.88b 99.68 ± 1.21a 144.76 ± 1.05b 1039.48 ± 10.96b

K 73.67 ± 4.82 56.83 ± 3.71c 56.70 ± 0.60c 72.28 ± 6.80 53.51 ± 3.75c 153.64 ± 6.71a

Fe 1.90 ± 0.02 1.20 ± 0.03 1.30 ± 0.04a 1.38 ± 0.12d 1.42 ± 0.04a 1.86 ± 0.03Zn ND ND ND ND ND 1.86 ± 0.30a

Mn 2.51 ± 0.10 0.79 ± 0.007a 1.14 ± 0.01a 1.03 ± 0.001a 0.53 ± 0.02b 0.59 ± 0.02b

Cu 0.14 ± 0.03 0.29 ± 0.02d 0.31 ± 0.01d 0.42 ± 0.02a 0.53 ± 0.04a 2.38 ± 0.16a

Cr ND 0.43 ± 0.05a 0.35 ± 0.01 0.26 ± 0.08c 1.39 ± 0.08a 2.00 ± 0.03b

Pb ND 0.20 ± 0.02a 0.35 ± 0.10a 0.48 ± 0.04a 1.31 ± 0.03b 0.25 ± 0.07c

Ni ND ND ND ND ND 1.50 ± 0.01Cd ND 0.60 ± 0.02b 0.41 ± 0.02b 0.35 ± 0.02a 0.58 ± 0.01 1.26 ± 0.01

All values are mean of three replicate ± SD. ND, not detected. Students t-test (one tailed as compared to control).a p < 0.05.b p < 0.02.c p < 0.01.d p < 0.001.

Table 6NaNO3 extractable metals (mg kg�1 dw) in different amendments of tannery sludge and control soil before the growth of S. indicum

Metals Amendments

C 10% TS 25% TS 35% TS 50% TS 100% TS

Na 15529 ± 141 15298 ± 90 16929 ± 52 16330 ± 126 15992 ± 11a 17594 ± 21b

K 64.30 ± 2.07 34.23 ± 0.13c 46.49 ± 0.20c 44.86 ± 0.04c 56.08 ± 0.77d 106.89 ± 1.8c

Fe 9.01 ± 0.66 2.94 ± 0.12 3.16 ± 0.09 1.98 ± 0.35 5.90 ± 0.39 8.93 ± 0.14Zn 0.33 ± 0.01 0.69 ± 0.03 0.54 ± 0.05 0.45 ± 0.02 0.30 ± 0.03 0.19 ± 0.03Mn 0.70 ± 0.04 0.40 ± 0.01 0.36 ± 0.02 0.28 ± 0.02 0.21 ± 0.01 0.08 ± 0.01Cu 0.52 ± 0.03 0.34 ± 0.01a 0.33 ± 0.02a 0.27 ± 0.01c 0.32 ± 0.01c 0.63 ± 0.04Cr ND 0.50 ± 0.01 0.32 ± 0.03c 0.23 ± 0.01 0.93 ± 0.02b 2.52 ± 0.03b

Pb ND 0.28 ± 0.01c 0.19 ± 0.02c 0.11 ± 0.01 0.21 ± 0.01 0.35 ± 06c

Ni 0.33 ± 0.02 0.56 ± 0.04c 0.38 ± 0.01 0.34 ± 0.01 0.20 ± 0.01c 0.22 ± 0.01a

Cd ND 0.15 ± 0.02c 0.09 ± 0.003 0.09 ± 0.02a 0.12 ± 0.03d 1.61 ± 0.05b

All the values are mean of three replicates ± SD. ND, not detected. Students t-test (one tailed as compared to control).a p < 0.05.b p < 0.02.c p < 0.01.d p < 0.001.

166 A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173

these metals are associated with Fe–Mn fraction in controlsoil.

In different amendments of tannery sludge, Mn and Zn(Fig. 1) were mostly associated with Fe–Mn oxide fractionand their percentage increased with increase in sludge ratio.The percentage of Cr (Fig. 1) bound with Fe–Mn fractionin different amendments decreased (83.0–56.66%) withincrease in sludge ratio of pseudo total metals. In loweramendments (10% and 25% TS), Fe (Fig. 1) was mostlyconcentrated (about 55.03%) in the OM bound fraction,whereas, in higher amendments, maximum amount(90.31% and 86.13% in 35 and 50% TS, respectively) wasassociated with RES fraction. The percentage of Pb inRES fraction (Fig. 1) decreased (66.23–36.18%) withincrease in sludge ratio from 10% to 50% TS. In all thesludge amendments and control soil, Cd (Fig. 1) was notdetected in RES fraction. In 100% TS, maximum percent-age (69.36%) was found in Fe–Mn fraction.

Most of the Na (Fig. 2) in 100% TS (78.08%) and con-trol soil (74.83%) was found in CAR bound form. Maxi-mum percentage of K and Ni (Fig. 2) was found in RESfraction. Copper (Fig. 2) was mostly found in OM boundfraction in all the amendment and control soil. An increasein sludge amendment ratio decreased EXC fraction of Cuwhich ranged between 3.65% and 0.78%.

Sequential extraction procedures have commonly beenused to study metal mobility and availability in soils. Thereare many reports (Alvarez et al., 2002; Fuentes et al., 2004)on sequential extraction of metals from sewage sludge;however, no work has so far been carried out on extractionof metals from tannery sludge. In the present study, Cu ismainly associated with the OM fraction due to the forma-tion of stable complexes and maximum extraction percent-age was obtained in the �oxidizable� fraction of tannerysludge (Sims and Kline, 1991; Scancar et al., 2000; Fuenteset al., 2004). The level of Zn in the tannery sludge showed

Fig. 1. Fractionation of Mn, Cr, Zn, Fe, Pb, Cd in different amendments of tannery sludge and control soil before the growth of S. indicum.

A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173 167

least degree of mobility as evident from the high propor-tion of metal extracted in the Fe–Mn oxide fraction (Alva-rez et al., 2002; Su and Wong, 2003).

Organic matter is one of the factors that may reduce theavailability of metals to the plant due to metal–organiccomplexation. Udom et al. (2004) reported that metalorganic complexation decreased heavy metal mobility insoils at low pH. Van Erp and Van Lune (1991) also empha-sized that Pb and Cu strongly bound to OM and would bereleased slowly over time as OM of the sludge is decom-posed, whereas, Cd and Zn are not bound as strongly toOM.

Walter and Cuevas (1999) reported that maximumamount of Pb was found in INOR and RES fractionsand Cd in CAR fraction, which are in agreement withthe present findings. They also reported that Cr was foundin Fe–Mn oxide and RES fractions. However, these resultsdiffer from those reported by other authors (Tokaliogluet al., 2000; Alvarez et al., 2002), where it was principallydistributed between OM and RES fractions. In contrast,the higher percentage of Cd was reported in the EXC frac-tion of the soil (Sims and Kline, 1991; Berti and Jacobs,

1996). There are various reports (Sims and Kline, 1991;Fuentes et al., 2004) on sewage sludge showing maximumamount of Ni in RES fraction. In the present study, the sig-nificant increase in the level of Ni in RES fraction may bedue to alkaline stabilization process. Metals confined in theRES fractions are usually not expected to release over shortperiods of time under the conditions usually encountered innature (Chlopecka et al., 1996; Su and Wong, 2003).

3.4. Vegetative growth response to tannery sludge

amendments

The fresh weight and number of leaves in treated plantsincreased significantly with increase in sludge amendmentsup to 50% TS at 60 d, whereas, no marked change wasobserved in root length (Figs. 3 and 4). However, shootlength of the plants increased significantly up to 35% TSfollowed by decrease at 50% and 100% TS. Our findingsthat tannery sludge favors the growth of the plants at loweramendment rates are in agreement as reported by Singhet al. (2004b). They also reported that shoot length andnumber of leaves increased in the plants of Helianthus

0

20

40

60

80

100

120

140

C 10 25 35 50 100

Ro

ot

sh

oo

t le

ng

th

Root length

Shoot length/

Fig. 3. Effect on root and shoot lengths (cm) of S. indicum grown ondifferent amendments of tannery sludge. All the values are mean of threereplicates ± SD.

0

10

20

30

40

50

60

70

C 10 25 35 50 100

No

. of

leaf

an

d f

resh

wei

gh

t

No. of leaf

Fresh weight

Fig. 4. Effect on no. of leaf and fresh weight (g) of S. indicum grown ondifferent amendments of tannery sludge. All the values are mean of threereplicates ± SD.

Fig. 2. Fractionation of Na, K, Ni and Cu in different amendments of tannery sludge and control soil before the growth of S. indicum.

168 A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173

annuus grown on soil amended with different amount oftannery sludge.

3.5. Metal accumulation

The data (Figs. 5 and 6) showed that the accumulationof essential and non-essential metals was found more inlower parts of the plant except K. The total (upper andlower parts) accumulation of metals (Cr and Cd) increasedwith increase in sludge amendments ratio, whereas, theaccumulation of Mn and Pb decreased with increase insludge amendments. The total accumulation of Fe and Niincreased in the plants grown in 25% TS followed bydecrease. No marked change was observed in case of Naand K accumulation.

Minimum accumulation of Mn, Zn, Cu, Cr, Pb and Niin the studied plant may be due to their maximum affinitywith more stable fraction (Fe–Mn oxide, OM, RES frac-tions). The estimation of bioavailable fraction is moreappropriate for the estimation of short and medium termrisks involved due to metal accumulation in plants (Tackand Verloo, 1996). Although, there has been much debateabout which part of metals in soils is the �bioavailable� frac-tion. Considerable efforts have been made to measure accu-rately extractable trace metal concentrations in soil samples(Quevauviller, 1998). The results of single extractant usedin the present experiment showed that the metal extract-ability was found to differ among the five extractants used.The accumulation of metals in the plants grown on tannerysludge amended soils has been correlated with extractablefraction of the metals in the soil which provide relatively

Fig. 5. Accumulation (mg kg�1 dw) of Pb, Cr, Cd, Ni, Na and K in the lower and upper parts of S. indicum grown on different amendments of tannerysludge after 60 d of exposure. All the values are mean of three replicates ± SD.

Fig. 6. Accumulation (mg kg�1 dw) of Fe, Zn, Mn and Cu in the lower and upper parts of S. indicum grown on different amendments of tannery sludgeafter 60 d of exposure. All the values are mean of three replicates ± SD.

A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173 169

170 A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173

quick and simple chemical extraction in order to describethe mobility and bioavailability of heavy metals. EDTAwas expected to extract more heavy metal due to its lowpH than DTPA and NaNO3 irrespective of the soil pH inthis study.

Abdel-Saabour and Abo El-Seoud (1996) studied theeffects of organic waste compost on sesame growth andmetal accumulation. Recently, significantly high accumula-tion of metals in different parts of the plants of Lycopers-

icon esculentum (Singh et al., 2004a) and Helianthus

annuus (Singh et al., 2004b) grown on soil amended withtannery sludge has been reported. Brooks and Robinson(1998) suggested that suitable plants for rhizofiltrationinclude oil-yielding plants, Indian mustard and sunflower.Zayed et al. (1998) reported that different plants vary intheir ability to accumulate Cr in their tissues, however,highest Cr concentration was recorded in the members ofthe Brassicaceae family. Shahandeh and Hossner (2000)also compared the accumulation of Cr in thirty-six plantspecies and found that Indian mustard and sunflower accu-mulated more Cr than other agricultural plant species fromthe soil. Other researchers used the plants as hyperaccumu-lators for the phytoremediation of heavy metals (Salt et al.,1995; Ebbs and Kochian, 1997; Blaylock and Huang,2000).

3.6. Correlation between the extractable metal fraction

and metal accumulation in plants

One of the most important applications of single extrac-tion (EDTA, DTPA, CaCl2, NH4NO3, NaNO3) of metalsin the soils is to assess the bioavailability of metals. In thepresent study, correlation analysis (r) was performed inorder to investigate the relationship between the extractablemetals content in different amendments of the tannerysludge and the total accumulation of metal in the plantparts (Table 7). Significant (p < 0.01) negative correlation(r = �0.972) was found between DTPA extractable Feand plant accumulated metals, whereas, significant positive

Table 7Correlation coefficient (r) between metal accumulation in the plant of S.

indicum and extractable metals

Metals Different extractants

EDTA DTPA CaCl2 NH4NO3 NaNO3

Na �0.760 0.740 0.701 0.740 0.738K �0.211 �0.486 0.190 �0.271 �0.136Fe 0.851* �0.972** 0.330 �0.592 �0.629Zn 0.925** 0.991** – 0.981** �0.617Mn �0.012 0.936** 0.904* 0.817* 0.912*

Cu �0.004 �0.170 0.571 �0.177 �0.320Cr 0.506 0.952** 0.951** 0.889* 0.968**

Pb 0.591 �0.062 0.954** 0.212 0.900*

Ni 0.954** 0.326 – 0.326 0.073Cd 0.834* 0.696 0.655 0.822* 0.755

The level of Zn and Ni in CaCl2 fraction was not detected.* Significant at the level p < 0.05.

** Significant at the level p < 0.01.

(p < 0.05) correlation (r = 0.851) was observed with EDTA.In the case of Cu, Ni, Pb and Na, non-significant positiveand negative correlation was emerged. Zinc showed bettercorrelation with all the three extractants (DTPA, NH4NO3,EDTA) whereas, in case of Mn, all the extractants haveshown significant correlation coefficient except EDTAwhere, it was negative correlated. Chromium accumulationshowed significant correlation with different extractantsand the order is as follows NaNO3 > DTPA > CaCl2 >NH4NO3 whereas, Cd accumulation showed significantcorrelation with EDTA followed by NH4NO3. Overallanalysis of the correlation data showed that there is no sin-gle extractant which have shown positive correlation withall the tested metals. However, EDTA extractions haveshown better correlation coefficient (r) with most of thetested metals.

Bhogal et al. (2003) found better correlation betweencrop yields and NH4NO3 extractable metals concentrationin sewage sludge application sites. Pueyo et al. (2004) usedthree extraction procedures (CaCl2, NaNO3, NH4NO3)for predicting trace metal mobility (Cd > Zn > Cu > Pb)in the contaminated soil. They found that out of three,CaCl2 extractions procedure seems to be the most suitablemethod for bioavailability of these metals. However in thepresent study, the metals extracted with EDTA have shownmost efficient extractants for the bioavailability of metals.Nyamangara and Mzezewa (1999) also reported the use ofEDTA-extractable metals as indication of bioavailable frac-tion in sewage sludge contaminated area.

3.7. Correlation between the metal accumulation and

physico-chemical properties

All the physico-chemical parameters (pH, EC, CEC,OC, OM) have shown significant correlation with Zn, Crand Cd accumulation in the plants except non-significantpositive correlation (r = 0.740) in case of Cd with EC(Table 8). In case of Fe, accumulation in the plants, onlypH was found to be significantly correlated (r = 0.857).

Table 8Correlation coefficients (r) of various parameters studied in the differentamendments and metals accumulation in the plants of S. indicum

Metals Physico-chemical parameters

pH EC CEC OC OM

Na 0.471 0.768 0.769 0.723 0.724K �0.746 �0.474 �0.526 �0.499 �0.499Fe 0.857* �0.964** �0.959** �0.982** �0.982**

Zn 0.847* 0.980** 0.995** 0.990** 0.990**

Mn �0.863* �0.755 �0.798* �0.773 �0.773Cu 0.329 �0.238 �0.196 �0.132 �0.134Cr 0.916** 0.957** 0.975** 0.978** 0.978**

Pb 0.134 �0.046 �0.024 �0.068 �0.068Ni 0.617 0.377 0.429 0.389 0.389Cd 0.944** 0.740 0.793* 0.785* 0.785*

* Significant at the level p < 0.05.** Significant at the level p < 0.0.

A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173 171

Significant negative correlation was observed in all phys-ico-chemical parameters studied and K and Mn accumula-tion in the plants. In contrast, Na and Ni have shownpositive correlation (non-significant) with the physico-chemical properties.

3.8. Correlation between the physico-chemical properties

and EDTA extractable metals

In present study, EDTA extractant was found one ofthe most efficient extractant for the bioavailability ofmetals. Thus, correlation analysis (r) was performedbetween physico-chemical properties (pH, EC, CEC, OCand OM) and EDTA extractable metals (Table 9). Correla-tion data showed significant positive correlation of EDTAextractable metals (Zn, Cu, Pb, Cd) with physico-chemicalproperties, whereas, negative correlation was observedwith Na, K, Fe and Mn. It is evident from the correla-tion analysis of the results that physico-chemical proper-ties play vital role in bioavailability of the metals.Udom et al. (2004), also reported that Zn, Cu, Pb, Cdshowed significant positive correlation with OM in thesewage soil.

Table 9Correlation coefficients (r) of various parameters studied in the differentamendments and EDTA extractable metals

Metals Physico-chemical parameters

pH EC CEC OC OM

Na �0.753 �0.996** �0.988** �0.988** �0.988**

K �0.457 �0.663 �0.629 �0.684 �0.684Fe �0.794* �0.693 �0.707 �0.758 �0.757Zn 0.899* 0.856* 0.898* 0.886* 0.886*

Mn �0.322 �0.570 �0.539 �0.586 �0.586Cu 0.942** 0.921* 0.952** 0.946** 0.946**

Cr 0.703 0.289 0.389 0.351 0.351Pb 0.701 0.822* 0.828* 0.796* 0.797*

Ni 0.387 0.254 0.292 0.237 0.237Cd 0.884* 0.982** 0.993** 0.995** 0.995**

* Significant at the level p < 0.05.** Significant at the level p < 0.0.

Fig. 7. Dendrogram of hierarchical cluster analysis between physico-chemicagrown on different amendments of tannery sludge.

3.9. Cluster analysis

Cluster analysis is a statistical tool and used to group thedata having identical and similar behavior. In presentstudy, the information about the relationship among thedifferent physico-chemical parameters (pH, EC, CEC, OCand OM), metal accumulation in the plants and the levelof metals extracted with different extractants from differentsludge amendments along with control soil were analyzedusing cluster analysis. The cluster diagram (Fig. 7) showedtwo main clusters; A includes 100% TS, cluster B containall other amendments (10%, 25%, 35% and 50%) with con-trol as well. These dendrograms explain the grouping ofamendments of similar or nearly identical, physico-chemi-cal properties and metal behavior. Thus, 100% TS haveshown different behavior than other amendments.

4. Conclusion

It is advisable to adopt a single procedure for the deter-mination of trace metal mobility and/or availability toplants in the present set of conditions and the use of theEDTA extracting solution led, in general, to greater testedmetals extractability. However, CaCl2 showed betterextraction efficiency with K in comparison to rest of theextractants. The results obtained from sequential extrac-tion schemes indicate that K, Cr and Ni from differentamendments are most abundant in the RES fraction while,Na and Cd are bound to CAR fraction. The pools of Zn,Fe, Cu and Pb are changed with the addition of tannerysludge. No such change was observed in case of Mn.

Finally, it must be pointed out that small fraction ofpseudo total metals (Pb, Ni, Cr, Mn, Zn, Cu) present in dif-ferent sludge amendments accumulated by the plants asthese metals are firmly bound with more stable fraction.Further, some physical–chemical parameters are modifiedand such modifications, too, may affect metal bioavailabil-ity. Overall, lower amendments (25%) of tannery sludgewere found suitable for phytoremediation of metals (Na,K, Fe, Cr, Ni) as evident from the results of growth para-meters and metal accumulation.

l parameters, different extractants and metal accumulations in the plants

172 A.K. Gupta, S. Sinha / Chemosphere 64 (2006) 161–173

Acknowledgements

We thank the Director, National Botanical ResearchInstitute, Lucknow (India) for providing required researchfacilities. Amit K. Gupta is grateful to NRCD, Ministry ofEnvironment and Forests, Government of India, New Del-hi for financial assistance. We wish to thank Mr. SureshKumar Sharma and Mr. Rakesh Kumar for their assis-tance in this work.

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