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Bioresource Technology 98 (2007) 442446

Short Communication

Phytoextraction capacity of the Chenopodium album L. grown on soil amended with tannery sludge
A.K. Gupta, S. Sinha

Ecotoxicology and Bioremediation, National Botanical Research Institute, Lucknow 226 001, India Received 24 October 2005; received in revised form 12 January 2006; accepted 18 January 2006 Available online 15 March 2006

Abstract The metal accumulation potential of Chenopodium album L. grown on various amendments of tannery sludge (TS) was studied after 60 days of sapling planted. The analysis of the results showed that the levels of pH, cation exchange capacity, organic carbon, organic matter and DTPA extractable metals (except Mn) of amendments increased by the addition of tannery sludge ratio. Shoot length of the plant increased by the addition of sludge, whereas, no marked change was observed in root length, fresh and dry weight of the plant. Accumulation of the metals in the plants was found in the order; Fe > Mn > Zn > Cr > Cu > Pb > Ni > Cd. Translocation of toxic metals (Cr, Pb, Cd) in diVerent parts of the tested plant was found in the order; leaves > stems > roots. An increase in the photosynthetic pigments, carotenoid and leaf protein contents of the plants were found to increase with increase in sludge amendments. Correlation analysis between metal accumulation in the plants with DTPA extractable metals emphasized that Mn, Ni, Cr, Pb and Cd showed positive correlation (p < 0.05), whereas, Fe, Zn and Cu showed negative correlation. Transfer factor analysis emphasized that 10% TS amendments were suitable for phytoextraction of Cr. Overall analysis of the data exhibited that the plants may be used for phytoextraction of Cr from tannery waste contaminated soil as most of the metal was accumulated in harvestable part which is a matter of serious concern, whenever used for edible purposes. 2006 Elsevier Ltd. All rights reserved.
Keywords: Tannery sludge; Chenopodium album; Phytoextraction; DTPA; Bioavailability

1. Introduction Production of sludge is increasing day by day as a result of wastewater treatment, which is generally bulky with high moisture contents, and from highly organic to mineral depending on their origin in nature. There is an increasing interest in the agricultural application of sludge obtained from wastewater treatment plants due to the possibility of recycling valuable components: organic matter, N, P and other plant nutrients (Wong et al., 2001). Although, tannery sludge has been shown to increase crop production (Singh and Sinha, 2004a,b) at lower amendment, it may contain cer* Corresponding author. Tel.: +91 522 205831 35x221; fax: +91 522 205839/836. E-mail address: sinha_sarita@rediVmail.com (S. Sinha).

tain trace elements at level injurious to plants and magniWed (Singh et al., 2004a,b). Addition of organic matter (OM) amendments, such as sludge, compost together with lime to raise the soil pH is a common practice for immobilization of heavy metals and soil amelioration, to facilitate re-vegetation of contaminated soils (Clemente et al., 2003). The eVects of OM amendments on heavy metal bioavailability depends on the nature of organic matter, their microbial degradability, salt content and eVect on soil pH and redox potential, as well as on the particular soil type and metals concerned (Walker et al., 2004). A number of studies have been carried out in the last few years on the risk associated with the application of metal loaded sludge on agricultural land (Sinha et al., 2006). Phytoremediation of heavy metal-contaminated soil is an emerging technology that aims to extract or inactivate metals in soils. It has attracted attention in recent years for its low

0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.01.015

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cost of implementation and environmental beneWts (Garbisu and Alkorta, 2001). In addition to these ex situ techniques, there are in situ methods using phytoextraction (Salt et al., 1998). Soil contaminated with multiple heavy metals may pose a diYcult challenge for both the approaches of phytoextraction. Although, some hyperaccumulator plants appear to be capable of accumulating elevated concentrations of several heavy metals. Simultaneously, there is still considerable speciWcity in metal hyperaccumulation (Chaney et al., 2005). Such plants are compatible with routine agricultural practices and allow repeated planting and harvesting of the metal-rich tissues. India is one of the largest producers of leather in the world and there are at present more than 3000 tanneries with annual processing capacity of 0.7 million tones of hides and skins (Chhonkar et al., 2000). Metals in tannery waste occur in complex form and vary widely in their availability to the plants. There are several reports on the accumulation of metals in the plants growing on soils receiving tannery waste (Singh et al., 2004a,b; Sinha et al., 2006). Experimental design of present study was inXuenced with Walker et al. (2004) results, which emphasized that Chenopodium album L. plant was found to be one of the initial plant species colonizing in mine spill area (heavy metal contaminated site). In the present study, the plants of C. album L. were grown on soil amended with tannery sludge. The accumulation and translocation pattern of metals in diVerent parts of the plant was studied in order to assess the phytoextraction potential of the plant grown on tannery waste contaminated soil. 2. Methods Dewatered anaerobically digested tannery sludge cakes were collected from Jajmau wastewater treatment plant, Jajmau, Kanpur (UP, India) in large plastic bags and brought to the Weld laboratory. The uncontaminated garden sub soil was collected from National Botanical Research Institute, Lucknow. The tannery sludge (TS) and garden soil (GS) were air-dried, Wnely powdered and sieved with 2 mm mesh size before use. The various amendments of tannery sludge (10% and 25% TS) were prepared using garden soil as a control. The saplings of C. album L. were evenly planted in pots of 12 inches in diameter, each in three replicates. Pots were placed in a green house in a randomized block design at an average diurnal temperature of 2545 C. The tap water was provided to the pots to keep the topsoil moist and avoid leaking from the pots. The plants were harvested after 60 days after sapling planted. After harvesting, the length of roots and shoots of all the plants was measured, while the number of leaves was recorded manually. 2.1. Physico-chemical properties, DTPA and photosynthetic pigments and protein content The physico-chemical parameters of diVerent amendments of the tannery sludge were estimated using manual of

Kalra and Maynard (1991). DTPA extractable metals were estimated by the method of Lindsay (1978). Chlorophyll content in the fresh leaves of the plant was estimated following the method of Arnon (1949). Carotenoid content was determined by the method of Duxbury and Yentsch (1956). Protein content in the leaves of plants was estimated using BSA as a standard protein (Peterson, 1977). 2.2. Digestion of samples for metal estimation Plant parts were separated manually and oven dried (70 C). After dry weight determination, the samples were ground and digested in HNO3 (70%) using Microwave Digestion System MDS 2000 and metal contents were estimated using Atomic Absorption Spectrophotometer (GBC Avanta ). 2.3. Quality control and quality assurance The standard reference material of metals (E-Merck, Germany) was used for the calibration and quality assurance for each analytical batch. Analytical data quality of metals was ensured through repeated analysis (n D 6) of EPA quality control samples (Lot TMA 989) for metals (Cd, Cr, Cu, Pb) in water and the results were found to be within 4.8% of certiWed values. Method validation (accuracy and repeatability) was performed by analyzing the certiWed materials reference solution (BND 1101.02) of multi-elements (Zn, Fe, Cu) and single element reference solution, BND 102.03 (Pb); BND 402.02 (Cr) and BND 1001.02 (Ni) provided by National Physical Laboratory (NPL), New Delhi, India, and the results were found to be within 1.70% of certiWed values (n D 10). 2.4. Statistical analysis The experiment was performed in a completely randomized block design. To conWrm the variability of the data and validity of the results, Students t-test (one tailed) and Pearsons correlation coeYcients, a measure of the linear association of two variables, were determined for plant metal concentration versus DTPA-extractable metals from diVerent amendments (n D 3). 3. Results and discussion Table 1 shows the physico-chemical properties of diVerent amendments of tannery sludge and garden soil. With an increase in tannery sludge amendments, an increase in pH, cation exchange capacity (CEC), organic carbon (OC) and organic matter (OM) was recorded, whereas, EC was maximum in 10% TS and minimum in GS. The most important beneWts of sludge addition to soil are related to the increase in the level of OM content and biological activity. Karaca (2004) reported that organic matter from sludge acted as a nutrient pool and improved nutrient cycling. However, Yoo and James (2002) observed that high organic matter content

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Table 1 Physico-chemical properties of tannery sludge and garden soil Parameters pH (1:2.5) EC ( s cm1) CEC Cmol (p+) kg1 OC (%) OM (%) GS 6.7 0.02 461.0 0.01 32.2 2.6 0.4 0.1 0.7 0.01 10% TS 6.9 0.01 716.2 0.5 42.2 3.0 0.7 0.02 1.0 0.01 12.6 0.3 7.1 1.2 15.1 0.3 2.7 0.1 3.6 0.1 2.7 0.3 1.3 0.1 1.2 0.1 1.0 0.0 25% TS 7.2 0.01 617.0 0.01 50.3 1.2 0.8 0.01 1.4 0.01 16.7 0.2 13.6 0.4 12.3 0.2 3.5 0.0 5.6 0.1 3.4 0.1 2.9 0.0 1.6 0.0 2.2 0.1

DTPA extractable metals (mg kg1 dw) Fe 10.0 0.2 Zn 5.2 0.6 Mn 20.0 1.0 Cu 1.8 0.2 Cr BDL Pb 0.7 0.1 Ni BDL Cd BDL Co BDL

All the values are mean of three replicates SD. BDL D below detection limits, Students t-test (two tailed as compared to GS). p < 0.05. p < 0.02. p < 0.01. p < 0.001.

in soil increased the level of CEC, buVering capacity, reduce compaction, and improve soil physical properties. In case of DTPA extractable metals (Table 1), it was observed that the level of metals (Fe, Zn, Cu, Cr, Pb, Ni, Cd) increased with increase in sludge ratio, whereas the level of Mn decreased. In GS, the level of Cr, Ni, Cd and Co was below detection limits (BDL). The analysis of the data showed high concentrations of DTPA extractable metals in soil amended with tannery sludge than GS. The analysis of the data (Fig. 1A) showed that the shoot length of the plant increased with increase in sludge amendment ratio, whereas no marked change was observed in case of root length, fresh and dry weight of the plant. Maximum increase of 20.24% was recorded in shoot length of the plants grown in 25% TS as compared to GS. Similarly Wndings were recorded in the plants of Convolvulua arvensis grown for

15 days on agar based medium containing 20 mg l1 Cu (Gardea-Torresdey et al., 2004). The plants showed signiWcantly high accumulation of toxic metals (Cr, Pb, Cd) in upper parts (Table 2) and found in the order; leaves > stems > roots. The accumulation of essential metals (Fe, Mn, Zn, Cu) was also signiWcantly high in all parts of the plants grown on sludge-amended soil. However, Mn and Zn accumulation was found maximum in roots followed by leaves, whereas, Fe accumulation was found maximum in leaves followed by roots. The availability and plant uptake of Pb were reduced by the addition of sludge amendments. This could be the consequence of the immobilization of Pb by humiWed organic matter. Metal concentration recorded in diVerent parts of the plant was found positively (p < 0.05) correlated with DTPA extractable metals (Mn, Ni, Cr, Pb, Cd) content in the medium. Further, the accumulation of metals (Cr, Pb) in the plants showed signiWcant (p < 0.05) positive correlation with pH and OM, however, CEC showed signiWcant positive correlation (p < 0.05) with Ni, Cr and Pb. Several studies demonstrated that metal concentration in the plant tissue was a fraction of the heavy metal content in the growing environment (Lee et al., 2002). Recently, Sinha et al. (2006) also reported that long-term irrigation with treated tannery wastewater enhanced the accumulation of metals in the vegetables/crops growing in contaminated soil and it also aVected the environmental quality. It has been well documented that the accumulation of metals was more in lower part of the plant grown on contaminated soil than upper part (Singh et al., 2004a,b; Sinha and Gupta, 2005). The transport of metals from roots to shoots included long distance translocation in the xylem and storage in the vacuole of leaf cells and these processes aVected by many factors (Yang et al., 1997). The high accumulation of Mn and Zn particularly in the root tissues could be due to complexation of metals with the sulphydryl groups resulting into less translocation of metals to upper part of the plant, which varied from one metal to another. It is known that P and Cr

Table 2 Accumulation of metals (mg kg1 dw) in the plants grown on soil amended with tannery sludge Amendments Plants parts Metals accumulation (mg kg1 dw) Fe GS Roots Stems Leaves Roots Stems Leaves Roots Stems Leaves 499.5 47.8 313.8 13.7 1321.8 93.5 366.3 53.3 264.8 23.7 859.5 66.9 410.5 31.2 269.1 28.4 440.1 61.3 Mn 210.2 11.4 53.6 5.9 79.8 3.2 77.5 8.7 38.0 5.2 44.6 6.9 70.5 7.9 25.7 5.4 36.0 7.2 Zn 97.6 9.7 44.7 11.0 41.9 2.2 76.3 4.5 40.9 3.6 48.5 4.3 71.5 3.4 41.4 2.7 55.3 4.5 Cu 17.0 3.2 11.9 3.2 15.5 1.6 15.8 0.6 9.8 1.7 19.1 1.7 15.4 3.6 12.2 2.8 15.6 1.9 Ni 2.5 0.3 1.1 0.6 3.9 0.3 7.1 1.2 6.6 1.1 8.8 2.5 9.1 2.0 5.7 0.6 5.8 0.7 Cr 4.0 0.6 1.3 0.0 11.5 1.8 21.4 1.9 15.4 1.7 272.6 19.2 20.9 1.8 34.1 3.3 219.5 26.4 Pb 1.0 0.1 1.2 0.0 3.9 0.5 8.5 2.5 11.0 2.7 11.2 2.0 9.4 1.7 8.3 0.8 9.9 1.8 Cd 1.1 0.1 BDL BDL 2.8 0.3 1.3 0.3 8.8 1.4 1.9 0.4 1.3 0.6 4.1 0.7

10% TS

25% TS

All the values of mean of three replicate SD, BDL D below detection limits, Students t-test (one tailed as compared to control). p < 0.05. p < 0.02. p < 0.01. p < 0.001.

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are competitive for surface sites and Fe, S and Mn are also known to compete with Cr for transport binding (Wallace et al., 1976). In contrast to the present Wndings, several reports are available for poor translocation of Cr in upper parts of the plants (Singh et al., 2004a,b). Soil-to-plant transfer ratio is one of the key components of phytoextraction. The values of transfer factor (TF) for all the tested metals varied from one metal to another. Maximum TF values for toxic elements were found in the plants grown in 10% TS (28.64, Cr; 3.79, Pb; 5.78, Ni; 3.58, Cd), whereas essential micro elements showed maximum TF values in the plants grown in GS (72.92, Fe; 11.81, Zn; 5.73, Mn, 8.22, Cu). Estimation of chlorophyll content (Fig. 1B) is often accomplished to assess the impact of most environmental stresses as the pigment content is linked to the visual symptoms and photosynthetic plant productivity. The level of total chlorophyll, chlorophyll a, b, carotenoid and leaf protein content of the plants grown on soil amended with sludge increased in both the amendments as compared to GS. The Wndings of our results are in agreement with the studies reported earlier (Singh and Sinha, 2005) in the plant grown on tannery sludge amended soil, which may be attributed due to the presence of essential metal ions in tannery waste required for chlorophyll biosynthesis. Carotenoid, a non-enzymatic antioxidant, is a part of photosynthetic pigment. It plays an important role in the protection of chlorophyll pigment under stress conditions by quenching the photodynamic reactions, replacing peroxidation and collapsing of membrane in chloroplasts (Kenneth et al., 2000). In agreement to our results, an increase in carotenoid content was also observed in the plants (Helianthus annus) grown on diVerent amendment of tannery sludge (Singh

et al., 2004b) and Sesbania cannabina grown on diVerent amendments of Xy ash (Sinha and Gupta, 2005). Singh and Sinha (2005) reported increase in leaf protein content in the plant of Brassica juncea grown on sludge amended soil, which strengthen the Wndings of the present study. 4. Conclusion From the results it could be concluded that C. album accumulated signiWcantly high quantities of toxic metals in the upper parts of the plant. The results of correlation analysis also showed signiWcant (p < 0.05) positive correlation with Ni, Cr, Pb and Cd. Maximum TF values for toxic elements were found in the plants grown in 10% TS. High accumulation of metals in the upper part of the plant, correlation analysis and transfer factor (TF) strengthened that the plant could be used for phytoextraction of Cr from tannery waste contaminated sites and was a matter of serious concern, whenever used as edible purposes. Acknowledgements We thank the Director, National Botanical Research Institute, Lucknow (India) for providing required research facilities and keen interest in the work. Amit K. Gupta is grateful to NRCD, Ministry of Environment and Forests, Government of India, New Delhi for providing Wnancial assistance. References
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Fig. 1. Ecophysiological parameters of plant grown on soil amended with tannery sludge. All the values are mean of three replicate SD. BDL below detection limits. Students t-test (one tailed as compared to control), (a D p < 0.05, b D p < 0.02, c D p < 0.01, d D p < 0.001).

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A.K. Gupta, S. Sinha / Bioresource Technology 98 (2007) 442446 Singh, S., Sinha, S., 2005. Accumulation of metals and its eVect in Brassica juncea L. Czern. (var. rohini) grown on various amendments of tannery waste. Ecotox. Environ. Saf. 62, 118127. Sinha, S., Gupta, A.K., 2005. Translocation of metals from Xy ash amended soil in the plant of Sesbania cannabina L. Ritz: eVect on antioxidants. Chemos. 61, 12041214. Sinha, S., Gupta, A.K., Bhatt, K., Pandey, K., Rai, U.N., Singh, K.P., 2006. Distribution of metals in the edible plants grown at Jajmau, Kanpur (India) receiving treated tannery wastewater: relation with physicochemical properties of the soil. Environ. Monit. Assess (March Issue). Walker, D.J., Clements, R., Bernal, M.P., 2004. Contrasting eVects of manure and compost on soil pH heavy metal availability and growth of Chenopodium album L. in a soil contaminated by pyrite mine waste. Chemos. 57, 215224. Wallace, A., SouW, S.M., Cha, J.W., 1976. Some eVects of chromium toxicity on bush bean plants grown in soil. Pl. Soil. 44, 471473. Wong, J.W.C., Li, K., Fang, M., Su, D.C., 2001. Toxicity evaluation of sewage sludge in Hong Kong. Environ. Int. 27, 373380. Yoo, M.S., James, B.R., 2002. Zinc extractability as a function of pH in organic waste-contaminated soils. Soil Sci. 167, 246259. Yang, X.E., Baligar, V.C., Foster, J.C., Martens, D.C., 1997. Accumulation and transport of nickel in relation to organic acids in ryegrass and maize grown with diVerent nickel levels. Pl. Soil. 196, 271276.

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