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    Uptake and translocation of metals in Spinacia oleracea L. grownon tannery sludge-amended and contaminated soils: Effect on lipid

    peroxidation, morpho-anatomical changes and antioxidants

    Sarita Sinha *, Shekhar Mallick, Rohit Kumar Misra, Sarita Singh, Ankita Basant,Amit Kumar Gupta

    Ecotoxicology and Bioremediation Group, National Botanical Research Institute, Lucknow 226 001, India

    Received 9 May 2006; received in revised form 7 August 2006; accepted 23 August 2006Available online 13 November 2006

    Abstract

    The plants ofSpinacia oleracea L. grown on contaminated soil (CS) and different amendments of tannery sludge (TS) have shownhigh accumulation of metals in its edible part. The accumulation of toxic metal (Cr) in the leaves of the plants grown on CS was recordedas 40.67lg g1 dw. However, the leaves of the plants grown on 100% TS have accumulated about two times (70.80 lg g1 dw) higher Crthan the 10% TS (31.21lg g1 dw). Among growth parameters, the root length was more affected at 90 d than the shoot length, numberof leaves and leaf area. The study of scanning electron micrographs showed 29.31% increase in stomatal length in the leaves of the plantsgrown on CS as compared to garden soil (GS), which served as control, however it decreased in the plants grown on higher amendmentsof TS. The decrease in MDA content at initial period of exposure and lower amendment was recorded in the leaves, whereas, significantincrease (>10% TS onward) was observed with increase in tannery sludge ratio at 90 d as compared to GS. A coordinated increase in all

    the studied antioxidants (cysteine, non-protein thiol, ascorbic acid, carotenoid contents) was found up to 75 d of growth. At 90 d, most ofthe antioxidant decreased as compared to 75 d causing oxidative stress as evidenced by increased level of lipid peroxidation anddecreased chlorophyll and protein contents. Maximum increase of 181.43% in MDA content and maximum decrease of 53.69% in totalchlorophyll content was recorded in the leaves of the plants grown on 100% TS after 90 d of growth. The plants grown on CS have shownan increase in shoot length, number of leaves, leaf area, photosynthetic pigments and protein contents and in all the studied antioxidants.Thus, these plants are able to combat stress involving defense mechanism, resulting in healthy growth of the plants. The results are wellcoordinated as there is no change in the MDA content as compared to the plants grown on GS. In view of high Cr accumulation in ediblepart ofS. oleraceagrown on CS after irrigation with tap water, it is not advisable to use these plants for edible purposes. Summing up, itis recommended that the level of metals in the edible part should be checked instead of healthy growth as deciding parameter for con-sumption. It is demonstrated through this study that metal enriched plants have detoxification mechanism and grow well on organicmatter enriched contaminated soil. 2006 Elsevier Ltd. All rights reserved.

    Keywords: Spinacia oleracea; Tannery waste; Metals; Antioxidants; Lipid peroxidation; Scanning electron micrographs

    1. Introduction

    Heavy metal pollution in and around industrial sites ofany city in developing countries has become a common

    phenomenon, due to the lack of proper waste disposalpractices. Among all the industries, the tannery industryis one of the notorious in terms of heavy metal contamina-tion to the soil. The leachate from the dried tannery sludgecakes and sludge itself is posing a potential hazard of heavymetals contamination, particularly hexavalent chromium(Cr+6) into the soil, on which it is dumped. In addition,in the peri-urban and rural areas of most of the developing

    0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved.

    doi:10.1016/j.chemosphere.2006.08.026

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

    E-mail address: [email protected](S. Sinha).

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    Chemosphere 67 (2007) 176187

    mailto:[email protected]:[email protected]
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    countries, the use of sewage and treated industrial waste-water for irrigating the crops is a common practice (Nanand Cheng, 2001; Wong et al., 2001; Sinha et al., 2006)which leads to cumulative contamination of heavy metalsto the soil. Chromium is one of the major heavy metal,which is predominantly being released through the waste

    from the tannery industry, used for chrome tanning ofraw leather hides. Chromium merits a special referencefor its extreme toxicity due to interaction of its compoundswith living cells (Cieslak-Golonka, 1995; Costa, 1997)Besides Cr, Fe is an another major element present in thetannery sludge and comes from animals hides and ferrousaluminum sulphate, which is used for precipitation of sus-pended solids during wastewater treatment. However,excessive concentrations of Fe have also been reported tobe toxic to the plants (Sinha et al., 1997; Sinha and Saxena,2006). Metals in tannery waste occur in complex forms andvary widely in their availability to the plants.

    The generation of reactive oxygen species is stimulated

    in the presence of metals which can seriously disturbnormal metabolism through oxidative damage to cellularcompartments.To counteract this damage, highly efficientantioxidant defense mechanism in its cells can scavenge ordeactivate metal stress-generated by reactive oxygen radi-cals. Antioxidant substances like cysteine, ascorbic acid,non-protein thiol (sulfhydryl) and antioxidant enzymesplay a vital role in providing cellular defense towards oxi-dative stress (Sinha et al., 1997, 2005a; Halliwell and Gut-teridge, 2004; Sinha and Saxena, 2006). There are manyreports on metal-induced oxidative stress and response ofantioxidants in the plants grown on contaminated soil

    (Singh et al., 2004a,b; Singh and Sinha, 2005).Metal accumulation in the leaves of the plants grown on

    contaminated soil has shown various morphological andstructural changes such as wider opening of stomata,increase in the stomata size, thinning of wax depositionand elevation of stomatal complex. Lesser wax depositionhas been attributed to increased lipid peroxidation inducedby metal toxicity (Rai et al., 2005).

    Due to the crisis of the arable land compounded by thecontamination of fertile soil with improper disposal of tan-nery waste, farmers are forced to grow food crops on landscontaminated by heavy metals. Among all the parts of theplant, maximum accumulation of metals was found in theroots in most of the plants. However, some of the metalsare translocated to the edible part. Therefore, entry of met-als into the food-chain through plants grown on contami-nated soils is a matter of serious concern. Sinha et al.(2006) reported metal accumulation in various parts ofthe plants grown on soil receiving treated tannery wastewa-ter, maximum being in leafy vegetables than fruits bearingvegetables/crops. Further, the plants have shown bettergrowth in the plants grown on contaminated soil than nor-mal soil. However, no work has been carried out to studythe toxicity and defense mechanism of leafy vegetablesgrown on contaminated agricultural soil. Based on our ear-

    lier work (Sinha et al., 2006), where high accumulation of

    toxic metal (Cr) in the leaves of Spinacia oleracea wasobserved, the present study is undertaken with a view (i)to assess the accumulation of metals in the leaves of theplants collected from Jajmau (Kanpur, India) irrigatedwith treated tannery wastewater; (ii) to assess the transloca-tion of metals in the plants grown on contaminated soil in

    pots irrigated with tap water and to investigate the effectson its physiological and biochemical parameters; and (iii)to compare the translocation of metals in the plants grownon various amendments of tannery sludge with the plantsgrown on contaminated soil and physiological and bio-chemical effects induced in these plants. The outcome ofthe results should be helpful to elucidate metal detoxifica-tion mechanism in S. oleracea which have shown healthygrowth when grown on tannery waste contaminated soil.

    2. Material and methods

    In India, Jajmau (Kanpur) is a major industrial townand lies in the Indo-Gangetic plains between the parallelsof 2628 0N and 8024 0E. It is one of the major centers toprocess raw hides. The discharge from these industries istreated in an Up-flow Anaerobic Sludge Blanket (UASB)treatment plant before releasing. The treated wastewateris being used by the local farmers for irrigation of ediblecrops/vegetables in the adjacent agricultural fields (2100acre).

    2.1. Collection of plants from agricultural field

    Plants ofSpinacia oleracea L. (spinach) were collected intwo consecutive years (June 2002, January 2003, June2003) from the agricultural field of Jajmau (Kanpur) andbrought to the laboratory to check the level of metals inthe edible part of the plant. The agricultural land is beingirrigated with treated tannery wastewater since last manydecades.

    2.2. Experimental design

    Air dried tannery sludge (TS) was collected from thewastewater treatment plant at Jajmau, Kanpur (Uttar Pra-desh, India) and were brought to the laboratory. Uncon-taminated garden soil (GS) was collected from NationalBotanical Research Institute (NBRI) and was used as acontrol. The tannery sludge and the garden soil were finelygrounded and passed through 2 mm sieve to get a uniformsize, before filling up in terracotta earthen pots (35 cm indiameter). Different amendments (10%, 20%, 35%, 50%and 100%) of the TS were prepared using GS. In anotherset, the contaminated soil (CS) was also collected fromJajmau, Kanpur which is being irrigated with treated tan-nery wastewater in the agricultural fields (Jajmau, Kanpur)and sieved soil were filled in terracotta earthen pots (35 cmin diameter). The experiment was carried out in these pots

    for three months, with three harvests.

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    Seeds ofS. oleracea were sterilized in 3% formalin for5 min followed by washing with double distilled waterand soaked in water overnight. These seeds were sown ina nursery bed. After the seeds germinated and the saplingsgrew to 10 cm, they were transplanted to earthen pots filledwith different amendments (10%, 25%, 35%, 50%, and

    100%) of TS in triplicates along with one set GS and CS.The plants were allowed to grow in the field at NationalBotanical Research Institute in a randomized block design,at an average diurnal temperature of 2535 C. The plantswere watered by normal tap water and harvested after 60,75 and 90 d of growth.

    2.3. Metal accumulation

    Harvested plants were washed thoroughly with distilledwater and blotted dry. Different parts were separatedmanually, cut in small pieces and oven dried at 70 C tillconstant weight. The dried samples were ground (> 2 mm)

    and digested in HNO3(70%) in Microwave DigestionSystem (MDS 2000) and analyzed for metals contentusing Atomic Absorption Spectrophotometer (GBC,AvantaR).

    2.4. Estimation of various physiological and biochemical

    parameters

    Fresh weight, root length, shoot length, leaf area of theplant were recorded immediately after harvesting. Freshleaves were used for the estimation of various parameters.Chlorophyll content in the fresh leaves of the plant

    (100 mg) was estimated following the method of Arnon(1949). Protein content in the leaves and roots of the plantswere determined using BSA as standard protein (Lowryet al., 1951). Lipid peroxidation in the plant tissue wasmeasured indirectly in terms of malondialdehyde (MDA)content, determined by thio barbituric acid (TBA) reaction(Heath and Packer, 1968). Cysteine content was estimatedby the method ofGaitonde (1967). Non-protein thiol (acidsoluble thiol) content was measured (Ellman, 1959) usingEllmans reagent (5,5 0-dithiobis 2-nitrobenzoic acid). Ascor-bic acid content in the leaves was estimated as per methodofKeller and Schwager (1977).

    2.5. Scanning electron microscopy

    Central part of the leaves from each set of the treatmentwas collected for studying the surface structure throughScanning Electron Microscopy. The leaves were kept in2.5% gluteraldehyde overnight for fixation followed bydehydration in ethanol series (30%, 50%, 70%, 90% and100%). Further dehydration was done in BAL-TECCPD-030 critical point drier using liquid CO2 as carriergas. The leaves were mounted on a stub and were coatedwith 15lm conductive gold, in an ion sputter coater(TFC 1100). Coated specimens were mounted on Philips

    XL-20 Scanning Electron Microscope.

    2.6. Data analysis and interpretation

    The experiment was performed in completely random-ized block design involving six amendments of TS withGS, in triplicates, for three time interval. All the datasetobtained from the experiment, were subjected to two way

    analysis of variance (ANOVA) using Microsoft Excel2000 followed by least significant difference (LSD) calcula-tion (Gomez and Gomez, 1984). Studentt-test (two tailed)was applied between the data obtained from CS as com-pared to GS.

    2.7. Quality control and quality assurance

    The standard reference material of metals (E-Merck,Germany) was used for calibration and quality assurancefor each analytical batch. The reference solution (BND1101.02) of multi-elements (Zn, Fe, Cu) was also usedfor calibration of analytical equipment and validation of

    test methods provided by National Physical Laboratory(NPL), New Delhi (India) and the results were found tobe within 1.50% of certified values. EPA quality controlsamples (Lot TMA 989) for metals (Cd, Cr, Cu, Pb) wasused in order to ensure analytical data quality in waterand the results were found to be within 2.79% of certifiedvalues. The recoveries of metals from the plant tissues werefound to be more than 98.5% as determined by digestingthree 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.

    3. Results and discussion

    3.1. Physico-chemical properties of different substrates

    The results of physico-chemical analysis of GS and itsdifferent amendments along with CS (Table 1) revealed thatpH, salinity, EC, CEC, OC and OM of both CS and TSwere significantly higher than the level of respective param-eters in GS. The level of metals (Cr, Zn, Mn and Cu) wassignificantly high in CS and TS as compared to the GS. Incontrast, the level of Fe was higher in GS as compared tothe CS and TS, which is due to the presence of Fe as oneof the major constituent in earth crust. The level of allthe physico-chemical parameters was found high in con-taminated soil as it is receiving treated tannery wastewaterfor irrigation except Fe (Sinha et al., 2006).

    3.2. Level of metals in S. oleracea collected from

    contaminated agricultural field

    The accumulation of metals in the leaves ofS. oleraceagrown on contaminated soil was recorded for the period(20022003), which was receiving treated tannery wastewa-ter since last few decades at Jajmau, Kanpur (Table 2). Thelevel of toxic metal (Cr) was found high in the leaves of the

    plants. Variation in metal accumulation in the plant from

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    year to year can be attributed to the varying physico-chem-

    ical parameters of the substrate and other edaphic factors.Recently, Sinha et al. (2006) carried out extensive studieson the accumulation of metals in the edible part of vegeta-bles/crops growing on treated tannery wastewater contam-inated soil. They reported that agricultural land is notsuitable for the cultivation of leafy vegetables due to highaccumulation of metals in the edible part. Although, theplants grown on these soil have shown healthy growth pre-sumably due to the presence of essential nutrients andorganic matter. Similarly, the use of metal contaminatedwastewater for irrigation of vegetables and crops mayresults in elevated level of metals in the soil. Metals eventu-ally get translocated to the plants which affect health andagricultural and environmental quality (Singh et al.,2004c). Plants take up metals via roots, which depend uponthe physico-chemical characteristics of the soil, concentra-tion, solubility, species, cultivar age and organ of the plant.In addition, strict measures have been taken in the area torestrict the cultivation of leafy vegetables in the recent past.Recently,Sinha et al. (2005b)reported accumulation of Cr(3.048.55lg g1 dw) in the leaves ofS. oleracea grown inthe area which is being irrigated with river water. As perreport of NIN (Anonymous, 1982), on metal levels inIndian leafy vegetables, roots and tubers, metal content(lg g1 dw) in green leafy vegetables ranged from 16 to

    95 for Zn, 8 to 96 for Mn, 1.9 to 18 for Cu and 0.52 to

    4.37 for Cr. In general, Fe content ranged 50250

    lg g1

    dw in the plants.

    3.3. Metal accumulation

    In pot experimental studies, the accumulation of metalsin the leaves and roots of the plant after 90 d of growth ondifferent substrates, exhibited partitioning of the metals inboth parts of the plant (Table 3). Overall metal accumula-tion in the leaves was found to be in the order ofFe > Zn > Mn > Cr > Cu. The accumulation of essentialmetals (Zn, Mn, Cu) was found more in the leaves thanroots except in case of Zn and Cu in the plants grown on10% TS. In contrast, the accumulation of toxic metal(Cr) was found more in the roots than in the leaves exceptfor the plants grown on 10% TS. In case of Fe, the accumu-lation in the leaves was recorded low at higher amendments(35% TS onwards) than lower amendments. Leaves ofS. oleracea grown on 25% TS had the highest Fe contentand in roots grown on 10% TS after 90 d of growth.Recently,Gupta and Sinha (2006)observed that Fe accu-mulation bySesamum indicum(L.) var. T55 decreased withincrease in tannery sludge amendments beyond 25% TS,this was attributed to low level of metals in TS. They havealso reported that the level of Fe extracted with EDTA(bioavailable) decreased with increase in TS amendments

    (>25% TS). In the present study, low accumulation of Fe

    Table 1Physico-chemical properties of different substrates

    Parameters Substrates

    GS CS TS

    pH (1:2 ratio) 6.63 0.04 7.86 0.005 7.84 0.005Salinity (&) 0.2 1.3 7.5

    EC (lS m

    1

    ) 708 1.6 2506 5.47 3044 11.4CEC (C mol (p+) kg1) 55.69 0.83 83.8 1.2 146.25 1.87OC (%) 0.48 0.04 1.27 0.07 5.47 0.27OM (%) 0.793 0.121 2.18 0.01 9.39 0.49

    Total concentration of metals (lg g1 dw)Fe 38862 3684 22898 1740 19401.44 61Cr 5.1 0.34 145.87 21.51 7489.76 203Zn 45.15 0.86 217.03 22.81 340.69 11.10Mn 238.93 15.3 310.76 68.8 294.19 15.50Cu 18.67 0.85 18.67 0.85 188.09 5.81

    All the values are mean of three replicates SD.

    Table 2Levels of metal content in the leaves ofS. olarace collected from agriculturala field of Jajmau, Kanpur for two consecutive years

    Study periods Metals (lg g1 dw)

    Fe Cr Zn Mn Cu

    June 2002 1646.15 232 35.19 6.34 67.74 9.86 43.67 4.34 21.74 18.0January 2003 850.16 79.20 30.38 1.78 182.07 19.07 70.83 5.56 16.04 1.133June 2003 1246.15 132 25.19 4.34 69.74 9.01 47.07 1.34 25.74 2.8

    All values are mean of three replicates SD.a Tannery wastewater contaminated soil.

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    at higher amendments of TS may be attributed due to lowlevel of EDTA extractable metal.

    Pot experimental studies were also conducted on con-taminated soil (CS) collected from the field in order toassess the metal accumulation potential of S. oleraceaunder controlled conditions using tap water for irrigation(Table 3). High accumulation of toxic metal (Cr) in theleaves of the plants irrigated with tap water was observed,however, it is almost same as recorded in the leaves of theplants collected from Jajmau, Kanpur (Table 2) which isbeing irrigated from treated tannery wastewater. In view

    of high Cr accumulation in edible part ofS. oleraceaafterirrigation with tap water, it is not advisable to use suchplants for edible purposes. Further, the accumulation ofmost of the metals in the leaves was recorded almost sameas in the plants grown on 25% TS.

    The leaves to roots ratio of metal accumulation, weremostly >1 except for Fe, Cr. Translocation of metals(Cu, Zn, Mn) was found to be higher in the leaves thanits roots. The translocation of toxic metal chromium wasfound less at higher amendments (25% TS onwards) of tan-nery sludge, retaining most of the chromium in the roots ofthe plant. Recently, Sinha et al. (2005a,b) reported thatmost of the chromium in the plant, Pistia stratiotes wasfound in the roots, which is probably due to binding ofmetals to the ligands and thus reducing its mobility fromroots to aerial parts. It is a common strategy of the plantsto restrict metal translocation to the above ground parts asreported recently in the plants grown on tannery sludge-amended soil (Singh et al., 2004a,b; Singh and Sinha,2005; Gupta and Sinha, 2006).

    3.4. Morphological parameters

    The effect on growth of the plants grown on differentsubstrates (tannery waste contaminated) expressed as root

    length, shoot length, leaf area and number of leaves, are

    shown inFig. 1. The plants grown on all the TS amend-ments have shown significant increase in shoot length andleaf area at all the exposure periods as compared to GS(Fig. 1). At 75 and 90 d, the number of leaves of the plantsgrown on all the amendments has shown an increase ascompared to their respective plants grown on GS. Thus,maximum increase (p< 0.05) of 79.71% in shoot length,165.4% in leaf area and 232.7% in number of leaves (com-pared to GS) was observed in the plants grown on 35% TSafter 90 d of growth. On contrary to these growth param-eters, the roots exhibited no definite trend, however, it

    was recorded less in the plants grown on higher amend-ments of TS. The root length significantly (p< 0.05)decreased in the plants grown on 50% and 100% TS at 75and 90 d of exposure, respectively.

    The growth parameters was also recorded in the plantsgrown on contaminated soil (Fig. 1) collected from Jajmau,Kanpur. The analysis of the results revealed that significant(p < 0.02) increase of 67.81% in shoot length, 123.29% innumber of leaves and 130.04% in leaf area of the plantswas recorded after 90 d of growth as compared to GS.No marked difference was observed in the roots of theplants grown on contaminated soil.

    The changes observed in the growth ofS. oleraceawereconsistent with the results reported recently by Gupta andSinha (2006). They also observed no change in root length,however, shoot length increased at initial period of growthand lower amendments of tannery sludge. In other studies,it was reported that the shoot length ofBrassica junceavar.rohiniand Helianthus annuusgrown on 75% tannery sludgeamendment have shown an increase in its length as com-pared to control, however, root length of the exposedplants increased up to 35% tannery sludge as comparedto control. Other growth parameters increased in the plantsgrown on 50% tannery sludge at 90 d. In the present study,the analysis of the results of growth parameters have

    shown reduced growth in the plants grown on 50% TS

    Table 3Levels of various metals (lg g1 dw) in different parts ofS. oleracea grown on different substrate types after 90 d of growth

    Substrate types (%) Metals (lg g1 dw)

    Fe Cr Mn Zn Cu

    10 1265.41 30.11 29.90 0.68 67.40 1.32 72.12 1.44 14.94 1.02(1019.78 100.47) (31.21 3.49) (21.04 2.88) (34.13 3.66) (25.15 2.79)

    25 921.40 35.05 63.12 5.78 51.74 3.08 65.17 7.49 9.06 1.68(1411.86 147.8)9 (36.90 3.78) (70.20 7.89) (159.18 7.57) (15.40 0.07)

    35 787.44 71.55 78.26 8.99 41.80 5.80 61.7 7.29 14.02 1.11(393.08 19.30) (17.84 2.14) (58.36 6.01) (172.00 7.69) (26.73 1.07)

    50 899.96 84.65 68.70 7.29 36.89 1.24 39.76 3.30 7.19 0.13(419.32 16.02) (29.90 3.01) (56.52 5.54) (108.78 25.7) (15.42 0.03)

    100 682.93 65.47 205.95 22.45 44.39 4.97 70.54 8.70 17.04 0.78(395.38 30.92) (70.80 7.88) (50.24 0.78) (195.99 0.44) (32.88 1.14)

    CS 850 49 34.21 19.26 49.83 23.65 60.51 6.30 11.09 2.68(1511 152) (40.67 21.2) (138.6 41.8) (143.6 4.3) (21.74 18)

    The values in the parenthesis are of leaves. All values are mean of three replicates SD.

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    (Singh and Sinha, 2004a,b). The soil contaminated withtreated tannery wastewater offers the potential for recycling

    of nutrients present in the organic matter which serve asplant nutrients.

    Scanning electron micrographs of the leaf surface ofS. oleracea (Fig. 2AJ) after 90 d of growth, showed anincrease in stomatal length (Fig. 3) in the plants grownon 25% TS and decrease at 50% and 100% TS as comparedto GS. Further, the length of the stomata of the leaves ofthe plants grown on CS has shown an increase of 29.31%in stomatal length as compared to GS. Some of the sto-mata were found closed in the leaves of the plants grownon different TS amendments.

    The metal ions seems to attack various cellular compo-nents including cell wall and membranes resulting indifferential alterations that ultimately lead to their disorga-nization and mechanical injury i.e. necrosis. Stomatalopening was thought to be due to either metal-inducedinhibition of an energy system or the alterations of K+

    channel activities in guard cells. Maurel (1997) reportedthat aquaporins are present in guard cells and these toxinsinterfere with the polymerization and depolymerization ofactin filaments altering K+ channel activities in guard cells(Hwang et al., 1997).Singh and Sinha, 2004areported anincrease in stomatal size in the plants of Brassica grownon 50% amendment of tannery sludge. The decrease in sto-matal aperture may be due to rapid and preferential

    absorption of metals by subsidiary cells followed by

    changes in membrane permeability causing decrease in cellturgor as reported in Cd treated Phyllanthus amarus (Rai

    et al., 2005). The closure of the stomata may also be dueto a strategy of the plants to prevent the water loss throughtranspiration as the translocation of water and solutes getdisturbed in the presence of metals. Bondada and Oos-terhuis (2000) reported that closed stomata of the leafresult in a slower rate of diffusion due to greater diffusiongradient of water vapour. The decrease in size of stomatalaperture in the leaves is in the line with the hypothesis thatmetals induce water stress (Singh and Sinha, 2004b).

    Among other surface features, there is a marked increasein surface roughness in the leaves of the plants grown ondifferent amendments of TS, as compared to control. Inthe leaves of the plants grown on TS amended soil, stoma-tal frequency increased in the leaves of exposed plants ascompared to GS. Slight elevation in the stomatal complexis also observed in the plants growing on higher amend-ment of tannery sludge. Guard cells were slightly swollenin the leaves growing in higher amendment of TS.

    3.5. Photosynthetic pigment

    The total chlorophyll, chlorophyll a and b contents(Table 4) increased in the plants grown at all the amend-ments at 60 d and up to 25% TS after 75 d as compared tothe plants grown on GS. The plants grown on 100% TS

    have shown significant (p< 0.05) increase of 54.82% in

    Fig. 1. All values are mean of three replicates SD. The effect on the levels of growth parameters in the plants ofS. oleraceagrown on different substrates.*p< 0.05 as compared to GS based on LSD calculations. CS data not included in LSD calculations. t-test (two tailed as compared to GS) ap< 0.05,bp< 0.02, cp< 0.01.

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    total chlorophyll and 61.94% in chlorophyll a contents at60 d and significant decrease (p < 0.05) at 75 and 90 d.Maximum decrease of 53.69% in total chlorophyll and50.54% in chlorophyll a content were observed at 90 din the plants grown on 100% TS. The increase (non-signif-

    icant) in chlorophyll b content at 60 d and significant

    decrease of 70.17% in the leaves of the plant grown on100% TS was found after 90 d as compared to GS. Theanalysis of the results revealed that decrease in chloro-phyll b was more than chlorophyll a content in the leavesof the plants grown on 100% TS at the same exposure

    period.

    Fig. 2. SEM photographs of leaf surface ofS. oleracea (AJ) after 90 d of growth: (A) leaf epidermis of control plant (500 ); (B) magnified view ofstomata in plate A (2000); (C) leaf epidermis of plant grown on 10% TS (500); (D) magnified view of stomata of plate C (2000); (E) leaf epidermis ofplant grown on 50% TS (500); (F) magnified view of stomata in plate E (2000); (G) view of leaf epidermis of plants grown on 100% TS(500); (H)magnified view of stomata in plate G (2000); (I) view of leaf epidermis of plants grown on CS (500); (J) magnified view of stomata in plate I (2000).

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    At initial period of growth (60 and 75 d), the carotenoidcontent in the leaves of the plants grown on differentamendments of TS increased with increase in TS amend-

    ments, however, increase was significant (p < 0.05) athigher amendments at 35% and 25% TS after 60 and75 d, respectively as compared to GS. However, no changein carotenoid content in the leaves of the plants grown on

    35% TS was recorded followed by significant decrease ascompared to GS at 90 d of growth. The maximum increaseof 62.22% at 75 d and maximum decrease of 34% at 90 dwas observed in carotenoid content in the leaves of theplants grown on 100% TS.

    The total chlorophyll, chlorophyll a, chlorophyll b and

    carotenoid contents in the leaves of the plants grown onCS has shown significant (p< 0.05) increase at 60 d andnon-significant increase at 90 d as compared to theirrespective GS.

    The initial increase in the chlorophyll content can be dueto the availability of essential elements in abundance incomparison to GS. However, these metals,which were act-ing as nutrients, crossed the threshold limit and becametoxic agent with the advance of growth. Therefore, thedecline in the chlorophyll concentration can be attributedto the interference of heavy metals present in the substratein the formation of chlorophyll (Van Assche and Clijsters,1990). The level of photosynthetic contents in the plants

    grown on TS amended soil decreased which is consideredas one of the sensitive parameter in metal exposedplants (Singh et al., 2004a,b). Besides, lipid peroxidationalso causes degradation of the photosynthetic pigments

    0

    5

    10

    15

    20

    GS 10% 25% 50% 100% CS

    Substrate types

    Stomataldimension(m)

    length

    breath

    Fig. 3. Graphical representation of the stomatal dimension (lm) of S.oleraceagrown on different amendment of TS and CS after 90 d of growth.

    Table 4Levels of plant pigments (chlorophylls and carotenoid) in the leaves ofS. oleracea grown on different substrate types

    Substrate types (%) Exposure periods (d) Photosynthetic pigments (mg g1 fw)

    60 75 90

    GS 1.97 0.08 2.37 0.20 2.98 0.28 Total chlorophyll1.34 0.13 1.49 0.15 1.84 0.06 Chlorophyll a0.64 0.08 0.88 0.02 1.14 0.04 Chlorophyll b

    0.41 0.07 0.45 0.02 0.50 0.03 Carotenoid

    10 2.21 0.10* 3.12 0.18* 3.22 0.15* Total chlorophyll1.46 0.08 1.95 0.21* 2.06 0.06* Chlorophyll a0.75 0.10* 1.18 0.18* 1.16 0.15* Chlorophyll b0.47 0.02 0.50 0.01 0.53 0.02 Carotenoid

    025 2.44 0.08* 3.44 0.12* 2.60 0.06* Total chlorophyll1.63 0.04* 2.20 0.08* 1.47 0.09* Chlorophyll a0.81 0.08* 1.25 0.12* 1.14 0.04 Chlorophyll b0.52 0.10* 0.57 0.04* 0.53 0.01 Carotenoid

    35 2.6 0.11* 2.15 0.27 2.0 0.12* Total chlorophyll1.75 0.09* 1.40 0.29 1.11 0.12* Chlorophyll a0.85 0.11* 1.04 0.27* 0.60 0.004* Chlorophyll b0.63 0.02* 0.67 0.01* 0.52 0.12 Carotenoid

    50 2.84 0.06* 1.80 0.18* 1.55 0.03* Total chlorophyll1.91 0.07* 1.18 0.14* 1.06 0.03* Chlorophyll a0.94 0.05* 0.73 0.08* 0.37 0.004* Chlorophyll b0.67 0.01* 0.70 0.01* 0.34 0.02* Carotenoid

    100 3.05 0.05* 1.60 0.41* 1.38 0.02* Total chlorophyll2.17 0.02* 1.04 0.30* 0.91 0.05* Chlorophyll a0.88 0.05* 0.70 0.04* 0.34 0.06* Chlorophyll b0.71 0.04* 0.73 0.01* 0.33 0.02* Carotenoid

    CS 2.86 0.1c 3.35 0.04a 3.32 0.32 Total chlorophyll1.93 0.05b 2.26 0.21a 2.1 0.15 Chlorophyll a0.98 0.0.03a 1.19 0.19 1.24 0.2 Chlorophyll b0.75 0.02b 0.61 0.02b 0.65 0.06 Carotenoid

    All values are mean of triplicates SD.*p< 0.05 as compared to GS based on LSD calculations. CS data not included in LSD calculations. t-test (two

    tailed as compared to GS) a

    p< 0.05, b

    p< 0.02, c

    p< 0.01.

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    (Somashekaraiah et al., 1992). In this study,increased lipidperoxidation was observed in S. oleracea which mightcause degradation in photosynthetic pigments.

    The plants of S. oleracea grown under stress conditionalso shown increase in carotenoid content which served asaccessory pigments for photosynthesis. Carotenoids, which

    are important constituents of chloroplast membranes,quench singlet oxygen rapidly and can therefore, help toprotect chlorophyll and membrane against damage. Carote-noids are also able to absorb energy from, and so diminishthe concentration of, those exited states of chlorophyll thatlead to singlet oxygen species formation. Hence they have adual role: decreasing the formation of singlet oxygen in vivo,and helping to remove any singlet oxygen does happen to beformed. They may also react directly with peroxy and alk-oxy radicals, and so interfere with the chain reaction in lipidperoxidation. (Halliwell and Gutteridge, 2004).Thus, carot-enoid acts as a non-enzymatic antioxidant, and plays animportant role in protection of chlorophyll under stress

    condition (Kenneth et al., 2000). Increase in the carotenoidcontent is considered as a plants defense mechanismtowards metal stress; however, it is observed in the presentstudy that carotenoid content was found to decrease after75 d of growth. Thus, it may be inferred that, excessiveaccumulation of metals may cause toxicity.

    It has been observed that, the effect on chlorophyll bcontent was observed in the plants ofS. oleracea by metaltoxicity in comparison to chlorophyll a as it can be inferredfrom the increasing trend of chl a/b ratio at 90 d. There arevarious reports where chl a/b ratio was recorded more inmetal treated plants (Larsen et al., 1998; Rai et al., 2005).

    The carotenoid content increased significantly in all theamendment except at 10% TS at initial period of harvesting(60 d, 75 d), however, at 90 d of growth, it decreased athigher amendments (50% and 100% TS). This patterncould be due to initial availability of all the metals andorganic matter in abundance in the substrate but prolongedexposure to the substrate led to toxic effect and subsequentdecline in carotenoid levels.

    3.6. Effect on protein content

    The protein content (Table 5) in the roots and leaves ofthe plants grown on various amendments of TS increasedat all the TS amendments at all the growth periods exceptnon-significant decrease at higher amendments at 90 d inboth parts of the plant. In both parts, significant increasein protein content was recorded in the plants grown onhigher amendments (>10% TS) except non-significantincrease in the leaves at 25% TS after 60 d. The maximumincrease was recorded as 132.58% in the roots 136.72% inthe leaves of the plants grown on 100% TS at 75 d. As com-pared to control, the protein content increased in the rootsof the plants grown on 35% TS at 90 d, however, in theleaves up to 50% TS.

    Growth and development occur as a result of overall

    balance between protein synthesis and biogenesis, and pro-

    teolysis which is associated to oxidative stress promoted byreactive oxygen species (Solomon et al., 1999; Palma et al.,2002). The increase in protein content in this study may bedue to no induction in MDA content at 60 d and loweramendments at 75 d, thus, the protein degradation is lowerdown in the plants grown on tannery sludge amendments.There are other reports (Singh et al., 2004a,b; Singh and

    Sinha, 2005) showing an increase in protein content.

    3.7. Malondialdehyde content

    Malondialdehyde (MDA) is a major cytotoxic productof lipid peroxidation and acts as indicator of free radicalproduction and therefore, formation of MDA is consideredas measure of lipid peroxidation (Singh and Sinha, 2005).Malondialdehyde content in the leaves ofS. oleracea wasobserved to decrease with increase in sludge amendmentat 60 d, however, it was observed to increase signifi-cantly (p< 0.05) at 90 d of exposure except non-significantincrease in the plants grown at 10% TS. The increase of181.43% in MDA content was recorded in the leaves ofthe plants grown on 100% TS as compared to GS after90 d of growth (Table 6).

    The role of redox metals in the onset of peroxidationof membrane lipids in the plants have been realized dueto induction of toxic oxygen species. The formation ofmalondialdehyde (MDA) was considered as a measure oflipid peroxidation (Halliwell and Gutteridge, 2004). Similarto these reports, excessive accumulation of metals wasrecorded in the plants of S. oleracea at 90 d of growthwhich resulted an increase in leaves MDA content. The roleof redox metal in the induction of MDA content was

    reported in the plants treated with Zn (Chaoui et al.,

    Table 5Levels of protein content (mg g1 fw) in roots and leaves ofS. oleraceagrown on different substrate types

    Substrate types(%)

    Exposure periods (d)

    60 75 90

    GS 18.71 0.70 19.15 0.65 20.05 0.71

    (21.29 0.22) (22.11 2.14) (24.46 1.97)10 21.33 1.37* 23.56 0.66* 27.10 1.22*

    (23.02 0.01) (25.81 1.33*) (26.19 2.39)

    25 26.38 1.53* 30.81 1.32* 26.66 0.49*

    (25.88 1.94*) (31.34 2.97*) (39.20 3.74*)

    35 30.58 0.82* 38.31 0.95* 24.29 1.05*

    (31.18 5.14*) (40.52 1.57*) (32.92 4.94*)

    50 35.21 1.32* 40.88 .59* 19.86 .49(36.88 0.83*) (42.38 1.62*) (30.60 3.56*)

    100 40.70 2.44* 44.54 2.45* 16.73 1.01*

    (39.77 2.49*) (52.34 10.08*) (22.27 0.36*)

    CS 25.35 2.46a 31.01 1.25b 25.56 0.39b

    All values are mean of three replicates SD. Values in parenthesis areprotein content in leaves. *p< 0.05 as compared to GS based on LSDcalculations. CS data not included in LSD calculations. t-test (two tailedas compared to GS) ap< 0.05, bp< 0.01.

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    1997), with Fe (Sinha et al., 1997; Sinha and Saxena, 2006),with Cr (Sinha et al., 2005a), with Cu (Jouili and Ferjani,2003). There are various reports (Singh et al., 2004a,b;

    Singh and Sinha, 2005) which conferred similar conclu-sions showing an increase in the level of MDA content inthe plants grown on treated tannery sludge amendmentsdue to the presence of redox metals.

    3.8. Antioxidants

    A coordinated increase was observed in all the studiedantioxidant parameters. At 60 and 75 d of growth, cys-teine, non-protein thiol, ascorbic acid and proline content

    in the leaves increased as compared to GS, however,increased significantly (p< 0.05) at higher amendments(Fig. 4AD). At 60 d, cysteine, non-protein thiol, ascorbicacid and proline contents in the leaves increased withincrease in TS amendments with maximum increase of233.31%, 336.80%, 71.48% and 396.16%, respectively in

    the plants grown at 100% TS. As compared to GS, theseantioxidants increased at all the amendments after 75 dof growth. At 90 d of growth, cysteine and non-proteinthiol (Fig. 4A and B) contents responded in similar patternshowing significant increase in the leaves of the plantsgrown up to 25% TS with maximum increase of 142.84%and 128.65%, respectively. However, the level of ascorbicacid and proline increased significantly in the leaves ofthe plants grown on 10% TS followed by decrease. Overallanalysis of the results revealed that, the non-proteinthiol and cysteine contents have shown increase in theleaves of the plants grown on 100% TS as comparedto GS. Whereas, proline and ascorbic acid contents

    increased in the leaves of the plants grown on 50% TSand 25% TS, respectively after 90 d of growth followedby decrease.

    The plants grown on contaminated soil have shown sig-nificant increase in all the antioxidant parameters studied.The maximum increase of 134.70% (90 d), 145.3% (75 d),61.82% (60 d) and 251.33% (75 d) in cysteine, non-proteinthiol, ascorbic acid and proline contents, respectively wasrecorded as compared to the plants grown on GS(Fig. 4AD).

    Table 6Levels of MDA content (lmol g1 fw) in the leaves ofS. oleraceagrownon different substrate types

    Substrate types (%) Exposure periods (d)

    60 75 90

    GS 4.91 0.15 4.97 0.25 5.01 0.47

    10 3.80 0.24*

    4.15 0.35*

    5.86 0.54*

    25 3.07 0.16* 3.77 0.06* 8.27 0.53*

    35 2.97 0.08* 5.94 0.5* 9.41 0.28*

    50 2.52 0.07* 7.92 2.64* 12.12 0.51*

    100 2.35 0.07* 10.49 2.30* 14.10 1.50*

    CS 4.76 0.17 5.00 0.81 5.11 0.59

    All the values are means of triplicates SD. *p< 0.05 as compared to GSbased on LSD calculations. CS data not included in LSD calculations.

    t-test (two tailed) as compared to GS.

    Fig. 4. All values are mean of three replicates SD. The effect on the levels of cysteine (nmol g1 fw) (A), non-protein thiol (lmol g1 fw) (B), ascorbicacid (lg g1 fw) (C), free proline (lmol g1 fw), (D) contents in the plants ofS. oleraceagrown on different substrates. *p< 0.05 as compared to GS based

    on LSD calculations. CS data not included in LSD calculations. t-test (two tailed as compared to GS) a

    p< 0.05, b

    p< 0.01, c

    p< 0.001.

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    As it is evident from r2 values presented inTable 7thatthe accumulation of metals has shown positive correlationwith MDA content after 90 d of growth in the leaves of theplants with all the metals except Fe. However, the accumu-

    lation of metals has shown negative correlation with all thegrowth parameters and antioxidants except Fe. There is apositive correlation between Fe accumulation and antioxi-dants in 90 d old plants. This is due to decreasing pattern ofboth Fe and cysteine in the plant, as it has been discussedearlier in this paper that Fe content in TS is lower than thatin GS, therefore, with increasing proportion of TS in thesubstrate, Fe concentration is expected to fall. This couldbe due to the consumption of all the antioxidants, againstproviding defense mechanism to the plants, posed by oxi-dative stress. The correlation calculation (data not shown)was also performed between MDA content and various

    antioxidants which showed negative correlation at 90 d.Thus, with increase in MDA content, the level of antioxi-dant was expected to fall.

    The mechanism of metal detoxification adopted by theplants to scavenge free radicals and peroxides include sev-eral antioxidant substances. These non-enzymatic cellularentities include cysteine, non-protein thiol, ascorbic acid,carotenoid etc. which play an important role in inducingresistance to metals by protecting macromolecules againstfree radicals which are formed during various metabolicreactions leading to oxidative stress. In the present study,the enhanced level of cysteine and non-protein thiol sug-gests, its active participation in detoxification of toxic oxy-gen species. The stressed leaves of the plants showedincrease in the level of antioxidants in varying degree andprovide endogenous protection effects. Higher level of anti-oxidants at 60 d of growth might be related with low levelsof MDA which may be due to efficient defense mechanismagainst the stress-generated by metals. High thiol contentmight enable metabolites to function in the detoxificationof reactive oxygen species, which are detoxified by oxida-tion of sulfhydryl moieties to disulphides under metalstress.

    Ascorbic acid plays an important role in a-tocopherolregeneration which has been reported to act as the primary

    antioxidant. Besides this, ascorbate plays many other roles

    in the antioxidant metabolism. In plants, the level of pro-line enhanced in response to toxic metal exposures whichmight be attributed to the strategies adapted to cope upwith toxicity as main function of metal-induced prolineaccumulation may be associated with osmoregulation,and enzyme protection against dehydration rather than

    metal sequestration (Sharma et al., 1998). During the pres-ent study, an increase in ascorbic acid and proline contentsin the leaves of S. oleracea at initial period of exposuremay be considered as defense strategy to combat metalstress in the plant grown on tannery waste contaminatedsoil as reported earlier (Singh et al., 2004b; Singh andSinha, 2005).

    4. Conclusion

    In conclusion, the morphological parameters of theplants increased up to 50% TS showing that the tannery

    sludge supports the growth of the mature plants at loweramendments of sludge. Further, SEM micrograph revealedthat increased amendments of tannery sludge led to closureof stomata, increase in their frequency and degeneration. Acoordinated increase in antioxidants was noted withincrease in metal concentrations in the leaves of the plantsat initial period of growth. This indicates that the plantshave a detoxification mechanism to cope with such a highconcentrations of metals and no effect on MDA content.However, at higher amendment in mature plants, it experi-enced stress conditions due to increase in MDA contentwith increase in sludge amendments and the antioxidantsystem of the plant was not sufficient to revert the stressof a prolonged period of metal exposition. The plantsgrown on contaminated soil are able to combat stressinvolving defense mechanism, resulting in healthy growthof the plants as evidenced by the increase in growth param-eters photosynthetic pigments and antioxidants. Theresults are well coordinated as there is no change in theMDA content as compared to the plants grown on GSdue to increase in the level of all the antioxidants studied.

    Summing up it is recommended that the emphasisshould be given to the level of metals in the edible partof the plants instead of healthy growth as metal enrichedplants have detoxification mechanism and grow well on

    organic matter enriched contaminated soil. In view of highCr accumulation in edible part of S. oleracea grown oncontaminated soil after irrigation with tap water, it is notadvisable to use these plants for edible purposes; however,these plants have shown healthy growth. Thus, it can beconcluded that the contaminated soil which is receivingtreated tannery wastewater should not be used for the cul-tivation of leafy vegetables on the basis of these studies.

    Acknowledgements

    We thank the Director, National Botanical Research

    Institute, Lucknow (India), for providing required research

    Table 7Correlation factors (r2) between metals and various parameters in theleaves of in S. oleraceaafter 90 d of growth

    Parameters Fe Cr Mn Zn Cu

    Shoot length 0.21 0.90 0.34 0.03 0.39Root length 0.93 0.14 0.24 0.34 0.06No. of leaves 0.49 0.06 0.77 0.86 0.06

    Leaf area 0.11 0.81 0.03 0.14 0.07Total chl 0.78 0.41 0.49 0.68 0.04Carotenoid 0.65 0.61 0.11 0.30 0.04MDA 0.70 0.62 0.40 0.66 0.04Cysteine 0.79 0.55 0.37 0.07 0.60Proline 0.72 0.42 0.56 0.76 0.19Non-protein thiol 0.75 0.71 0.07 0.35 0.54Ascorbic acid 0.61 0.31 0.73 0.84 0.08

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    facilities. We are also thankful to Sri V.K. Lal for helpingus in electron microscopy.

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