Uptake and translocation of metals in Spinacia oleracea L. grown on tannery sludge-amended and contaminated soils: Effect on lipid peroxidation, morpho-anatomical changes and antioxidants

Chemosphere 67 (2007) 176–187 www.elsevier.com/locate/chemosphere

Uptake and translocation of metals in Spinacia oleracea L. grown on 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 2006 Available online 13 November 2006

Abstract The plants of Spinacia oleracea L. grown on contaminated soil (CS) and different amendments of tannery sludge (TS) have shown high accumulation of metals in its edible part. The accumulation of toxic metal (Cr) in the leaves of the plants grown on CS was recorded as 40.67 lg g1 dw. However, the leaves of the plants grown on 100% TS have accumulated about two times (70.80 lg g1 dw) higher Cr than the 10% TS (31.21 lg g1 dw). Among growth parameters, the root length was more affected at 90 d than the shoot length, number of leaves and leaf area. The study of scanning electron micrographs showed 29.31% increase in stomatal length in the leaves of the plants grown on CS as compared to garden soil (GS), which served as control, however it decreased in the plants grown on higher amendments of TS. The decrease in MDA content at initial period of exposure and lower amendment was recorded in the leaves, whereas, significant increase (>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 of the antioxidant decreased as compared to 75 d causing oxidative stress as evidenced by increased level of lipid peroxidation and decreased chlorophyll and protein contents. Maximum increase of 181.43% in MDA content and maximum decrease of 53.69% in total chlorophyll content was recorded in the leaves of the plants grown on 100% TS after 90 d of growth. The plants grown on CS have shown an 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 well coordinated as there is no change in the MDA content as compared to the plants grown on GS. In view of high Cr accumulation in edible part of S. oleracea grown on CS after irrigation with tap water, it is not advisable to use these plants for edible purposes. Summing up, it is recommended that the level of metals in the edible part should be checked instead of healthy growth as deciding parameter for consumption. It is demonstrated through this study that metal enriched plants have detoxification mechanism and grow well on organic matter 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 of any city in developing countries has become a common *

Corresponding author. Tel.: +91 522 205831 35x221; fax: +91 522 205839, +91 522 205836. E-mail address: sinha_sarita@rediffmail.com (S. Sinha). 0045-6535/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.08.026

phenomenon, due to the lack of proper waste disposal practices. Among all the industries, the tannery industry is one of the notorious in terms of heavy metal contamination to the soil. The leachate from the dried tannery sludge cakes and sludge itself is posing a potential hazard of heavy metals 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

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countries, the use of sewage and treated industrial wastewater for irrigating the crops is a common practice (Nan and Cheng, 2001; Wong et al., 2001; Sinha et al., 2006) which leads to cumulative contamination of heavy metals to 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 of raw leather hides. Chromium merits a special reference for its extreme toxicity due to interaction of its compounds with living cells (Cieslak-Golonka, 1995; Costa, 1997) Besides Cr, Fe is an another major element present in the tannery sludge and comes from animal’s hides and ferrous aluminum sulphate, which is used for precipitation of suspended solids during wastewater treatment. However, excessive concentrations of Fe have also been reported to be toxic to the plants (Sinha et al., 1997; Sinha and Saxena, 2006). Metals in tannery waste occur in complex forms and vary widely in their availability to the plants. The generation of reactive oxygen species is stimulated in the presence of metals which can seriously disturb normal metabolism through oxidative damage to cellular compartments.To counteract this damage, highly efficient antioxidant defense mechanism in its cells can scavenge or deactivate metal stress-generated by reactive oxygen radicals. Antioxidant substances like cysteine, ascorbic acid, non-protein thiol (sulfhydryl) and antioxidant enzymes play a vital role in providing cellular defense towards oxidative stress (Sinha et al., 1997, 2005a; Halliwell and Gutteridge, 2004; Sinha and Saxena, 2006). There are many reports on metal-induced oxidative stress and response of antioxidants 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 and structural changes such as wider opening of stomata, increase in the stomata size, thinning of wax deposition and elevation of stomatal complex. Lesser wax deposition has been attributed to increased lipid peroxidation induced by metal toxicity (Rai et al., 2005). Due to the crisis of the arable land compounded by the contamination of fertile soil with improper disposal of tannery waste, farmers are forced to grow food crops on lands contaminated by heavy metals. Among all the parts of the plant, maximum accumulation of metals was found in the roots in most of the plants. However, some of the metals are translocated to the edible part. Therefore, entry of metals into the food-chain through plants grown on contaminated soils is a matter of serious concern. Sinha et al. (2006) reported metal accumulation in various parts of the plants grown on soil receiving treated tannery wastewater, maximum being in leafy vegetables than fruits bearing vegetables/crops. Further, the plants have shown better growth in the plants grown on contaminated soil than normal soil. However, no work has been carried out to study the toxicity and defense mechanism of leafy vegetables grown on contaminated agricultural soil. Based on our earlier work (Sinha et al., 2006), where high accumulation of

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toxic metal (Cr) in the leaves of Spinacia oleracea was observed, the present study is undertaken with a view (i) to assess the accumulation of metals in the leaves of the plants collected from Jajmau (Kanpur, India) irrigated with treated tannery wastewater; (ii) to assess the translocation of metals in the plants grown on contaminated soil in pots irrigated with tap water and to investigate the effects on its physiological and biochemical parameters; and (iii) to compare the translocation of metals in the plants grown on various amendments of tannery sludge with the plants grown on contaminated soil and physiological and biochemical effects induced in these plants. The outcome of the results should be helpful to elucidate metal detoxification mechanism in S. oleracea which have shown healthy growth when grown on tannery waste contaminated soil. 2. Material and methods In India, Jajmau (Kanpur) is a major industrial town and lies in the Indo-Gangetic plains between the parallels of 2628 0 N and 8024 0 E. It is one of the major centers to process raw hides. The discharge from these industries is treated in an Up-flow Anaerobic Sludge Blanket (UASB) treatment plant before releasing. The treated wastewater is being used by the local farmers for irrigation of edible crops/vegetables in the adjacent agricultural fields (2100 acre). 2.1. Collection of plants from agricultural field Plants of Spinacia oleracea L. (spinach) were collected in two consecutive years (June 2002, January 2003, June 2003) from the agricultural field of Jajmau (Kanpur) and brought to the laboratory to check the level of metals in the edible part of the plant. The agricultural land is being irrigated with treated tannery wastewater since last many decades. 2.2. Experimental design Air dried tannery sludge (TS) was collected from the wastewater treatment plant at Jajmau, Kanpur (Uttar Pradesh, India) and were brought to the laboratory. Uncontaminated garden soil (GS) was collected from National Botanical Research Institute (NBRI) and was used as a control. The tannery sludge and the garden soil were finely grounded and passed through 2 mm sieve to get a uniform size, before filling up in terracotta earthen pots (35 cm in diameter). Different amendments (10%, 20%, 35%, 50% and 100%) of the TS were prepared using GS. In another set, the contaminated soil (CS) was also collected from Jajmau, Kanpur which is being irrigated with treated tannery wastewater in the agricultural fields (Jajmau, Kanpur) and sieved soil were filled in terracotta earthen pots (35 cm in diameter). The experiment was carried out in these pots for three months, with three harvests.

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Seeds of S. oleracea were sterilized in 3% formalin for 5 min followed by washing with double distilled water and soaked in water overnight. These seeds were sown in a nursery bed. After the seeds germinated and the saplings grew to 10 cm, they were transplanted to earthen pots filled with 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 National Botanical Research Institute in a randomized block design, at an average diurnal temperature of 25–35 C. The plants were watered by normal tap water and harvested after 60, 75 and 90 d of growth.

2.6. Data analysis and interpretation The experiment was performed in completely randomized block design involving six amendments of TS with GS, in triplicates, for three time interval. All the dataset obtained from the experiment, were subjected to two way analysis of variance (ANOVA) using Microsoft Excel 2000 followed by least significant difference (LSD) calculation (Gomez and Gomez, 1984). Student t-test (two tailed) was applied between the data obtained from CS as compared to GS. 2.7. Quality control and quality assurance

2.3. Metal accumulation Harvested plants were washed thoroughly with distilled water and blotted dry. Different parts were separated manually, cut in small pieces and oven dried at 70 C till constant weight. The dried samples were ground (> 2 mm) and digested in HNO3(70%) in Microwave Digestion System (MDS 2000) and analyzed for metals content using Atomic Absorption Spectrophotometer (GBC, AvantaR). 2.4. Estimation of various physiological and biochemical parameters Fresh weight, root length, shoot length, leaf area of the plant were recorded immediately after harvesting. Fresh leaves 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 plants were determined using BSA as standard protein (Lowry et al., 1951). Lipid peroxidation in the plant tissue was measured indirectly in terms of malondialdehyde (MDA) content, determined by thio barbituric acid (TBA) reaction (Heath and Packer, 1968). Cysteine content was estimated by the method of Gaitonde (1967). Non-protein thiol (acid soluble thiol) content was measured (Ellman, 1959) using Ellmans reagent (5,5 0 -dithiobis 2-nitrobenzoic acid). Ascorbic acid content in the leaves was estimated as per method of Keller and Schwager (1977). 2.5. Scanning electron microscopy Central part of the leaves from each set of the treatment was collected for studying the surface structure through Scanning Electron Microscopy. The leaves were kept in 2.5% gluteraldehyde overnight for fixation followed by dehydration in ethanol series (30%, 50%, 70%, 90% and 100%). Further dehydration was done in BAL-TEC CPD-030 critical point drier using liquid CO2 as carrier gas. The leaves were mounted on a stub and were coated with 15 lm conductive gold, in an ion sputter coater (TFC 1100). Coated specimens were mounted on Philips XL-20 Scanning Electron Microscope.

The standard reference material of metals (E-Merck, Germany) was used for calibration and quality assurance for each analytical batch. The reference solution (BND 1101.02) of multi-elements (Zn, Fe, Cu) was also used for calibration of analytical equipment and validation of test methods provided by National Physical Laboratory (NPL), New Delhi (India) and the results were found to be within ±1.50% of certified values. EPA quality control samples (Lot TMA 989) for metals (Cd, Cr, Cu, Pb) was used in order to ensure analytical data quality in water and the results were found to be within ±2.79% of certified values. The recoveries of metals from the plant tissues were found to be more than 98.5% as determined by digesting three samples each from an untreated plant with known amount of metals. The blanks were run in triplicate to check 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 its different amendments along with CS (Table 1) revealed that pH, salinity, EC, CEC, OC and OM of both CS and TS were significantly higher than the level of respective parameters in GS. The level of metals (Cr, Zn, Mn and Cu) was significantly high in CS and TS as compared to the GS. In contrast, the level of Fe was higher in GS as compared to the CS and TS, which is due to the presence of Fe as one of the major constituent in earth crust. The level of all the physico-chemical parameters was found high in contaminated soil as it is receiving treated tannery wastewater for 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 of S. oleracea grown on contaminated soil was recorded for the period (2002–2003), which was receiving treated tannery wastewater since last few decades at Jajmau, Kanpur (Table 2). The level 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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Table 1 Physico-chemical properties of different substrates Parameters

Substrates GS

CS

TS

pH (1:2 ratio) Salinity (&) EC (lS m1) CEC (C mol (p+) kg1) OC (%) OM (%)

6.63 ± 0.04 0.2 708 ± 1.6 55.69 ± 0.83 0.48 ± 0.04 0.793 ± 0.121

7.86 ± 0.005 1.3 2506 ± 5.47 83.8 ± 1.2 1.27 ± 0.07 2.18 ± 0.01

7.84 ± 0.005 7.5 3044 ± 11.4 146.25 ± 1.87 5.47 ± 0.27 9.39 ± 0.49

Total concentration of metals (lg g1 dw) Fe Cr Zn Mn Cu

38862 ± 3684 5.1 ± 0.34 45.15 ± 0.86 238.93 ± 15.3 18.67 ± 0.85

22898 ± 1740 145.87 ± 21.51 217.03 ± 22.81 310.76 ± 68.8 18.67 ± 0.85

19401.44 ± 61 7489.76 ± 203 340.69 ± 11.10 294.19 ± 15.50 188.09 ± 5.81

All the values are mean of three replicates ± SD.

Table 2 Levels of metal content in the leaves of S. 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 January 2003 June 2003

1646.15 ± 232 850.16 ± 79.20 1246.15 ± 132

35.19 ± 6.34 30.38 ± 1.78 25.19 ± 4.34

67.74 ± 9.86 182.07 ± 19.07 69.74 ± 9.01

43.67 ± 4.34 70.83 ± 5.56 47.07 ± 1.34

21.74 ± 18.0 16.04 ± 1.133 25.74 ± 2.8

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

year to year can be attributed to the varying physico-chemical parameters of the substrate and other edaphic factors. Recently, Sinha et al. (2006) carried out extensive studies on the accumulation of metals in the edible part of vegetables/crops growing on treated tannery wastewater contaminated soil. They reported that agricultural land is not suitable for the cultivation of leafy vegetables due to high accumulation of metals in the edible part. Although, the plants grown on these soil have shown healthy growth presumably due to the presence of essential nutrients and organic matter. Similarly, the use of metal contaminated wastewater for irrigation of vegetables and crops may results in elevated level of metals in the soil. Metals eventually get translocated to the plants which affect health and agricultural and environmental quality (Singh et al., 2004c). Plants take up metals via roots, which depend upon the physico-chemical characteristics of the soil, concentration, solubility, species, cultivar age and organ of the plant. In addition, strict measures have been taken in the area to restrict the cultivation of leafy vegetables in the recent past. Recently, Sinha et al. (2005b) reported accumulation of Cr (3.04–8.55 lg g1 dw) in the leaves of S. oleracea grown in the area which is being irrigated with river water. As per report of NIN (Anonymous, 1982), on metal levels in Indian 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 50–250 lg g1 dw in the plants. 3.3. Metal accumulation In pot experimental studies, the accumulation of metals in the leaves and roots of the plant after 90 d of growth on different substrates, exhibited partitioning of the metals in both parts of the plant (Table 3). Overall metal accumulation in the leaves was found to be in the order of Fe > Zn > Mn > Cr > Cu. The accumulation of essential metals (Zn, Mn, Cu) was found more in the leaves than roots except in case of Zn and Cu in the plants grown on 10% TS. In contrast, the accumulation of toxic metal (Cr) was found more in the roots than in the leaves except for the plants grown on 10% TS. In case of Fe, the accumulation in the leaves was recorded low at higher amendments (35% TS onwards) than lower amendments. Leaves of S. oleracea grown on 25% TS had the highest Fe content and in roots grown on 10% TS after 90 d of growth. Recently, Gupta and Sinha (2006) observed that Fe accumulation by Sesamum indicum (L.) var. T55 decreased with increase in tannery sludge amendments beyond 25% TS, this was attributed to low level of metals in TS. They have also 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

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Table 3 Levels of various metals (lg g1 dw) in different parts of S. 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 (1019.78 ± 100.47)

29.90 ± 0.68 (31.21 ± 3.49)

67.40 ± 1.32 (21.04 ± 2.88)

72.12 ± 1.44 (34.13 ± 3.66)

14.94 ± 1.02 (25.15 ± 2.79)

25

921.40 ± 35.05 (1411.86 ± 147.8)9

63.12 ± 5.78 (36.90 ± 3.78)

51.74 ± 3.08 (70.20 ± 7.89)

65.17 ± 7.49 (159.18 ± 7.57)

9.06 ± 1.68 (15.40 ± 0.07)

35

787.44 ± 71.55 (393.08 ± 19.30)

78.26 ± 8.99 (17.84 ± 2.14)

41.80 ± 5.80 (58.36 ± 6.01)

61.7 ± 7.29 (172.00 ± 7.69)

14.02 ± 1.11 (26.73 ± 1.07)

50

899.96 ± 84.65 (419.32 ± 16.02)

68.70 ± 7.29 (29.90 ± 3.01)

36.89 ± 1.24 (56.52 ± 5.54)

39.76 ± 3.30 (108.78 ± 25.7)

7.19 ± 0.13 (15.42 ± 0.03)

100

682.93 ± 65.47 (395.38 ± 30.92)

205.95 ± 22.45 (70.80 ± 7.88)

44.39 ± 4.97 (50.24 ± 0.78)

70.54 ± 8.70 (195.99 ± 0.44)

17.04 ± 0.78 (32.88 ± 1.14)

CS

850 ± 49 (1511 ± 152)

34.21 ± 19.26 (40.67 ± 21.2)

49.83 ± 23.65 (138.6 ± 41.8)

60.51 ± 6.30 (143.6 ± 4.3)

11.09 ± 2.68 (21.74 ± 18)

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

at higher amendments of TS may be attributed due to low level of EDTA extractable metal. Pot experimental studies were also conducted on contaminated soil (CS) collected from the field in order to assess the metal accumulation potential of S. oleracea under controlled conditions using tap water for irrigation (Table 3). High accumulation of toxic metal (Cr) in the leaves of the plants irrigated with tap water was observed, however, it is almost same as recorded in the leaves of the plants collected from Jajmau, Kanpur (Table 2) which is being irrigated from treated tannery wastewater. In view of high Cr accumulation in edible part of S. oleracea after irrigation with tap water, it is not advisable to use such plants for edible purposes. Further, the accumulation of most of the metals in the leaves was recorded almost same as in the plants grown on 25% TS. The leaves to roots ratio of metal accumulation, were mostly >1 except for Fe, Cr. Translocation of metals (Cu, Zn, Mn) was found to be higher in the leaves than its roots. The translocation of toxic metal chromium was found less at higher amendments (25% TS onwards) of tannery sludge, retaining most of the chromium in the roots of the plant. Recently, Sinha et al. (2005a,b) reported that most of the chromium in the plant, Pistia stratiotes was found in the roots, which is probably due to binding of metals to the ligands and thus reducing its mobility from roots to aerial parts. It is a common strategy of the plants to restrict metal translocation to the above ground parts as reported recently in the plants grown on tannery sludgeamended 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 different substrates (tannery waste contaminated) expressed as root length, shoot length, leaf area and number of leaves, are

shown in Fig. 1. The plants grown on all the TS amendments have shown significant increase in shoot length and leaf area at all the exposure periods as compared to GS (Fig. 1). At 75 and 90 d, the number of leaves of the plants grown on all the amendments has shown an increase as compared 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 (compared to GS) was observed in the plants grown on 35% TS after 90 d of growth. On contrary to these growth parameters, the roots exhibited no definite trend, however, it was recorded less in the plants grown on higher amendments of TS. The root length significantly (p < 0.05) decreased in the plants grown on 50% and 100% TS at 75 and 90 d of exposure, respectively. The growth parameters was also recorded in the plants grown 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% in number of leaves and 130.04% in leaf area of the plants was recorded after 90 d of growth as compared to GS. No marked difference was observed in the roots of the plants grown on contaminated soil. The changes observed in the growth of S. oleracea were consistent with the results reported recently by Gupta and Sinha (2006). They also observed no change in root length, however, shoot length increased at initial period of growth and lower amendments of tannery sludge. In other studies, it was reported that the shoot length of Brassica juncea var. rohini and Helianthus annuus grown on 75% tannery sludge amendment have shown an increase in its length as compared to control, however, root length of the exposed plants increased up to 35% tannery sludge as compared to control. Other growth parameters increased in the plants grown 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

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Fig. 1. All values are mean of three replicates ± SD. The effect on the levels of growth parameters in the plants of S. 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 < 0.02, c p < 0.01.

(Singh and Sinha, 2004a,b). The soil contaminated with treated tannery wastewater offers the potential for recycling of nutrients present in the organic matter which serve as plant nutrients. Scanning electron micrographs of the leaf surface of S. oleracea (Fig. 2A–J) after 90 d of growth, showed an increase in stomatal length (Fig. 3) in the plants grown on 25% TS and decrease at 50% and 100% TS as compared to GS. Further, the length of the stomata of the leaves of the plants grown on CS has shown an increase of 29.31% in stomatal length as compared to GS. Some of the stomata were found closed in the leaves of the plants grown on different TS amendments. The metal ions seems to attack various cellular components including cell wall and membranes resulting in differential alterations that ultimately lead to their disorganization and mechanical injury i.e. necrosis. Stomatal opening was thought to be due to either metal-induced inhibition of an energy system or the alterations of K+ channel activities in guard cells. Maurel (1997) reported that aquaporins are present in guard cells and these toxins interfere with the polymerization and depolymerization of actin filaments altering K+ channel activities in guard cells (Hwang et al., 1997). Singh and Sinha, 2004a reported an increase in stomatal size in the plants of Brassica grown on 50% amendment of tannery sludge. The decrease in stomatal aperture may be due to rapid and preferential absorption of metals by subsidiary cells followed by

changes in membrane permeability causing decrease in cell turgor as reported in Cd treated Phyllanthus amarus (Rai et al., 2005). The closure of the stomata may also be due to a strategy of the plants to prevent the water loss through transpiration as the translocation of water and solutes get disturbed in the presence of metals. Bondada and Oosterhuis (2000) reported that closed stomata of the leaf result in a slower rate of diffusion due to greater diffusion gradient of water vapour. The decrease in size of stomatal aperture in the leaves is in the line with the hypothesis that metals induce water stress (Singh and Sinha, 2004b). Among other surface features, there is a marked increase in surface roughness in the leaves of the plants grown on different amendments of TS, as compared to control. In the leaves of the plants grown on TS amended soil, stomatal frequency increased in the leaves of exposed plants as compared to GS. Slight elevation in the stomatal complex is also observed in the plants growing on higher amendment of tannery sludge. Guard cells were slightly swollen in 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 amendments at 60 d and up to 25% TS after 75 d as compared to the plants grown on GS. The plants grown on 100% TS have shown significant (p < 0.05) increase of 54.82% in

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Fig. 2. SEM photographs of leaf surface of S. oleracea (A–J) after 90 d of growth: (A) leaf epidermis of control plant (500·); (B) magnified view of stomata 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 of plant 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·).

total chlorophyll and 61.94% in chlorophyll a contents at 60 d and significant decrease (p < 0.05) at 75 and 90 d. Maximum decrease of 53.69% in total chlorophyll and 50.54% in chlorophyll a content were observed at 90 d in the plants grown on 100% TS. The increase (non-significant) in chlorophyll b content at 60 d and significant

decrease of 70.17% in the leaves of the plant grown on 100% TS was found after 90 d as compared to GS. The analysis of the results revealed that decrease in chlorophyll b was more than chlorophyll a content in the leaves of the plants grown on 100% TS at the same exposure period.

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Stomatal dimension (µm)

20

length breath

15

10

5

0 GS

10%

25% 50% Substrate types

100%

CS

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

At initial period of growth (60 and 75 d), the carotenoid content in the leaves of the plants grown on different amendments of TS increased with increase in TS amendments, however, increase was significant (p < 0.05) at higher amendments at 35% and 25% TS after 60 and 75 d, respectively as compared to GS. However, no change in carotenoid content in the leaves of the plants grown on

183

35% TS was recorded followed by significant decrease as compared to GS at 90 d of growth. The maximum increase of 62.22% at 75 d and maximum decrease of 34% at 90 d was observed in carotenoid content in the leaves of the plants grown on 100% TS. The total chlorophyll, chlorophyll a, chlorophyll b and carotenoid contents in the leaves of the plants grown on CS has shown significant (p < 0.05) increase at 60 d and non-significant increase at 90 d as compared to their respective GS. The initial increase in the chlorophyll content can be due to the availability of essential elements in abundance in comparison to GS. However, these metals,which were acting as nutrients, crossed the threshold limit and became toxic agent with the advance of growth. Therefore, the decline in the chlorophyll concentration can be attributed to the interference of heavy metals present in the substrate in 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 considered as one of the sensitive parameter in metal exposed plants (Singh et al., 2004a,b). Besides, lipid peroxidation also causes degradation of the photosynthetic pigments

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

Photosynthetic pigments (mg g1 fw)

Exposure periods (d) 60

75

90

GS

1.97 ± 0.08 1.34 ± 0.13 0.64 ± 0.08 0.41 ± 0.07

2.37 ± 0.20 1.49 ± 0.15 0.88 ± 0.02 0.45 ± 0.02

2.98 ± 0.28 1.84 ± 0.06 1.14 ± 0.04 0.50 ± 0.03

Total chlorophyll Chlorophyll a Chlorophyll b Carotenoid

10

2.21 ± 0.10* 1.46 ± 0.08 0.75 ± 0.10* 0.47 ± 0.02

3.12 ± 0.18* 1.95 ± 0.21* 1.18 ± 0.18* 0.50 ± 0.01

3.22 ± 0.15* 2.06 ± 0.06* 1.16 ± 0.15* 0.53 ± 0.02

Total chlorophyll Chlorophyll a Chlorophyll b Carotenoid

025

2.44 ± 0.08* 1.63 ± 0.04* 0.81 ± 0.08* 0.52 ± 0.10*

3.44 ± 0.12* 2.20 ± 0.08* 1.25 ± 0.12* 0.57 ± 0.04*

2.60 ± 0.06* 1.47 ± 0.09* 1.14 ± 0.04 0.53 ± 0.01

Total chlorophyll Chlorophyll a Chlorophyll b Carotenoid

35

2.6 ± 0.11* 1.75 ± 0.09* 0.85 ± 0.11* 0.63 ± 0.02*

2.15 ± 0.27 1.40 ± 0.29 1.04 ± 0.27* 0.67 ± 0.01*

2.0 ± 0.12* 1.11 ± 0.12* 0.60 ± 0.004* 0.52 ± 0.12

Total chlorophyll Chlorophyll a Chlorophyll b Carotenoid

50

2.84 ± 0.06* 1.91 ± 0.07* 0.94 ± 0.05* 0.67 ± 0.01*

1.80 ± 0.18* 1.18 ± 0.14* 0.73 ± 0.08* 0.70 ± 0.01*

1.55 ± 0.03* 1.06 ± 0.03* 0.37 ± 0.004* 0.34 ± 0.02*

Total chlorophyll Chlorophyll a Chlorophyll b Carotenoid

100

3.05 ± 0.05* 2.17 ± 0.02* 0.88 ± 0.05* 0.71 ± 0.04*

1.60 ± 0.41* 1.04 ± 0.30* 0.70 ± 0.04* 0.73 ± 0.01*

1.38 ± 0.02* 0.91 ± 0.05* 0.34 ± 0.06* 0.33 ± 0.02*

Total chlorophyll Chlorophyll a Chlorophyll b Carotenoid

CS

2.86 ± 0.1c 1.93 ± 0.05b 0.98 ± 0.0.03a 0.75 ± 0.02b

3.35 ± 0.04a 2.26 ± 0.21a 1.19 ± 0.19 0.61 ± 0.02b

3.32 ± 0.32 2.1 ± 0.15 1.24 ± 0.2 0.65 ± 0.06

Total chlorophyll Chlorophyll a Chlorophyll b 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 lipid peroxidation was observed in S. oleracea which might cause degradation in photosynthetic pigments. The plants of S. oleracea grown under stress condition also shown increase in carotenoid content which served as accessory pigments for photosynthesis. Carotenoids, which are important constituents of chloroplast membranes, quench singlet oxygen rapidly and can therefore, help to protect chlorophyll and membrane against damage. Carotenoids are also able to absorb energy from, and so diminish the concentration of, those exited states of chlorophyll that lead to singlet oxygen species formation. Hence they have a dual role: decreasing the formation of singlet oxygen in vivo, and helping to remove any singlet oxygen does happen to be formed. They may also react directly with peroxy and alkoxy radicals, and so interfere with the chain reaction in lipid peroxidation. (Halliwell and Gutteridge, 2004).Thus, carotenoid acts as a non-enzymatic antioxidant, and plays an important role in protection of chlorophyll under stress condition (Kenneth et al., 2000). Increase in the carotenoid content is considered as a plant’s defense mechanism towards metal stress; however, it is observed in the present study that carotenoid content was found to decrease after 75 d of growth. Thus, it may be inferred that, excessive accumulation of metals may cause toxicity. It has been observed that, the effect on chlorophyll b content was observed in the plants of S. oleracea by metal toxicity in comparison to chlorophyll a as it can be inferred from the increasing trend of chl a/b ratio at 90 d. There are various reports where chl a/b ratio was recorded more in metal treated plants (Larsen et al., 1998; Rai et al., 2005). The carotenoid content increased significantly in all the amendment except at 10% TS at initial period of harvesting (60 d, 75 d), however, at 90 d of growth, it decreased at higher amendments (50% and 100% TS). This pattern could be due to initial availability of all the metals and organic matter in abundance in the substrate but prolonged exposure to the substrate led to toxic effect and subsequent decline in carotenoid levels. 3.6. Effect on protein content The protein content (Table 5) in the roots and leaves of the plants grown on various amendments of TS increased at all the TS amendments at all the growth periods except non-significant decrease at higher amendments at 90 d in both parts of the plant. In both parts, significant increase in protein content was recorded in the plants grown on higher amendments (>10% TS) except non-significant increase in the leaves at 25% TS after 60 d. The maximum increase was recorded as 132.58% in the roots 136.72% in the leaves of the plants grown on 100% TS at 75 d. As compared to control, the protein content increased in the roots of the plants grown on 35% TS at 90 d, however, in the leaves up to 50% TS. Growth and development occur as a result of overall balance between protein synthesis and biogenesis, and pro-

Table 5 Levels of protein content (mg g1 fw) in roots and leaves of S. oleracea grown on different substrate types Substrate types (%)

Exposure periods (d) 60

75

90

GS

18.71 ± 0.70 (21.29 ± 0.22)

19.15 ± 0.65 (22.11 ± 2.14)

20.05 ± 0.71 (24.46 ± 1.97)

10

21.33 ± 1.37* (23.02 ± 0.01)

23.56 ± 0.66* (25.81 ± 1.33*)

27.10 ± 1.22* (26.19 ± 2.39)

25

26.38 ± 1.53* (25.88 ± 1.94*)

30.81 ± 1.32* (31.34 ± 2.97*)

26.66 ± 0.49* (39.20 ± 3.74*)

35

30.58 ± 0.82* (31.18 ± 5.14*)

38.31 ± 0.95* (40.52 ± 1.57*)

24.29 ± 1.05* (32.92 ± 4.94*)

50

35.21 ± 1.32* (36.88 ± 0.83*)

40.88 ± .59* (42.38 ± 1.62*)

19.86 ± .49 (30.60 ± 3.56*)

100

40.70 ± 2.44* (39.77 ± 2.49*)

44.54 ± 2.45* (52.34 ± 10.08*)

16.73 ± 1.01* (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 are protein content in leaves. * 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.

teolysis which is associated to oxidative stress promoted by reactive oxygen species (Solomon et al., 1999; Palma et al., 2002). The increase in protein content in this study may be due to no induction in MDA content at 60 d and lower amendments at 75 d, thus, the protein degradation is lower down 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 product of lipid peroxidation and acts as indicator of free radical production and therefore, formation of MDA is considered as measure of lipid peroxidation (Singh and Sinha, 2005). Malondialdehyde content in the leaves of S. oleracea was observed to decrease with increase in sludge amendment at 60 d, however, it was observed to increase significantly (p < 0.05) at 90 d of exposure except non-significant increase in the plants grown at 10% TS. The increase of 181.43% in MDA content was recorded in the leaves of the plants grown on 100% TS as compared to GS after 90 d of growth (Table 6). The role of redox metals in the onset of peroxidation of membrane lipids in the plants have been realized due to induction of toxic oxygen species. The formation of malondialdehyde (MDA) was considered as a measure of lipid peroxidation (Halliwell and Gutteridge, 2004). Similar to these reports, excessive accumulation of metals was recorded in the plants of S. oleracea at 90 d of growth which resulted an increase in leaves MDA content. The role of redox metal in the induction of MDA content was reported in the plants treated with Zn (Chaoui et al.,

S. Sinha et al. / Chemosphere 67 (2007) 176–187 Table 6 Levels of MDA content (lmol g1 fw) in the leaves of S. oleracea grown on different substrate types Substrate types (%)

GS 10 25 35 50 100 CS

Exposure periods (d) 60

75

90

4.91 ± 0.15 3.80 ± 0.24* 3.07 ± 0.16* 2.97 ± 0.08* 2.52 ± 0.07* 2.35 ± 0.07* 4.76 ± 0.17

4.97 ± 0.25 4.15 ± 0.35* 3.77 ± 0.06* 5.94 ± 0.5* 7.92 ± 2.64* 10.49 ± 2.30* 5.00 ± 0.81

5.01 ± 0.47 5.86 ± 0.54* 8.27 ± 0.53* 9.41 ± 0.28* 12.12 ± 0.51* 14.10 ± 1.50* 5.11 ± 0.59

All the values are means 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.

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 conclusions showing an increase in the level of MDA content in the plants grown on treated tannery sludge amendments due to the presence of redox metals. 3.8. Antioxidants A coordinated increase was observed in all the studied antioxidant parameters. At 60 and 75 d of growth, cysteine, non-protein thiol, ascorbic acid and proline content

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in the leaves increased as compared to GS, however, increased significantly (p < 0.05) at higher amendments (Fig. 4A–D). At 60 d, cysteine, non-protein thiol, ascorbic acid and proline contents in the leaves increased with increase in TS amendments with maximum increase of 233.31%, 336.80%, 71.48% and 396.16%, respectively in the plants grown at 100% TS. As compared to GS, these antioxidants increased at all the amendments after 75 d of growth. At 90 d of growth, cysteine and non-protein thiol (Fig. 4A and B) contents responded in similar pattern showing significant increase in the leaves of the plants grown up to 25% TS with maximum increase of 142.84% and 128.65%, respectively. However, the level of ascorbic acid and proline increased significantly in the leaves of the plants grown on 10% TS followed by decrease. Overall analysis of the results revealed that, the non-protein thiol and cysteine contents have shown increase in the leaves of the plants grown on 100% TS as compared to GS. Whereas, proline and ascorbic acid contents increased in the leaves of the plants grown on 50% TS and 25% TS, respectively after 90 d of growth followed by decrease. The plants grown on contaminated soil have shown significant 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-protein thiol, ascorbic acid and proline contents, respectively was recorded as compared to the plants grown on GS (Fig. 4A–D).

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), ascorbic acid (lg g1 fw) (C), free proline (lmol g1 fw), (D) contents in the plants of S. oleracea grown 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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Table 7 Correlation factors (r2) between metals and various parameters in the leaves of in S. oleracea after 90 d of growth Parameters

Fe

Cr

Mn

Zn

Cu

Shoot length Root length No. of leaves Leaf area Total chl Carotenoid MDA Cysteine Proline Non-protein thiol Ascorbic acid

0.21 0.93 0.49 0.11 0.78 0.65 0.70 0.79 0.72 0.75 0.61

0.90 0.14 0.06 0.81 0.41 0.61 0.62 0.55 0.42 0.71 0.31

0.34 0.24 0.77 0.03 0.49 0.11 0.40 0.37 0.56 0.07 0.73

0.03 0.34 0.86 0.14 0.68 0.30 0.66 0.07 0.76 0.35 0.84

0.39 0.06 0.06 0.07 0.04 0.04 0.04 0.60 0.19 0.54 0.08

As it is evident from r2 values presented in Table 7 that the accumulation of metals has shown positive correlation with MDA content after 90 d of growth in the leaves of the plants with all the metals except Fe. However, the accumulation of metals has shown negative correlation with all the growth parameters and antioxidants except Fe. There is a positive correlation between Fe accumulation and antioxidants in 90 d old plants. This is due to decreasing pattern of both Fe and cysteine in the plant, as it has been discussed earlier in this paper that Fe content in TS is lower than that in GS, therefore, with increasing proportion of TS in the substrate, Fe concentration is expected to fall. This could be due to the consumption of all the antioxidants, against providing defense mechanism to the plants, posed by oxidative 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 antioxidant was expected to fall. The mechanism of metal detoxification adopted by the plants to scavenge free radicals and peroxides include several antioxidant substances. These non-enzymatic cellular entities include cysteine, non-protein thiol, ascorbic acid, carotenoid etc. which play an important role in inducing resistance to metals by protecting macromolecules against free radicals which are formed during various metabolic reactions leading to oxidative stress. In the present study, the enhanced level of cysteine and non-protein thiol suggests, its active participation in detoxification of toxic oxygen species. The stressed leaves of the plants showed increase in the level of antioxidants in varying degree and provide endogenous protection effects. Higher level of antioxidants at 60 d of growth might be related with low levels of MDA which may be due to efficient defense mechanism against the stress-generated by metals. High thiol content might enable metabolites to function in the detoxification of reactive oxygen species, which are detoxified by oxidation of sulfhydryl moieties to disulphides under metal stress. Ascorbic acid plays an important role in a-tocopherol regeneration 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 proline enhanced in response to toxic metal exposures which might be attributed to the strategies adapted to cope up with toxicity as main function of metal-induced proline accumulation may be associated with osmoregulation, and enzyme protection against dehydration rather than metal sequestration (Sharma et al., 1998). During the present study, an increase in ascorbic acid and proline contents in the leaves of S. oleracea at initial period of exposure may be considered as defense strategy to combat metal stress in the plant grown on tannery waste contaminated soil as reported earlier (Singh et al., 2004b; Singh and Sinha, 2005). 4. Conclusion In conclusion, the morphological parameters of the plants increased up to 50% TS showing that the tannery sludge supports the growth of the mature plants at lower amendments of sludge. Further, SEM micrograph revealed that increased amendments of tannery sludge led to closure of stomata, increase in their frequency and degeneration. A coordinated increase in antioxidants was noted with increase in metal concentrations in the leaves of the plants at initial period of growth. This indicates that the plants have a detoxification mechanism to cope with such a high concentrations of metals and no effect on MDA content. However, at higher amendment in mature plants, it experienced stress conditions due to increase in MDA content with increase in sludge amendments and the antioxidant system of the plant was not sufficient to revert the stress of a prolonged period of metal exposition. The plants grown on contaminated soil are able to combat stress involving defense mechanism, resulting in healthy growth of the plants as evidenced by the increase in growth parameters photosynthetic pigments and antioxidants. The results are well coordinated as there is no change in the MDA content as compared to the plants grown on GS due to increase in the level of all the antioxidants studied. Summing up it is recommended that the emphasis should be given to the level of metals in the edible part of the plants instead of healthy growth as metal enriched plants have detoxification mechanism and grow well on organic matter enriched contaminated soil. In view of high Cr accumulation in edible part of S. oleracea grown on contaminated soil after irrigation with tap water, it is not advisable to use these plants for edible purposes; however, these plants have shown healthy growth. Thus, it can be concluded that the contaminated soil which is receiving treated tannery wastewater should not be used for the cultivation of leafy vegetables on the basis of these studies. Acknowledgements We thank the Director, National Botanical Research Institute, Lucknow (India), for providing required research

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