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Feasibility Study of Phragmites karka and Christella dentata Grown in West Bengal as Arsenic Accumulator a

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Anshita Raj , Sarah Jamil , Pankaj Kumar Srivastava , Rudra Deo Tripathi , Yogesh Kumar b

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Sharma & Nandita Singh a

CSIR-National Botanical Research Institute, Council of Scientific and Industrial Research, Lucknow, India b

Department of Botany, University of Lucknow, Lucknow, India Accepted author version posted online: 01 Dec 2014.

Click for updates To cite this article: Anshita Raj, Sarah Jamil, Pankaj Kumar Srivastava, Rudra Deo Tripathi, Yogesh Kumar Sharma & Nandita Singh (2015) Feasibility Study of Phragmites karka and Christella dentata Grown in West Bengal as Arsenic Accumulator, International Journal of Phytoremediation, 17:9, 869-878, DOI: 10.1080/15226514.2014.964845 To link to this article: http://dx.doi.org/10.1080/15226514.2014.964845

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International Journal of Phytoremediation, 17: 869–878, 2015 C Taylor & Francis Group, LLC Copyright  ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2014.964845

Feasibility Study of Phragmites karka and Christella dentata Grown in West Bengal as Arsenic Accumulator ANSHITA RAJ1, SARAH JAMIL1, PANKAJ KUMAR SRIVASTAVA1, RUDRA DEO TRIPATHI1, YOGESH KUMAR SHARMA2, and NANDITA SINGH1 1

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CSIR-National Botanical Research Institute, Council of Scientific and Industrial Research, Lucknow, India Department of Botany, University of Lucknow, Lucknow, India

A survey was undertaken, in arsenic (As) contaminated area of the Nadia district, West Bengal, India, to find native As accumulator plants. As was determined both in soil and plant parts. The results showed that the mean translocation factor of Pteris vittata L, Phragmites karka (Cav.) Trin. Ex. Steud and Christella dentata Forssk were higher than 1. It thus appeared that these plants can be efficient accumulators of As. Phytoremediation ability of C. dentata and P. karka was evaluated and compared with known As-hyperaccumulators -P. vittata and Adiantum capillus veneris L. Plants were grown in the As spiked soil (25, 50, 75 and 100 mg kg−1). As accumulation was found to be highest in P. vittata, 117.18 mg kg−1 in leaf at 100 mg kg−1 As treatment, followed by A. capillus veneris, P. karka and C. dentata being 74, 83.87 and 40.36 mg kg−1, respectively. Lipid peroxidation increased after As exposure in all plants. However, the antioxidant enzyme activity and molecules concentration also increased which helped the plants to overcome As-induced oxidative stress. The study indicates that P. karka and C. dentata could be considered as As-accumulators and find application for As-phytoextraction in field conditions. Keywords: arsenic, Pteris vittata, Adiantum capillus veneris, Phragmites karka, Christella dentata

Introduction Arsenic (As) a non-essential element for plants and animals occurs naturally in the environment through anthropogenic and geological activities (Zhao et al. 2010). Millions of people have suffered from As poisoning from consumption of As contaminated ground water and foods all over the world, especially in the South East Asia (Zhu et al. 2008; Pal et al. 2009). Phytoremediation, a plant based green technology, has received increasing attention as an effective, ecofriendly cleanup option for As-contaminated soil and water (Kertulis –Tartar et al. 2006; Yong et al. 2010). The evolution of physiological and molecular mechanisms of phytoremediation, together with recently developed biological and engineering strategies, has helped to improve the performance of both heavy metal phytoextraction and phytostabilization (Kramer 2005). The amount of As translocated from roots to shoots indicates the phytoextraction efficiency of that plant. Few plants have this ability. However, Chinese brake fern (Pteris vittata L.) has shown to accumulate and translocate As from roots to shoots (Ma et al. 2001). Many plants have been reported as As hy-

Address correspondence to Dr. Nandita Singh, Scientist & Group Leader, Eco-auditing Group, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India. E-mail: [email protected]

peraccumulators. Xie et al. (2009) have listed ferns as naturally evolved As accumulators, and the majority of them are members of the Pteris genus. Beside these, efforts have led to the identification of other potential As-accumulator aquatic and terrestrial plants (Singh et al. 2010; Ozturk et al. 2010; Tripathi et al. 2012). However, there is a need to screen more plants to find the best suited option for a particular area. The use of indigenous plants with high tolerance and accumulation capacity for As could be a very convenient approach to Asphytoremediation. From phytoremediation perspective, plants should display, (i) high uptake rate; (ii) tolerance to high concentration of As; (iii) high translocation to shoot system; (iv) efficient system to tolerate high As level in plant parts (Singh et al. 2006, 2010; Tripathi et al. 2007). Besides exploring for new phytoremediators for As, studying the mechanism of tolerance and detoxification is also important. It is well documented that plant exposure to AsV or AsIII induces production of reactive oxygen species (ROS) such as superoxide (O2 ·−) hydroxyradical (·OH) and H2 O2 (Finnegan and Chen 2012). ROS can damage proteins amino acids, nucleotides and induce peroxidation of membrane lipids (Finnegan and Chen 2012). If ROS support does not get controlled then oxidative chain reaction will start. Plants usually respond to this stress by increasing activities of antioxidant enzymes, like superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione-s- transferase. Besides the antioxidative

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enzymes, plants possess antioxidant molecules for defence like ascorbate and glutathione. The altered activity of these antioxidant enzymes and molecules in plants due to As- stress has been observed during investigating for As- hyperaccumulators (Kertulis-Tartar et al. 2009; Singh et al. 2010; Lyubenova and Schroder 2011; Srivastava et al. 2011). The present study aims (i) to study the plants grown in As contaminated sites to identify As resistant plants; (ii) to grow the selected As resistant plants in As spiked soil under greenhouse conditions and compare their accumulation and defence abilities with known As–hyperaccumulators. (iii) to explore the defence mechanism in the identified plants, with respect to antioxidative enzymes and molecules which provide them efficient tolerance against As-treatment.

Materials and Methods Field Study Study area The Birnagar village (latitude 72◦ 51 E; longitude 22◦ 41 N) Nadia District of West Bengal, India is highly contaminated with As. Three sites, covering around 10 ha, were chosen for the survey. In this area, the As level in groundwater ranges from 0.16 to 0.4 mg l−1, which exceeds the WHO permissible water limit (0.01 mg l−1) (WHO 1992) and As concentration in soil is 15 mg kg−1 (Dwivedi et al. 2010).. Extensive survey was undertaken to collect some ferns and other wetland plants which are growing well at all the three sites. The samples of plants and soils were collected and brought to the laboratory for identification and analysis. Plant identification and analysis Four ferns and five wetland plants were collected and kept in plastic bags. Herbarium sheets of unidentified plants were prepared and brought to lab for identification using the taxonomic keys given by Duthie (1960). The As concentration in plant parts was estimated following the method given in Section 2.2.3. Greenhouse Experiment The rationale of the experimental set-up was to compare As accumulation and tolerance of field identified As tolerant plantsPhragmites karka (Cav.) Trin. Ex. Steud and Christella dentata Forssk with known As-hyperaccumulators – Pteris vittata L. and Adiantum capillus veneris L. exposed to a range of varying As concentrations, higher to generally present in the As-contaminated sites of West Bengal (India). For the experiment around four months old plants of P. vittata, C. dentata, A. capillus veneris and P. karka (two months old) were taken from NBRI fernery and nursery. Experimental setup The soil used in the study was classified as sandy loam and was collected from the CSIR-NBRI garden. The soil was devoid of As. Other soil characteristics are given in Table 1.

A. Raj et al. Table 1. Physico-chemical properties of As contaminated soil from different sites of Nadia District, West Bengal Site 1 pH EC μS cm−1 TOC (%) Available N (%) Available P (%) WHC (%) MBC (μg g−1) Texture Total As (mg kg−1)

Site 2

Site 3

7.24 ± 0.14 7.63 ± 0.10 6.77 ± 0.66 63.87 ± 0.75 54.67 ± 1.24 127.37 ± 24.26 2.14 ± 0.16 2.75 ± 0.46 1.47 ± 0.26 0.05 ± 0.01 0.04 ± 0.02 0.02 ± 0.0 71.95 ± 12.36 71.75 ± 20.42 86.42 ± 14.34 86.42 ± 20.34 85.26 ± 12.34 84.27 ± 18.4 636.6 ± 85.09 411.99 ± 88.85 524.27 ± 26.42 Clayloam Clayloam Clayloam 15.54 ± 1.32 12.59 ± 1.827 10.43 ± 1.536

Pots (20 cm dia.) were taken and filled with 2.5 kg of soil. As using Sodium arsenate was mixed with the soil to give 25, 50, 75 and 100 mg As kg−1 soil per pot. Garden soil, without As amendment, was taken as the control. Plants were raised under greenhouse conditions during the experiment with the ambient temperature ranging between 30–40◦ C and a natural light regime. The soil was kept at a constant water holding capacity (WHC) of 30% by maintaining a water supply in the pots throughout the experiment, and care was taken to avoid the contact of shoots with the soil. The As-exposed pots were randomized with changing the pots position at weekly interval. Plants were harvested six month after plantation. At harvest, plants were removed from the pots and rhizome and roots were cleaned to remove the adhering soil. The plants were analysed for lipid peroxidation, antioxidant enzymes and molecules to assess responses to As exposure. On harvesting, portion of plant samples were frozen in liquid nitrogen and stored at –80◦ C for enzyme analysis and biochemical parameters and rest plant samples were dried at 65◦ C in oven for 48 h for dry biomass and As estimation. Soil samples were collected, air dried and sieved (1mm mesh) for further analyses. Physico-chemical properties of the soil The Garden soil (GS) was collected from NBRI, Lucknow (India). After 5 days of incubation, physico-chemical analysis of treated garden soil was done by procedures described by Kalra and Maynard (1991). All the chemicals used were of analytical reagent grade. As estimation in soil and plant tissue After harvesting, plants parts were separated and washed extensively with distilled water. The roots were kept in EDTA (20 mM) solution for 15 min to remove the adhering metal on the root surface. Samples were oven dried at 75◦ C till constant weight was achieved and powdered. The powdered soil and plant material (0.1 g DW) were digested in BURGHOFspeedwave- MSW-3+ microwave digestion unit with 5ml of 60% nitric acid and 1 ml of 40% hydrofluoric acid and filtered through Whatman filter paper no. 44. As in the digested samples was determined by inductively coupled plasma mass spectrometry [ICP-MS] (Agilent-7500 cx).

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Table 2. Screening of native ferns and other plants, from Nadia district, West Bengal, for As resistance Average As Concentration (mg kg−1)

No.

Plant Species Ferns

Family

1 2 3 4

Pteris vittata Amphilopteris punctatum Christella dentata Microsorum punctatum

1 2 3 4 5

Angiosperms Phragmites karka Vetiveria zizinoides Vernonia cineraria Colocasia formasa Typha latifolia

Root

Rhizome

Frond

Pteridaceae Theypteridaceae Theypteridaceae Polypodiaceae

21.47 ± 3.24 24.16 ± 4.28 26.42 ± 5.32 5.33 ± 9.34

34.28 ± 10.26 5.32 ± 12.26 18.75 ± 14.37 2.98 ± 7.14

52.46 ± 18.36 26.24 ± 12.47 31.74 ± 7.82 6.41 ± 8.32

Poaceae Poaceae Asteraceae Colocasceae Typhaceae

Root 17.64 ± 10.27 15.36 ± 2.17 5.24 ± 8.32 2.17 ± 4.72 4.24 ± 5.31

Shoot 16.84 ± 8.17 21.46 ± 8.42 1.42 ± 2.47 0.64 ± 1.27 2.17 ± 4.26

Leaf 26.42 ± 6.71 14.46 ± 4.22 1.06 ± 2.34 0.24 ± 0.16 1.37 ± 4.18

Quality control and quality assurance The standard reference material of As (E-Merck, Germany) was used for each analytical batch. Analytical data quality was ensured with repeated analysis of quality control samples and the results were within 92–98% limit of the certified values. Standard AA03N-3 (Accustandard, USA) was used as a matrix reference material which was spiked with known concentration (0–50 μg L−1 As) of standard reference material, and the recovery of total As were within 85.3 to 89.5%. Phytoextraction ability The phytoextraction ability of plants was assessed using both the translocation factor (TF) and the bioaccumulation factor (BF) as follows: TF = [As](frond/leaf) /[As]root BCF = [As](leaf+root) /[As]soil Determination of lipid peroxidation The level of lipid peroxidation in plant tissues was determined as 2-thiobarbituric acid (TBA) reactive metabolites mainly malondialdehyde (MDA) (Heath and Packer 1968). Plant tissues (0.5 g) were extracted in 2.5 ml of 5% trichloroacetic acid (TCA) and centrifuged at 10,000x g for 15 min. To 1ml of aliquot of the supernatant, 1ml of 0.5% TBA in 20% TCA ◦ were added and incubated at 95 C for 25 min and then quickly cooled on ice. The solutions were centrifuged at 10,000 x g for 5 min and the absorbance was measured at 532 nm, using a UV-VIS Spectrophotometer (Thermo-Helios-ß UVB131312). The value for non-specific turbidity was made by subtracting the absorption value taken at 600 nm from the one measured at 532 nm. The level of lipid peroxidation was expressed as nmol of MDA formed using an extinction coefficient of 155 mM cm−1 at 532 nm. Assay of antioxidant enzymes Superoxide dismutase (SOD) activity was measured with the reduction of nitroblue tetrazolium (NBT) to form formazon (Beyer and Fridovich 1987). The samples were homogenized in 5 ml extraction buffer consisting of 50 mM phosphate, pH

7.5 containing 1mM dithiothreitol (DTT) and 1mM ethylenediamine tetra acetic acid (EDTA), and centrifuged at 20,000 ◦ x g for 15 min at 4 C. The assay mixture contained 50 mM phosphate (pH 7.8), 1% (w/v) Triton X-100, 0.0044% (w/v) riboflavin, 57 μM NBT and 9.9 mM L- methionine. The photoreduction of NBT (formation of purple formazon) was measured at 560 nm. One unit of SOD activity is defined as the amount of enzyme that gave 50% inhibition of NBT reduction in one minute. For the assay of catalase (CAT) activity, 0.5 g of plant samples were homogenized in 5ml extraction solution, containing 50 mM phosphate buffer (pH 7.0) and 1 mM DTT. CAT activity was assayed in 50 mM phosphate buffer (pH 7.0) by monitoring the production of dioxygen from hydrogen peroxide (1%) at 240 nm (del Rio et al. 1977). Glutathione Reductase (GR) was assayed from 0.5 g plant tissues extracted in 0.1M phosphate buffer (pH 7.5) containing 0.5 mM EDTA, the extract was centrifuged at 20,000 x g for 15 min at 4◦ C and GR activity was monitored by following the increase in absorbance at 412 nm, when DTNB (5,5 - dithiobis2-nitrobenzoic acid) was reduced by glutathione (GSH) to form TNB (5-thio-2-nitrobenzoic acid) (Smith et al. 1988). The reaction mixture was 0.2M phosphate buffer (pH 7.5) containing 1 mM EDTA, 3 mM DTNB in 0.01 M phosphate buffer (pH 7.5), 2 mM NADPH and 20 mM GSSG (oxidised glutathione). Glutathione-S-Transferase (GST) activity was determined with 1 g plant material extracted in 5 ml extraction solution consisting of 50 mM phosphate buffer (pH 7.5), 1 mM EDTA and 1 mM DTT. The enzyme activity was assayed in a reaction mixture containing 50 mM phosphate buffer (pH 7.5), 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and extract equivalent to 100 μg of protein. The reaction was initiated by the addition of 1 mM GSH and formation of S-(2,4-dinitrophenyl) glutathione (DNP-GS) was monitored as an increase in absorbance at 334 nm to calculate the GST specific activity (Li et al. 1995). Ascorbate peroxidase (APX) activity was determined with 0.5 g plant samples extracted with 2.5 ml of 100 mM phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 0.1 mM ascorbate and 2% (v/v) β- mercaptoethanol. The rate of

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hydrogen peroxide-dependent oxidation of ascorbic acid was determined in a reaction mixture that contained 50 mM phosphate buffer (pH 7.0), 0.6 mM ascorbic acid and enzyme extract (Chen and Asada 1989). The reaction was initiated by addition of 10% (v/v) H2 O2 and the oxidation rate of ascorbic acid was estimated by following the decrease in absorbance at 290 nm for 3 min.

Test (DMRT). A two-tailed (α = 2) probability p < 0.05 was considered statistically significant.

Glutathione determination Total glutathione level was monitored by extracting plant tissues (1 g) homogenised in 5 ml of ice-cold 6% (w/v) mphosphoric acid (pH 2.8) containing 1 mM EDTA. The homogenate was centrifuged at 22,000 x g for 15 min. Total glutathione was measured in a reaction mixture consisting of 400 ml of solution A [110 mM Na2 PO4 .7H2 O, 40 mM NaH2 PO4 . H2 O, 15 mM EDTA, 0.3 mM 5,5 -dithiobis-(2nitrobenzoic acid)(DTNB) and 0.4 ml l−1 BSA], 320 ml of solution B (1 mM EDTA, 50 mM imidazole, 0.2 ml l −1 BSA and an equivalent of 1.5 units of glutathione reductase), and 400 ml of a 1:50 dilution of the extract in 5% (w/v) Na2 HPO4 (pH 7.5) and 80 ml of 3 mM NADPH. The reaction rate was measured spectro photometrically by following the change in absorbance at 412 nm for 4 min. For oxidized glutathione (GSSG), 1 ml of the 1:50 extract dilution was initially incubated with 40 ml of 2 vinylpyridine at 25◦ C for 1 h and assayed as described above. A standard curve was developed by preparing solutions of 0.5–16 mM reduced glutathione (GSH) and analyzing them in the same manner as the extracts. GSH was estimated as the difference between total glutathione and GSSG (Gossett et al. 1994).

As content of the soil samples taken from the study area, of Nadia District, were found to be on average 13.1 ± 2.1 mg kg−1 (Table 1). The soil type was clay loam with pH ranging between 6.7 to 7.6. Other soil properties as given in Table 1 show similarity within the sites. As concentration in the different parts of sampled plants are given in Table 2. Mean As value in the root, rhizome and frond for P. vittata were 21.4, 34.2 and 52.4 mg kg−1, respectively. P. vittata accumulated highest concentration of As in its plant parts (root and shoot) at all the study sites located at Birnagar village, Nadia district, West Bengal, India. In all plants, the As concentrations in the plants were higher than the normal As concentrations reported in plants from this area (Tripathi et al. 2012). The As concentrations in leaves/fronds of P. vittata, C. dentata and P. karka were invariably higher than that in root (Translocation factor >1). This indicates a special ability of the plant to absorb and transport metals from soil and to store these in their above ground parts (Zhao et al. 2010). The results show that C. dentata and P. karka have the ability to uptake high concentration of As and accumulate in the frond/leaf. Further as these plants were growing well in the As contaminated soil they are supposed to have the ability to detoxify As efficiently at least up to 15.5 ±1.3 mg kg−1. To confirm whether these plants may be classified as Ashyperaccumulator, experiments were undertaken in controlled greenhouse conditions using As spiked soil and comparing with known As-hyperaccumulators – P. vittata and A. capillus veneris, under same experiments.

Ascorbate determination Reduced ascorbate (AA), dehydroascorbate (DHA) and total ascorbate AsC (AA+ DHA) were determined by following the method of Hodges et al. (1969). About 200 mg of plant sample was extracted in 2 ml of 10% Trichloro acetic acid and 50 μM phosphate buffer (pH 7.4) containing 3 mM EDTA and 1 mM DTT. For the assay of reduced ascorbate, distilled water was used instead of DTT (dissolved in phosphate buffer pH 7.4). The extract was centrifuged at 14,000 x g for 15 min. The aliquot was incubated at 25◦ C for 10 min. In 2 ml of this incubated aliquot, the color was developed with the addition of N-ethyl malemide (NEM) 0.5% (w/v) in ethanol, 0.61 M TCA, 0.8 M H3 PO4 , 65 mM α- α bipyridyl and 110 mM ferric chloride. The reaction mixtures were incubated in a water bath at 55◦ C for 10 min OD taken at 525 nm, using UV-VIS Spectrophotometer (Thermo-Helios-ß UVB-131312). A standard curve was developed by preparing solutions of ascorbate, 1–50 μmol dissolved in 10% TCA, and analyzing them in the same manner as the extracts. DHA was estimated as the difference between total ascorbate and AA. Statistical Analysis Data were summarized as Mean ± SD. Groups were compared by two factor (treatments and plants) analysis of variance (ANOVA) and the significance of mean difference within and between the groups was done by Duncan Multiple Range

Results Field Study

Greenhouse Experiment Pot experiment was performed to compare the As tolerance of P. karka and C. dentata in comparison with known As hyperacumulators - P. vittata and A. capillus veneris. Although, As is not an essential nutrient for plants, but in all the plants, even after its exposure up to 100 mg kg−1 As for 6 months there was no visual toxicity symptoms. It might be possible that all the plant species under the study, might have evolved an efficient defence system to mitigate As-induced oxidative stress, as reported in P. vittata and A. capillus veneris (Singh et al. 2010). Interestingly, the biomass of all plants increased with the increasing As in soil, in comparison to the control (Fig. 1A) . As accumulation To evaluate the As accumulation capacity of the studied plants, the As concentration was measured in roots and shoot (leaf/frond) of individual plants growing in As-spiked soil. As accumulation was concentration-dependent. The As content in the plant parts significantly increased with its concentration

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Fig. 2. Content of MDA (μmol g−1 fwt) in the frond/leaf and root of plants when grown in As spiked soil. Vertical bars indicate means ± S.D. of three replicates. Means followed by same letter were not significantly different at p < 0.05 within the species, in same category, according to DMRT.

(p < 0.05) from As accumulation in root. The ability to translocate As from roots to shoots was also high in case of P. vittata. However, P. karka also showed high translocation factor quite comparable with A. capillus veneris (Fig. 1C). In case of C. dentata the TF is lowest, but >1. The metal accumulation efficiency in plants can be evaluated using the bio-concentration factor (BCF), which is defined as the ratio of metal concentration in the plant biomass to metal concentration in the soil. The BCF of all the plant species was > 1 as depicted in figure (Fig. 1D). In case of C. dentata BCF was lowest at 100 mg kg−1.

Fig. 1. (A) Dry Biomass (mg g−1), (B) As concentration in plant parts, (C) Translocation factor (root to frond/leaf) (D) Bioconcentration Factor (As leaf + root/ soil) of plant species when grown in As spiked soil. Vertical bars indicate means ± S.D. of three replicates. Means followed by same letter were not significantly different at p < 0.05 within the species, in same category, according to DMRT.

in soil (p < 0.01). Out of the four tested species, P. vittata had the highest level of As both in roots and fronds (Fig. 1B). This was followed by A. capillus veneris, P. karka. C. dentata accumulated least in fronds which was not significantly different

Oxidative stress Oxidative stress occurred in the As exposed plants. The level of MDA, the final decomposition product of lipid peroxidation, in the frond/leaf and root increased in plants growing in Asspiked soil (Fig. 2). The increment varied between the plants but it was not dose-dependent. The rate of lipid peroxidation was higher in the frond/leaf in comparison to roots. Further, the increment was highest in case of C. dentata, where the per cent increase in MDA content of frond was between 81.91and 87.84, compared with the control (Fig. 2). SOD activity which is responsible for the scavenging of oxygen species increased due to As-treatment. The SOD activity of the frond/leaf and root tissues in all the plants increased significantly upon exposure of the plants to different As concentrations (Fig. 3A). In the case of frond/leaf, the increase was in the range of 16 to 46% in case of P.vittata, 8 to 11% in A. capillus veneris, 32 to 79% in P. karka and 13 to 24% in C. dentata. Similarly, CAT activity also showed 3–4 fold increase in As-exposed plants both in frond/leaf and root (Fig. 3B), in comparison to the control. This increment was prominently higher in the roots of P.vittata and P. karka as compared to the other two species. The APX activity also showed remarkable increase upon As-exposure in all species (Fig. 3C). In P. karka, the APX was significantly higher (p < 0.01) than that of the As hyperaccumulor P. vittata both in leaves and roots. Similar results were obtained in A. capillus veneris. However, in the case of

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significantly according to the As-dose but the GSH concentration was always much higher maintaining a high GSH/GSSG ratio in all the plant tissues. The activity of GR was significantly (p < 0.05) higher in the roots of P. vittata and P. karka compared to the fronds/leaves tissue (Fig. 4E). Although in C. dentata, the GR activity in fronds and roots did not show any significant difference compared to control. The GR activity showed slight increase with increasing As-exposure, but the change was not significant except in P. vittata. The GST activity in all plant species and in all tissues (root and frond/leaf) increased significantly (p < 0.01) with the As dose (Fig. 4F) and As levels in plant tissues. As in soil Figure 5A shows the residual As upon harvesting of plants, after 6 months. The concentration of As decreased in the soil of all plants corresponding to the As-spiked in soil. Significant differences (p > 0.05) were observed in the As concentration in soils between the initial and final contents, before plantation and after harvesting, respectively. As expected, the highest per cent depletion was in the case of P. vittata which removed around 40–60% of soil As, followed by A. capillus veneris and P. karka where the removal was between 25–50%. Although, the least amount was depleted (17–28%) in case of C. dentata (Fig. 5B) .

Discussion

Fig. 3. Activities of antioxidant enzymes, A. SOD, B. CAT and C. APX, in the frond/leaf and root of plants when grown in As spiked soil. Vertical bars indicate means ± S.D. of three replicates. Means followed by same letter were not significantly different at p < 0.05 within a species according to DMRT.

C. dentata, a significant increase (p < 0.01) in the activity of APX was found in the root tissue of As – exposed, but not to that extent in the fronds. Figure 4A shows a significant increase (p < 0.01) of AA in the frond/leaf and root of As exposed plants, in comparison to control. Whereas, the DHA showed variation in different plants (Fig. 4B). In P. vittata the DHA decreased both in leaf and root with the increasing As-exposure. Its reduction was more in the roots. In case of A. capillus veneris and C. dentata, the AA and DHA were significantly higher (p < 0.05) in the roots than the fronds. As exposure increased the DHA significantly (p < 0.01) in A. capillus veneris and C. dentata, but there was no effect due to the variation in As concentrations. In P. karka, the AA concentration was constantly higher than DHA in both leaves and roots. The GSH content (Fig 4C) in all plants increased with increasing As concentration in soil and was found to be highest in P. karka. Although GSSG (Fig 4D) also increased

Since phytoremediation is being recognized as a very efficient, inexpensive and environment friendly alternative to the traditional technologies for remediation of contaminated soil, the search for new suitable indigenous species and study their unique metabolism have gained momentum. The plants (P. karka and C. dentata) characterized as promising for phytoremediation in the field study showed the same response when grown in As spiked soil. The pattern of As accumulation was same. When growing in contaminated soil of West Bengal, P. karka accumulated high concentration of As in leaf compared to root, whereas, C. dentata accumulated high As equally both in root and fronds. Similar results were observed when these plants were grown in As spiked soil. In this study, the P. karka and C. dentata exhibited the characteristics of an As-hyperaccumulator, i.e. they were tolerant to high concentration of As as P. vittata and A. capillus veneris. In P. vittata and A. capillus veneris, along with some other ferns of family Pteridaceae, it is reported that As is beneficial (Ma et al. 2001; Singh et al. 2010). However, fern C. dentata, belonging to family Thelypteridaceae, can be considered as new report as As hyperaccumulator. Whereas, Phragmites sp. has been considered to be resistant to As (Ghassemzadeh et al. 2008; Rahman and Hasegawa 2011), but increase in biomass on As exposure has not been reported earlier. The translocation factor (TF) of plant species helps to classify into As hyperaccumulator, accumulator and excluders (Vithange et al. 2012). P. karka showed a substantial accumulation of As in leaves, whereas C. dentata showed equal

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Native Ferns as Arsenic Accumulator

Fig. 4. Changes in the ascorbate concentration, A. Reduced (AA); B. Oxidised (DHA); and glutathione pools, C. Reduced (GSH); D. Oxidised (GSSG); E. Glutathione reductase activity (GR) and F. Glutathione-S-Transferase activity (GST), in the frond/leaf and root of plants when grown in As spiked soil. Vertical bars indicate means ± S.D. of three replicates. Means followed by same letter were not significantly different at p < 0.05 within the species, in same category, according to DMRT.

accumulation in root and frond. Both the plants accumulated As to high concentration with shoot (frond/leaf) to root quotient of metal concentration (TF) greater than one. Although As has not been shown to be an essential plant nutrient, but no visual symptom of toxicity were observed in all the four plants after As exposure up to 100 mg kg−1 for 6 months. This may be due to the active defence system in plants. Though BCF all the plant species was >1, except C. dentata at 100 mg kg−1 but it was able to combat As toxicity by elevating its defence system. The level of lipid peroxidation that was determined by malondialdehyde (MDA) content showed to be concentration

dependent. Generally, significant differences were observed between the control and As spiked plants (Fig. 2). The rate of lipid peroxidation was highest in the fronds of C. dentata. Although each plant species might have a unique mechanism against metals, their biochemical responses are complex and several defence strategies including antioxidative responses have been suggested. The As toxicity was minimized by the antioxidative enzymes (SOD, APX and CAT) and the nonenzymatic antioxidants which include the major cellular redox buffers like ascorbate and glutathione (Apel and Hirt 2004). The super oxide radicals, produced due to As-treatment, are immediately dismuted by SOD and resulting H2 O2 is

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Fig. 5. As in soil after 6 months of plant growth. A. As concentration (mg kg−1), B. As percentage reduction in soil. Vertical bars indicate means ± S.D. of three replicates. Means followed by same letter were not significantly different at p < 0.05 within the species, in same category, according to DMRT.

scavenged by APX and CAT (Pandey et al. 2010). A significant increase in the levels of SOD in all the plants was found. However, the highest level of SOD generation was observed in the fronds and root of A. capillus veneris (Fig. 3A), followed by P. vittata, C. dentata and P. karka. Similar increase in SOD activity in response to heavy metal stress has been reported earlier (Singh et al. 2006, 2010; Diwan et al. 2007). In the present study, CAT showed active participation in the H2 O2 reduction as its activity increased with dose in all the plants. CAT activity was significantly higher in P. vittata and P. karka as compared to A. capillus veneris and C. dentata. The high CAT activity mediated the removal of H2 O2 and toxic peroxides in these plants, which helped them to survive even at high As in experimental soils (Kertulis-Tartar et al. 2009; Singh et al. 2010). Beside CAT, the greater activity of H2 O2

removing enzyme ascorbic peroxidase (APX), especially in chloroplast, can further detoxify H2 O2 . Upregulation of APX activity has been reported in rice seedlings (Shri et al. 2009) and mung bean (Singh et al. 2007), exposed to As. In parallel, the non-enzymatic antioxidants such as ascorbate (AA) and glutathione (GSH) also increased in As-treated plants, which are comparable to earlier observations on P. vittata, A. capillus verneris (Singh et al. 2006, 2010), rice (Shri et al. 2009), mung bean (Malik et al. 2012) plants treated with As. In all experimental plants the AA and GSH concentration increased both in root and frond/leaf with increasing As in soil. The ROS produced during As treatment typically induces an increase in the oxidation state of the redox pools of GSH and ascorbate (DHA) over the more reduced GSH and hydroascorbate (AA) (Singh et al. 2006). In our

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Native Ferns as Arsenic Accumulator experiments, the rise in GSSG (oxidised GSH) was also dependent on the As-treatment dose but within the plants the concentration was minimum in the P. karka root and leaf. Whereas, DHA (Oxidised AA) increased in A. capillus veneris, C. dentata and P. karka on As exposure however, the increase was independent of As dose. The glutathione reductase (GR) activity also increased upon As exposure. This enzyme (GR) efficiently recycles oxidized GSH to allow further cycles of H2 O2 reduction. The enzymes involve in the recycling of oxidised GSH and ascorbate have been found to increase upon exposure of plants to As (Ahsan et al. 2008; Khan et al. 2009, Raj et al. 2011). Thus, the inter-dependent ascorbateGSH cycle, when it can be established, has an important role in maintaining ROS balance in plants (Foyer and Shigeoka 2011). Like SOD and CAT, glutathione-S-transferase (GST) activity also increased in all the plants growing in As spiked soil. It has been observed that As induces the GST activity in mesquite, maize and rice (Mokgalaka-Matlala et al. 2009; Chakrabarty et al. 2009; Ahsan et al. 2008). The study shows that, despite accumulation of As in plants parts and induction of MDA formation in all plants subjected to As-exposure, there was no apparent phytotoxicity in any part of all the plants. This indicates the ameliorative role of oxidative stress enzymes and molecules which are induced in As-resistant plants. P. vittata and A. capillus veneris are known As-hyperaccumulators (Ma et al. 2001; Singh et al. 2010) and P. karka and C. dentata have also shown the property of hyperaccumulator, in this study. Phytoremediation Ability of the Plants As shown in Fig. 5B the soil As decreased after growing these plants for 6 months. In situ study conducted by Kertulis-Tartar et al. (2006) to determine the efficiency of P. vittata for removal of As from a CCA site, showed a reduction of approximately 19.3 g from the soil in two years. Whereas, Gonzaga et al. (2008) observed 6.4–13% reduction of As from soil by P. vittata in a greenhouse experiment. The present study of six months showed that P. vittata reduced 40–60% As depending on the As spiked in soil. Other plants A. capillus veneris, P. karka and C.dentata also removed 20–50% As from the soil but the removal was more in case of lower As spiked soil. This shows that all for plants have As phytoremediation capability especially C. dentata and P. karka appears promising As hyperaccumulators.

Conclusions The present study shows that there was no As toxicity observed in P. karka and C. dentata. There is seemingly high degree of adaptive strategy, which provided C. dentata and P. karka to have the abilities to grow in As contaminated sites. The study suggests that As accumulation and translocation to shoot are well developed in both the plants, making them capable for As hyperaccumulation. In addition, the higher antioxidative enzyme activities and elevated ascorbate and GSH

877 levels, counteracted each other in these plants and helped them to grow in high As contaminated soils.

Acknowledgments The authors are grateful to the Director, CSIR-NBRI for keen interest and continuous encouragement to conduct the experiments. AR thankfully acknowledges CSIR for awarding SRF grant.

Funding This work was carried out under the network project (NWP19 and INDEPTH) and supported by the Council of Scientific and Industrial Research, Govt. of India.

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Feasibility Study of Phragmites karka and Christella dentata Grown in West Bengal as Arsenic Accumulator.

A survey was undertaken, in arsenic (As) contaminated area of the Nadia district, West Bengal, India, to find native As accumulator plants. As was det...
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