Phytoremediation of hexavalent chromium by Triticum aestivum L.

Scientia Agriculturae www.pscipub.com/SA E-ISSN: 2310-953X / P-ISSN: 2311-0228 DOI: 10.15192/PSCP.SA.2015.9.1.1622

Sci. Agri. 9 (1), 2015: 16-22 © PSCI Publications

Phytoremediation of hexavalent chromium by Triticum aestivum L. A K Nayak1*, R C Jena1, S Jena1, R. Bhol2, H. K. Patra1 1. P.G. Department of Botany, Utkal University, Bhubaneswar-751004, Odisha 2. Department of Biotechnology, Rama Devi Women’s (Auto) College, Bhubaneswar-751022, Odisha Corresponding Author: A K Nayak Paper Information

ABSTRACT Chromium is considered a serious environmental pollutant due to Received: 21 October, 2014 its wide industrial use. Contamination of soil and water by chromium (Cr) is of recent concern. Toxicity of Cr to plants Accepted: 16 November, 2015 depends on its valence state: Cr (VI) is highly toxic and mobile whereas Cr(III) is less toxic. Present study deals with Published: 20 January, 2015 phytoremediation of chromium by wheat seedlings and results indicated that there is significant reduction of biomass of the plant with increased dosage of chromium. The study were designed to compare Citation the seed germination and growth parameters of Tritic um aestivum Nayak AK, Jena RC, Jena S, Bhol R, Patra HK. 2015. L on several concentration of hexavalent chromium without Phytoremediation of hexavalent chromium by Triticum chelating agent (5 µm, 10 µm, 100 µm) and chromium with aestivum L. Scientia Agriculturae, 9 (1), 16-22. Retrieved chelating agents like Cr +6 +EDTA, Cr +6 +CA and Cr +6 +Zn +2 at 5 from www.pscipub.com (DOI: µM. Effect of hexavalent chromium with and without chelating 10.15192/PSCP.SA.2015.9.1.1622) agents were assessed in hydroponically grown seedlings. It was noted that the growth parameters like root and shoot length, root and shoot fresh weight, root and shoot dry weight and total chlorophyll content deteriorated inclination with increasing chromium concentrations without chelating agents. However average hexavalent chromium bio-availabilities were increased from shoots to roots with increase hexavalent chromium concentration at 5 µM and 10 µM. When the seedlings were grown with Cr +6 +EDTA,Cr +6 +CA,Cr +6 +Zn +2 at 5 µM Cr +6 +EDTA was demonstrated highest growth rate of root and shoot length, root and shoot fresh weight, root and shoot dry weight, chlorophyll, chromium content of root and shoot among the other chelating agents. Phytoremediation is a low cost and effective soil treatment for bioremediation has gained interest in recent years. © 2015 PSCI Publisher All rights reserved. Key words: Triticum aestivum L., Hytoremediation, Chromium, Chelating agent, Citric acid, EDTA, ZnSO 4

Introduction Heavy metals are toxic pollutants released into the environment by a large number of industrial operations such as electroplating, chromate manufacturing, dyes and pigment manufacturing, wood preservation, leather tanning industry, manufacture of alloys and as corrosion inhibitor in conventional and nuclear power plants. Chromium is the second most common heavy-metal pollutant (Kar et al., 2008; Ogundiran and Afolabi, 2008; Jun et al., 2009). In India, about 2,00032,000 tons of elemental Cr per annum escapes into the environment from tanneries only (Dey et al., 2009). Chromium and its compounds are categorized in Group 1 by the International Agency for research on Cancer i.e., carcinogenic to humans and animals (Yadav et al., 2005). Chromium in trace amount is considered as an essential nutrient for many organisms. It is a micronutrient for humans, for it has role in fat and glucose metabolism and proper functioning of insulin but its importance in plants has not yet been reported. In nature, chromium exists in valence states ranging from -2 to +6 out of which Cr (III) and Cr (VI) are more stable in natural environment. Compound of Cr (III) are 10 to 100 times less toxic than those of Cr (VI) (Ruotolo et al., 2011). It is less mobile, less toxic and is mainly found bound to organic substances in the environment (Becquer et al., 2003; Dey et al., 2009). Hexavalent Cr usually occurs associated with oxygen as chromate (CrO 42-) or dichromate (Cr2O72- ) oxyaniona and is therefore, highly soluble in water. It is highly mobile and considered as the most toxic form of Cr. Hexavalent chromium causes lung cancer, chromate ulcer, perforation of nasal septum and kidney damage in humans, teratogenesis and is also toxic to other organisms (Smith et al., 2002). Cr (VI) has a high tendency to bind with oxygen (Thacker et al., 2006). The chromate ion causes oxidative DNA damage by the production of free radicals. Cr (VI) induced inactivation of mitochondrial electron transport and superoxide generation has been observed in higher plants.

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Reactive oxygen species such as singlet oxygen, hydroxyl ions and hydrogen peroxide in cells cause oxidative stress and hence symptoms of toxicity are observed in whole plant (Shanker et al., 2004). Some crops are not affected by low Cr concentration (3.8 X 104 µM) (Huffman and Allaway, 1973) but Cr is toxic to most higher plants at 100 µM kg -1dry weight (Davies et al., 2002; Shanker et al., 2004). Excess amount of chromium in the plant can inhibit seed germination, cause loss of photosynthetic pigments, reduction in growth, leaves chlorosis, tissue necrosis, decreases enzyme activity, membrane damage, low yield and other physiological anomalies (Bercelo and Poschenrieder, 1997; Shanker et al., 2004; Dey et al., 2009) Chromium remediation is a challenge to the environment. Phytoremediation is a cost effective and safe technology that exploits a plants ability to accumulate a variety of chemical element and transport them from the substrate to above ground parts (Dushenkov et al., 1997). This approach has arisen because plants have a remarkable ability to extract, concentrate and metabolic materials from air, water and soil. The plants respond to soil contaminants in three wages; they can act as contaminant accumulation, indicator, or excluder, based in the way they take up and translocation constituents above the ground biomass (Baker et al., 1981). Accumulators are plants that survive despite concentrating contaminants in their aerial tissue. Indicator plants have a mechanism that controls the translocation of contaminants from the root to this shoots. Excluder restrict contaminant uptake to the biomass. Most applications to date have focused on the remediation of nutrients, trace metals and organics. It is emerging as attractive methods due to its simplicity; relatively low cost and in situ ‘green clean approach’. Remediation of inorganic contaminant involves either physical removal or conversion into biologically inert forms (Cunningham, 1996). To optimize phytoremediation, cheating agents are often used (Huang et al., 1997) Ingestion of plant derived chromium containing foods provides a major share of the daily chromium intake. It is therefore essential to determine the harmfulness effects of free and chelated chromium on toxicological, physiological and biochemical changes during the seedling growth of wheat plants. Wheat is a most essential cereal for the world population including India. Like many plants wheat at seedling germination and seedling stages are sensitive to environmental influences. The current phytoremediation techniques are based on the toxic effects of chromium and have been optimized by the use of chelating agents (Hung et al., 1997; Mohanty and Patra, 2012). Unfortunately, information on wheat using different chelated chromium compounds on plant growth, physiological and biochemical compounds on plant growth is not only inadequate but also there is no report, to date. The present investigation is, therefore, aimed at describing the phytotoxic effects of Cr+6 and the role of chelated compounds in wheat seedlings. The attempts have been made to investigate and study the varying degrees of effect at different concentrations of Cr+6 and chelated chromium compounds like EDTA, Citric acid and Zinc on chromium accumulation and seedling growth, physiological and biochemical activities of wheat. Materials and Methods Plant Material Garden dry seeds of wheat Triticum aestivum L. were obtained from Orissa University of Agriculture and Technology (OUAT), Bhubaneswar, Odisha. The seeds were stored in dark and cool place for experimental purpose, uniform seeds were selected and surfaces sterilized by soaking in 0.1%. HgCl2 for about five minutes and then washed several times with distilled water. The surface sterilized petriplates over saturated cotton pads for germination. 30ml of distilled water (control) and solutions of potassium dichromate (K2Cr2O7) containing specific concentrations of chromium (VI) were poured into control and treated petriplates respectively. The seeds were germinated under controlled condition at 25°C in darkness for two days. Emergence of 2 mm primary root was used as the operational definition at germination. Nutrient culture experiment The seedling was grown in different Cr (VI) concentrations (5M, 10M and 100M) with or without chelating agents. The pH of nutrient solution was adjusted to 6.8 with help of pH meter. For chromium bio-availability study, the seedlings were grown in different concentrations of Cr separately and with chelating agents at 5M (Cr+6+EDTA, Cr+6+CA, Cr+6+Zn2+). After 7-days treatment, chromium bioavailability on seedlings was measured by Atomic absorption spectrometer. For seedling growth in different concentration of Cr (VI) the pH of the nutrient solution was adjusted to 7.0. The seedlings were also grown in different concentrations of Cr (VI) (5M, 10M, 100M) with or without chelating agents and the growth parameters and chlorophyll content of seedlings were determined. A control pot was also run side by side which was provided with nutrient solution without chromium supply and without chelating agents. Seedling Growth After two days of germination, the seeds were transferred to well aired Hoogland’s nutrient solutions (full strength) placed in glass culture vessels. The seedlings were grown in growth chamber with 7 hr/16hr light and dark period. The white light was provided by filtered cool white fluorescence tubes (36 W Philips TLD) with a photon flux density of 52 E m-2s-1; PAR. (Mohanty and Patra, 2012). 17

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Growth Parameter study The growth parameters like root length, shoot length, fresh root matter and dry shoot matter of 5-7 days old seedlings were used for study. Different Cr (VI) concentrations (5M, 10M, 100M) with chelating agents (EDTA, Citric acid, ZnSO4) or without chelating agent were used during growth parameter study. For study of root and shoot length at 5-7 days old wheat seedlings, the roots and shoots were first detached from each plant. Individual length of root and shoot were measured in centimeter. Ten numbers of seedlings (triplicate) were taken and washed thoroughly with distilled water and soak by using blotting papers. Fresh matter content of both control and treated samples were noted with the help of electric balance. These seedlings were then kept in an oven at 80°C for a period of three days or more for dry weight determination. Then constant dry weight of root and shoot biomass was measured. Both fresh and dry weight of seedlings was expressed in mg/seedling basis. Chlorophyll analysis Chlorophyll analysis were done in both control and treated seedlings growth in different toxic concentrated at Cr (VI) (5M,10M and 100M) and chromium with chelating agent at 50 µm ( Cr+6+EDTA, Cr+6+CA, Cr+6+Zn) for determination of chlorophyll. Chlorophyll analysis was done from 7 days old primary leaves of wheat. The seedlings were grown in different concentrations of Cr (VI) like (5M, 10M and 100M), without chelating agents and with chelating agents at 5 µm (Cr+6+EDTA, Cr+6+CA, Cr+6+Zn). Chlorophyll content was calculated by help of SPAD-meter and using acetone as solvent (Arnon et al., 1949). The pH value of different Cr+6 solutions were calculated by help of pH meter. Chromium analysis Hydroponically grown 7-day old wheat seedlings (control and treated) were oven dried at 80°C for five days and grinded to fine powders. The nitric acids (HNO3) and perchloric acid (HClO4) in the ratio of 10:1 were added and volume was made up to 30 ml by adding distilled water to the weighted and grinded plant powder samples (roots and shoots separately) and kept for 24 hours. Then the acid mixed plant samples were digested until a clear solution was obtained. Then the acid digested solutions were filtered and the final volume was made up to 25 ml. Then total chromium content of roots and shoots were analyzed with the help of an Atomic absorption spectrophotometer. Statistical Analysis All experiments were set up in a completely randomized design (CRD). Each treatment consisted of 3 replicates. Each experiment was repeated three times. Data were analysed using analysis of variance (ANOVA) for a completely randomized design. Duncan’s new multiple range test (DMRT) was used to separate the mean of significant effect. Results Plant growth The treatment with different Chromium (VI) concentrations (5 m,10m,100m ) and in presence of chelating agents at 5 µM (EDTA, CA and Zn2+) showed marked changes in the different growth parameters of 7 days old wheat seedlings (Table 1, Figure 1 & 2). The respective growth parameters of the seedlings were affected by Cr (VI) along its interaction with chelating agents. The root length of wheat seedlings grown under different conditions of chromium treatment was observed. The root length increased with increase in growth period of the seedlings but decreased (13.46, 10.24 and 3.53) markedly with increase in Cr (VI) concentrations (5, 10 and 100 µM). The root length of seedlings treated with Cr+6 (100m) had smaller roots (3.53) than treated with Cr+6 (5 m). The root length of control (15.79) was maximum as compared to all other Cr (VI) treated seedlings (13.46, 10.24 and 3.53) and chromium with chelating agents (14.44, 12.16 and 13.18). Among different conditions of treatments, the seedlings treated with Cr+6+EDTA (5M) had the maximum length (14.44) of roots as compared to other treatments of chromium with chelating agents Cr+2+CA (12.16) and Cr+Zn+2) and (13.18) respectively. The shoot length increased with increasing in the growth period. The shoot length of control seedlings was higher (18.07) as compared to other conditions. The shoot length (15.85, 12.53 and 6.70) decreased with increase of Cr+6 concentrations with 100 µm treated seedlings showing minimum growth. The seedlings with chelating agent Cr+6+EDTA (5m) had highest length (16.45) and Cr+6+CA had lowest length (13.21) of root. The change in fresh root weight of wheat seedlings grown under nutrient condition was noted. The root fresh matter increased with increase in the growth period, but it gradually decreased (0.605, 0.575 and 0.183) with increase Cr+6 concentrations (5, 10, 100 µm). A marked increase in root fresh weight (0.646) was observed in the seedlings treated with Cr+6+EDTA (5m) as compared to other treatment (Cr+6+CA,Cr+6+Zn+2). The shoot fresh matter increased with increase in the growth period. The seedlings under control had the maximum value (1.235). The fresh shoot-weight gradually decreased (1.204, 0.876 and 0.439) with increase in Cr+6 concentrations (5, 10 and 100 µm). Among the seedlings treated with chelating agents Cr+6- EDTA (5M) had maximum shoot fresh weight (0.880) and Cr+6+CA had minimum (0.817). The change in dry root weight of wheat seedlings grown under hydroponics conditions was observed. The dry root matter of the seedlings was found to be highest grown under control condition. The dry root matters showed a decreasing order (0.054, 0.053 and 0.028) with increase in Cr+6 concentrations (5, 10 and 100 µm). The dry root weight of the seedlings was highest (0.309) grown under supplemented with Cr+6+EDTA (5m). 18

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Table 1. Effect of Cr (VI) treatments on shoot length, root length, fresh weight and dry weight of 7 days old Growth parameters

Control

RL (cm) SL (cm) FRwt (mg) FSwt (mg) DRwt (mg) DSwt (mg)

15.79 b 18.07 a 0.693 cd 1.235 c 0.391 de 0.530 def

Cr (VI) treatment 5 m 10 m

Cr (VI)+Chelating agents (5m) Cr 6 +EDTA Cr 6 +CA (5m) (5m) 13.46 b 10.24 b 3.53 b 14.44 b 12.16 b a a a a 15.85 12.53 6.70 16.45 13.21 a cd cd cd cd 0.605 0.575 0.183 0.646 0.627 cd c c c c 1.204 0.876 0.439 0.836 0.817 c def cdef cdef cdef 0.054 0.053 0.028 0.309 0.213 cdef de cde cde cde 0.160 0.130 0.082 0.392 0.300 cde wheat seedlings grow in nutrient solution. 100 m

Cr 6 +Zn 2+ (5 m) 13.18 b 14.52 a 0.629 cd 0.880 c 0.266 cdef 0.304 cde

Data pooled from a total of 3 separate experiments each comprising of 3 replicate pots containing 10 plants per pot. Mean values within column with different superscript alphabets are significantly different (p< 0.05; Duncan’s New Multiple Range Test) Chlorophyll Content Treatment of different Chromium (VI) concentrations (5m,10m and 100m) in absence or presence of chelating agent at the concentration of 5 µm (edta, CA and Zn2+) showed marked changes in the chlorophyll content of 7 days old wheat seedlings grown in hydroponics conditions. A marked increase in chlorophyll-a-content was found in the seedlings grown in controlled condition with increase in growth period but with increase in Cr (VI) concentration Chlorophyll content decreased. Among the seedlings treated with chelating agent (EDTA, CA and Zn2+) the Chlorophylla, Chlorophyll-b and total Chlorophyll was maximum in the Cr+6 supplemented with EDTA i.e. 1.19±0.01, 0.67 and 1.99 respectively but minimum chlorophyll level was observed with Cr+6+CA at the same concentration. The total chlorophyll content decreased 2.23, 1.11 and 1.04 with increase in Cr (VI) concentration (Table 2). Table 2. Effect of Cr +6 treatments on chlorophyll contents (mg/ g fresh weight) of 7 days old wheat seedlings grown in hydroponics condition Chlorophyll Control Cr +6 (m) Cr +6 : Chelating gents (5m) content Cr 6 : EDTA Cr 6 : CA Cr 6 : Zn 2+ 5 m 10 m 100 m Chlorophyll-a 1.42±0.01 1.21±0.01 0.63±0.01 0.60±0.01 1.19±0.01 0.54±0.01 0.86±0.02 Chlorophyll-b 0.77±0.02 0.75±0.02 0.52±0.02 0.50±0.01 0.67±0.01 0.49±0.01 0.53±0.01 Total chlorophyll 2.35±0.01 2.23±0.03 1.11±0.03 1.04±0.02 1.99±0.02 0.93±0.02 1.98±0.02 Results were expressed as mean ± standard error of mean of three different replicates Table 3. Bioavailability of chromium in root and shoot of Triticum aestivum L. after 7 days of seedlings Treatments pH (Before seed pH (After seed Cr +6 content in Chromium content (mg/kg germination) germination) nutrient solution dry weight) (ppm) Root Shoot Control 7.0 7.3 Nil Nil Nil 7.2 7.7 0.504 ± 0.03 31.05 ± 2.79 15.08 ± 0.67 Cr +6 -5 M 7.3 6.9 0.225 ± 0.008 62.5 ± 3.08 31.75 ± 2.03 Cr +6 +10 M 5.3 5.2 0.960 ± 0.05 25.71 ± 2.76 32.24 ±3.02 Cr +6 +EDTA (5M) +6 5.5 7.5 0.934 ± 0.008 15.03 ± 0.91 9.9 ± 0.44 Cr +CA (5M) +6 7.5 5.5 0.868 ± 0.075 18.02 ± 1.03 25 .83 ± 0.62 Cr +Znso 4 (5M) Results were expressed as mean ± standard error of mean of three different replicates pH value pH value of normal solution varies with different chromium concentration with and without chelating agents. After 7 days seedlings growth, the pH value varies with different concentration of solution (Table 3, Figure 3). Chromium content nutrient solution after seedlings growth Distribution of chromium in the plant cell and cellular level varies with the chemical form added to the nutrient medium. The stable chromium levels were used in a preliminary experiment to determine the forms of chromium to be 19

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added to the media in order to avoid any toxicity symptoms in wheat plants. The Chromium (Cr+6) content in the nutrient solution after 7 days seedlings growth varies remarkably. In the solution of Cr+6 (10M) the bioavailability of Chromium was found to be maximum and in a Cr+6 supplemented with Zn (5 M) solution the content was found to be minimum. Chromium bioavailability in root and shoot The Chromium (Cr+6) content (5 µM and 10 µM) in roots as well as shoots of the seedlings increased with increase in Cr+6 concentrations (31.05, 62.5 and 15.08, 31.75) with respect to control were expressed in mg /kg of dry matter respectively. It was observed that roots immobilized more chromium than the shoots of the dry matter and also root containing plant supplemented with chelating agent (Cr+6+EDTA, Cr+6+CA andCr+6+Zn+2) showed average lower value (25.71, 15.03, 18.02) than the shoot (32.24, 9.9, 25.83) of the dry matter. Discussion Plants are exposed to various kinds of stresses in environment in which heavy metal stress is one type of emerging stress caused due to natural as well as man-made activities (Mohanty and Patra, 2011). Toxicity of metals is caused either by natural soil properties, agricultural practices, manufacturing mining and waste disposal practices occurs environmental hazards (Zayed et al., 1998; Zayed and Terry, 2003; Mohanty and Patra, 2011). Heavy metals toxicity receives considerable attention due to its occurrence in nature as well as for mining activities. Phytotoxic effect with respect to chromium was reported by many researchers (Panda and Patra, 1997; Zayed and Terry 2003; Mahanty et al., 2008; Mahanty and Patra, 2012). Chromium is one of the major toxic heavy metals found in nature, exists in two forms viz Cr (III) and Cr (VI), Cr (VI) is found to be more toxic to plants than Cr (III) (Hauschild et al., 1993). Phytoremediation of chromium results in reduced rate of growth, damaged cell walls and membranes and changes in metabolic status of the plants (Nayak et al., 2004, Shanker et al., 2004; Mohanty and Patra, 2012). Present study demonstrates the toxic effects of hexavalent chromium and its uptake in presence different chelating agents like EDTA, CA, and Zn2+. Basically chelators are substances that render insoluble cations soluble which making them available to plants (Norvell, 1972; Lindsay, 1974).,in which less toxic multi-dentate chelating agents such as EDTA are used to enhance the bioavailability of heavy metals for plants uptake (Leyol et al., 1995; Turnace, 1998; Shanker et al., 2004). Our result showed the enhancement of chromium uptake by different chelators that differs from each other. The effects of the interaction of Cr (VI) with chelating agents on the changes in growth parameters (root length, shoot length, fresh weight and dry weight) and physiochemical parameter (chlorophyll) are demonstrated. Wheat seedlings exhibited growth retardation at increasing chromium concentrations (5 M, 10 M, 100M), 5M concentration shows favorable growth as compared to controlled treatment. When chromium (5M) associated with EDTA shows high growth rate than all the treatments. Earlier reports has shown the similar findings by Bonet et al., 1991 who studied the inhibitory effect of higher chromium concentration on bush bean (Phaseolus vulgaria L.) plants was also confirmed by other researchers (Cervantes et al., 2001; Nayak et al., 2004; Mohanty and Patra, 2012). Earlier studies reported by many researches that 90% of the supplied chromium was incorporated in roots in barley and wheat plants with poor translocation to aerial parts and this was reflected in the present study. Chromium bio-accumulation at higher level takes place in plants treated with high hexavalent chromium concentration. Chromium translocation to the aerial parts (shoots) was less as compared to the roots (Hulfman and Allaway, 1973). Chlorophyll-biosynthesis with respect to chromium stress studies was further extended. Higher concentration of Cr+6 supply (i.e.100 µM), corroborates with the findings of Bonet et al., 1991 on Fedeficient plants of bush bean in which significant positive correlation supplied with the chlorophyll concentration in primary and first trifoliate leaves of Fe-deficient plants.

Figure 1. Effect of chromium and chelating agent on root and shoot length of 7 days old wheat seedlings.

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Figure 2. Effect of chromium and chelating agent on root and shoot dry weight of 7 days old wheat seedlings.

Figure 3. Effect of chelating agents for chromium bioavailability on roots and shoots after 7 days of seedlings

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