New natural chelating agents with modulator effects on copper phytoextraction

New natural chelating agents with modulator effects on copper phytoextraction
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   Environmental Engineering and Management Journal    February 2012, Vol.11, No. 2, 487-491 “Gheorghe Asachi” Technical University of Iasi, Romania NEW NATURAL CHELATING AGENTS WITH MODULATOR EFFECTS ON COPPER PHYTOEXTRACTION Irina Volf, Alina Stîngu   , Valentin I. Popa “Gheorghe Asachi” Technical University of Iasi, Faculty of Chemical Engineering and Environmental Protection,  Environmental Engineering and Management Department, 73 Prof. dr. Doc. Dimitrie Mangeron Street, 700050, Iasi, Romania Abstract Polyphenols are a widespread group of secondary metabolites found in woody species, including softwood. Higher amounts of these bioactive compounds occurring in bark, heartwood, roots, branch bases and wound tissues and have a number of important roles to play in viticulture and ecology, including UV protection, disease resistance, pollination, color, and defense against  predation in plants, plant growth regulation and bioremediation. The use of synthetic chelates has been strongly criticized because of excessive amount of metals solubilized, associated with risk of groundwater contamination through metal leaching. This study evaluate the possibility of using natural bioactive compounds, characteristic to grape seeds extracts, in phytoextraction as an alternative to synthetic chelators. The results obtained shown an improving trend in the bioaccumulation process of heavy metals into the rape plant. It may be properly mentioned that grape seeds natural polyphenolic extract present an important role in  phytoextraction process.  Key words:  catechine, copper, grape seeds polyphenolic extract, phytoextraction  Received: September, 2011; Revised final: January, 2012; Accepted: February, 2012    Author to whom all correspondence should be addressed: e-mail:; Phone + 40724349991 1. Introduction Heavy metal contamination soil is one of the most concerning problem of our days since they  persist into to medium and caused several damages to the entire ecosystem. Phytoremediation has been developed as an economically and environmentally attractive approach to clean up heavy metal-polluted soils. Phytoextraction use plants to extract metals from soil and translocate them to shoots. The aim of  phytoextraction is to reduce the concentration of metals in contaminated soils to regulatory levels within a reasonable time frame ( Nascimento, 2006). Two approaches have been proposed to reach such goal: the use of plants with exceptionally high, natural metal-accumulating capacity, named hyperaccumulators, and the utilization of high- biomass crop plants, such as corn or  Indian mustard in association with a chemically enhanced method of  phytoextraction (Lombi et al., 2001; Chen et al.,  2004). Achievement of hyperaccumulator status is  based on the ability to uptake and retain within the shoot (stem and leaves) one of the following metals at a minimum of: 10 mg/ g of Zn or Mn, 1 mg /g of Ni, Co, Cr, Cu or Pb, 0.1 mg/ g of As or Cd (Turgut et al., 2005). One approach to increase the uptake of heavy metals is the use of chelators to mobilize the metals to the root zone (Madrid et al., 2003). Synthetic chelates have been successfully used to induce accumulation of metals by high biomass plants but they pose serious environmental drawbacks regarding excessive amount of metals solubilized and being poorly  biodegraded in the soils (Luo et al., 2006). It can decrease plant biomass, destroy the physiological  barriers of the root, or inactivate transporter proteins to the extent that its metal mobilizing and translocation benefits are minimized (Nacimento et al., 2006; Luo et al., 2006).   Volf et al./Environmental Engineering and Management Journal 11  (2012), 2, 487-491 488 The use of easily biodegradable chelates has  been thus proposed to enhance the metal soil availability and accumulation in plants while avoiding leaching risks but do not provide long term effectiveness since they are easily biodegraded (Turgut et al., 2005; Nacimento et al., 2006; Evangelou et al., 2006). It has been shown that vanillic and gallic acids, applied to heavy metal contaminated soils in 20 mM/ kg dose, solubilized significant amounts of Zn, Ni, and Cd from soil ( Nascimento et al., 2006). Increasing interest in the replacement of synthetic chelators has determined to research into natural sources of chelating agents, especially using  plants as raw materials. This will allow a possibility to valorize waste industrial products which represent an important challenge of sustainable development  based on environmentally friendly processes correlated with complex processing of biomass. The aim of this study was to evaluate the influence of grape seeds aqueous extract and catechine, the main component of the extract, on  Brassica napus  plant growth, development and copper bioaccumulation. 2. Material and methods 2.1. Extraction procedure and characterization of  grape seed aqueous extract The aqueous extraction was carried out on 20g of dried material using 125 mL of distilled water at 70°C for 45 min. The extraction was repeated three times and extracts were cumulated to a volume of 500 mL using distilled water (Ignat et al., 2011)   Total content of polyphenols, tannins, flavonoids, flavonols and antocyanins were determined using different colorimetric methods. The total polyphenolic content was determined using Folin Ciocalteau method and the concentrations were expressed as the number of equivalents of gallic acid (GAE) (Bao et al., 2005). The total content of tannins was determined using a method based on the  precipitation of tannins with casein. The tannins content was determined using FC method and expressed as the difference between the initial content of polyphenols and the content after the precipitation with casein (El-Sayed, 2009). The contents of flavonoids and flavonols were determined by aluminium chloride method using rutin as a reference compound according to Makris et al. (2008), respectively El-Sayed (2009). The antocyanins content was determined using pH differential method described by Ribereau-Gayon (1972).   2.2. Germination tests experiment Germination tests experiment was carried out in Petri dishes each one containing 10 rape seeds pre-disinfected with NaOCl 1% for 15 min and 10mL tested solution. It was tested two different concentrations of copper ions (2.5 and 25 µg/mL CuSO 4 ) with and without grape seed aqueous extract addition. The tested solutions were suggestively noted as: Cu-2.5, Cu-25 for copper salts; Cu-2.5 / Cu-25 Grape s. extract, in the case of grape seed extract (GS) addition and Cu-2.5 Cu 25 Catech 1, Cu-2.5 / Cu 25 Catech 10, Cu-2.5 / Cu 25 Catech 50; Cu-2.5 / Cu 25 Catech 100 in the case of Catechine solution addition in four different concentration (Popa et al., 2010). The Petri dishes were incubated in a thermostatic chamber at 25-27 °C for seven days. After 168 h, rape seedling were exposed to day light for 48 h to promote chlorophyll pigments  bioshynthesis. The experiment was carried out in triplicate and the average was reported. 2.3. Plant analysis 2.3.1. Plant growth and development To evaluate the influence of natural amendments on plant growth and development in different copper stress conditions, after ten days,  Brassica napus  plants were separated into roots, stems and primary leaves, followed by biometric measurements of plant elongation and quantitative determinations of biomass. 2.3.2. Pigments assimilation assay The chlorophyll was extracted in 80% acetone and spectrophotometrically determinated by reading the absorbances at fixed wavelength of 470, 646, 663 nm. The concentrations of chlorophyll pigments were calculated by using the specific coefficients suggested  by Lichtenthaler and Wellburn (1983). The results were expressed in % comparing with control for a  better evaluation of stimulatory/ inhibitory effects of grape seeds extract/catechine addition in a copper contaminated medium on pigment photo-assimilation  process. 2.3.3. Determination of heavy metal concentration Individual oven dried plant sample was digested, for at least 5h on a hot plate, using HNO 3 (65%) and H 2 O 2 (30%) followed by copper concentration (µg/mL) determination using a GBC Atomic Absorption Spectrometer (Smith et al., 2008; Stingu et al., 2009). The concentrations of copper were determined by calibration curves (y= 0.059X +0.0016, R  2  = 0.9996) obtained using standards solutions of pure metal ions (Fisher Scientific). Calibration curves were generated using three replicates per metal concentration (0.02-5.00 µg/mL)  by reading the absorbance at fixed wavelength (324.70 nm). The determination of copper concentrations into different parts of rape plant permitted the quantification of the following parameters: Bioaccumulation coefficient = metal ions concentration into the plant / metal concentration into the growth medium (Stingu et al., 2009). Translocation factor = metal ions concentrations into the shoots / metal ions concentration into the root   (Sun et al., 2008).    New natural chelating agents with modulator effects on copper phytoextraction   4893. Results and discussions The total polyphenolic content in the case of grape seeds extract was 506.25 mg GAE /100g, while the total tannins concentration was 198.38 mg GAE /100g. The total content of flavonoids was 27.73 mg RE/100g and the content of flavonols was about 7.11 mg RE/100g. The yield of total antocyanins was 18.52 mg/100g.  Nascimento (2006) discussed the potential of natural, aromatic or aliphatic low molecular weight organic acids in providing metal concentrations in solution that are both environmentally safe and high enough to increase plant uptake up to levels adequate to phytoextraction. It was demonstrated that gallic acid applied at 10 mM/ kg could remove as much Cd, Zn, Cu, and Ni from soil as EDTA without increasing the leaching risk ( Nascimento et al., 2006). Polyphenolic profile of grape seeds aqueous extract was characterized by the presence of catechine (44.36 mg/100g), which according to numerous evidences is the main component of this extract beside gallic acid (6.12 mg/100g) and epicatechine (Balas and Popa, 2007; Ignat et al., 2011). The evaluation of rape plant elongation and  biomass quantification allow us to observe the influence of grape seeds polyphenolic extract on plant growth and development and also to elucidate if catechine is responsible for registered effects.  Brassica napus  plant growth was inhibited in the presence of copper stress conditions, both in 2.5 and 25µg/mL Cu (II) contamination level (Fig.1). Plant elongation decreased with increasing copper concentrations in the growth medium. The inhibitory effects, comparing with control, varied between 21.82 %- 55.91 % in 2.5µg/mL Cu (II) and with 70.61 – 77.73 % in 25µg/mL Cu (II) stress conditions, even when the growth medium was supplemented with grape seeds extracts and catechine in 1-100µM concentrations. The presence of grape seeds extract reduce the inhibitory effect down to 21.82 %, reported to control, comparing with the values observed in a 2.5 µg/mL Cu (II) contamination level when the inhibitory effects was 55.42 %, reported to control.  Not the same thing was observed in the case of 25µg/mL Cu (II) stress conditions when, the supplementation of the artificially contaminated growth medium, with grape seeds extract amplify the inhibitory effect on plant growth up to 77.73% comparing with control (Fig. 1). Biomass accumulation was also inhibited in the presence of copper ions. The inhibitory effect (18.49% comparing with control) registered in 2.5µg/mL Cu (II) contamination level, was reduced with the addition of grape seeds polyphenolic extract and catechine (1-100µM) into the growth medium (Fig. 2). The same trend was also observed for 25µg/mL Cu (II) stress conditions. The modulator effects of grape seeds polyphenolic extracts on rape  plant growth and development in a copper polluted environment depends on metal contamination level and on bioactive compounds concentration. The only stimulatory effect (1.90% comparing with control) on  biomass accumulation process was obtained in the  presence of catechine (100µM) in the case of 2.5 µg/mL Cu (II) contaminated medium, meanwhile for 25µg/mL Cu (II) stress condition the stimulatory effect was observed only in the presence of grape seeds polyphenolic extracts (9.64% reported to control sample) (Fig. 2). Fig. 1.  Variation of  Brassica napus  length growth under heavy metal stress conditions and grape seeds extract/ catechine treatments Fig. 2.  Variation of  Brassica napus  biomass under heavy metal stress conditions and grape seeds extract/ catechine treatments The leaves of rape plant were significantly affected by Cu, which resulted in a decline of chlorophyll content (Fig. 3). Cu inhibits chlorophyll and carotene biosynthesis, and retards the incorporation of these pigments in photo systems. The chlorophyll (a + b) content of the leaves for rape plant decreased under copper ions stress, but not in a significant way with increasing Cu concentration. Additionally, total chlorophyll content in the presence of 2.5µg/mL Cu (II) decreased about 23.27% while this decrease was around 43.93% for rape plant, at highly toxic Cu level compared to the control. The supplementation of growth medium with grape seeds extracts promoted chlorophyll assimilation with 20.79% in the case of 2.5µg/mL Cu (II) and with 54.55% under 25µg/mL Cu (II) stress condition. Similar effects (60.51%, 15.02%, 25.70%) were registered in the case of 10, 50, 100µM catechine addition into 25µg/mL Cu (II) contamination level.   Volf et al./Environmental Engineering and Management Journal 11  (2012), 2, 487-491 490 The highest carotene content was measured in the presence of 50 µM catechine for both copper contamination level. The presence of grape seeds extracts accentuated the decreasing trend for carotene assimilation with increasing Cu concentration (39.59%, 59.47% comparing with control). The obtained results have shown that catechine, even if it is the main bioactive compounds of grape seeds polyphenolic extracts, it was not the main responsible for the effects observed for  pigments assimilation in the presence of grape seeds extract (Fig. 3). The effect of grape seeds extract and different concentration of catechine on metal accumulation in different parts of rape plant was also investigated in the present study. The accumulation of metals in various parts of plants is often accompanied by an induction of variety of cellular changes, some of which directly contribute to metal tolerance capacity of plants. The Cu content of rape plant decreased with increasing Cu concentration into the growth medium (Fig. 4). Fig. 3.  Variation of pigments assimilation into  Brassica napus leaves under heavy metal stress conditions and grape seeds extract/ catechine treatments The bioaccumulation of copper ions into rape  plant was considerable higher (512.69) in the  presence of grape seeds polyphenolic extracts, with 81.44% comparing with the values registered for 2.5 µg/mL Cu contamination level (282.57) and with 101.94% reported to 25µg/mL Cu (II) stress conditions (Fig. 4). Heavy metal bioaccumulation  process was also stimulated in the presence of catechine (50, 100µM), with 51.16%, 8.90% in the case of 2.5µg/mL Cu (II) contamination level and with 3.49%, 7.31% in the case of 25µg/mL Cu (II). In our study, it was observed a relative reduction of shoot growth and remarkable increase in copper bioaccumulation in  Brassica napus  leaves for catechine (1, 50µM) supplementation of 2.5µg/mL Cu (II) contaminated environment. On the other hand, the increase of Cu concentration up to 25µg/mL Cu (II) induced a decrease in root length; being more  pronounced in the case of grape seeds extracts (92.05% compared with control) addition. The  presence of grape seeds extract promoted the  bioaccumulation of copper ions with 67.02%, 50.01% in roots, both in 2.5 and 25µg/mL Cu (II) stresses conditions.   Fig. 4.  The influence of grape seeds extract/ catechine treatments on copper bioaccumulation into  Brassica napus Analyzing the modulators effects of natural extracts and catechine it was observed that the  bioaccumulation capacity which represent the  proportion between (µg metal/g dry weight plant) and (µg metal/ mL solution) registered in the presence of grape seeds extract shown approximately the same values as the ones registered in the presence of 50µM catechine into the medium. The roots could play an important role for the retention of the metal, preventing its excessive accumulation in shoots. Roots act as a barrier against heavy metals translocation to the tops. The highest values for translocation factor (0.27, 0.56) were registered in the presence of grape seeds aqueous extract, for both copper contamination level. The  presence of catechine into a copper contaminated growth conditions, promoted also the translocation of heavy metals to the aerial parts of rape plant (Fig. 5). Fig. 5.  The influence of grape seeds extract/ catechine treatments on translocation factor variation 4. Conclusions Grape seeds polyphenolic extracts and catechine have shown modulator effects on copper  bioaccumulation into rape plant and promoted the  bioaccumulation of heavy metals to the aerial parts of  plant. Improving the bioaccumulation of heavy metals into the rape plant, it may be properly mentioned that grape seeds natural polyphenolic    New natural chelating agents with modulator effects on copper phytoextraction   491 makes the use of these compounds in phytoextraction a viable alternative to synthetic chelates application. Catechine present proximately the same effects as grape seeds polyphenolic extracts, but even so, it could not be concluded that cathechine is the one responsible for registered effects. Further investigation will analyze the influence of gallic acid, another important component of grape seeds extracts, on copper bioaccumulation in  Brassica napus, for a better understanding of  polyphenolic extracts modulator effects. Acknowledgements This paper was realized with the support of BRAIN “Doctoral scholarships as an investment in intelligence”  project, financed by the European Social Found and Romanian Government.   References Balas A., Popa V.I., (2007), Bioactive aromatic compounds-The influences of natural aromatic compounds on the development of  Lycopersicon esculentum  plantlets,  BioResources , 2 , 363-370. Bao J.S., Cai Y., Sun M., Wang G.Y., Corke H., (2005), Anthocyanins, flavonols, and free radical scavenging activity of Chinese bayberry (  Myrica rubra ) extracts and their color properties and stability,  Journal of   Agricultural and Food Chemistry , 53 , 2327-2332. Chen Y., Xiangdong L., Shen Z., (2004), Leaching and uptake of heavy metals by ten different species of   plants during an EDTA-assisted phytoextraction  process, Chemosphere , 57 , 187-196. El-Sayed S.A.H., (2009), Total phenolic contents and free radical scavenging activity of certain Egyptian  ficus  species leaf samples,  Food Chemistry , 114 , 1271-1277. Evangelou M., Ebel M., Scaeffer A., (2006), Evaluation of  the effect of small organic acids on phytoextraction of  Cu and Pb from soil with tobacco  Nicotiana tabacum ,  Chemosphere , 63 , 996-1004. Lichtenthaler H.K., Wellburn A.R., (1983), Determinations of total carotenoids and chlorophylls a and b of leaf  extracts in different solvents,  Biochemical Society Transactions , 11 , 591-598. Lombi E., Zhao F.J., Dunham S.J., Mcgrath S.P., (2001), Phytoremediation of heavy-metal contaminated soils: natural hyperaccumulation versus chemically enhanced  phytoextraction,  Journal of Environmental Quality , 30 , 1919-1926. Luo C., Shen Z., Lou L., Li X., (2006), EDDS and EDTA-enhanced phytoextraction of metals from artificially contaminated soils and residual effects of chelant compounds,  Environmental Pollution , 144 , 862–871. 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