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INTRODUCTION
Throughout history, human settlement has led to the emergence of hotspots of different forms of pollution involving organic and inorganic compounds. Among the inorganic compounds, heavy metals may come from alkaline batteries, electronic equipment, fossil fuel incineration, gases released from industrial activities and mining waste solids (Raskin et al. 1994). In addition, low concentrations of heavy metals in domestic wastewater can cause highly toxic symptoms in humans and ecosystems, and their accumulation over time can eventually affect quality of life (Cheng et al. 2005, Singh et al. 2004). Heavy metals are present in both soils and streams as natural components (Raskin et al. 1994), and although plants have the capacity to degrade or sequester many toxic compounds, they are also sensitive to many of them (Davis et al. 2002). Plants' stress response to the presence of pollutants, including metals, can alter their capacity to control the uptake of such pollutants, and indeed in some cases may lead them to increase their uptake, which may seriously threaten plant viability (Almeida et al. 2009, Stepniewska et al. 2005). However, these effects depend on the pollutant, plant species, concentration and time of exposure to the pollutant (Davis et al. 2002). For example, copper (Cu) is an essential micronutrient and necessary for all organisms (Weser et al. 1979), but becomes toxic at elevated levels (Flemming & Trevors 1989). In plants, copper-induced iron deficiency is considered a typical copper toxicity symptom (Marchner 1995). In addition, at toxic levels Cu affects photosystem II (PSII) by modifying the electron chain during photosynthesis at the plastocyanin and superoxide dismutase levels, causing a decrease in the photosynthetic rate and in effective photosynthesis (Epstein & Bloom 2005). On the other hand, cadmium (Cd) is considered the pollutant most toxic to organisms, and its release into the environment is associated with human activities (Das et al. 1997; Nriagu & Pacyna 1988; Sanità di Toppi & Gabbrielli 1999). Once Cd is inside the plant cell, it causes chlorosis by inhibiting FeIII-reductase and generating ion deficiency, which affects photosynthesis (Alcantara et al. 1994). It also has a harmful effect on the photosynthetic apparatus, mainly in the harvest electron complex II and in PSI and PSII (Krupa 1988, Siedlecka & Baszynsky 1993, Siedlecka & Krupa 1996).
Phytoremediation, a new biotechnology intended to reduce heavy metal concentrations, has emerged as a useful tool for the remediation of water, soil and air (Raskin et al. 1997). It works with the plant's metabolic activity, which accumulates heavy metals on the tissue (McGrath et al. 2002). The phytoremediation of polluted water, or rhizofiltration, is a relatively new technology (Dushenkov et al. 1997). The process, also referred to as phytofiltration, is based on a hydroponically grown plant medium that has been shown to be efficient in removing heavy metals from water (Raskin et al. 1997). However, some of these elements play no known physiological role (Lasat 2002, McGrath & Zhao 2003), and only 0.2% of the total angiosperms have been reported to have phytoremediation potential; but the use of chelated compounds could increase the number of species useful for this process (McGrath & Zhao 2003). Classic phytoremediation is defined as the circumstance in which the pollutants were present as ions in the solution or environment, whereas enhanced phytoremediation establishes that chemical modifications of the rhizosphere through the addition of chelated compounds can improve the plant accumulation of ions (Evangelou et al. 2007).
Ethylenediaminetetraacetic acid (EDTA) is the chelate most often used for enhanced phytoremediation of soils, but its properties and interactions in the aquatic environment have been little studied (Bonfranceschi et al. 2009, Chen et al. 2010, January et al. 2008, Li et al. 2009). In soil, it is known that EDTA can change metal speciation and thereby affect the metal's bioavailability in the soil, but this has not been studied in aquatic systems. EDTA's ability to increase metal concentration in the soil solution depends on multiple factors, including metal and EDTA concentration, the presence of competitor ions, metallic species, distribution in the soil fraction, soil pH, adsorption of free ions or complexes by the soil particle and the constant of complex formation (McGrath et al. 2002, Saifullah et al. 2009). EDTA is suggested as one of most effective chelating agents in assisting phytoextraction, which can increase metal mobility in the soil solid phase, thus enhancing the concentrations of heavy metals in plant shoot tissue (Hong et al. 1999, Meers et al. 2005, Wong et al. 2004). The assumed reason for this is that P-Type ATPases are responsible for the translocation of both necessary (e.g. Cu2+, Zn2+, Mn2+) and nonessential metals (e.g. Cd2+, Pb2+, Hg2+) through the biological membranes (Ghestem & Bermond 1998, Rensing et al. 1998, Williams et al. 2000). EDTA may induce the activation of ATPases in the plasma membrane, producing changes on ion transport through the membrane. Additionally, EDTA regulates a protein membrane that is related to Pb transport function, and thus Pb can easily be translocated from roots to aerial parts of the plant through the prevention of cell wall retention (Ghestem & Bermond 1998). In general, the chelant-enhanced uptake of Cu in plant shoots has been found to be minimal (Kayser et al. 2000, Kulli et al. 1999, Lombi et al. 2001, Römkens et al. 2002, Shen et al. 2002, Thayalakumaran et al. 2003, Wenzel et al. 2003), while EDTA application reportedly reduced Cd concentrations in some plant species (Luo et al. 2006).
The increased accumulation of heavy metals in plants is a multifactorial phenomenon, with physiological response depending upon the kinds of heavy metals, chemical specificity, the pH of the growth medium and other factors.
The Photosynthetic efficiency (Fv/Fm) is often used as a stress indicator, and describes the potential yield of the photochemical reaction (Björkman & Demmig 1987) due to the location of the heavy metals in the Photosystem II (PSII) and Photosystem I (PSI); the Fv/Fm decreased in plants when they were exposed to a toxic level of the heavy metals. Sánchez-Viveros et al. (2010) evaluated the effects of exposure to Cu2+ in Azolla caroliniana Willd. and A. filiculoides Lam. in the Fv/Fm, where the Cu2+ presence has a negative effect on the Fv/Fm; a similar effect was found in other plants (Küpper et al. 2002, Sivaci et al. 2008). The reduction of the Fv/Fm may be explained by the Cu2+ (and other divalent ions) affecting the photochemical reactions in the PSII, where the electron transport is blocked (Tyystjärvi 2008).
The plant genus most studied for heavy metal accumulation is Azolla, which has been investigated for use in the remediation of different kinds of pollutants, including organic and inorganic compounds, and in different growth media (Arora et al. 2004, Dai et al. 2006, Rai 2008, Sela et al. 1989, Sela et al. 1988, Stepniewska et al. 2005). Azolla filiculoides is a small aquatic fern that has a symbiotic relationship with the heterocystous blue-green alga, Anabaena azollae Strasburger (Lumpkin & Plucknett 1980). In regard to its distribution, it is a cosmopolitan species growing in freshwater streams with low levels of mineral nutrients, and its ability to fix nitrogen from the atmosphere allows it to grow under a variety of conditions (Sood et al. 2011). Azolla's ability to accumulate heavy metals has been studied with different elements (e.g. Ag, Cd, Cu(II), Cr III, Cr(IV), Cr(VI), Hg, Ni (II), Pb, Zn) on live and immobilized Azolla tissues (Arora et al. 2004, Bennicelli et al. 2004, Elmachliy et al. 2011, Fogarty et al. 1999, Khosravi et al. 2005, Mashkani & Ghazvini 2009, Stepniewska et al. 2005, Valderrama et al. 2013, Zhao & Duncan 1997). The response of A. filiculoides to EDTA complex exposure has not been studied previously, and research on this topic has provided knowledge of the species's heavy metal accumulation capability and the physiological consequences of it. As both heavy metals, Cu and Cd, are toxic and harmful to humans and plants, this investigation sought to evaluate the induced Cd and Cu rhizofiltration of Azolla filiculoides.
The aims of this study were: i) to assess the capacity of A. filiculoides Lam. to accumulate Cd and Cu, in the form of EDTA complexes, in an induced rhizofiltration system (Hernández-Allica et al. 2007, Kari & Giger 1996); ii) to determine the stress effect in an induced rhizofiltration system through the stress indicator of PSII photochemistry (Fv/Fm); and iii) to correlate the accumulation of heavy metals (Cd and Cu) by A. filiculoides with the Fv/Fm as physiological responses, because the exact tolerance and physiological mechanisms of Cd and Cu toxicity as EDTA complex have been scarcely studied in an induced phytoremediation with A. filiculoides.
MATERIALS AND METHODS
PLANT MATERIAL
Plant material was obtained from the Lircay River (35°23'34"S, 71°36'49.4"W) in Chile's Maule Region. The plants were identified and the voucher was deposited in the herbarium of the Universidad de Concepción (CONC 171639).
EXPERIMENTAL CONDITIONS
The rhizofiltration system was based on the International Rice Research Institute (IRRI) nutrient solution, as proposed by Watanabe et al. (1992). Experimental conditions were controlled in a laboratory, with a temperature range of 20-25ºC, a mixed light source (fluorescent tubes and tungsten bulb), light intensity of 135 μmol m2 s-1 at the level of the plants, a 16h:8h photoperiod (light:dark) on an automatic timer and continuous aeration at the bottom of the containers using an aquarium pump (Elite 800, 1200 mL min-1 and 2.5 psi); on each treatment 50 g fresh weight collected in the Lircay River was used, having previously been acclimatized by 10 days in the experimental conditions.
The enhanced rhizofiltration treatments were formulated by adding a Cd-EDTA or Cu-EDTA complex, which was prepared by adding a 6.2 mM EDTA solution to a 1 N KOH solution. The final Cd-EDTA and Cu-EDTA solutions were 0.12 mM and 0.082 mM, respectively. Cd-EDTA and Cu- EDTA were evaluated at concentrations of 0.03, 0.30, 0.70, 1.35, 2.00, and 2.70 mg L-1, and 0.10, 0.25, 0.50, 0.80, 1.00, 1.60 and 2.60 mg L-1, respectively (Table I). The control treatments were established without the addition of Cd- EDTA or Cu-EDTA. In all cases, six replicates per treatment were evaluated, including the control systems, with seven days of exposure.
PHOTOSYNTHETIC EFFICIENCY
As an indicator of physiological performance, the maximum quantum yield of PSII (Fv/Fm) was determined in fully expanded leaves of A. filiculoides in three randomly selected plants from each container of rhizofiltration treatments, and was measured using an open gas-exchange system with an integrated fluorescence chamber (Li-6400; Li-Cor, Inc., Lincoln, NE). The Fv/Fm was estimated as the ratio of the variable (Fv) to maximum fluorescence (Fm) of dark- adapted leaves as (Fv/Fm=[Fm-Fo]/Fm), where Fo is the minimum or initial fluorescence to about 0.5 μmol photon m-2 s-1 of light, and Fm is the maximum fluorescence after the application of a saturating flash of about 10,000 μmol photon m-2 s-1 and 0.8 s duration (Maxwell & Johnson 2000).
METAL DETERMINATION IN PLANT TISSUES
Whole plants were harvested and rinsed with deionized water and then dried in an oven at 106 °C until a constant weight was achieved (~500 mg dry weight). These were then ground up in a porcelain mortar. The Cd determination was made by wet digestion and the Cu determination was made by calcination digestion (Allen et al. 1986). Heavy metal content was measured in a flame atomic absorption spectrophotometer (Unicam Solaar mod. 969). To evaluate experimental reproducibility, sampling analyses were repeated six times and chemical analysis was run in triplicate. Each data set was calculated at a 95% confidence level (p<0.05) to determine margins of error (Long & Winefordner 1983). A correlation coefficient for a calibration curve of 0.9994 or greater was obtained for both copper and cadmium. In addition, the cadmium measurement included a deuterium background corrector. The limits of detection for cadmium and copper were 0.083 and 0.094 mg L-1, respectively. The quantitation limit for the analyses and the measured conditions of Cd and Cu were 11.100 and 23.317 μg kg-1, respectively. Certified standard reference materials for both metals were used for calibration and quality assurance on each analytical batch (SRM-1570, spinach, National Institute of Standards and Technology). Blank reagents and analytical duplicates were also used with each chemical treatment to ensure accuracy and precision in the analysis. The recovery rates of the reference materials for cadmium and copper were 103% and 89.7%, respectively.
STATISTICAL ANALYSIS
The data were analysed using one-way analysis of variance (ANOVA), in which the statistical significance of the treatments was 95%, with the least statistical difference (LSD) equal to a p value of <0.05, and the Multiple Range test was used to compare the means. All data analyses were performed with Statgraphics Centurion XV software, and the correlation analysis was carried out using JMP 8 software; statistical significance was determined when p was <0.05. The bivariate correlation was made by fit equation with better r2 value, where the values between 0.25 and 0.50 have a low to moderate correlation; between 0.50 and 0.75 have a moderate to significant correlation; and between 0.75 and 1.00 have a very significant correlation (Salkind 1999).
RESULTS
CADMIUM ACCUMULATION
The Cd levels in the control plants indicated unpolluted conditions in the source (Lircay River) and in the rhizofiltration system (Table I). The accumulation capability of A. filiculoides under EDTA rhizofiltration conditions was lower than under classic rhizofiltration conditions (Valderrama et al. 2013). When A. filiculoides was exposed at equal Cd concentrations in the grown medium with 1.0 and 2.5 mg L-1 of Cd, the Cd accumulation was 188.70 and 673.53 mg kg-1, respectively. In this investigation at the maximum concentrations, 2.70 mg L-1 of Cd-EDTA in the growth media, the highest accumulation achieved was 93.11±10.07 mg kg-1, suggesting some effects of EDTA in the accumulation capability. These results were confirmed by the ANOVA analysis yielding a p value less than 0.05, which implies a statistically significant difference between the treatments and confirms the adequacy of the experimental design.
COPPER ACCUMULATION
Exposure of Azolla filiculoides to Cu-EDTA has not been previously reported or widely studied, and water-enhanced rhizofiltration was developed only recently with other species (Sun et al. 2009; Zhao et al. 2010). The highest copper accumulation was achieved at 2.60 mg L-1 in the growth medium and accumulate 1169.45±204.93 mg kg-1, which are better than the classic rhizofiltration (Valderrama et al. 2013). The ANOVA analyses showed a statistical significance, as p values between treatments were less than 0.05.
PHYSIOLOGICAL RESPONSE
The concentrations of Cd and Cu achieved in the EDTA phytoremediation treatments indicated a decrease in the Fv/ Fm (Table I). These results suggest that heavy metals have a highly toxic effect on photosynthetic performance.
CORRELATION ANALYSIS
The correlation analysis confirmed the toxicity and physiological effect of cadmium and copper (Fig. 1). The relationship between cadmium and copper accumulation with Fv/Fm, respectively, is explained by a polynomial quadratic equation, where (r2=0.484, p value= 0.0062) and (r2=0.417, p value=0.0027), respectively. The graph explains the influence on the photosynthetic apparatus and confirms the harmfulness of cadmium and copper for the plants.
In addition, the polynomial quadratic equation explained three phases of response by the plants; the graph explains the influence on the photosynthetic apparatus and confirms the harmfulness of cadmium and copper for the plants. There were three phases of response by the plants. First, non-harmful concentrations of cadmium or copper were observed, and the Fv/Fm were not affected by the presence of heavy metals in this tissue. However, when the concentrations of metal-EDTA are higher, the concentrations in the tissue were harmful and the Fv/Fm observed were less; at these points, the heavy metals interfered with the PSII and PSI. Finally, the plants' response was to start an exclusion process of heavy metals; the concentrations inside the plants were now at their highest and did not allow the entrance of more cadmium or copper ions. Because of the exclusion process, the Fv/Fm were slightly higher in the third phase than in the second.
FIGURE 1 Correlation analysis between the cadmium and copper accumulation capability of Azolla filiculoides in induced rhizofiltration systems associated with the physiological responses, as Fv/Fm. (A) Relationship between Cd accumulation (mg kg-1) and Fv/Fm; (B) Relationship between Cu accumulation (mg kg-1) and Fv/Fm. FIGURA 1. Análisis de correlación entre la capacidad de acumulación de cadmio y cobre de Azolla filiculoides en sistema de rizofiltración inducido asociado a la respuesta fisiológica, como Fv/Fm. (A) Relación entre acumulación de Cd (mg kg-1) y Fv/Fm; (B) Relación entre acumulación de Cu (mg kg-1) y Fv/Fm.
TABLE I Induced-EDTA rhizofiltration of cadmium and copper by Azolla filiculoides in hydroponic systems. TABLA I. Rizofiltración EDTA-inducida de cadmio y cobre en sistemas hidropónicos de Azolla filiculoides.
d.w. dry weight, †statistical significance was calculated by the Multiple Range test; different letters indicate significant differences between the treatment p<0.05. / d.w. peso seco, † Significancia estadística fue calculada con la Prueba de Ranqueo Múltiple; letras diferentes indican diferencias significativas entre los tratamientos con valor p<0,05.
DISCUSSION
January et al. (2008) showed that the capability of EDTA to accumulate chelated heavy metals depends on the heavy metals and the species studied, and the presence of EDTA alters metal speciation and metal phytotoxicity (Chen et al. 2010). Although A. filiculoides is reportedly a species with phytoremediator potential, when exposed to Cd-EDTA complex, the accumulation of the ions was not increased. If the present results are compared with Cd-classic rhizofiltration with Azolla species, the natural accumulation potential of the genus is seen to be indisputable; however, none of the historical results is lower than the Cd-EDTA complex obtained in this investigation (Arora et al. 2004, Sela et al. 1989, Stepniewska et al. 2005). Sela et al. (1989) exposed A. filiculoides to 10 mg L-1 of Cd in the medium and found that the cadmium content was highest in the dark grains located in the xylem cells and in the lower part of the stem. This phenomenon could explain the plant's lack of a favourable response to enhanced rhizofiltration of cadmium, because the EDTA complex modifies the toxicity effects of the cadmium in the plant cell, owing to a higher metabolic cost to metabolize than that produced by pure cadmium ions (Nörtemann 1999).
In the case of copper, the response obtained in A. filiculoides coincides with the assumption that exposure to the metal-EDTA complex increases the accumulation of the metal in plants (Evangelou et al. 2007). In the present research, when exposed to Cu-EDTA complex in rhizofiltration treatment, A. filiculoides accumulated more Cu than in classic rhizofiltration (Valderrama et al. 2013). When A. filiculoides was exposed to 0.10 mg L-1 of Cu-EDTA, it accumulated 500.59±106.34 kg kg-1, similar to when it was exposed to 1.0 mg L-1 of Cu in the medium (Table I).
However, the use of EDTA could present risk to the plant, including a reduction in growth or in biomass production, necrosis, and/or chlorosis (Epstein & Bloom 2005, Huang et al. 1997, Jiang et al. 2003, Luo et al. 2005). Azolla filiculoides displayed Cu selectivity in its response to Cu-EDTA exposure, and its ability to remediate water polluted with Cu was confirmed (Fogarty et al. 1999, Sela et al. 1989). In both cases, the photosynthetic effect confirmed the foliar allocation of the ions and the EDTA-induced mobility changes caused by increased translocation from the root to the stems or leaves (Chen & Cutright 2001, Jiang et al. 2003). Although this investigation did not evaluate the allocation of cadmium or copper in the plant tissue, it is clear that these ions allocated in the leaf tissues at the chloroplast level and affected the photosynthetic reactions by reducing Fv/Fm.
Cadmium is the element most toxic to plants (Das et al. 1997), and the Cd ions inhibited the formation of chlorophyll by interfering with photochlorophyllide reduction and the synthesis of aminoevulinic acid, resulting in the inhibitionof photosynthetic CO2 fixation (Mohan & Hosetti 1997, Weigel 1985). On the other hand, copper can cause oxidative stress by generating reactive oxygen species (ROS) such as superoxide radicals (O-), which can be further converted to hydrogen peroxide and the hydroxyl radical (OH-) (Cho & Seo 2005, Hall 2002). ROS affect the photosynthetic apparatus indirectly by inhibiting the repair of crucial PSII proteins (Murata et al. 2007). Although Cu is an essential micronutrient for plants, it can be a strong photosynthesis inhibitor at high levels, and the decrease in the Fv/Fm could be caused by peroxidation of chloroplast membranes or may result in a decrease in the electron transfer sites consequent to its binding to those sites (Frankfart et al. 2002, Maksymiec 1998, Mal et al. 2002, Sandmann & Böger 1980, Vavilin et al. 1995).
Our results showed a marked decrease in the Fv/Fm in cadmium treatment when compared to Cu, and was confirmed in other aquatic macrophytes in hydroponic conditions, including Azolla pinnata R. Br., Lemna minor L., Pistia stratioides L., Spirodela polyrhiza (L.) Schleid. and Eichhornia crassipes (Mart.) Solms (Hou et al. 2007, Mishra et al. 2008, Sarkar & Jana 1986). These results showed the toxic effects of these ions at high concentrations in the rhizofiltration systems and in the entire plants. In both analyses, the r2 was not high, but the p value determined that the results were statistically significant.
CONCLUSION
The novel part of this investigation was the use of the EDTA complex in the rhizofiltration system using Azolla filiculoides, which allowed the analysis of each metal (cadmium and copper) and their effects in the physiological response. Cd-EDTA rhyzofiltration was not able to increase the accumulation of Cd in the plant tissue beyond that obtained in classic Cd-rhyzofiltration, and even the small quantity of Cd in the plant was strongly harmful to photosynthetic metabolism. In comparison, Cu-EDTA rhyzofiltration increased Cu accumulation in the plant, while the photosynthetic response showed an effect, but not a critical one. Different concentrations of Cd-EDTA and Cu-EDTA in the growth medium showed a marked effect on the accumulation of the ions and altered the performance of A. filiculoides when the heavy metal-EDTA complexes were absorbed. The physiological plant responses were evaluated using Fv/Fm as an indicator of stress in the photosynthetic metabolism, and the correlation furthered understanding of the complexity of plant systems. In the future, it is hoped to evaluate the plant physiological response with additional tools to build a comprehensive view of the plants' response to the medium.
