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PHYTOREMEDIATION OF Cd, Ni, Pb AND Zn BY Salvinia minima a
Danilo Sinhei Iha & Irineu Bianchini Jr.
a
a
UFSCar - Universidade Federal de São Carlos, Departamento de Hidrobiologia, 13565-905 – Brazil - SP Accepted author version posted online: 07 Apr 2015.
Click for updates To cite this article: Danilo Sinhei Iha & Irineu Bianchini Jr. (2015): PHYTOREMEDIATION OF Cd, Ni, Pb AND Zn BY Salvinia minima, International Journal of Phytoremediation, DOI: 10.1080/15226514.2014.1003793 To link to this article: http://dx.doi.org/10.1080/15226514.2014.1003793
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ACCEPTED MANUSCRIPT PHYTOREMEDIATION OF Cd, Ni, Pb AND Zn BY Salvinia minima DANILO SINHEI IHA* and IRINEU BIANCHINI JR. UFSCar - Universidade Federal de São Carlos, Departamento de Hidrobiologia, 13565-905 – Brazil - SP *[email protected]
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ABSTRACT Most metals disperse easily in environments and can be bioconcentrated in tissues of many organisms causing risks to the health and stability of aquatic ecosystems even at low concentrations. The use of plants to phytoremediation has been evaluated to mitigate the environmental contamination by metals since they have large capacity to adsorb or accumulate these elements. In this study we evaluate Salvinia minima growth and its ability to accumulate metals. The plants were cultivated for about 60 days in different concentrations of Cd, Ni, Pb and Zn (tested alone) in controlled environmental conditions and availability of nutrients. The results indicated that S. minima was able to grow in low concentrations of selected metals (0.03 mg L-1 Cd, 0.40 mg L-1 Ni, 1.00 mg L-1 Pb and 1.00 mg L-1 Zn) and still able to adsorb or accumulate metals in their tissues when cultivated in higher concentrations of selected metals without necessarily grow. The maximum values of removal metal rates (mg m² day-1) for each metal (Cd = 0.0045, Ni = 0.0595, Pb = 0.1423 e Zn = 0.4046) are listed. We concluded that S. minima may be used as an additional tool for metals removal from effluent. KEYWORDS Aquatic macrophytes; effluent treatment; metals; bioaccumulation; continuous flow.
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ACCEPTED MANUSCRIPT 1. INTRODUCTION Metals have toxic potential even at low concentrations causing risks to human health. Due to their high dispersal ability metals can accumulate in the environment and in different organisms tissues (Yokel et al., 2006). In recent decades there was an increase of metal concentrations in many regions of the world and the industrial, domestic and agricultural effluents are the most important sources of aquatic environments contamination (Jordão et al.,
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2005). The metals are removed from effluent by wetlands and usually it happen on tertiary treatment where significant decrease occurs in various elements concentration (Hadad et al., 2006; Maine et al., 2006; Sperling & Chernicharo, 2005). Among the techniques for metals removal the plants use (phytoremediation) has emerged as a promising alternative (Moshiri, 1993). In recent decades several studies have demonstrated the effectiveness of aquatic macrophytes in wastewater treatment (Casabianca, 1995; Henry-silva et al., 2006; Moshiri, 1993; Stottmeister et al., 2003). Macrophytes not only accumulate metals in their tissues but also promote a variety of chemical and biochemical reactions that decrease pollution by increasing environmental diversity in the roots region (Jenssen et al., 1993). In this regard Salvinia minima has rapid growth and is found in different parts of the world (Bianchini Jr. et al., 2010; Tryon & Tryon, 1982). It is a floating aquatic macrophyte, native from South America, has great potential to remove metals from contaminated areas and high capacity to absorb cadmium and lead (Ali et al., 2013; Espinoza-Quiñones et al., 2005; Olguín et al., 2002; Oliveira et al., 2001).
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ACCEPTED MANUSCRIPT Plant growth models are mathematical representations of physiological processes associated to its metabolism (Best, 1990). Growth models have been considered important tool for understanding aquatic ecosystems processes and especially for predicting about the behavior of these systems (Thomaz & Bini, 2003). This study had as main aim the evaluate Salvinia minima growth in order to use it to phytoremediation of effluents contaminated with metals. The selection of this species was based on studies that test its ability to assimilate metals and its easy
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handling, (Ali et al., 2013; Hadad et al., 2006; Olguín et al., 2005; 2002; Santos, 2009; Santos, 2006).
2. MATERIAL AND METHODS The growth of Salvinia minima Baker cultivated in solutions with different concentrations of Cd, Ni, Pb and Zn was evaluated in triplicate. In total 720 individuals of similar size and stage of development were collected (21°59'17" S e 47°53'2" W; datum WGS84) and washed in a solution of sodium hypochlorite (NaClO 0.5%) for 5 min. Plants were cultivated for about 60 days in polyethylene recipients (217 x 145 x 70 mm) in germination chambers (COLDLAB) under controlled conditions (20 ºC, photoperiod: 12h and light intensity: 4.31x10-3 µmol cm-2 s-1). The culture system was kept under continuous flow (volume: 1.0L; flow: 0.35 mL min-1; residence time: 2 days) through peristaltic pump (MasterFlex, 07524-40, 12 channels) (Figure 1). All solutions were prepared with 15% culture medium Hoagland-Arnon (Hoagland and Arnon, 1950) and the pH was adjusted to 6.5 with sodium hydroxide (NaOH 50%) or hydrochloric acid (HCl 18%) and subsequently added different metals concentrations.
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ACCEPTED MANUSCRIPT To evaluate the growth of S. minima without metals exposed (control group), 180 individuals was cultivated in 12 recipients with 15 individuals each. Forty five individuals (cultivated in three recipients with 15 individuals each) were exposed to 90 L of solution with the following metals concentrations tested alone: 0.003, 0.030 and 0.300 mg L-1 Cd (CdCl2.2,5H2O); 0.02, 0.20 and 0.40 mg L-1 Ni (NiCl2.6H2O); 0.01, 0.10 and 1.00 mg L-1 Pb (PbCl2); and finally,
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0.1, 1.0 and 10.0 mg L-1 Zn (ZnSO4.7H2O). For the amount of Zn (0.008 mg L-1), normally present in the nutrient solution, did not interfere in the experiments that assessed the macrophyte growth cultivated with this metal only these experiments the culture medium was prepared without addition of ZnSO4.7H2O. The growths or decrease (death) of macrophytes were assessed twice a week and determined through measurements of leaf area by photographic
images using the Spring®
software (Iha & Bianchini Jr, 2013). The relationship between leaf area (cm²) and dry mass is described by the following mathematical expression: dry mass (g) = 0.001888 x leaf area; N=18; R² = 0.97. The values of leaf area and the time were fitted to a logistic model (Vogels et al., 1975; Eq. 1) and the growth coefficient values (rm) were obtained. =
(
)
(Eq. 1),
where: N = leaf área (cm²); t = time (days); K = maximum area (cm²); a = constant of integration and rm = growth coefficient (day-1).
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ACCEPTED MANUSCRIPT After the last day of cultivation the plants were removed from the solution, rinsed with deionized water and dried (at 45 °C) until constant weight. The dried material was subsequently digested and analyzes of the metal concentration in plants tissue were carried out (it was considered the metals content in the macrophyte the metal adsorbed, or adhered to the outer wall tissue of the plant and the accumulated metal that has been assimilated by the plant and is found in the intracellular or interstitial region), the method used was optical emission spectrometry with
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inductively coupled plasma using equipment ICP-OES, Optima model 8000 Perkin Elmer® (ref. SMEWW 21 3030 E). Concentrations of metals in solution culture were also determined by the same method and equipment. These collections were performed once a week during the experimental period. To assess the bioaccumulation of metals in plant tissue the bioconcentration factor (BCF) was calculated according to Zayed et al. (1998); (Eq. 2). (
=
) (
)
Eq. 2,
To evaluate the tissue metal content according to the concentration of metal in the solution the Eq. 3 was used (Monod,1949). R=
Eq. 3,
where, R = metal content in plant tissue (mg g-1); C= metal concentration in the solution (mg L-1); RK = maximum plant tissue content (mg g-1); C1= metal concentration for 50% of RK.
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ACCEPTED MANUSCRIPT The calculation of removal metal rate can be used to determine the efficiency of phytoremediation by S. minima at different growth stages depending on the growing tank area, the density and the solution metal concentration. Based on these calculations can be suggested guidelines to identify when these plants can be used as a tool to reduce the wastewater metals concentration. The removal metal rate in milligrams per day is given by Eq. 4, adapted from
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Gutiérrez et al., (2001) and Mahujchariyawong and Ikeda, (2001): =
×(
−
²) ×
Eq. 4,
where, Txa = removal metal rate (mg day-1); R= metal content in plant tissue (mg g-1); D = 529.7 cm² (area occupied by 1 g of dry mass basis of S. minima); rm= growth coefficient (day-1); b= percentage of the tank area covered by S. minima (%); A = total area of cultivation tank (cm²). All parameters of this study (Eq. 1 and 3) were obtained by non-linear regressions, calculated with the algorithm interactive Levenberg-Marquardt (Press et al., 1993). The calculations were presented where the statistical probability of random data are correlated (p) is less than 5% (p0.05, Table 2). This result was expected, since the experimental strategy was just to assess the growth of S. minima in constant concentrations. The Zn concentration was lower than expected, maybe because this metal is an essential macro nutrient. However, aiming the use of this species to phytoremediation, the cultivation tank size should be adjusted to the flows and concentrations of
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metals for the residence time associated with metal removal rate (Txa) and growth coefficient (rm) allow increased efficiency in treating wastewater contaminated. The values of rm were lower where added any concentrations of Cd, Ni and Pb on solution comparing to the control group (rmcontrol= 0.0567+0.0100 dia-1). Excess of Cd interferes in the synthesis of compounds rich in thiol groups, causes a decrease in photosynthesis, reducing the absorption and transport of nutrients, damage to membranes, hormonal disorders and abnormal activity of several enzymes resulting in eventual death of plants (Barceló & Poschenrieder, 1990; Oliveira et al., 2001). In most cases, the inhibition by Cd and Pb results on the interaction between metals and enzymes sulphydryl groups (-SH), blocking these enzymes inhibits the activity of over 100 enzymes, these interactions negatively affect the function of these enzymes as alter the stability of its tertiary structure, and affect the absorption of essential nutrients such as N, K, among others (Seregin & Ivanov, 2001). Sen and Bhattacharyya (1994) cultivated S. natans in different concentrations of Ni and concluded that concentrations of 0.010 mg L-1 are toxic to this species, photosynthesis and chlorophyll-a decreases as a result of the conversion of chlorophyll to pheophytin, this reduction may be related to inactivation of the enzymes sulphydryl groups needed for the synthesis of fatty acids. In this study there was low
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ACCEPTED MANUSCRIPT growth of S. minima cultivated at 0.40 and 0.20 mg L-1 Ni (Figures 1 and 2). Reductions in photosynthetic pigments when exposed to metals have been reported by several authors for others Salvinia species (Al-Hamdani & Blair, 2004; Hadad et al., 2007; Nichols et al., 2000). With respect to Zn, the rm values increased until the concentration of 1.0 mg L-1 (rm= 0.0571+0.0034 dia-1). This result may be related to the fact that Zn is an essential
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micronutrient for the growth of various plant species and plays an important role in the activation of specific enzymes such as peptidases, dehydrogenases, proteinases and fosfohidrolases (Mehra & Farago, 2008). No growth occurred for cultivation at 10 mg L-1 Zn and therefore this concentration is toxic to S. minima. Dhir et al., (2008) evaluated changes in photochemical activities of Salvinia natans when exposed to concentrations of 50 mg L-1 Zn and observed changes in potential carbon assimilation by ribulose-1, 5 carboxylase / oxygenase activity, indicating that Zn at concentrations near to these can be toxic to Salvinia. As the plant grows it is able to adsorb and accumulate metals, so the growth rate of plants is related to the amount of metal that can be removed from the environment. Olguín et al., (2002) evaluating bioconcentration in S. minima observed that macrophytes cultured for 5 days in solution with 4.00 mg L-1 Cd assimilated 10.930 mg g-1 in the tissues and obtained a BCF = 2718. The value found by the authors is close to calculated in our study (BCF0.300mg L Cd= 2,676 and BCF0.030mg
L Cd=
2,876). The highest removal rate of Cd occurred
when S. minima was cultivated in lower concentration (0.003 mg L-1). Also, the higher value of rm was observed compared to others concentrations of Cd. Analyzing the data in Figure 3, it can be affirmed that even at low concentrations, Cd is toxic to S. minima and despite the metal
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ACCEPTED MANUSCRIPT content in their tissues have increased along with the concentration of this metal in the environment (Figure 4) the plant is so sensitive to this metal that Txa not increased (Table 1). Regarding the cultures performed with Ni, the highest Txa (0.0595 mg m² dia-1) was observed when the concentration was 0.0200 mg L-1. The rm and Txa decreased even more when the Ni concentration was highest than 0.200 mg L-1 (Figure 3 and Table 1). Although growth
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coefficient have decreased as the Ni concentration increased a little growth was observed in cultured with 0.400 mg L-1 Ni (rm=0.0113 day-1). Ceribasi and Yetis, (2004) studied the absorption of Ni by a fungus (Phanerochaete chrysosporium) associated with the macrophyte roots and found high levels of Ni removal by these microorganisms (77.96 mg g-1, with 50 mg L1
Ni in solution). The use of S. minima aiming the Ni removal from industrial effluents should be
better studied, and preferably in consortium with other macrophytes or microorganisms that are able to remove this metal before passing through the S. minima cultivation. An alternative would be the consortium with Brassica juncea or Brassica carinata (Panwar et al., 2002); where the authors found the assimilation of 28.410 mg g-1 Ni. Once electroplating effluents have nickel, the presence of this element may limit the use of S. minima in order to treat such effluents. The phytochelatin cytoplasmic protein is involved in the mechanisms of metals bioaccumulation by cells of S. minima, the concentration of Pb causes higher production of this protein, they bind to toxic metals and form complexes that are stored within vacuoles and cellular compartments such as chloroplasts and thus the deleterious effects of toxic metals is reduced (Estrella-Gómez et al., 2009). The highest Pb removal rate was obtained when S. minima grown in 1.0 mg L-1 of this metal, despite the low growth (lower rm) higher accumulation
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ACCEPTED MANUSCRIPT capacity and adsorption of Pb allows larger Txa. Olguín et al. (2002), evaluating S. minima bioconcentration cultivated for 5 days in solution with 3.00 mg L-1 Pb, observed accumulation of 9.780 mg g-1 in tissues and obtained BCF = 3,304; these values are much higher than those observed in this study, when the same species was grown in 1.00 mg L-1 (R= 1.786 mg g-1 ; BCF = 1,786), other tests are needed to find the maximum factor bioaccumulation of Pb by this macrophyte; perhaps the difference between the values found by the two works lies in metal
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concentration in solution culture and time of contact with the contaminated medium, since in this experiment the plants were grown for about 60 days and no plant growth was observed and on the Olguín et al. work the plant remained in contact with the solution for 5 days and the system was not continuous flow. Olguín et al., (2005) cultivated S. minima in solutions with 12.9 mg L-1 Pb and found 27.473 mg g-1 accumulated metal in their tissue. These authors also found Pb adhered to the external tissues of the plant (adsorbed) and intracellular and interstitial (accumulated) region and concluded that S. minima can remove Pb from the environment through these two processes. The metals adsorption by macrophytes were also described by (Schneider & Rubio, 1999), the authors suggest that the external metal adsorption plant tissue is related to the ion exchange mechanisms and may be related to the carboxylic groups present in the cell wall. This mechanism becomes even more favorable to the use of S. minima aiming remove metals because the connections are made in the outer plant tissue, preventing the metal to become toxic by interfering in physiological processes.
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ACCEPTED MANUSCRIPT It was observed that the maximum metal removal rate (Txa) was found when S. minima was grown in 1 mg L-1 Zn (Txa = 0.4046 mg m-2 dia-1), from these data it is expected that at least one tank of the A = 2.472 m² (Eq. 4) has the ability to remove 1.0 mg per day of Zn if the concentration in the culture solution was 1.0 mg L-1 Zn and the residence time of 2 days. The same calculations can be made for other metals to determine the minimum area required for
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removal of Cd, Pb and Ni effluent considering the same experimental conditions (Eq. 3 and 4). As described, all rm values increased when the solution concentrations of Cd, Ni and Pb were lower. These results corroborate the indication of the macrophytes use for treatment of effluents contaminated with these metals since the macrophyte is able to reduce the concentrations of metals in the environment and also increase their ability to grow. According to the mathematical model prediction, the metal concentration in the culture medium greatly decreases the ability of S. minima growing, it is suggested to treat contaminated effluent by Cd, Ni and Pb the plants are first grown in a tank uncontaminated (thus plants could grow in the first tank). In a second phase, to be introduced in the second tank (contaminated) and withdrawn when they reach the maximum concentration of accumulation or adsorption in their tissues (RK). Olguín et al. (2005) suggest that the maximum capacity for metal accumulation by S. minima occurs in a 24 hour period. In a hypothetical situation, in an environment where the concentration of Zn is close to 10 mg L-1 does not occur growth of S. minima, the system will have greater efficiency at removing Zn if individuals are introduced covering the whole extent and removed from the tank when the content of Zn is near R = 10 mg g-1. In this case, the 100% cover of the tank is better than putting
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ACCEPTED MANUSCRIPT a smaller amount of plants to which the Zn is removed during plant growth. Therefore, the use of S. minima to phytoremediation on treatment plant sewage must consider the environmental conditions, available area, flow and concentration of nutrients and metals and other factors such as economic and ecological viability. In this context, additional tests are required to evaluate the growth of S. minima when more than one metal is present in the cultivation solution, because the interactions between metals may alter the ability of this plant act as phytoremediation and grow.
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Chemical options sewage treatment and concomitant use of chemical processes with biological (phytoremediation) should not be refused; the use of both can improve the ability to remove metals.
5. CONCLUSIONS S. minima was able to grow at concentrations of 0.030 mg L-1 Cd, 0.40 mg L-1 Ni, 1.00 mg L-1 Pb and 1.00 mg L-1 Zn tested alone. It was not necessary growth of S. minima occur to remove metals from the environment, i.e. when placed in contaminated environments they can adsorb and accumulate the metal from solution. This species can be used to phytoremediate wastewater contaminated by Cd, Ni, Pb e Zn. The rate of metal removal depends on the cultivation area and there is a direct relationship between this area and its efficiency.
6. ACKNOWLEDGMENTS The authors thanks the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (processes 556092/2010-4; 144436/2010-8), Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (process 2010/15728-1).
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ACCEPTED MANUSCRIPT Santos, G. A. dos. (2006). Crescimento e respostas antioxidantes de macrófitas aquáticas submetidas ao arsênio. Universidade Federal de Viçosa.
Schneider, I. A. H., & Rubio, J. (1999). Sorption of heavy metal ions by the nonliving biomass of freshwater macrophytes. Environmental Science & Technology, 33(13), 2213–2217. doi:10.1021/es981090z
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Sen, A. K., & Bhattacharyya, M. (1994). Studies of uptake and toxic effects of NI (II) on Salvinia natans. Water, Air, & Soil Pollution, 78(1-2), 141–152. doi:10.1007/BF00475673
Seregin, I. V., & Ivanov, V. B. (2001). Physiological aspects of cadmium and lead toxic effects on higher plants. Russian Journal of Plant Physiology, 48(4), 523–544. Sperling, M. von, & Chernicharo, C. A. de L. (2005). Biological Wastewater Treatment in Warm Climate Regions (p. 1460). Stottmeister, U., Wiebner, A., Kuschk, P., Kappelmeyer, U., Kästner, M., Bederski, O., … Moormann, H. (2003). Effects of plants and microorganisms in constructed wetlands for wastewater treatment. Biotechnology Advances, 22(1-2), 93–117. doi:10.1016/j.biotechadv.2003.08.010 Thomaz, S. M., & Bini, L. M. (2003). Ecologia e Manejo de Macrófitas Aquáticas (EDUEM., p. 341). Maringá. Tryon, R. M., & Tryon, A. F. (1982). Ferns and allied plants: with special reference to tropical America (Springer-V., p. 857).
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ACCEPTED MANUSCRIPT Vogels, M., Zoeckler, R., Stasiw, D. M., & Cerny, L. C. (1975). P. F. Verhulst’s “notice sur la loi que la populations suit dans son accroissement” from correspondence mathematique et physique. Journal of Biological Physics, 3(4), 183–192. doi:10.1007/BF02309004
Yokel, R., Lasley, S. M., & Dorman, D. C. (2006). The speciation of metals in mammals influences their toxicokinetics and toxicodynamics and therefore human health risk
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assessment. Journal of Toxicology and Environmental Health. Part B, Critical Reviews, 9(1), 63–85. doi:10.1080/15287390500196230 Zar, J. H. (2010). Biostatistical Analysis (5th ed., p. 947). New Jersey: Pearson Prentice Hall.
Zayed, A., Gowthaman, S. & Terry N. (1998). Phytoaccumulation of Trace Elements by Wetland Plants: I. Duckweed. Journal of Environmental Quality. 27:715-721. doi:10.2134/jeq1998.00472425002700030032x
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ACCEPTED MANUSCRIPT Table 1. Assimilation rate (Txa), maximum harvest of plants in fresh mass and bioconcentration factor (BCF) according to the metal concentration (C) in culture solution.
Metal
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Cd
Ni
Pb
Zn
C (mg L-1) Control - 0.000 0.003 0.030 0.300 Control - 0.000 0.020 0.200 0.400 Control - 0.00 0.01 0.10 1.00 Control - 0.00 0.10 1.00 10.00
Txa R rm (mg m-² day(mg g-1) (day-1) 1 ) 0.0000 0.0432 0.0000 0.0371 0.0257 0.0045 0.0863 0.0067 0.0027 0.8028 0.0000 0.0000 0.0021 0.0432 0.0004 0.1296 0.0235 0.0143 0.6772 0.0186 0.0595 0.3600 0.0113 0.0192 0.0045 0.0472 0.0010 0.0244 0.0356 0.0041 1.2571 0.0198 0.1173 1.7856 0.0169 0.1423 0.4806 0.0169 0.0383 0.6249 0.0210 0.0618 1.9856 0.0432 0.4046 10.4639 0.0000 0.0000
Maximum harvest (gF m-² day-1) 4.2198 2.5131 0.6588 0.0000 4.2198 2.2932 1.8210 1.1046 4.6137 3.4759 1.9325 1.6510 1.6500 2.0498 4.2198 0.0000
BCF
12353 2876 2676 6480 3386 900 2439 12571 1786 6249 1986 1046
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Table 2. Means of metals in effluents in mg L-1. Cd 0.003
International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
PHYTOREMEDIATION OF Cd, Ni, Pb AND Zn BY Salvinia minima a
Danilo Sinhei Iha & Irineu Bianchini Jr.
a
a
UFSCar - Universidade Federal de São Carlos, Departamento de Hidrobiologia, 13565-905 – Brazil - SP Accepted author version posted online: 07 Apr 2015.
Click for updates To cite this article: Danilo Sinhei Iha & Irineu Bianchini Jr. (2015): PHYTOREMEDIATION OF Cd, Ni, Pb AND Zn BY Salvinia minima, International Journal of Phytoremediation, DOI: 10.1080/15226514.2014.1003793 To link to this article: http://dx.doi.org/10.1080/15226514.2014.1003793
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ACCEPTED MANUSCRIPT PHYTOREMEDIATION OF Cd, Ni, Pb AND Zn BY Salvinia minima DANILO SINHEI IHA* and IRINEU BIANCHINI JR. UFSCar - Universidade Federal de São Carlos, Departamento de Hidrobiologia, 13565-905 – Brazil - SP *[email protected]
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ABSTRACT Most metals disperse easily in environments and can be bioconcentrated in tissues of many organisms causing risks to the health and stability of aquatic ecosystems even at low concentrations. The use of plants to phytoremediation has been evaluated to mitigate the environmental contamination by metals since they have large capacity to adsorb or accumulate these elements. In this study we evaluate Salvinia minima growth and its ability to accumulate metals. The plants were cultivated for about 60 days in different concentrations of Cd, Ni, Pb and Zn (tested alone) in controlled environmental conditions and availability of nutrients. The results indicated that S. minima was able to grow in low concentrations of selected metals (0.03 mg L-1 Cd, 0.40 mg L-1 Ni, 1.00 mg L-1 Pb and 1.00 mg L-1 Zn) and still able to adsorb or accumulate metals in their tissues when cultivated in higher concentrations of selected metals without necessarily grow. The maximum values of removal metal rates (mg m² day-1) for each metal (Cd = 0.0045, Ni = 0.0595, Pb = 0.1423 e Zn = 0.4046) are listed. We concluded that S. minima may be used as an additional tool for metals removal from effluent. KEYWORDS Aquatic macrophytes; effluent treatment; metals; bioaccumulation; continuous flow.
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ACCEPTED MANUSCRIPT 1. INTRODUCTION Metals have toxic potential even at low concentrations causing risks to human health. Due to their high dispersal ability metals can accumulate in the environment and in different organisms tissues (Yokel et al., 2006). In recent decades there was an increase of metal concentrations in many regions of the world and the industrial, domestic and agricultural effluents are the most important sources of aquatic environments contamination (Jordão et al.,
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2005). The metals are removed from effluent by wetlands and usually it happen on tertiary treatment where significant decrease occurs in various elements concentration (Hadad et al., 2006; Maine et al., 2006; Sperling & Chernicharo, 2005). Among the techniques for metals removal the plants use (phytoremediation) has emerged as a promising alternative (Moshiri, 1993). In recent decades several studies have demonstrated the effectiveness of aquatic macrophytes in wastewater treatment (Casabianca, 1995; Henry-silva et al., 2006; Moshiri, 1993; Stottmeister et al., 2003). Macrophytes not only accumulate metals in their tissues but also promote a variety of chemical and biochemical reactions that decrease pollution by increasing environmental diversity in the roots region (Jenssen et al., 1993). In this regard Salvinia minima has rapid growth and is found in different parts of the world (Bianchini Jr. et al., 2010; Tryon & Tryon, 1982). It is a floating aquatic macrophyte, native from South America, has great potential to remove metals from contaminated areas and high capacity to absorb cadmium and lead (Ali et al., 2013; Espinoza-Quiñones et al., 2005; Olguín et al., 2002; Oliveira et al., 2001).
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ACCEPTED MANUSCRIPT Plant growth models are mathematical representations of physiological processes associated to its metabolism (Best, 1990). Growth models have been considered important tool for understanding aquatic ecosystems processes and especially for predicting about the behavior of these systems (Thomaz & Bini, 2003). This study had as main aim the evaluate Salvinia minima growth in order to use it to phytoremediation of effluents contaminated with metals. The selection of this species was based on studies that test its ability to assimilate metals and its easy
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handling, (Ali et al., 2013; Hadad et al., 2006; Olguín et al., 2005; 2002; Santos, 2009; Santos, 2006).
2. MATERIAL AND METHODS The growth of Salvinia minima Baker cultivated in solutions with different concentrations of Cd, Ni, Pb and Zn was evaluated in triplicate. In total 720 individuals of similar size and stage of development were collected (21°59'17" S e 47°53'2" W; datum WGS84) and washed in a solution of sodium hypochlorite (NaClO 0.5%) for 5 min. Plants were cultivated for about 60 days in polyethylene recipients (217 x 145 x 70 mm) in germination chambers (COLDLAB) under controlled conditions (20 ºC, photoperiod: 12h and light intensity: 4.31x10-3 µmol cm-2 s-1). The culture system was kept under continuous flow (volume: 1.0L; flow: 0.35 mL min-1; residence time: 2 days) through peristaltic pump (MasterFlex, 07524-40, 12 channels) (Figure 1). All solutions were prepared with 15% culture medium Hoagland-Arnon (Hoagland and Arnon, 1950) and the pH was adjusted to 6.5 with sodium hydroxide (NaOH 50%) or hydrochloric acid (HCl 18%) and subsequently added different metals concentrations.
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ACCEPTED MANUSCRIPT To evaluate the growth of S. minima without metals exposed (control group), 180 individuals was cultivated in 12 recipients with 15 individuals each. Forty five individuals (cultivated in three recipients with 15 individuals each) were exposed to 90 L of solution with the following metals concentrations tested alone: 0.003, 0.030 and 0.300 mg L-1 Cd (CdCl2.2,5H2O); 0.02, 0.20 and 0.40 mg L-1 Ni (NiCl2.6H2O); 0.01, 0.10 and 1.00 mg L-1 Pb (PbCl2); and finally,
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0.1, 1.0 and 10.0 mg L-1 Zn (ZnSO4.7H2O). For the amount of Zn (0.008 mg L-1), normally present in the nutrient solution, did not interfere in the experiments that assessed the macrophyte growth cultivated with this metal only these experiments the culture medium was prepared without addition of ZnSO4.7H2O. The growths or decrease (death) of macrophytes were assessed twice a week and determined through measurements of leaf area by photographic
images using the Spring®
software (Iha & Bianchini Jr, 2013). The relationship between leaf area (cm²) and dry mass is described by the following mathematical expression: dry mass (g) = 0.001888 x leaf area; N=18; R² = 0.97. The values of leaf area and the time were fitted to a logistic model (Vogels et al., 1975; Eq. 1) and the growth coefficient values (rm) were obtained. =
(
)
(Eq. 1),
where: N = leaf área (cm²); t = time (days); K = maximum area (cm²); a = constant of integration and rm = growth coefficient (day-1).
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ACCEPTED MANUSCRIPT After the last day of cultivation the plants were removed from the solution, rinsed with deionized water and dried (at 45 °C) until constant weight. The dried material was subsequently digested and analyzes of the metal concentration in plants tissue were carried out (it was considered the metals content in the macrophyte the metal adsorbed, or adhered to the outer wall tissue of the plant and the accumulated metal that has been assimilated by the plant and is found in the intracellular or interstitial region), the method used was optical emission spectrometry with
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inductively coupled plasma using equipment ICP-OES, Optima model 8000 Perkin Elmer® (ref. SMEWW 21 3030 E). Concentrations of metals in solution culture were also determined by the same method and equipment. These collections were performed once a week during the experimental period. To assess the bioaccumulation of metals in plant tissue the bioconcentration factor (BCF) was calculated according to Zayed et al. (1998); (Eq. 2). (
=
) (
)
Eq. 2,
To evaluate the tissue metal content according to the concentration of metal in the solution the Eq. 3 was used (Monod,1949). R=
Eq. 3,
where, R = metal content in plant tissue (mg g-1); C= metal concentration in the solution (mg L-1); RK = maximum plant tissue content (mg g-1); C1= metal concentration for 50% of RK.
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ACCEPTED MANUSCRIPT The calculation of removal metal rate can be used to determine the efficiency of phytoremediation by S. minima at different growth stages depending on the growing tank area, the density and the solution metal concentration. Based on these calculations can be suggested guidelines to identify when these plants can be used as a tool to reduce the wastewater metals concentration. The removal metal rate in milligrams per day is given by Eq. 4, adapted from
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Gutiérrez et al., (2001) and Mahujchariyawong and Ikeda, (2001): =
×(
−
²) ×
Eq. 4,
where, Txa = removal metal rate (mg day-1); R= metal content in plant tissue (mg g-1); D = 529.7 cm² (area occupied by 1 g of dry mass basis of S. minima); rm= growth coefficient (day-1); b= percentage of the tank area covered by S. minima (%); A = total area of cultivation tank (cm²). All parameters of this study (Eq. 1 and 3) were obtained by non-linear regressions, calculated with the algorithm interactive Levenberg-Marquardt (Press et al., 1993). The calculations were presented where the statistical probability of random data are correlated (p) is less than 5% (p0.05, Table 2). This result was expected, since the experimental strategy was just to assess the growth of S. minima in constant concentrations. The Zn concentration was lower than expected, maybe because this metal is an essential macro nutrient. However, aiming the use of this species to phytoremediation, the cultivation tank size should be adjusted to the flows and concentrations of
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metals for the residence time associated with metal removal rate (Txa) and growth coefficient (rm) allow increased efficiency in treating wastewater contaminated. The values of rm were lower where added any concentrations of Cd, Ni and Pb on solution comparing to the control group (rmcontrol= 0.0567+0.0100 dia-1). Excess of Cd interferes in the synthesis of compounds rich in thiol groups, causes a decrease in photosynthesis, reducing the absorption and transport of nutrients, damage to membranes, hormonal disorders and abnormal activity of several enzymes resulting in eventual death of plants (Barceló & Poschenrieder, 1990; Oliveira et al., 2001). In most cases, the inhibition by Cd and Pb results on the interaction between metals and enzymes sulphydryl groups (-SH), blocking these enzymes inhibits the activity of over 100 enzymes, these interactions negatively affect the function of these enzymes as alter the stability of its tertiary structure, and affect the absorption of essential nutrients such as N, K, among others (Seregin & Ivanov, 2001). Sen and Bhattacharyya (1994) cultivated S. natans in different concentrations of Ni and concluded that concentrations of 0.010 mg L-1 are toxic to this species, photosynthesis and chlorophyll-a decreases as a result of the conversion of chlorophyll to pheophytin, this reduction may be related to inactivation of the enzymes sulphydryl groups needed for the synthesis of fatty acids. In this study there was low
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ACCEPTED MANUSCRIPT growth of S. minima cultivated at 0.40 and 0.20 mg L-1 Ni (Figures 1 and 2). Reductions in photosynthetic pigments when exposed to metals have been reported by several authors for others Salvinia species (Al-Hamdani & Blair, 2004; Hadad et al., 2007; Nichols et al., 2000). With respect to Zn, the rm values increased until the concentration of 1.0 mg L-1 (rm= 0.0571+0.0034 dia-1). This result may be related to the fact that Zn is an essential
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micronutrient for the growth of various plant species and plays an important role in the activation of specific enzymes such as peptidases, dehydrogenases, proteinases and fosfohidrolases (Mehra & Farago, 2008). No growth occurred for cultivation at 10 mg L-1 Zn and therefore this concentration is toxic to S. minima. Dhir et al., (2008) evaluated changes in photochemical activities of Salvinia natans when exposed to concentrations of 50 mg L-1 Zn and observed changes in potential carbon assimilation by ribulose-1, 5 carboxylase / oxygenase activity, indicating that Zn at concentrations near to these can be toxic to Salvinia. As the plant grows it is able to adsorb and accumulate metals, so the growth rate of plants is related to the amount of metal that can be removed from the environment. Olguín et al., (2002) evaluating bioconcentration in S. minima observed that macrophytes cultured for 5 days in solution with 4.00 mg L-1 Cd assimilated 10.930 mg g-1 in the tissues and obtained a BCF = 2718. The value found by the authors is close to calculated in our study (BCF0.300mg L Cd= 2,676 and BCF0.030mg
L Cd=
2,876). The highest removal rate of Cd occurred
when S. minima was cultivated in lower concentration (0.003 mg L-1). Also, the higher value of rm was observed compared to others concentrations of Cd. Analyzing the data in Figure 3, it can be affirmed that even at low concentrations, Cd is toxic to S. minima and despite the metal
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ACCEPTED MANUSCRIPT content in their tissues have increased along with the concentration of this metal in the environment (Figure 4) the plant is so sensitive to this metal that Txa not increased (Table 1). Regarding the cultures performed with Ni, the highest Txa (0.0595 mg m² dia-1) was observed when the concentration was 0.0200 mg L-1. The rm and Txa decreased even more when the Ni concentration was highest than 0.200 mg L-1 (Figure 3 and Table 1). Although growth
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coefficient have decreased as the Ni concentration increased a little growth was observed in cultured with 0.400 mg L-1 Ni (rm=0.0113 day-1). Ceribasi and Yetis, (2004) studied the absorption of Ni by a fungus (Phanerochaete chrysosporium) associated with the macrophyte roots and found high levels of Ni removal by these microorganisms (77.96 mg g-1, with 50 mg L1
Ni in solution). The use of S. minima aiming the Ni removal from industrial effluents should be
better studied, and preferably in consortium with other macrophytes or microorganisms that are able to remove this metal before passing through the S. minima cultivation. An alternative would be the consortium with Brassica juncea or Brassica carinata (Panwar et al., 2002); where the authors found the assimilation of 28.410 mg g-1 Ni. Once electroplating effluents have nickel, the presence of this element may limit the use of S. minima in order to treat such effluents. The phytochelatin cytoplasmic protein is involved in the mechanisms of metals bioaccumulation by cells of S. minima, the concentration of Pb causes higher production of this protein, they bind to toxic metals and form complexes that are stored within vacuoles and cellular compartments such as chloroplasts and thus the deleterious effects of toxic metals is reduced (Estrella-Gómez et al., 2009). The highest Pb removal rate was obtained when S. minima grown in 1.0 mg L-1 of this metal, despite the low growth (lower rm) higher accumulation
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ACCEPTED MANUSCRIPT capacity and adsorption of Pb allows larger Txa. Olguín et al. (2002), evaluating S. minima bioconcentration cultivated for 5 days in solution with 3.00 mg L-1 Pb, observed accumulation of 9.780 mg g-1 in tissues and obtained BCF = 3,304; these values are much higher than those observed in this study, when the same species was grown in 1.00 mg L-1 (R= 1.786 mg g-1 ; BCF = 1,786), other tests are needed to find the maximum factor bioaccumulation of Pb by this macrophyte; perhaps the difference between the values found by the two works lies in metal
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concentration in solution culture and time of contact with the contaminated medium, since in this experiment the plants were grown for about 60 days and no plant growth was observed and on the Olguín et al. work the plant remained in contact with the solution for 5 days and the system was not continuous flow. Olguín et al., (2005) cultivated S. minima in solutions with 12.9 mg L-1 Pb and found 27.473 mg g-1 accumulated metal in their tissue. These authors also found Pb adhered to the external tissues of the plant (adsorbed) and intracellular and interstitial (accumulated) region and concluded that S. minima can remove Pb from the environment through these two processes. The metals adsorption by macrophytes were also described by (Schneider & Rubio, 1999), the authors suggest that the external metal adsorption plant tissue is related to the ion exchange mechanisms and may be related to the carboxylic groups present in the cell wall. This mechanism becomes even more favorable to the use of S. minima aiming remove metals because the connections are made in the outer plant tissue, preventing the metal to become toxic by interfering in physiological processes.
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ACCEPTED MANUSCRIPT It was observed that the maximum metal removal rate (Txa) was found when S. minima was grown in 1 mg L-1 Zn (Txa = 0.4046 mg m-2 dia-1), from these data it is expected that at least one tank of the A = 2.472 m² (Eq. 4) has the ability to remove 1.0 mg per day of Zn if the concentration in the culture solution was 1.0 mg L-1 Zn and the residence time of 2 days. The same calculations can be made for other metals to determine the minimum area required for
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removal of Cd, Pb and Ni effluent considering the same experimental conditions (Eq. 3 and 4). As described, all rm values increased when the solution concentrations of Cd, Ni and Pb were lower. These results corroborate the indication of the macrophytes use for treatment of effluents contaminated with these metals since the macrophyte is able to reduce the concentrations of metals in the environment and also increase their ability to grow. According to the mathematical model prediction, the metal concentration in the culture medium greatly decreases the ability of S. minima growing, it is suggested to treat contaminated effluent by Cd, Ni and Pb the plants are first grown in a tank uncontaminated (thus plants could grow in the first tank). In a second phase, to be introduced in the second tank (contaminated) and withdrawn when they reach the maximum concentration of accumulation or adsorption in their tissues (RK). Olguín et al. (2005) suggest that the maximum capacity for metal accumulation by S. minima occurs in a 24 hour period. In a hypothetical situation, in an environment where the concentration of Zn is close to 10 mg L-1 does not occur growth of S. minima, the system will have greater efficiency at removing Zn if individuals are introduced covering the whole extent and removed from the tank when the content of Zn is near R = 10 mg g-1. In this case, the 100% cover of the tank is better than putting
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ACCEPTED MANUSCRIPT a smaller amount of plants to which the Zn is removed during plant growth. Therefore, the use of S. minima to phytoremediation on treatment plant sewage must consider the environmental conditions, available area, flow and concentration of nutrients and metals and other factors such as economic and ecological viability. In this context, additional tests are required to evaluate the growth of S. minima when more than one metal is present in the cultivation solution, because the interactions between metals may alter the ability of this plant act as phytoremediation and grow.
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Chemical options sewage treatment and concomitant use of chemical processes with biological (phytoremediation) should not be refused; the use of both can improve the ability to remove metals.
5. CONCLUSIONS S. minima was able to grow at concentrations of 0.030 mg L-1 Cd, 0.40 mg L-1 Ni, 1.00 mg L-1 Pb and 1.00 mg L-1 Zn tested alone. It was not necessary growth of S. minima occur to remove metals from the environment, i.e. when placed in contaminated environments they can adsorb and accumulate the metal from solution. This species can be used to phytoremediate wastewater contaminated by Cd, Ni, Pb e Zn. The rate of metal removal depends on the cultivation area and there is a direct relationship between this area and its efficiency.
6. ACKNOWLEDGMENTS The authors thanks the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (processes 556092/2010-4; 144436/2010-8), Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (process 2010/15728-1).
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ACCEPTED MANUSCRIPT 7. REFERENCES
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ACCEPTED MANUSCRIPT Hadad, H. R., Maine, M. A., & Bonetto, C. A. (2006). Macrophyte growth in a pilot-scale constructed wetland for industrial wastewater treatment. Chemosphere, 63(10), 1744–1753. doi:10.1016/j.chemosphere.2005.09.014
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ACCEPTED MANUSCRIPT Table 1. Assimilation rate (Txa), maximum harvest of plants in fresh mass and bioconcentration factor (BCF) according to the metal concentration (C) in culture solution.
Metal
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Cd
Ni
Pb
Zn
C (mg L-1) Control - 0.000 0.003 0.030 0.300 Control - 0.000 0.020 0.200 0.400 Control - 0.00 0.01 0.10 1.00 Control - 0.00 0.10 1.00 10.00
Txa R rm (mg m-² day(mg g-1) (day-1) 1 ) 0.0000 0.0432 0.0000 0.0371 0.0257 0.0045 0.0863 0.0067 0.0027 0.8028 0.0000 0.0000 0.0021 0.0432 0.0004 0.1296 0.0235 0.0143 0.6772 0.0186 0.0595 0.3600 0.0113 0.0192 0.0045 0.0472 0.0010 0.0244 0.0356 0.0041 1.2571 0.0198 0.1173 1.7856 0.0169 0.1423 0.4806 0.0169 0.0383 0.6249 0.0210 0.0618 1.9856 0.0432 0.4046 10.4639 0.0000 0.0000
Maximum harvest (gF m-² day-1) 4.2198 2.5131 0.6588 0.0000 4.2198 2.2932 1.8210 1.1046 4.6137 3.4759 1.9325 1.6510 1.6500 2.0498 4.2198 0.0000
BCF
12353 2876 2676 6480 3386 900 2439 12571 1786 6249 1986 1046
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Table 2. Means of metals in effluents in mg L-1. Cd 0.003
