Evaluation of the potential of Atriplex halimus stem cuttings for phytoremediation of metal-polluted soils

1 Evaluation of the potential of Atriplex halimus stem cuttings for phytoremediation of metal- 1 polluted soils 2 3 Juan M. Mancilla-Leytón, María José Navarro-Ramos, Sara Muñoz-Vallés, M. Enrique Figueroa 4 and Jesús Cambrollé* 5 6 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, 7 Apartado 1095, 41080 - Sevilla, España 8 9 *Corresponding author. 10 Postal address: Jesús Cambrollé Silva, Dpto. Biología Vegetal y Ecología, Facultad de Biología, 11 Universidad de Sevilla, Av. Reina Mercedes 6, 41012 Seville, Spain. 12 Tel.: +34-95-4557165; fax: +34-95-4615780. E-mail address: [email protected] 13 14 Note: J.M. Mancilla-Leytón and M.J. Navarro-Ramos contributed equally to this work. 15 16 17 18 19 20 21 22 23 24 25 26 2 Abstract 1 The use of cuttings in the restoration of degraded ecosystems presents important advantages 2 compared to seed sowing. The applicability of cuttings from some coastal plants for the 3 recovery of soils contaminated with heavy metals has been recently recognised. A greenhouse 4 experiment was carried out to analyse the effects of a concentration range of copper (0-9 5 mmol l-1) on the establishment, growth and photosynthetic performance of Atriplex halimus 6 cuttings, in order to determine the phytotoxicity threshold values of plants from cuttings and 7 assess the possible physiological effects of this metal. The cuttings from the study species 8 showed the ability to survive, root and grow under Cu concentrations of up to 9 mmol l-1. 9 Although no effects were observed in the photosynthetic apparatus, concentrations of 4.5 10 mmol l-1 and higher caused a decrease in growth, net photosynthetic rate and rooting 11 percentages. The present study allows concluding that the use of cuttings from Atriplex 12 halimus could be a valuable and efficient tool for the restoration of vegetation in Cu-polluted 13 coastal ecosystems. 14 15 16 17 18 19 20 21 Keywords: Heavy metals; phytoremediation; wetland restoration. 22 23 3 1. Introduction 1 The salt marshes, estuaries and nearby areas receive all the load of sediments and 2 pollutants that have been dragged along the river and its basin. The pollutants may have 3 different origins, even natural ones coming from the river basin itself. Salt marshes and 4 estuaries usually present high concentrations of pollutants, mainly heavy metals, due to 5 anthropologic activities that take place in estuaries or in contiguous areas, like mining, 6 refineries and oil stations, among others (Hong-yun et al., 2005; Sousa et al., 2008). At the 7 present time, eutrophication and chemical pollution are affecting salt marshes all over the 8 world. The main ecological issues resulting from pollution are the introduction of heavy metals 9 in the food cycle of marshland and estuary ecosystems, and bioamplification in the different 10 trophic levels (Luque et al., 1999). 11 There is a large number of studies focused on the presence of heavy metals in soils of 12 coastal areas (Almeida et al., 2004; Allen et al., 1990; Caçador et al., 1996; Mortimer and Rae, 13 2000). The sediments dragged by the rivers and accumulated in the marshlands contribute to 14 increase the pollution of these areas. This is the case of the pyrite belt in Sierra Morena, which 15 is linked to the marshes of the Odiel and Tinto rivers. The watercourse of these rivers has a 16 high concentration of heavy metals that might affect the local plants near the area. Factors 17 such as pH, soil texture, organic matter and clay content, and redox potential, influence the 18 bioavailability and solubility of metals in plants (Greguer, 2004). 19 Copper is one of the main heavy metals found in the marsh due to the high mining 20 activity in the local area, the processing of metals, and the use of fertilizers and fungicides, 21 among other actions (Kabata-Pendias and Pendias, 2001). Even though it is an essential trace 22 element, involved in many different biochemical and physiological processes, such as 23 photosynthesis and breathing (Barón et al., 1995; Pilon et al., 2006), it is toxic when in high 24 concentration for most plants (Dewez et al., 2005). It has been observed that Cu overload 25 4 affects growth, photosynthetic activity and even the respiration process (Nalewajko and 1 Olaveson, 1995). However, some plants are able to grow in soils polluted with Cu and 2 accumulate a high amount of it with no marked impact (Ernst et al., 2000; Kabata-Pendias and 3 Pendias, 2001). 4 Recent studies prove that some coastal plants present an inherent tolerance to heavy 5 metals (McCabe et al., 2001). Phytoremediation, the use of plants to extract, sequester and/or 6 detoxify pollutants, is widely viewed as the ecological alternative to the environmentally 7 destructive physical remediation methods currently practiced (Meagher, 2000; Garbisu et al., 8 2002). This technology requires knowledge of some biological aspects of the species to be 9 used. 10 Most of the dominant plant species in salt marshes are halophytic, which means they 11 are resistant to high concentrations of salt that are commonly found in the marshland systems. 12 Halimione portulacoides, Atriplex halimus, Salicornia ramosissima and Spartina maritima are 13 among these type of plants. Atriplex halimus is a perennial shrub, original from arid and semi- 14 arid zones of the Mediterranean, from the Chenopodiaceae family. This halophytic plant grows 15 in both emerged areas in marshlands (Fernández-Illescas et al., 2010b) and sandy soil (López 16 González, 2001), and it can tolerate high salinity levels (Bajji et al., 1998), light stress (Streb et 17 al., 1997) and drought (Martinez et al., 2005). Recent studies have proved that Atripex halimus 18 can tolerate and accumulate high concentrations of heavy metals in its tissues, such as Cd, Zn 19 (Lutts et al., 2004) and Cu (Mateos-Naranjo et al., 2013), which indicates that it could be viable 20 for phytoremediation techniques. 21 Most of the plants used in phytoremediation techniques are from greenhouses, where 22 they are spread from seeds. Recently, Andrades et al., (2013) tested, for the first time, the 23 capability of a plant species to grow under high concentrations of heavy metals, proving the 24 viability of stem cuttings to restore polluted soils. The use of stem cuttings in 25 5 phytoremediation might have potential advantages over the use of adult plants (such as less 1 time and resources required); however, some key factors regarding this method have not been 2 studied yet. 3 The main objective of this study was to evaluate Cu tolerance in stem cuttings of A. 4 halimus. The specific objectives were: (i) to determine the Cu phytotoxicity thresholds of stem 5 cuttings of the study species, by analyzing the establishment, survival and growth of plants in a 6 range of external Cu concentrations, from 0 to 9 mmol l-1; and (ii) to ascertain the extent to 7 which Cu determines plant performance in plants grown from stem cuttings, in terms of 8 influence on the photosynthetic apparatus (PSII chemistry) and gas exchange characteristics. 9 10 2. Materials and methods 11 2.1 Plant material and copper treatments 12 Seeds of A. halimus were collected in the salt marshes of "La Mata-Torrevieja" 13 (Alicante, SE Spain). The collected seeds were subsequently germinated in perlite moistened 14 with distilled water, and maintained at 25ºC for 30 days. The resulting seedlings were sown in 15 individual plastic pots (with a diameter of 11 cm) filled with perlite, and placed in a glasshouse 16 with minimum-maximun temperatures of 21-25 ºC, 40-60% relative humidity and natural 17 daylight (minimun and maximun ligth flux: 200 and 1000 μmol m -2 s -1, respectively). Pots 18 were carefully irrigated with 20% Hoagland´ solution (Hoagland y Arnon, 1938) as required. 19 After 5 months of growth, when seedlings were between 40 and 50 cm in height, 20 apical stem cuttings of 15 cm were taken from the seedlings, with 5 nodes (including the apical 21 node). The cuttings were allocated to individual plastic pots filled with perlite moistened and a 22 deep of 4 cm. 23 6 The pots were allocated to four different Cu concentration treatments: 0, 1.5, 4.5 y 9 1 mmol l-1, applied in shallow trays within the same glasshouse (ten pots per tray, one tray per 2 Cu treatment). Cu treatments were prepared by mixing 20% Hoagland´s solution with CuSO4 · 3 5H2O of the appropriate concentration. The control, 0 mmol l-1Cu treatment, in fact contained 4 0.0005 mmol l-1of Cu, since Hoagland´s solution contains a small amount of Cu as an essential 5 trade nutrient. These Cu concentrations were chosen in order to reflect the range of levels 6 found by several authors in studies of the salt-marshes of different metal polluted estuaries 7 (Cambrollé et al., 2008; Mateos-Naranjo et al., 2013; Sáinz y Ruiz, 2006), as well as in previous 8 experiments to determine the phytotoxicity threshold of stem cutting of A. halimus. 9 At the beginning of the experiment, a 3 L volume of the appropriate solution was 10 placed in each of the trays to a market depth of 1 cm. Throughout the experiment, solution 11 levels in the trays were monitored and topped up to the marked level with 20% Hoagland´s 12 solution, (with no addition CuSO4 · 5H2O ), in order to limit the change in Cu concentration due 13 to evaporation of the water in the nutrient solution. In addition, the entire solution (including 14 Cu- CuSO4 · 5H2O) was changed on a weekly basis. 15 16 2.2 Growth 17 At the beginning of the experiment, 10 stem cuttings were taken and dried at 80ºC for 18 48 h and then weighed in order to obtain the initial dry mass. At the end of the experiment 19 (i.e. after 50 days of treatment), all the plants from each treatment were taken and the same 20 methodology was followed in order to obtain the final dry mass. 21 The relative growth rate (RGR) of whole plants was calculated using the formula: 22 RGR = (ln Bf – ln Bi) · D-1 (g g-1day-1) 23 7 where: 1 Bf =final dry mass; Bi =initial dry mass ; D =duration of experiment (days). 2 Plant development was monitored by a weekly count of the number of leaves. Plant 3 height was measured from the base of the stem to the tip of the uppermost leaf. Leaf area was 4 determined from the projected area by scanning and digitalising the leaves (Epson V30, Seiko 5 Epson Corp., Nagano, Japan), and using appropriate software (MideBMP v. 4.2.; Ordiales-Plaza, 6 2000) for processing and analysis. 7 8 2.3 Chlorophyll fluorescence 9 Chlorophyll fluorescence was measured in randomly selected, fully developed leaves 10 (n = 10; one measurement per plant for each treatment) using a portable modulated 11 fluorimeter (FMS-2, Hansatech Instruments Ltd., England) at the end of the experimental 12 period, following the methods described in Cambrollé et al. (2012). Values of variable 13 fluorescence (Fv = Fm - F0) and maximum quantum efficiency of PSII photochemistry (Fv/Fm) 14 were calculated from F0 and Fm. This ratio of variable to maximal fluorescence correlates with 15 the number of functional PSII reaction centres, and dark adapted values of Fv/Fm can be used 16 to quantify photoinhibition (Krivosheeva et al., 1996). 17 18 2.4 Gas exchange 19 Gas exchange measurements were taken from randomly selected, fully expanded 20 leaves (n = 10, one measurement per plant), following 50 days of treatment, using an infrared 21 gas analyzer in an open system (LI-6400, LI-COR Inc., Neb., USA). Net photosynthetic rate (A) 22 was determined at an ambient CO2 concentration of 400 µmol mol-1, temperature of 20/25ºC, 23 8 50 ± 5% relative humidity and a photon flux density of 1000 µmol m-2 s-1. Values of the 1 parameters were calculated using the standard formulae of Von Caemmerer and Farquhar 2 (1981). 3 4 2.5 Photosynthetic pigments 5 At the end of the experimental period, photosynthetic pigments were extracted from 6 fully expanded leaves from plants grown under each treatment, using 0.05 g of fresh plant 7 material in 10 ml of 80% aqueous acetone (n = 10, one measurement per plant). After filtering, 8 1 ml of the suspension was diluted with a further 2 ml of acetone, and the chlorophyll a (Chl a) 9 and chlorophyll b (Chl b) contents were each determined with a Hitachi U-2001 10 spectrophotometer (Hitachi Ltd, Japan), using three wavelengths (663.2, 646.8 and 470.0 nm). 11 Pigment concentrations (µg g-1 fwt) were calculated following the method of Lichtenthaler 12 (1987). 13 14 2.6 Statistical analysis 15 Statistical analysis was carried out using Statistica v. 6.0 (Statsoft Inc.). Pearson and 16 Spearman coefficients were calculated to assess the correlation between different variables. 17 Data were analyzed using one- and two-way analysis of variance (F-test). Data were tested for 18 normality with the Kolmogorov-Smirnov test and for homogeneity of variance with the Brown- 19 Forsythe test. Tukey tests were applied to significant test results for identification of important 20 contrasts. 21 22 23 3. Results 24 9 3.1 Survival 1 The survival rate of the stem cuttings in the control treatment and under 2 concentrations of 1.5 and 4.5 mmol l-1 Cu was 100%. However, this rate decreased to 60% with 3 a concentration of 9 mmol l-1 Cu. Symptoms of chlorosis and leaf necrosis were observed in the 4 highest Cu treatments from the second half of the experimental period. 5 6 3.2 Growth analysis 7 The Relative Growth Rate (RGR) decreased as the concentration of Cu increased above 8 1.5 mmol l-1 Cu (r=-0.567, p<0.01). The reductions of RGR registered in the treatments of 1.5, 9 4.5 and 9 mmol l-1 Cu were 6, 33 and 70%, respectively, compared to the control treatment. No 10 relevant differences between the control treatment and 1.5 mmol l-1 Cu were observed (t-test, 11 p>0.05; Fig. 1A). The plant height values of 0 and 1.5 mmol l-1 Cu treatments did not show 12 relevant differences between them, as well as between the treatments of 4.5 and 9 mmol l-1 13 Cu (ANOVA, Tukey test, p>0.05). Height values of 4.5 and 9 mmol l-1 Cu were significantly lower 14 than the values in 0 and 1.5 mmol l-1 Cu (ANOVA, Tukey test, p<0.05; Fig. 1B). 15 Shoot and root dry weight showed a tendency to decrease with increasing external 16 concentration of Cu, with no significant differences between the recorded values in the control 17 treatment and 1.5 mmol l-1 Cu (ANOVA, Tukey test, p> 0.05 in both cases). 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