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Evaluation of the potential of Atriplex halimus stem cuttings for phytoremediation of metal-polluted soils
Ecological Engineering 97 (2016) 553–557
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Evaluation of the potential of Atriplex halimus stem cuttings for phytoremediation of metal-polluted soils ˜ Juan M. Mancilla-Leytón 1 , María José Navarro-Ramos 1 , Sara Munoz-Vallés, M. Enrique Figueroa, Jesús Cambrollé ∗ Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080, Sevilla, Spain
a r t i c l e
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Article history: Received 15 June 2016 Received in revised form 5 September 2016 Accepted 11 October 2016 Available online 18 October 2016 Keywords: Heavy metals Phytoremediation Wetland restoration
a b s t r a c t The use of cuttings in the restoration of degraded ecosystems presents important advantages compared to seed sowing. The applicability of cuttings from some coastal plants for the recovery of soils contaminated with heavy metals has been recently recognised. A greenhouse experiment was carried out to analyse the effects of a concentration range of copper (0–9 mmol l−1 ) on the establishment, growth and photosynthetic performance of Atriplex halimus cuttings, in order to determine the phytotoxicity threshold values of plants from cuttings and assess the possible physiological effects of this metal. The cuttings from the study species showed the ability to survive, root and grow under Cu concentrations of up to 9 mmol l−1 . Although no effects were observed in the photosynthetic apparatus, concentrations of 4.5 mmol l−1 and higher caused a decrease in growth, net photosynthetic rate and rooting percentages. The present study allows concluding that the use of cuttings from Atriplex halimus could be a valuable and efficient tool for the restoration of vegetation in Cu-polluted coastal ecosystems. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The salt marshes, estuaries and nearby areas receive all the load of sediments and pollutants that have been dragged along the river and its basin. The pollutants may have different origins, even natural ones coming from the river basin itself. Salt marshes and estuaries usually present high concentrations of pollutants, mainly heavy metals, due to anthropologic activities that take place in estuaries or in contiguous areas, like mining, refineries and oil stations, among others (Hong-Yun et al., 2005; Sousa et al., 2008). At the present time, eutrophication and chemical pollution are affecting salt marshes all over the world. The main ecological issues resulting from pollution are the introduction of heavy metals in the food cycle of marshland and estuary ecosystems, and bioamplification in the different trophic levels. There is a large number of studies focused on the presence of heavy metals in soils of coastal areas (Almeida et al., 2004; Allen et al., 1990; Cac¸ador et al., 1996; Mortimer and Rae, 2000). The sediments dragged by the rivers and accumulated in the marshlands contribute to increase the pollution of these areas. This is the case of the pyrite belt in Sierra Morena, which is linked to the marshes
∗ Corresponding author at: Dpto. Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Av. Reina Mercedes 6, 41012 Seville, Spain. E-mail address: [email protected] (J. Cambrollé). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ecoleng.2016.10.062 0925-8574/© 2016 Elsevier B.V. All rights reserved.
of the Odiel and Tinto rivers. The watercourse of these rivers has a high concentration of heavy metals that might affect the local plants near the area. Factors such as pH, soil texture, organic matter and clay content, and redox potential, influence the bioavailability and solubility of metals in plants (Greger, 2004). Copper is one of the main heavy metals found in the marsh due to the high mining activity in the local area, the processing of metals, and the use of fertilizers and fungicides, among other actions (Kabata-Pendias and Pendias, 2001). Even though it is an essential trace element, involved in many different biochemical and physiological processes, such as photosynthesis and breathing (Barón et al., 1995; Pilon et al., 2006), it is toxic when in high concentration for most plants (Dewez et al., 2005). It has been observed that Cu overload affects growth, photosynthetic activity and even the respiration process (Nalewajko and Olaveson, 1995). However, some plants are able to grow in soils polluted with Cu and accumulate a high amount of it with no marked impact (Ernst et al., 2000; Kabata-Pendias and Pendias, 2001). Recent studies prove that some coastal plants present an inherent tolerance to heavy metals (McCabe et al., 2001). Phytoremediation, the use of plants to extract, sequester and/or detoxify pollutants, is widely viewed as the ecological alternative to the environmentally destructive physical remediation methods currently practiced (Meagher, 2000; Garbisu et al., 2002). This technology requires knowledge of some biological aspects of the species to be used.
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Most of the dominant plant species in salt marshes are halophytic, which means they are resistant to high concentrations of salt that are commonly found in the marshland systems. Halimione portulacoides, Atriplex halimus, Salicornia ramosissima and Spartina maritima are among these type of plants. Atriplex halimus is a perennial shrub, original from arid and semi-arid zones of the Mediterranean, from the Chenopodiaceae family. This halophytic plant grows in both emerged areas in marshlands (FernándezIllescas et al., 2010) and sandy soil (López González, 2001), and it can tolerate high salinity levels (Bajji et al., 1998), light stress (Streb et al., 1997) and drought (Martínez et al., 2005). Recent studies have proved that Atripex halimus can tolerate and accumulate high concentrations of heavy metals in its tissues, such as Cd, Zn (Lutts et al., 2004) and Cu (Mateos-Naranjo et al., 2013), which indicates that it could be viable for phytoremediation techniques. Most of the plants used in phytoremediation techniques are from greenhouses, where they are spread from seeds. Recently, Andrades-Moreno et al. (2013) tested, for the first time, the capability of a plant species to grow under high concentrations of heavy metals, proving the viability of stem cuttings to restore polluted soils. The use of stem cuttings in phytoremediation might have potential advantages over the use of adult plants (such as less time and resources required); however, some key factors regarding this method have not been studied yet. The main objective of this study was to evaluate Cu tolerance in stem cuttings of A. halimus. The specific objectives were: (i) to determine the Cu phytotoxicity thresholds of stem cuttings of the study species, by analysing the establishment, survival and growth of plants in a range of external Cu concentrations, from 0 to 9 mmol l−1 ; and (ii) to ascertain the extent to which Cu determines plant performance in plants grown from stem cuttings, in terms of influence on the photosynthetic apparatus (PSII chemistry) and gas exchange characteristics.
Ruiz, 2006Sáinz y Ruiz, 2006), as well as in previous experiments to determine the phytotoxicity threshold of stem cutting of A. halimus. At the beginning of the experiment, a 3 l volume of the appropriate solution was placed in each of the trays to a market depth of 1 cm. Throughout the experiment, solution levels in the trays were monitored and topped up to the marked level with 20% Hoagland´ıs solution, (with no addition CuSO4 · 5H2 O), in order to limit the change in Cu concentration due to evaporation of the water in the nutrient solution. In addition, the entire solution (including CuCuSO4 · 5H2 O) was changed on a weekly basis.
2. Materials and methods
2.3. Chlorophyll fluorescence
2.1. Plant material and copper treatments
Chlorophyll fluorescence was measured in randomly selected, fully developed leaves (n = 10; one measurement per plant for each treatment) using a portable modulated fluorimeter (FMS-2, Hansatech Instruments Ltd., England) at the end of the experimental period, following the methods described in Cambrollé et al. (2012). Values of variable fluorescence (Fv = Fm − F0 ) and maximum quantum efficiency of PSII photochemistry (Fv /Fm ) were calculated from F0 and Fm . This ratio of variable to maximal fluorescence correlates with the number of functional PSII reaction centres, and dark adapted values of Fv /Fm can be used to quantify photoinhibition (Krivosheeva et al., 1996).
Seeds of A. halimus were collected in the salt marshes of “La Mata-Torrevieja” (Alicante, SE Spain). The collected seeds were subsequently germinated in perlite moistened with distilled water, and maintained at 25 ◦ C for 30 days. The resulting seedlings were sown in individual plastic pots (with a diameter of 11 cm) filled with perlite, and placed in a glasshouse with minimummaximun temperatures of 21–25 ◦ C, 40–60% relative humidity and natural daylight (minimun and maximun ligth flux: 200 and 1000 mol m−2 s−1 , respectively). Pots were carefully irrigated with 20% Hoagland´ı solution (Hoagland and Arnon, 1938Hoagland y Arnon, 1938) as required. After 5 months of growth, when seedlings were between 40 and 50 cm in height, apical stem cuttings of 15 cm were taken from the seedlings, with 5 nodes (including the apical node). The cuttings were allocated to individual plastic pots filled with perlite moistened and a deep of 4 cm. The pots were allocated to four different Cu concentration treatments: 0, 1.5, 4.5 y 9 mmol l−1 , applied in shallow trays within the same glasshouse (ten pots per tray, one tray per Cu treatment). Cu treatments were prepared by mixing 20% Hoagland´ıs solution with CuSO4 · 5H2 O of the appropriate concentration. The control, 0 mmol l−1 Cu treatment, in fact contained 0.0005 mmol l−1 of Cu, since Hoagland´ıs solution contains a small amount of Cu as an essential trade nutrient. These Cu concentrations were chosen in order to reflect the range of levels found by several authors in studies of the salt-marshes of different metal polluted estuaries (Cambrollé et al., 2008; Mateos-Naranjo et al., 2013; Sáinz and
2.2. Growth At the beginning of the experiment, 10 stem cuttings were taken and dried at 80 ◦ C for 48 h and then weighed in order to obtain the initial dry mass. At the end of the experiment (i.e. after 50 days of treatment), all the plants from each treatment were taken and the same methodology was followed in order to obtain the final dry mass. The relative growth rate (RGR) of whole plants was calculated using the formula: RGR = (lnBf − lnBi) D−1 (g g−1 day−1 ) where: Bf = final dry mass; Bi = initial dry mass; D = duration of experiment (days). Plant development was monitored by a weekly count of the number of leaves. Plant height was measured from the base of the stem to the tip of the uppermost leaf. Leaf area was determined from the projected area by scanning and digitalising the leaves (Epson V30, Seiko Epson Corp., Nagano, Japan), and using appropriate software (MideBMP v. 4.2.; Ordiales-Plaza, 2000) for processing and analysis.
2.4. Gas exchange Gas exchange measurements were taken from randomly selected, fully expanded leaves (n = 10, one measurement per plant), following 50 days of treatment, using an infrared gas analyzer in an open system (LI-6400, LI-COR Inc., Neb., USA). Net photosynthetic rate (A) was determined at an ambient CO2 concentration of 400 mol mol−1 , temperature of 20/25 ◦ C, 50 ± 5% relative humidity and a photon flux density of 1000 mol m−2 s−1 . Values of the parameters were calculated using the standard formulae of Von Caemmerer and Farquhar (1981). 2.5. Photosynthetic pigments At the end of the experimental period, photosynthetic pigments were extracted from fully expanded leaves from plants grown under each treatment, using 0.05 g of fresh plant material in 10 ml
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of 80% aqueous acetone (n = 10, one measurement per plant). After filtering, 1 ml of the suspension was diluted with a further 2 ml of acetone, and the chlorophyll a (Chl a) and chlorophyll b (Chl b) contents were each determined with a Hitachi U-2001 spectrophotometer (Hitachi Ltd, Japan), using three wavelengths (663.2, 646.8 and 470.0 nm). Pigment concentrations (g g−1 fwt) were calculated following the method of Lichtenthaler (1987). 2.6. Statistical analysis Statistical analysis was carried out using Statistica v. 6.0 (Statsoft Inc.). Pearson and Spearman coefficients were calculated to assess the correlation between different variables. Data were analyzed using one- and two-way analysis of variance (F-test). Data were tested for normality with the Kolmogorov-Smirnov test and for homogeneity of variance with the Brown-Forsythe test. Tukey tests were applied to significant test results for identification of important contrasts. 3. Results 3.1. Survival The survival rate of the stem cuttings in the control treatment and under concentrations of 1.5 and 4.5 mmol l−1 Cu was 100%. However, this rate decreased to 60% with a concentration of 9 mmol l−1 Cu. Symptoms of chlorosis and leaf necrosis were observed in the highest Cu treatments from the second half of the experimental period. 3.2. Growth analysis The Relative Growth Rate (RGR) decreased as the concentration of Cu increased above 1.5 mmol l−1 Cu (r = −0.567, p < 0.01). The reductions of RGR registered in the treatments of 1.5, 4.5 and 9 mmol l−1 Cu were 6, 33 and 70%, respectively, compared to the control treatment. No relevant differences between the control treatment and 1.5 mmol l−1 Cu were observed (t-test, p > 0.05; Fig. 1A). The plant height values of 0 and 1.5 mmol l−1 Cu treatments did not show relevant differences between them, as well as between the treatments of 4.5 and 9 mmol l−1 Cu (ANOVA, Tukey test, p > 0.05). Height values of 4.5 and 9 mmol l−1 Cu were significantly lower than the values in 0 and 1.5 mmol l−1 Cu (ANOVA, Tukey test, p < 0.05; Fig. 1B). Shoot and root dry weight showed a tendency to decrease with increasing external concentration of Cu, with no significant differences between the recorded values in the control treatment and 1.5 mmol l−1 Cu (ANOVA, Tukey test, p> 0.05 in both cases). Shoot dry weight values at 4.5 and 9 mmol l−1 Cu were significantly lower than those of the control treatment (ANOVA, Tukey test, p <0.05). Root dry weight values at 9 mmol l−1 Cu were significantly lower than those recorded in the other treatments (ANOVA, Tukey test, p <0.01; Fig. 1C). 3.3. Temporal evolution of leaf area Under control and 1.5 mmol l−1 Cu treatments, leaf area showed a marked increase from the third week of the experimental period, reaching its maximum values at the end of the experiment (50–60 cm2 ). No significant differences were recorded between these treatments throughout the study period (two-way ANOVA, p > 0.05). In the treatment of 4.5 mmol l−1 Cu, the leaf area values showed little variations until the final stage of the experiment, where a significant increase was detected relative to the initial leaf area (ANOVA, Tukey test, p < 0.001). Since the area registered was exclusively of living leaves, in the case of 9 mmol l−1 Cu no
Fig. 1. Relative growth rate (A), plant height (B) and dry weight (C) in plants grown from stem cuttings of Atriplex halimus, in response to treatment with a range of external Cu concentrations for 50 days. Values represent mean ± SE, n = 10.
clear trend of variation was detected throughout the experimental period, with no significant differences between values recorded throughout the experiment (ANOVA, p > 0.05). The final values of leaf area in 4.5 and 9 mmol l−1 Cu treatments were significantly lower than those recorded in the control (ANOVA, Tukey test, p < 0.05 and p < 0.001, respectively; Fig. 2).
3.4. Gas exchange The net photosynthetic rate (A) decreased with increasing Cu concentration of up to 4.5 mmol l−1 (r = −0.762, p < 0.01). The lowest values were recorded in plants under 4.5 and 9 mmol l−1 Cu, with no significant differences between these treatments (ANOVA, Tukey test, p < 0.05; Fig. 3).
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Fig. 2. Temporal evolution of total leaf area in plants grown from stem cuttings of Atriplex halimus, in response to treatment with a range of external Cu concentrations for 50 days (0 mmol l−1 (䊉), 1.5 mmol l−1 (), 4.5 mmol l−1 () and 130 mmol l−1 ()). Values represent mean ± SE, n = 10.
Fig. 3. Net photosynthetic rate (A) in randomly selected, fully developed leaves of plants grown from stem cuttings of Atriplex halimus, in response to treatment with a range of external Cu concentrations for 50 days. Values represent mean ± SE, n = 10.
3.5. Chlorophyll fluorescence and photosynthetic pigments Maximum quantum efficiency of PSII (Fv /Fm ) measured at dawn showed no clear variations in the different treatments (ANOVA, Tukey test, p > 0.05, in all cases), with values ranging around 0.82. Pigment concentrations showed no significant differences between the different treatments tested (ANOVA, Tukey test, p > 0.05), with mean values ranging around 7.5 g g−1 . 4. Discussion Stem cuttings of Atriplex halimus showed high tolerance to Cu stress. Metal toxicity thresholds in plants can be difficult to determine due to complex interactions between the toxic metal and other nutrient elements, as well as to other complex biological and physical factors (Foy et al., 1978). However, the present study provides sufficient information to conduct a simplified approach to the determination of Cu phytotoxicity thresholds in stem cuttings of the study species. Previous studies have evaluated the effect
of Cu on the growth and physiological status of adult plants of A. halimus propagated from seeds (Mateos-Naranjo et al., 2013). According to that study, this species is highly tolerant to Cu stress, and the lethal concentration (LC50, the concentration of metal in the substrate causing death to 50% of the plants) ranges between 15 and 30 mmol l−1 . The results of the present study show that A. halimus can survive and establish from cuttings in substrates with high concentrations of Cu, since 100% survival was recorded in plants subjected to external concentrations of Cu up to 4.5 mmol l−1 and more than 50% survival was registered in stem cuttings under 9 mmol l−1 Cu. LC50 values close to 5 mmol l−1 after 60 d of treatment were recorded by Paschke and Redente (2002) for six grass species used in restoration activities. Growth inhibition and reduction of biomass are general responses to excess Cu in higher plants (Kabata-Pendias and Pendias, 2001). The effective concentration (EC50) can be defined as substrate metal concentration resulting in 50% biomass reduction. Mateos-Naranjo et al. (2013) found an EC50 value over 15 mmol l−1 Cu in A. halimus adult plants grown from seeds. The present study shows that the EC50 of cuttings of A. halimus was around 4.5 mmol l−1 , since at this external concentration, total biomass was reduced by approximately 50% compared to the control treatment. Also, reduction of the relative growth rate (RGR) at 4.5 and 9 mmol l−1 was 33 and 70%, respectively, compared to the control treatment. The increase in external concentration of Cu also led to 39% reduction in final plant height at 4.5 mmol l−1 and 51% at 9 mmol l−1 . The results of the present study indicate that high concentrations of Cu affect the growth and development of the study species. Several studies indicate that A. halimus develop different strategies to tolerate excess Cu, such as induced increases in the activity of certain antioxidant enzymes (Brahim and Mohamed, 2013) or different mechanisms to prevent the absorption of high amounts of Cu at the roots (Mateos-Naranjo et al., 2013). In that sense, it would be interesting to extend this study by analysing the concentrations of Cu in different plant tissues of plants grown from stem cuttings. Plant cuttings spend great amounts of energy in the development of an adequate root system and establishing in the medium, which explains why, in the present study, the cuttings of the control treatment and those under 1.5 mmol l−1 Cu produced scarce leaf biomass during the first weeks of the experiment. The cuttings under external Cu concentrations of 1.5 mmol l−1 showed development of aerial and subterranean biomasses similar to those shown by the cuttings of the control system. However, it should be noted that the cuttings subjected to 4.5 mmol l−1 Cu did not show increments in leaf surface until the last week of the experimental period, which indicates that these plants needed more time to develop a well established root system that would be sufficient so as to invest energy in the development of aerial biomass. The results of the present study indicate that Cu concentrations above 1.5 mmol l−1 would restrain root development in the study species. Under 9 mmol l−1 Cu, 40% of the cuttings died before developing roots, which indicates that such levels of metal concentration inhibit the development of root systems, thus preventing cutting survival. The effects of excess Cu on physiological functions have been intensively studied, and it is widely recognised that Cu toxicity induces inhibition of photosynthetic processes (Kabata-Pendias and Pendias, 2001). In the present study, the excess of Cu caused a negative effect on net photosynthetic rate (A), which was particularly pronounced at external concentrations of 4.5 mmol l−1 and higher. Numerous studies have reported a direct effect of Cu on the electron transport chain (Sandmann and Böger, 1980; Jegerschöld et al., 1995). However, in the present study, the maximum quantum efficiency of PSII (Fv /Fm ) measured at dawn showed values similar to the control parameters for unstressed plants (Björkman and
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Demmig, 1987), indicating that Cu did not cause irreversible effects on photosystem II. Likewise, the concentrations of photosynthetic pigments in the cuttings subjected to the different Cu treatments were similar to those found in the control treatment. Cambrollé et al. (2012) reported similar concentrations of chlorophyll a and b in the marine species Halimione portulacoides under Cu concentrations of up to 15 mmol l−1 . These results seem to confirm that the high concentrations of this metal did not cause drastic effects on the photosynthetic apparatus of A. halimus. The results of physiological parameters indicate that the decrease in A observed in plants from cuttings subjected to high concentrations of Cu could be related to stomatal limitations, and/or the possible effects of this metal on the activity of certain enzymes involved in the process of photosynthesis, such as RuBP-carboxylase. Previous studies have linked the decrease of photosynthetic rate to the effects of different heavy metals on the activity of RuBP-carboxylase (Siedlecka and Krupa, 2004; Cambrollé et al., 2011; among others). 5. Conclusions The use of plant cuttings in the restoration or recovery of ecosystems contaminated with heavy metals offers great advantages over seed sowing (Huiskes, 1979; Woodhouse, 1982; Gomes Neto et al., 2006). The present study is the first to test the survival and Cu toxicity limits on cuttings of Atriplex halimus. This study shows that the cuttings of this species are able to survive, root and grow under Cu concentrations of up to 9 mmol l−1 , and they show marked effects on growth and root percentages from 4.5 mmol l−1 . This study allows concluding that the use of cuttings from Atriplex halimus may be a valuable and efficient tool for the restoration of vegetation in Cu-polluted coastal ecosystems. Acknowledgements We are grateful to M. Navarro-Ramos and Mr. A. Serrano for revision of the English version of the manuscript. J. Mancilla-Leytón also thanks the University of Seville for a postdoctoral research contract (V Plan Propio de Investigación US, ref. II.5.B/2015). References Allen, J.R.L., Rae, J.E., Zanin, P.E., 1990. Metal speciation (Cu, Zn, Pb) and organic matter in an oxic salt marsh, Severn Estuary, Southwest Britain. Mar. Pollut. Bull. 21, 574–580. Almeida, C.M.R., Días, A.C., Mucha, A.P., Vasconcelos, M.T.S.D., 2004. Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant. Environ. Sci. Technol. 38, 3112–3118. Andrades-Moreno, L., Cambrollé, J., Figueroa, M.E., Mateos-Naranjo, E., 2013. Growth and survival of Halimione portulacoides stem cuttings in heavy metal contaminated soils. Mar. Pollut. Bull. 75, 28–32. Bajji, M., Kinet, J.M., Lutts, S., 1998. Salt stress effects on roots and leaves of Atriplex halimus and their corresponding callus cultures. Plant Sci. 137, 131–142. Barón, M., Arellano, J.B., Gorgé, L., 1995. Copper and photosystem II: a controversial relationship. Physiol. Plant. 94, 174–180. Björkman, O., Demmig, B., 1987. Photo yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170, 489–504. Brahim, L., Mohamed, M., 2013. Effects of copper stress on antioxidative enzymes, chlorophyll and protein content in Atriplex halimus. Afr. J. Biotechnol. 10, 10143–10148. Cac¸ador, I., Vale, C., Catarino, F., 1996. Accumulation of Zn, Pb, Cu and Ni in sediments between roots of the Tagus estuary salt marshes, Portugal. Estuar. Coast. Shelf Sci. 42, 393–403. Cambrollé, J., Redondo-Gómez, S., Mateos-Naranjo, E., Figueroa, M.E., 2008. Comparison of the role of two Spartina species in terms of phytostabilization and bioaccumulation of metals in the estuarine sediment. Mar. Pollut. Bull. 56, 2037–2042. Cambrollé, J., Mateos-Naranjo, E., Redondo-Gomez, S., Luque, T., Figueroa, M.E., 2011. Growth, reproductive and photosynthetic responses to copper in the yellow-horned poppy, Glaucium flavum Crantz. Environ. Exp. Bot. 71, 57–64. ˜ Cambrollé, J., Mancilla-Leytón, J.M., Munoz-Vallés, S., Luque, T., Figueroa, M.E., 2012. Zinc tolerance and accumulation in the salt-marsh shrub Halimione portulacoides. Chemosphere 86, 867–874.
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Evaluation of the potential of Atriplex halimus stem cuttings for phytoremediation of metal-polluted soils ˜ Juan M. Mancilla-Leytón 1 , María José Navarro-Ramos 1 , Sara Munoz-Vallés, M. Enrique Figueroa, Jesús Cambrollé ∗ Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080, Sevilla, Spain
a r t i c l e
i n f o
Article history: Received 15 June 2016 Received in revised form 5 September 2016 Accepted 11 October 2016 Available online 18 October 2016 Keywords: Heavy metals Phytoremediation Wetland restoration
a b s t r a c t The use of cuttings in the restoration of degraded ecosystems presents important advantages compared to seed sowing. The applicability of cuttings from some coastal plants for the recovery of soils contaminated with heavy metals has been recently recognised. A greenhouse experiment was carried out to analyse the effects of a concentration range of copper (0–9 mmol l−1 ) on the establishment, growth and photosynthetic performance of Atriplex halimus cuttings, in order to determine the phytotoxicity threshold values of plants from cuttings and assess the possible physiological effects of this metal. The cuttings from the study species showed the ability to survive, root and grow under Cu concentrations of up to 9 mmol l−1 . Although no effects were observed in the photosynthetic apparatus, concentrations of 4.5 mmol l−1 and higher caused a decrease in growth, net photosynthetic rate and rooting percentages. The present study allows concluding that the use of cuttings from Atriplex halimus could be a valuable and efficient tool for the restoration of vegetation in Cu-polluted coastal ecosystems. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The salt marshes, estuaries and nearby areas receive all the load of sediments and pollutants that have been dragged along the river and its basin. The pollutants may have different origins, even natural ones coming from the river basin itself. Salt marshes and estuaries usually present high concentrations of pollutants, mainly heavy metals, due to anthropologic activities that take place in estuaries or in contiguous areas, like mining, refineries and oil stations, among others (Hong-Yun et al., 2005; Sousa et al., 2008). At the present time, eutrophication and chemical pollution are affecting salt marshes all over the world. The main ecological issues resulting from pollution are the introduction of heavy metals in the food cycle of marshland and estuary ecosystems, and bioamplification in the different trophic levels. There is a large number of studies focused on the presence of heavy metals in soils of coastal areas (Almeida et al., 2004; Allen et al., 1990; Cac¸ador et al., 1996; Mortimer and Rae, 2000). The sediments dragged by the rivers and accumulated in the marshlands contribute to increase the pollution of these areas. This is the case of the pyrite belt in Sierra Morena, which is linked to the marshes
∗ Corresponding author at: Dpto. Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Av. Reina Mercedes 6, 41012 Seville, Spain. E-mail address: [email protected] (J. Cambrollé). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ecoleng.2016.10.062 0925-8574/© 2016 Elsevier B.V. All rights reserved.
of the Odiel and Tinto rivers. The watercourse of these rivers has a high concentration of heavy metals that might affect the local plants near the area. Factors such as pH, soil texture, organic matter and clay content, and redox potential, influence the bioavailability and solubility of metals in plants (Greger, 2004). Copper is one of the main heavy metals found in the marsh due to the high mining activity in the local area, the processing of metals, and the use of fertilizers and fungicides, among other actions (Kabata-Pendias and Pendias, 2001). Even though it is an essential trace element, involved in many different biochemical and physiological processes, such as photosynthesis and breathing (Barón et al., 1995; Pilon et al., 2006), it is toxic when in high concentration for most plants (Dewez et al., 2005). It has been observed that Cu overload affects growth, photosynthetic activity and even the respiration process (Nalewajko and Olaveson, 1995). However, some plants are able to grow in soils polluted with Cu and accumulate a high amount of it with no marked impact (Ernst et al., 2000; Kabata-Pendias and Pendias, 2001). Recent studies prove that some coastal plants present an inherent tolerance to heavy metals (McCabe et al., 2001). Phytoremediation, the use of plants to extract, sequester and/or detoxify pollutants, is widely viewed as the ecological alternative to the environmentally destructive physical remediation methods currently practiced (Meagher, 2000; Garbisu et al., 2002). This technology requires knowledge of some biological aspects of the species to be used.
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Most of the dominant plant species in salt marshes are halophytic, which means they are resistant to high concentrations of salt that are commonly found in the marshland systems. Halimione portulacoides, Atriplex halimus, Salicornia ramosissima and Spartina maritima are among these type of plants. Atriplex halimus is a perennial shrub, original from arid and semi-arid zones of the Mediterranean, from the Chenopodiaceae family. This halophytic plant grows in both emerged areas in marshlands (FernándezIllescas et al., 2010) and sandy soil (López González, 2001), and it can tolerate high salinity levels (Bajji et al., 1998), light stress (Streb et al., 1997) and drought (Martínez et al., 2005). Recent studies have proved that Atripex halimus can tolerate and accumulate high concentrations of heavy metals in its tissues, such as Cd, Zn (Lutts et al., 2004) and Cu (Mateos-Naranjo et al., 2013), which indicates that it could be viable for phytoremediation techniques. Most of the plants used in phytoremediation techniques are from greenhouses, where they are spread from seeds. Recently, Andrades-Moreno et al. (2013) tested, for the first time, the capability of a plant species to grow under high concentrations of heavy metals, proving the viability of stem cuttings to restore polluted soils. The use of stem cuttings in phytoremediation might have potential advantages over the use of adult plants (such as less time and resources required); however, some key factors regarding this method have not been studied yet. The main objective of this study was to evaluate Cu tolerance in stem cuttings of A. halimus. The specific objectives were: (i) to determine the Cu phytotoxicity thresholds of stem cuttings of the study species, by analysing the establishment, survival and growth of plants in a range of external Cu concentrations, from 0 to 9 mmol l−1 ; and (ii) to ascertain the extent to which Cu determines plant performance in plants grown from stem cuttings, in terms of influence on the photosynthetic apparatus (PSII chemistry) and gas exchange characteristics.
Ruiz, 2006Sáinz y Ruiz, 2006), as well as in previous experiments to determine the phytotoxicity threshold of stem cutting of A. halimus. At the beginning of the experiment, a 3 l volume of the appropriate solution was placed in each of the trays to a market depth of 1 cm. Throughout the experiment, solution levels in the trays were monitored and topped up to the marked level with 20% Hoagland´ıs solution, (with no addition CuSO4 · 5H2 O), in order to limit the change in Cu concentration due to evaporation of the water in the nutrient solution. In addition, the entire solution (including CuCuSO4 · 5H2 O) was changed on a weekly basis.
2. Materials and methods
2.3. Chlorophyll fluorescence
2.1. Plant material and copper treatments
Chlorophyll fluorescence was measured in randomly selected, fully developed leaves (n = 10; one measurement per plant for each treatment) using a portable modulated fluorimeter (FMS-2, Hansatech Instruments Ltd., England) at the end of the experimental period, following the methods described in Cambrollé et al. (2012). Values of variable fluorescence (Fv = Fm − F0 ) and maximum quantum efficiency of PSII photochemistry (Fv /Fm ) were calculated from F0 and Fm . This ratio of variable to maximal fluorescence correlates with the number of functional PSII reaction centres, and dark adapted values of Fv /Fm can be used to quantify photoinhibition (Krivosheeva et al., 1996).
Seeds of A. halimus were collected in the salt marshes of “La Mata-Torrevieja” (Alicante, SE Spain). The collected seeds were subsequently germinated in perlite moistened with distilled water, and maintained at 25 ◦ C for 30 days. The resulting seedlings were sown in individual plastic pots (with a diameter of 11 cm) filled with perlite, and placed in a glasshouse with minimummaximun temperatures of 21–25 ◦ C, 40–60% relative humidity and natural daylight (minimun and maximun ligth flux: 200 and 1000 mol m−2 s−1 , respectively). Pots were carefully irrigated with 20% Hoagland´ı solution (Hoagland and Arnon, 1938Hoagland y Arnon, 1938) as required. After 5 months of growth, when seedlings were between 40 and 50 cm in height, apical stem cuttings of 15 cm were taken from the seedlings, with 5 nodes (including the apical node). The cuttings were allocated to individual plastic pots filled with perlite moistened and a deep of 4 cm. The pots were allocated to four different Cu concentration treatments: 0, 1.5, 4.5 y 9 mmol l−1 , applied in shallow trays within the same glasshouse (ten pots per tray, one tray per Cu treatment). Cu treatments were prepared by mixing 20% Hoagland´ıs solution with CuSO4 · 5H2 O of the appropriate concentration. The control, 0 mmol l−1 Cu treatment, in fact contained 0.0005 mmol l−1 of Cu, since Hoagland´ıs solution contains a small amount of Cu as an essential trade nutrient. These Cu concentrations were chosen in order to reflect the range of levels found by several authors in studies of the salt-marshes of different metal polluted estuaries (Cambrollé et al., 2008; Mateos-Naranjo et al., 2013; Sáinz and
2.2. Growth At the beginning of the experiment, 10 stem cuttings were taken and dried at 80 ◦ C for 48 h and then weighed in order to obtain the initial dry mass. At the end of the experiment (i.e. after 50 days of treatment), all the plants from each treatment were taken and the same methodology was followed in order to obtain the final dry mass. The relative growth rate (RGR) of whole plants was calculated using the formula: RGR = (lnBf − lnBi) D−1 (g g−1 day−1 ) where: Bf = final dry mass; Bi = initial dry mass; D = duration of experiment (days). Plant development was monitored by a weekly count of the number of leaves. Plant height was measured from the base of the stem to the tip of the uppermost leaf. Leaf area was determined from the projected area by scanning and digitalising the leaves (Epson V30, Seiko Epson Corp., Nagano, Japan), and using appropriate software (MideBMP v. 4.2.; Ordiales-Plaza, 2000) for processing and analysis.
2.4. Gas exchange Gas exchange measurements were taken from randomly selected, fully expanded leaves (n = 10, one measurement per plant), following 50 days of treatment, using an infrared gas analyzer in an open system (LI-6400, LI-COR Inc., Neb., USA). Net photosynthetic rate (A) was determined at an ambient CO2 concentration of 400 mol mol−1 , temperature of 20/25 ◦ C, 50 ± 5% relative humidity and a photon flux density of 1000 mol m−2 s−1 . Values of the parameters were calculated using the standard formulae of Von Caemmerer and Farquhar (1981). 2.5. Photosynthetic pigments At the end of the experimental period, photosynthetic pigments were extracted from fully expanded leaves from plants grown under each treatment, using 0.05 g of fresh plant material in 10 ml
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of 80% aqueous acetone (n = 10, one measurement per plant). After filtering, 1 ml of the suspension was diluted with a further 2 ml of acetone, and the chlorophyll a (Chl a) and chlorophyll b (Chl b) contents were each determined with a Hitachi U-2001 spectrophotometer (Hitachi Ltd, Japan), using three wavelengths (663.2, 646.8 and 470.0 nm). Pigment concentrations (g g−1 fwt) were calculated following the method of Lichtenthaler (1987). 2.6. Statistical analysis Statistical analysis was carried out using Statistica v. 6.0 (Statsoft Inc.). Pearson and Spearman coefficients were calculated to assess the correlation between different variables. Data were analyzed using one- and two-way analysis of variance (F-test). Data were tested for normality with the Kolmogorov-Smirnov test and for homogeneity of variance with the Brown-Forsythe test. Tukey tests were applied to significant test results for identification of important contrasts. 3. Results 3.1. Survival The survival rate of the stem cuttings in the control treatment and under concentrations of 1.5 and 4.5 mmol l−1 Cu was 100%. However, this rate decreased to 60% with a concentration of 9 mmol l−1 Cu. Symptoms of chlorosis and leaf necrosis were observed in the highest Cu treatments from the second half of the experimental period. 3.2. Growth analysis The Relative Growth Rate (RGR) decreased as the concentration of Cu increased above 1.5 mmol l−1 Cu (r = −0.567, p < 0.01). The reductions of RGR registered in the treatments of 1.5, 4.5 and 9 mmol l−1 Cu were 6, 33 and 70%, respectively, compared to the control treatment. No relevant differences between the control treatment and 1.5 mmol l−1 Cu were observed (t-test, p > 0.05; Fig. 1A). The plant height values of 0 and 1.5 mmol l−1 Cu treatments did not show relevant differences between them, as well as between the treatments of 4.5 and 9 mmol l−1 Cu (ANOVA, Tukey test, p > 0.05). Height values of 4.5 and 9 mmol l−1 Cu were significantly lower than the values in 0 and 1.5 mmol l−1 Cu (ANOVA, Tukey test, p < 0.05; Fig. 1B). Shoot and root dry weight showed a tendency to decrease with increasing external concentration of Cu, with no significant differences between the recorded values in the control treatment and 1.5 mmol l−1 Cu (ANOVA, Tukey test, p> 0.05 in both cases). Shoot dry weight values at 4.5 and 9 mmol l−1 Cu were significantly lower than those of the control treatment (ANOVA, Tukey test, p <0.05). Root dry weight values at 9 mmol l−1 Cu were significantly lower than those recorded in the other treatments (ANOVA, Tukey test, p <0.01; Fig. 1C). 3.3. Temporal evolution of leaf area Under control and 1.5 mmol l−1 Cu treatments, leaf area showed a marked increase from the third week of the experimental period, reaching its maximum values at the end of the experiment (50–60 cm2 ). No significant differences were recorded between these treatments throughout the study period (two-way ANOVA, p > 0.05). In the treatment of 4.5 mmol l−1 Cu, the leaf area values showed little variations until the final stage of the experiment, where a significant increase was detected relative to the initial leaf area (ANOVA, Tukey test, p < 0.001). Since the area registered was exclusively of living leaves, in the case of 9 mmol l−1 Cu no
Fig. 1. Relative growth rate (A), plant height (B) and dry weight (C) in plants grown from stem cuttings of Atriplex halimus, in response to treatment with a range of external Cu concentrations for 50 days. Values represent mean ± SE, n = 10.
clear trend of variation was detected throughout the experimental period, with no significant differences between values recorded throughout the experiment (ANOVA, p > 0.05). The final values of leaf area in 4.5 and 9 mmol l−1 Cu treatments were significantly lower than those recorded in the control (ANOVA, Tukey test, p < 0.05 and p < 0.001, respectively; Fig. 2).
3.4. Gas exchange The net photosynthetic rate (A) decreased with increasing Cu concentration of up to 4.5 mmol l−1 (r = −0.762, p < 0.01). The lowest values were recorded in plants under 4.5 and 9 mmol l−1 Cu, with no significant differences between these treatments (ANOVA, Tukey test, p < 0.05; Fig. 3).
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Fig. 2. Temporal evolution of total leaf area in plants grown from stem cuttings of Atriplex halimus, in response to treatment with a range of external Cu concentrations for 50 days (0 mmol l−1 (䊉), 1.5 mmol l−1 (), 4.5 mmol l−1 () and 130 mmol l−1 ()). Values represent mean ± SE, n = 10.
Fig. 3. Net photosynthetic rate (A) in randomly selected, fully developed leaves of plants grown from stem cuttings of Atriplex halimus, in response to treatment with a range of external Cu concentrations for 50 days. Values represent mean ± SE, n = 10.
3.5. Chlorophyll fluorescence and photosynthetic pigments Maximum quantum efficiency of PSII (Fv /Fm ) measured at dawn showed no clear variations in the different treatments (ANOVA, Tukey test, p > 0.05, in all cases), with values ranging around 0.82. Pigment concentrations showed no significant differences between the different treatments tested (ANOVA, Tukey test, p > 0.05), with mean values ranging around 7.5 g g−1 . 4. Discussion Stem cuttings of Atriplex halimus showed high tolerance to Cu stress. Metal toxicity thresholds in plants can be difficult to determine due to complex interactions between the toxic metal and other nutrient elements, as well as to other complex biological and physical factors (Foy et al., 1978). However, the present study provides sufficient information to conduct a simplified approach to the determination of Cu phytotoxicity thresholds in stem cuttings of the study species. Previous studies have evaluated the effect
of Cu on the growth and physiological status of adult plants of A. halimus propagated from seeds (Mateos-Naranjo et al., 2013). According to that study, this species is highly tolerant to Cu stress, and the lethal concentration (LC50, the concentration of metal in the substrate causing death to 50% of the plants) ranges between 15 and 30 mmol l−1 . The results of the present study show that A. halimus can survive and establish from cuttings in substrates with high concentrations of Cu, since 100% survival was recorded in plants subjected to external concentrations of Cu up to 4.5 mmol l−1 and more than 50% survival was registered in stem cuttings under 9 mmol l−1 Cu. LC50 values close to 5 mmol l−1 after 60 d of treatment were recorded by Paschke and Redente (2002) for six grass species used in restoration activities. Growth inhibition and reduction of biomass are general responses to excess Cu in higher plants (Kabata-Pendias and Pendias, 2001). The effective concentration (EC50) can be defined as substrate metal concentration resulting in 50% biomass reduction. Mateos-Naranjo et al. (2013) found an EC50 value over 15 mmol l−1 Cu in A. halimus adult plants grown from seeds. The present study shows that the EC50 of cuttings of A. halimus was around 4.5 mmol l−1 , since at this external concentration, total biomass was reduced by approximately 50% compared to the control treatment. Also, reduction of the relative growth rate (RGR) at 4.5 and 9 mmol l−1 was 33 and 70%, respectively, compared to the control treatment. The increase in external concentration of Cu also led to 39% reduction in final plant height at 4.5 mmol l−1 and 51% at 9 mmol l−1 . The results of the present study indicate that high concentrations of Cu affect the growth and development of the study species. Several studies indicate that A. halimus develop different strategies to tolerate excess Cu, such as induced increases in the activity of certain antioxidant enzymes (Brahim and Mohamed, 2013) or different mechanisms to prevent the absorption of high amounts of Cu at the roots (Mateos-Naranjo et al., 2013). In that sense, it would be interesting to extend this study by analysing the concentrations of Cu in different plant tissues of plants grown from stem cuttings. Plant cuttings spend great amounts of energy in the development of an adequate root system and establishing in the medium, which explains why, in the present study, the cuttings of the control treatment and those under 1.5 mmol l−1 Cu produced scarce leaf biomass during the first weeks of the experiment. The cuttings under external Cu concentrations of 1.5 mmol l−1 showed development of aerial and subterranean biomasses similar to those shown by the cuttings of the control system. However, it should be noted that the cuttings subjected to 4.5 mmol l−1 Cu did not show increments in leaf surface until the last week of the experimental period, which indicates that these plants needed more time to develop a well established root system that would be sufficient so as to invest energy in the development of aerial biomass. The results of the present study indicate that Cu concentrations above 1.5 mmol l−1 would restrain root development in the study species. Under 9 mmol l−1 Cu, 40% of the cuttings died before developing roots, which indicates that such levels of metal concentration inhibit the development of root systems, thus preventing cutting survival. The effects of excess Cu on physiological functions have been intensively studied, and it is widely recognised that Cu toxicity induces inhibition of photosynthetic processes (Kabata-Pendias and Pendias, 2001). In the present study, the excess of Cu caused a negative effect on net photosynthetic rate (A), which was particularly pronounced at external concentrations of 4.5 mmol l−1 and higher. Numerous studies have reported a direct effect of Cu on the electron transport chain (Sandmann and Böger, 1980; Jegerschöld et al., 1995). However, in the present study, the maximum quantum efficiency of PSII (Fv /Fm ) measured at dawn showed values similar to the control parameters for unstressed plants (Björkman and
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Demmig, 1987), indicating that Cu did not cause irreversible effects on photosystem II. Likewise, the concentrations of photosynthetic pigments in the cuttings subjected to the different Cu treatments were similar to those found in the control treatment. Cambrollé et al. (2012) reported similar concentrations of chlorophyll a and b in the marine species Halimione portulacoides under Cu concentrations of up to 15 mmol l−1 . These results seem to confirm that the high concentrations of this metal did not cause drastic effects on the photosynthetic apparatus of A. halimus. The results of physiological parameters indicate that the decrease in A observed in plants from cuttings subjected to high concentrations of Cu could be related to stomatal limitations, and/or the possible effects of this metal on the activity of certain enzymes involved in the process of photosynthesis, such as RuBP-carboxylase. Previous studies have linked the decrease of photosynthetic rate to the effects of different heavy metals on the activity of RuBP-carboxylase (Siedlecka and Krupa, 2004; Cambrollé et al., 2011; among others). 5. Conclusions The use of plant cuttings in the restoration or recovery of ecosystems contaminated with heavy metals offers great advantages over seed sowing (Huiskes, 1979; Woodhouse, 1982; Gomes Neto et al., 2006). The present study is the first to test the survival and Cu toxicity limits on cuttings of Atriplex halimus. This study shows that the cuttings of this species are able to survive, root and grow under Cu concentrations of up to 9 mmol l−1 , and they show marked effects on growth and root percentages from 4.5 mmol l−1 . This study allows concluding that the use of cuttings from Atriplex halimus may be a valuable and efficient tool for the restoration of vegetation in Cu-polluted coastal ecosystems. Acknowledgements We are grateful to M. Navarro-Ramos and Mr. A. Serrano for revision of the English version of the manuscript. J. Mancilla-Leytón also thanks the University of Seville for a postdoctoral research contract (V Plan Propio de Investigación US, ref. II.5.B/2015). References Allen, J.R.L., Rae, J.E., Zanin, P.E., 1990. Metal speciation (Cu, Zn, Pb) and organic matter in an oxic salt marsh, Severn Estuary, Southwest Britain. Mar. Pollut. Bull. 21, 574–580. Almeida, C.M.R., Días, A.C., Mucha, A.P., Vasconcelos, M.T.S.D., 2004. Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant. Environ. Sci. Technol. 38, 3112–3118. Andrades-Moreno, L., Cambrollé, J., Figueroa, M.E., Mateos-Naranjo, E., 2013. Growth and survival of Halimione portulacoides stem cuttings in heavy metal contaminated soils. Mar. Pollut. Bull. 75, 28–32. Bajji, M., Kinet, J.M., Lutts, S., 1998. Salt stress effects on roots and leaves of Atriplex halimus and their corresponding callus cultures. Plant Sci. 137, 131–142. Barón, M., Arellano, J.B., Gorgé, L., 1995. Copper and photosystem II: a controversial relationship. Physiol. Plant. 94, 174–180. Björkman, O., Demmig, B., 1987. Photo yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170, 489–504. Brahim, L., Mohamed, M., 2013. Effects of copper stress on antioxidative enzymes, chlorophyll and protein content in Atriplex halimus. Afr. J. Biotechnol. 10, 10143–10148. Cac¸ador, I., Vale, C., Catarino, F., 1996. Accumulation of Zn, Pb, Cu and Ni in sediments between roots of the Tagus estuary salt marshes, Portugal. Estuar. Coast. Shelf Sci. 42, 393–403. Cambrollé, J., Redondo-Gómez, S., Mateos-Naranjo, E., Figueroa, M.E., 2008. Comparison of the role of two Spartina species in terms of phytostabilization and bioaccumulation of metals in the estuarine sediment. Mar. Pollut. Bull. 56, 2037–2042. Cambrollé, J., Mateos-Naranjo, E., Redondo-Gomez, S., Luque, T., Figueroa, M.E., 2011. Growth, reproductive and photosynthetic responses to copper in the yellow-horned poppy, Glaucium flavum Crantz. Environ. Exp. Bot. 71, 57–64. ˜ Cambrollé, J., Mancilla-Leytón, J.M., Munoz-Vallés, S., Luque, T., Figueroa, M.E., 2012. Zinc tolerance and accumulation in the salt-marsh shrub Halimione portulacoides. Chemosphere 86, 867–874.
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