2019
Chelate-assisted phytoextraction:
Growth and ecophysiological
responses by Salix schwerinii E.L Wolf grown in artificially polluted soils
Mohsin, Muhammad
Elsevier BV
Tieteelliset aikakauslehtiartikkelit
© Elsevier B.V.
CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
http://dx.doi.org/10.1016/j.gexplo.2019.106335
https://erepo.uef.fi/handle/123456789/7733
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Chelate-assisted phytoextraction: Growth and ecophysiological responses by Salix schwerinii E.L Wolf grown in artificially polluted soils
Muhammad Mohsin, Suvi Kuittinen, Mir Md Abdus Salam, Sirpa Peräniemi, Saila Laine, Pertti Pulkkinen, Erik Kaipiainen, Jouko Vepsäläinen, Ari Pappinen
PII: S0375-6742(17)30793-8
DOI: https://doi.org/10.1016/j.gexplo.2019.106335 Article Number: 106335
Reference: GEXPLO 106335
To appear in: Journal of Geochemical Exploration Received date: 15 November 2017
Revised date: 17 June 2019 Accepted date: 8 July 2019
Please cite this article as: M. Mohsin, S. Kuittinen, M.M.A. Salam, et al., Chelate-assisted phytoextraction: Growth and ecophysiological responses by Salix schwerinii E.L Wolf grown in artificially polluted soils, Journal of Geochemical Exploration, https://doi.org/
10.1016/j.gexplo.2019.106335
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Chelate-assisted Phytoextraction: Growth and Ecophysiological Responses by Salix Schwerinii E.L Wolf Grown in Artificially Polluted Soils
Muhammad Mohsin a*; Suvi Kuittinen a; Mir Md Abdus Salam a; Sirpa Peräniemi b; Saila Laine a; Pertti Pulkkinen c; Erik Kaipiainen a; Jouko Vepsäläinen b; Ari Pappinen a*
a School of Forest Sciences, University of Eastern Finland, Yliopistokatu 7, P.O. Box 111, 80100, Joensuu, Finland
b School of Pharmacy, Biocenter Kuopio, University of Eastern Finland, P.O. Box 1627, FIN- 70211 Kuopio, Finland
c Natural Resources Institute Finland (Luke), Haapastensyrjä Research Unit, Haapastensyrjäntie 34, FIN-12600, Läyliäinen, Finland
*Corresponding authors: ari.pappinen@uef.fi; muham@uef.fi
Abstract
The present study investigated the phytoextraction ability of Salix schwerinii E.L. Wolf enhanced with an application of the chelate N10O. Salix schwerinii were grown in garden soil that was also amended with Cu (400 mg kg-1), Ni (30 mg kg-1) and Zn (200 mg kg-1). Multiple doses of N10O were applied to the treatments as follows: Cu (3.45 g and 6.9 g), Ni (1.2 g and 2.4 g), and Zn (1.45 g and 2.9 g). Furthermore, N10O doses were also repeated with the control soil. The effect of N10O on height growth, biomass production, ecophysiological attributes, and the accumulation of metals (Cu, Ni, and Zn) in Salix in polluted soils was studied. Compared to the control, the total metal concentrations in S. schwerinii growing in the soils amended with N10O increased substantially by up to 895% for Cu, 324% for Ni and 722% for Zn. The translocation factor (TF) and bioconcentration factor (BF) values for S. schwerinii increased with the application of N10O and varied from 0.301.01 for Cu, 0.451.25 for Ni, and 4.405.89 for Zn, whereas, BF values varied from 0.601.15 for Cu, 0.801.50 for Ni, and 48 for Zn. This study indicated that S. schwerinii can be used for phytoextraction of Cu, Ni and Zn from contaminated soils. However, further research is needed to examine the phytoextraction potential of other Salix species using N10O to remediate soils polluted with various toxic metals.
Keywords: Metals; Phytoextraction; Soil remediation; Salix; Bisphosphonic acid; Photosynthesis
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2 1. Introduction
Metal contamination of soils is a serious threat to human health and to the environment (Tanhan et al., 2007; Yang et al., 2014). In natural ecosystems, low metal concentrations do not pose a significant toxic risk to living organisms (flora and fauna). However, the increase in metal accessibility in soil and water may have severe implications for wildlife, humans and plant life. In Europe, it is estimated that metal contamination affects about 60% of soils and water (Bernardini et al., 2016; Panagos et al., 2013). The main elements of soil pollution are metals that emerge through anthropogenic activities, such as mining, sewage production, and the incineration of fossil fuels, industrial processes and the extensive use of fertilizers (Bernardini et al., 2016). Soil polluted by Cu, Zn and Ni may cause severe hazards to humans and the environment, for example, by polluting underground water reserves, affecting food quality (by increasing the risk of metal induction in plants), and by reducing agricultural productivity (Wuana and Okieimen, 2011).
Metals such as Cu, Ni, Zn, Co, Fe and Mn are important for plant growth and development, although concentrations that exceed the desired level may cause oxidative stress and exchange these metals in pigments or enzymes that can disrupt functions and protein structure. A higher concentration of essential elements such as Cu and Zn can cause growth retardation, chlorosis, senescence, and oxidative stress in plants, and high Ni levels result in necrosis, nutrient and water imbalance, chlorosis, and disorder of cell membrane functions (Sunitha et al., 2013). Moreover, non-essential metals such as Cd and Pb can be poisonous even at low concentrations and could disturb the nutritional stability of the plant (Evlard et al., 2014). Furthermore, metals obstruct the physiological processes that take place in plants, such as photosynthesis, respiration and cell elongation, and can also influence mineral nutrition and the relationship between plants and water (Yasar et al., 2012). Accordingly, the physiology and leaf structure of plants are changed, as are respiration and photosynthesis rates. Consequently, plant metabolism is influenced by these changes together with transpiration rates and the movement of elements between the various plant organs (Shahid et al., 2015; Ying et al., 2010).
It has been previously reported that metals at elevated levels can result in distressed leaf function through direct and indirect means. For example, the accumulation of metals in plants at elevated levels can injure the leaf and roots as well as the cell wall. Furthermore, acclimatization of carbon dioxide in the plant during photosynthesis can be affected by metals that damage the somatic and
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photosynthetic apparatus and the water conducting system (Hermle et al., 2007). Some metals classified as redox-active (e.g. Fe, Cu, Co and Cr) are directly involved in reactive oxygen species (ROS) formation in the cell in the Haber-Weis and Fenton reaction. In contrast, redox-inactive metals, such as Cd, Zn, Ni and Al induce oxidative stress by interacting with the antioxidant defense system and the electron-transport chain (Evlard et al., 2014). Plants have developed a number of defensive systems to combat the stresses stimulated by the metals. These defensive systems alleviate the damage instigated by oxidative stress in order to sustain crop production and the cellular redox state (Sabir et al., 2015; Shahid et al., 2015).
The conventional physical-chemical technologies employed in soil remediation, including dig-and- dump, vitrification and soil washing or flushing, are generally quite expensive, energy consuming, difficult to implement and deleterious to soil properties (Chen et al., 2012; Lebrun et al., 2016).
Phytoremediation is defined as the remediation of polluted sites using plants in order to extract or stabilize the soil contaminants. Phytoremediation is an energy efficient, environmentally-friendly, cost-effective and attractable method (Lebrun et al., 2016; Moosavi and Seghatoleslami, 2013).
Phytoextraction is a sub-process of phytoremediation and is considered a potential method for cleaning polluted soils and wastewater by using high biomass producing plants to accumulate metals from the soil and water into plant harvestable parts (Afshan et al., 2015; Farooq et al., 2013).
Previously, Salicaceae has been proposed as a suitable phytoremediation plant (Lebrun et al., 2016).
The use of Salix species for the remediation of metal contaminated soils appears encouraging due to their rapid growth rates, high resistance to many pollutants, long vegetative season, resprouting capacity after the harvest of aboveground biomass, effective transpiration rates and high biomass production for energy generation (Kuzovkina et al., 2008; Salam et al., 2015). Plants of the Salix genus had shown potential in the phytoremediation of soil polluted with metals such as Ni, Cu and Zn due to its tolerance to specific metals, adaptation to soil and climate characteristics, and metal uptake capability (Jama and Nowak, 2012; Meers et al., 2007). However, the phytoextraction capability of Salix utilised in polluted and non-polluted areas is mainly dependent on soil type, choice of species, plant age, root development, and biomass production (Salam et al., 2016).
Moreover, successful phytoextraction is mainly dependent on the ability of the plant to produce high levels of biomass and take up large amounts of metals into the plant organs (Subhashini and Swamy, 2013). In a previous study, we have also found that Salix schwerinii E.L. Wolf can uptake and accumulate large amounts of metals (Cu, Zn, Ni, and Cr) into the harvestable parts of the plant (Salam et al., 2016).
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Previously, chelating agents such as EDTA, EDDS, and citric acid have been used as soil extractants, and some chelates have also proved to be a source of micronutrient fertilizers that uphold the solubility of micronutrients in hydroponic solutions (Alkorta et al., 2004). However, some plant roots release low molecular weight organic acids (LMWOA) in the rhizosphere where they act as a chelate and, as a result, create metal organic acid complexes with metal interactions and thus increase the uptake of metals (Lu et al., 2013). Moreover, chelates aid efficient metal phytoextraction but not their elimination, e.g. an increase in LMWOA concentration in the rhizosphere provides carbon sources for soil microorganisms that facilitate metal mobilization from the soil to the plant by (a) replacing adsorbed metals at the surface of soil particles through ligand- exchange reactions, and (b) developing metal-organic complexes (Kim et al., 2013).
The phytoextraction capacity of plants (uptake, accumulation, and translocation of metal ions from roots to shoots) could be significantly increased to enhance phytoextraction by pH adjustment, and the addition of fertilizer or chelating agents. Chelate-assisted phytoextraction is achieved through the addition of chelating agents to the soil in order to increase the bioavailability of the metals and their translocation from root to shoot (Pajević et al., 2016). However, the effect of adding bisphosphonate (BPs) chelating agents such as N10O into the soil to increase the phytoextraction process (by increasing the mobility of the metals in the soil and their subsequent uptake and translocation in plant) has received little attention. The metal-chelating ability of N10O, in combination with its short activity timespan in the soil due to rapid biodegradation, would indicate that it could be a promising soil amendment to increase phytoextraction processes (Alanne et al., 2014; Turhanen et al., 2015).
Bisphosphonates (BPs), such as N10O, are considered suitable metal chelates due to the high complex ability of the bisphosphonic moiety. Chemically, BPs are very stable molecules containing an O=P–C–P=O backbone (Alanne et al., 2014; Matczak-Jon and Videnova-Adrabinska, 2005).
Moreover, N10O is considered environmentally-friendly, easily biodegradable, and a low leaching risk. It is also sparingly soluble in water and has proven to be an excellent metal chelate (Turhanen et al., 2015). Thus, application of a biodegradable chelates such as N10O might minimize phytotoxicity and environmental problems. It has also been shown that BPs have a high competency to bind metal cations, although, due to their mobility and suppleness, the phosphonic functions may clasp several ionic radii (Dyba et al., 1996; Gumienna-Kontecka et al., 2002). Amino BPs are generally specified as nitrogen-containing BPs, which are an exciting class of P compounds that have been used for metal chelation. In addition, amino BPs have the capability to form chelating bonds by using phosphonate groups (P-O), a middle carbon OH group, and a lone pair on the N
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atom. As an alkyl chain of eleven carbon atoms together with an insoluble amino, BPs have been recognized as a productive choice for the chelation of Cd, Ni, Zn and Pb and producing stable complexes (Alanne et al., 2013; Alanne et al., 2014).
In many regions of Finland, mining practices have led to the degradation of soil and groundwater quality through increased pollutant levels, particularly Cu, Ni, and Zn (GTK, 2016). Other major activities that have created contaminated sites include landfills, impregnation plants, and shooting ranges. In these cases, the application of N10O in Salix plantations could assist in the remediation of polluted sites as well as increase the phytoextraction efficiency of the Salix plants.
However, to the best of our knowledge, enhanced phytoextraction of metals by S. schwerinii with N10O chelate has not been investigated in previous studies. Therefore, we conducted a pot experiment to evaluate the effect of N10O application on the growth, physiological attributes, and phytoextraction potential (e.g. metal translocation patterns, accumulation and uptake of Cu, Ni and Zn) of S. schwerinii grown under different metal treatments.
2. Materials and Methods
2.1 Soil Treatments
The phytoextraction experiment was conducted in a greenhouse at the School of Forest Sciences, University of Eastern Finland (UEF), Joensuu (63° 39ˊ N, 26° 2ˊ E) over a period of 180 days from January to June 2016. The experiment was carried out in an ambient environment where the photoperiod was set to 8 h dark/ 16 h light, the greenhouse temperature was set at 21–23 °C and relative humidity was set at approximately 70%. The garden soil used in this study was bought at a local Finnish market and comprises peat (70%), clay (15%) and sand (15%) and served as a control treatment (unpolluted). Before use, the soil was air-dried and passed through a 2-mm strainer. The amount of available trace elements in the garden soil was considerably lower than the threshold level set by the Ministry of Environment of Finland (2007) (Table 1).
The soil was artificially contaminated with three metals: Cu (400 mg kg-1 of soil), Zn (200 mg kg-1 of soil), and Ni (30 mg kg-1 of soil) as the salt in solid state of CuSO4, ZnSO4 and NiSO4, respectively (Table 2). These compounds were thoroughly mixed with the control soil to ensure a homogeneous mixture. The amount of chelate applied to each of the metal treatments was based on the relationship between the number of moles of the chelate and the relevant metal (Supplementary Material 1). The structure of the N10O used in this study is shown in Fig. 1.
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6 2.2 Trial period
A total of 96 S. schwerinii cuttings (approximately 20 cm long) were planted in 1.1 liters plastic pots (H = 17 cm, diameter = 13 mm) filled with 500 g of soil. Salix schwerinii E.L. Wolf is native to east Siberia and is found from Baikal in the east to Kamtschatka and Japan (Pohjonen, 1991).
These cuttings (diameter = 10-20 mm) were collected from the three-year old plantations (diameter 10-20 mm) in Siikasalmi, an experimental UEF area. The wood / (wood + bark) ratio of the plant material was 0.84.
During the growth phase and before planting, lime (20 g kg-1) was added to the soil in all the pots to stimulate plant growth in both the control and contaminated soils (Anderson et al., 2013; Salam et al., 2016). Two Finnish stick-based organic fertilizers were applied to enhance nutrient (NPK) concentrations in the soils. Biolan (Luonnonravinnepuikko) fertilizer was applied once in March and two sticks (1.778 g) were added to each pot. The nutritional value of the fertilizer was 4% N (2% water solubility), 1.2% P, 2% K, 10% moisture and 70% total organic material. In addition, Azet-Neudorff fertilizer was applied once in May and two sticks (6.25 g) were put in every plant pot. The elemental composition of the fertilizer was 8% N (4.2% water solubility), 0.87% P, 1.66 % K and 10% moisture.
The experimental design was fully randomized with six treatments of each metal, including the control with two repetitions. The locations of the pots were rotated within the greenhouse during the experimental period to avoid the effects of micro-climatic variation. The plants were irrigated daily with tap water and care was taken to avoid the leaching of water from the pots. Through visual observations, we neither observed any leaching of N10O from the soil pots nor any toxic and inhibitory chelate effects on the S. schwerinii growth.
2.3 Measurement of gas exchange parameters
Before harvesting, CO2 uptake (A; μmol CO2 m-2 s-1), transpiration rate (E; mmol H2Om-2 s-1), and chlorophyll fluorescence (Fv/Fm), which represents the efficiency of the photosystem after 20 minutes of dark adaptation on intact and fully-fledged leaves (Evlard et al., 2014), were measured using a portable Infrared Gas Exchange Analyzer equipped with an LED light source (LI- 6400 XT, Lincoln, USA). Instrument calibration was implemented daily according to the manufacturer’s guidelines. About three to four fully-mature, attached leaves from each treatment were measured, and the measurement was repeated to record five observations for each leaf sample, which were then averaged. During each measurement, the temperature in the leaf chamber was maintained at 23
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°C, relative humidity at 65%, photon flux density at approximately 1250 μmol m-2 s-1, and CO2 concentration at 400 μmol mol-1. In addition, the reference CO2 concentration was fixed at 400 μmol mol-1 (Andralojc et al., 2014). Lighting was provided by an LED light source to transfer photosynthetic active radiation (PAR) at 1500 µmol m-2 s-1. A stream of ambient air to the leaf chamber was provided by an air supply unit at a constant rate of 100 µmol s-1. All readings were taken by fixing the central slice of the leaf in the chamber of the gas analyser between 10:00 am and 2:00 pm to ensure constant photon flux density and temperature.
2.4 Determination of plant growth parameters
During the growth period, the mean absolute height of the S. schwerinii was measured for twenty- two weeks. The mean absolute height was calculated by subtracting the initial height of the plants from the final height. Plant growth parameters, such as shoot diameter, were measured with a digital caliper, and the leaf surface area (LSA) was measured with a Li-Cor Li-3000 leaf area meter.
Moreover, the number of leaves per plant and the dry biomass (leaves, shoots, and roots) were calculated. All the plant parts were brushed to remove impurities and washed with deionized water before they were placed in an oven. Subsequently, the leaves, shoots and roots were dried at 105 °C in the oven for 72 h and then weighed.
2.5 Metals analysis
The dry plant samples (leaves, shoots, and roots) were ground and then ashed at 500 °C in a muffle furnace and strained with a 1-mm sieve for chemical analysis of the metals. The dehydrated, ashed samples of the plant tissues were digested with 6 ml nitric acid (HNO3)+ 1 ml hydrogen peroxide (H2O2) in a microwave oven (Cem, MarsXpress) and brought up to 25-ml volume using microwave-assisted extraction method EPA 3051. We used similar method (EPA 3051) for the determination of extractable metals in the soil (EPA, 2007). Total reflection X-ray fluorescence (TXRF) spectrometry using Gallium (Ga) standard protocols (De La Calle et al., 2013; Towett et al., 2013) was used to determine the concentration of Cu, Zn and Ni in S. schwerinii tissues and soil.
2.6 Translocation factor (TF), bioconcentration factor (BF), and metal uptake percentage (%) Translocation factor (TF) was calculated to examine the metal translocation efficiency from the roots to the aerial parts of Salix as described by Hussain et al. (2017).
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Bioconcentration factor (BF) was calculated as described by Hussain et al. (2017) and Zhao et al.
(2003) used to investigate the capability of Salix to transport the metals from the soil (substrate) to the shoots.
BF =
The uptake percentage for total metal concentration (TMC) in the plant organs (leaves, shoots, and roots) was evaluated using the expression used by Karnib et al. (2014), Nazaralian et al. (2017), and Salam et al. (2016) as given below:
× 100 2.7 Statistical analysis
The analysis of co-variance (ANCOVA) procedure was performed to analyse the treatment effects on biomass, height, total metal concentration, and gas exchange parameters (p < 0.05; p < 0.001).
Before the analysis, Levene’s test was run to check the homogeneity of variance across all treatments. Wherever considerable differences were found among the treatments, a pairwise comparison was conducted using Tukey's test. All statistical analysis was carried out using the R- software tool (R development Core Team, 2017).
3. Results and Discussion
3.1 Mean absolute height
Salix schwerinii survival percentage was about 98% which characterizes its capacity to survive in polluted soils (Lebrun et al., 2016; Salam et al., 2016). The highest growth (72 cm) was found in the control + N10O 6.9 g treatment with 65% increase when compared to the control (Fig. 2A). When treatments Cu + N10O 3.45 g and Cu + N10O 6.9 g were compared with the control, no significant differences were observed. The high dose of N10O (6.9 g) in the control treatment (control + 6.9 g N10O) appeared to increase plant height but the medium dose of N10O (3.45 g) had no considerable effect on height in the Cu + N10O 3.45 g or in the control + N10O 3.45 g treatments (Fig. 2A). On the other hand, compared to the control, the treatment control + N10O 3.45 g represented significant variation (51%) in height. However, the addition of the N10O chelate did
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not enhance the height growth in the Cu, Cu + N10O 3.45 g and Cu + N10O 6.9 g treatments (Fig.
2A).
With soil polluted with Ni, no significant differences in the height of S. schwerinii were observed between the control and other treatments, except treatment Ni + N10O 1.2 g (Fig. 2B). The treatment Ni + N10O 1.2 g produced 33% lower height when compared to the control. More precisely, the increase in S. schwerinii height growth was more than 50 cm in all other treatments except the control (43 cm) and Ni + N10O 1.2 g (30 cm). Furthermore, in the treatment control + N10O 1.2 g, S. schwerinii showed a significant increase in height when compared with a similar dose (1.2 g) in the polluted soil treatment (Ni + N10O 1.2 g). However, in all treatments, the effect of N10O on plant height was observed non-significant (p > 0.05).
The increase in height of S. schwerinii in the Zn polluted and the control soils along with different doses of N10O was highest (68 cm) in the control + N10O 2.9 g treatment (Fig. 2C). In contrast, the lowest growth rate was observed in polluted soil treatment Zn + N10O 2.9 g. When compared with the control, these two soils (control + N10O 2.9 g and Zn + N10O 2.9 g) experienced a growth increase of about 55% and decrease of 20%, respectively. Gill et al. (2015) reported that a plant may reduce growth due to ultrastructural disruption in plant tissues, and Sharma and Dubey (2005) demonstrated that the inhibition of enzymatic activity, the distortion of nutrition, hormonal changes, and membrane permeability all may have adverse effects on the physiological processes of a plant.
In all treatments (Fig. 2A–C), an increase in height growth was observed in the control treatment irrespective of the N10O dose. However, N10O medium doses in Zn and Cu polluted soils and high doses in Ni polluted soils were observed favorable to increase growth. Normally, when the plants are grown in polluted environment, numerous factors such as soil pH, P concentration, organic matter content, metal concentrations (and subsequent soil interactions), and species tolerance may improve the growth of the plant (Kocoń and Jurga, 2017; Waterlot et al., 2011). There was no variation in plant growth between the metal-treated (Cu, Ni, and Zn) and the control soils (Fig. 2A–
C). Enzyme inhibition by 7-amino-1-hydroxyundecylidene-1, bisphosphonate (BP7) might be a factor for plant growth reduction, as bisphosphonates obstructs the assembly of a metal-dependent enzymes such as farnesyl pyrophosphate synthase (FPPS) (Alanne et al., 2014).
3.2 Dry biomass
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Biomass production can be considered as an important attribute to assess the ability of a plant to survive metal toxicity (Zárubová et al., 2012). The highest total mean dry biomass (sum of leaves, shoots and roots) 7 g and 6 g was found in the treatments control + N10O 3.45 g and control + N10O 6.9 g, respectively, representing 74% and 63% increase when compared to the control (Fig.
3A–C). The lowest biomass 2 g was found in the treatment Cu + N10O 6.9 g representing 33%
decrease when compared to the control. However, there was no effect of N10O observed in the Cu polluted soils to enhance the production of dry biomass (Fig. 3A). However, we found significant difference at (p < 0.05) among all the treatments (Supplementary Material 2).
Furthermore, in Ni polluted soils, control + N10O 1.2 g produced the highest biomass (9 g) and 135
% increase in biomass when compared to the control (Fig. 3B). At the same time, there was a slight decrease in the dry biomass found between Ni (4 g) and Ni + N10O 1.2 g (3 g) treatments and interestingly, the dry biomass of S. schwerinii increased by about 13% in the Ni treatment as compared to the control. The reason could be that the treatment received more nutrients, or Ni toxicity at low level of Ni concentration into soil does not have effect on biomass production.
Contrary to our result, the effect of Ni toxicity on plant biomass reduction has previously been observed, for example, for the Brassica juncea L (Ansari et al., 2015) and for the Solanum lycopersicum (Mosa et al., 2016). No significant differences were found among all the treatments, however, except the treatment control + N10O 2.9 g, which showed greater biomass production (6 g). In contrast, slight differences were found between the biomass production of treatment control + N10O 2.9 g (6 g) (+74%) and treatment control + N10O 1.45 g (5 g) (+36%), compared to the control (Fig. 3C).
When compared to the control treatment, S. schwerinii exhibited the equivalent biomass production in polluted (Cu, Ni and Zn) and polluted + N10O treated soils (p > 0.05). Nevertheless, S.
schwerinii did not produce an adequate amount of dry biomass in the control treatment. This could be due to the low amount of nutrients received by plant in the control treatment which is consistent with findings reported by Mohsin (2016) and Salam et al. (2016). Furthermore, Kuzovkina et al.
(2004) and Wang et al. (2016) also found reduction in the biomass yield with different Salix growing on polluted soils. The overall variation in dry biomass production in the S. schwerinii among all the treatments is presented in Supplementary Material 2. The chelate N10O application was found to be effective in enhancing height and biomass growth under polluted soils and significantly enhanced the total metal concentrations in the S. schwerinii. This could be due to the effect of N10O, which reduces metal phytotoxicity and increases biomass production and metal
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uptake and is in agreement with similar findings reported by Alanne et al. (2014) for Noccaea caerulescens grown in polluted soils amended with N10O. Differences in soil type, metals, and N10O concentrations could also be reasons for this effect since N10O contains N and P groups (Turhanen et al., 2015). Some chelates could have fertilizer properties as they can mobilize soil nutrients and enhance their bioavailability to the plants (Yeh and Pan, 2012).
When biomass production among all the treatments was compared, a considerable effect of N10O on dry biomass was observed in all N10O-amended control treatments. Salix schwerinii showed no difference in dry biomass when grown on the (without N10O) control and polluted soils (Fig. 3 AC). It has been reported that, Salix species has potential to produce good biomass in the Pb- polluted soil and to accumulate significant amounts of Pb into leaves, shoots and roots (Zhivotovsky et al. 2011). Salix species has potential to produce good biomass in Cu-, Ni-, and Zn- polluted soils have also been observed (Meers et al., 2007). Interestingly, N10O with multiple doses appeared to be a growth promoter when added in the control soil, and it could be used as a chelating agent. Alkorta et al. (2004) stated that some chelates have versatile properties that can be used as a micronutrient fertilizer and metal chelation. Thus, high biomass production by plants treated with a non-toxic chelate such as N10O could be a feasible choice for the removal of metals from soils.
3.3 Biometric analysis
Biometric analysis (shoot diameter, leaf surface area (LSA) and number of leaves per plant) can be a helpful tool to assess the productivity of a plant. The largest S. schwerinii shoot diameter (4 mm) was found in the control + N10O 3.45 g treatment, and it presented an increase of about 37% as compared to the control (Table 3a). Among the Cu polluted soils, the largest shoot diameter (3 mm) was observed in the Cu + N10O 3.45 g treatment, which represented 5% increase and 23% decrease when compared to the control and control + N10O 3.45 g treatments, respectively. The highest LSA value (191 cm2) was observed in the Cu treatment, representing 32% increase as compared to the control. The results revealed that S. schwerinii tended to maintain the same level of LSA, diameter growth, and number of leaves per plant under Cu treated soil and control soil. However, there were significant difference found on LSA with the treatment Cu + N10O 6.9 g (Table 3a).
When the soil was treated with Ni, no significant difference was found for shoot diameter among the treatments. On the other hand, significant differences were found in the case of LSA and the number of leaves (Table 3b). The number of leaves was significantly higher in both treatments Ni +
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N10O 1.2 g and Ni + N10O 2.4 g. The maximum LSA (264 cm2) was observed in the treatment Ni + N10O 2.4 g, which represented 55% increase in LSA when compared to the control.
With the treatments including Zn, the largest diameter (5 mm) was observed in the Zn + N10O 2.9 g treatment and the significant difference (p < 0.001) in comparison to other treatments was also found. Also, there was a significant difference found for LSA and the number of leaves with Zn polluted soils amended with N10O. The highest LSA (257 cm2) was found in Zn + N10O 2.9 g, with an increment of about 51% as compared the control. The number of leaves was significantly higher (p < 0.001) for both control and N10O treatments (Table 3c).
3.4 Gas exchange and chlorophyll fluorescence responses
The photosynthetic attributes (gas exchange and chlorophyll fluorescence) are used to examine the phytotoxicity of metals on plant growth in polluted soils (Iori et al., 2013). There were significant differences found among the treatments for photosynthesis (A) and transpiration (E), but not on fluorescence (Fv/Fm) (Table 4 ac). For the control treatment, the value of CO2 assimilation was (9 μmol CO2 m-2 s-1) and a robust inhibitory effect on photosynthesis was observed in S. schwerinii when exposed to Cu pollution (4 μmol CO2 m-2 s-1), which exhibited a reduction of about 53%
when compared to the control (Table 4a). Similar findings have been documented by Burkhead et al. (2009) and Heckathorn et al. (2004) with respect to a reduction in growth and photosynthesis activity, CO2 fixation, and photosystem reactions in Cu polluted soils. The higher concentration of Cu may influence the photosynthetic electron transport system in the plant (Shahid et al., 2015).
However, S. schwerinii exposed to the Cu pollution had reduced photosynthetic activity (CO2
uptake), which could be due to the reduction of chlorophyll content by toxic effects of Cu. Similar results were reported for Salix acmophylla in Cu and Ni polluted soils (Ali et al., 2003) and for hybrid S. purpurea × triandra × viminalis in Zn polluted soils by Borowiak et al. (2015). Results might be due to the formation of ROS that interferes with the thylakoid membrane of a chloroplast and to the negative impact of metals in plants, which has also been found for gas exchange activities (Almeida-Rodríguez et al., 2016).
Productive photosynthesis enhances the capability of a plant to produce metabolites for fortification and growth (Nikolić et al., 2015). The high dose of N10O positively influenced the photosynthetic efficiency (CO2 uptake) in the Cu polluted soil. In plant tissues, the availability of metals can reduce CO2 fixation through partial closure of the stomata (Pajević et al., 2009), which is consistent with results for the Cu-treated plants. However, following the addition of N10O, the highest CO2
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assimilation rate (11 μmol CO2 m-2 s-1) was found in the Cu + 6.9 g N10O treatment with an increment of 30% with respect to the control (Table 4a). Similarly, Hermle et al. (2007) reported increased photosynthetic rates in Salix viminalis grown in soils contaminated with metals.
Concerning to CO2 uptake, when compared with the control, a slight reduction was found in Ni and Zn treatment once amended with N10O doses. In contrast, in Ni and Zn treatments (without N10O) CO2 uptake increased by 36% and 37%, respectively, when compared to the control (Table 4 ac).
Pajević et al. (2009) documented that different metals might have a dissimilar influence on transpiration and growth in the same plant.
Chlorophyll fluorescence (Fv/Fm) parameter is normally used to evaluate stress on the metabolism of the plant (Evlard et al., 2014; Linger et al., 2005). And, any alteration in the plant photosystem (where protein is involved in photosynthesis) highlights impairment in the photosynthetic process (Torres Netto et al., 2009). Misra et al. (2012) found that Fv/Fm value between 0.78-0.84 indicates fitness of a plant. The attained ratio of Fv/Fm (0.80-0.84) (Table 4 ac) among all the treatments demonstrates that S. schwerinii remained healthy and had not been affected from any stress disorder resulted from metals concentration during the growth period (Bahri et al., 2015). The relative constancy of the fluorescence parameter Fv/Fm is characterizing the stability of the thylakoid structure as well as the flow of electrons in photosystems and is consistent with the results reported by Pajević et al. (2009). Similarly, Bernardini et al. (2016) also did not find disruption in Fv/Fm values for several Salix clones grown on contaminated soils. Notably, S. schwerinii did not exhibit metabolic disruption in the polluted soils as they produced an effective response with respect to CO2 uptake, transpiration, and photosystem efficiency.
3.5 Phytoextraction of the metals
The Cu, Ni, and Zn total metal concentrations (sum of dry leaves, dry shoots, and dry roots) in S.
schwerinii were determined to check the phytoextraction efficiency of the applied N10O (Fig. 4 AC). There was significant difference among the treatments for Cu accumulation (Supplementary Material 2). The highest accumulation (230 mg kg-1) was found in the Cu + N10O 6.9 g treatment indicating the effectiveness of N10O to extract Cu about 892% compared to the control. However, when the unamended (without N10O) Cu treatment was compared with the unamended control treatment, the total Cu concentration increased about 730%. Furthermore, when the unamended Cu treatment was compared with the amended Cu treatments (with N10O), the total Cu concentration considerably increased up to 68% in the high amended Cu treatment (Cu + N10O 6.9 g), however, decreased by 38% in the medium amended Cu treatment (Cu + N10O 3.45 g) (Fig. 4A). It can be
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concluded that Salix are Cu-tolerant plants as has been previously reported by Kuzovkina et al.
(2004), Meers et al. (2007) and Yang et al. (2014). Generally, the capability of the plants to accumulate Cu is contingent on certain factors including seedling age (Yang et al., 2014), and transpiration rate of the plant (higher transpiration rate increases metals uptake) (Mleczek et al., 2010).
Total Ni accumulation in S. schwerinii was highest (123 mg kg-1) in Ni + N10O 1.2 g treatment by an increase of 321% compared to the control (Fig. 4B). Nickel uptake was considerably increased by the addition of multiple N10O doses. Comparatively, no significant difference was found between Ni + N10O 1.2 g and Ni + N10O 2.4 g treatments, while considerable differences were found between all treatments (Supplementary Material 2). However, the use of N10O for the phytoremediation of Ni polluted soils should receive further investigation. Furthermore, the maximum total Zn concentration in S. schwerinii (4483 mg kg-1) was observed in Zn + N10O 1.45 g treatment. On the other hand, in Zn treatment (without N10O) the concentration of Zn in S.
schwerinii was 3695 mg kg-1 (Fig. 4C). Significant differences among the treatments (p < 0.001) were found, and it was observed that the medium dose of N10O (1.45 g) was much more effective than the higher dose (2.9 g) in terms of Zn accumulation.
Visual metal-related phytotoxicity symptoms, such as necrosis and chlorosis, were not observed during the experimental period. This could be due to the applied concentration of metals that was not found severely toxic to S. schwerinii. Similar observations were reported by Ji et al. (2011) and McIntosh (2014) who did not find symptoms of metal stress on Solanum nigrum L and different Salix species such as Salix dasyclados, Salix eriocephala, Salix miyabeana and Salix purpurea respectively, when grown in polluted soils. However, S. schwerinii accumulated Zn at higher rates, followed by Cu and Ni. The phytoremediation potential of S. schwerinii to tolerate and accumulate a considerable amount of Cu, Ni and Zn from polluted soils is consistent with previous studies (Desjardins et al., 2016; Evlard et al., 2014; Meers et al., 2007; Salam et al., 2016; Wang et al., 2016). Researchers assume that, the metal concentrations in the soil is generally absorbed by plant roots and is regulated by metabolic processes within the plant which consequently impacts development, growth, and drought resistance (Mandre, 2014). However, it is expected that the longer the plants can remain in situ and absorb metals, the greater the amount of metals will be extracted (do Nascimento et al., 2006).
3.6 Translocation factor (TF) bioconcentration factor (BF) and uptake percentage of metals
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The translocation factor and BF values can be used to evaluate the potential of a plant to accumulate metals from the soil and to translocate them to aerial parts (Hussain et al., 2017). At TF < 1 there is low metal transfer and at TF > 1 there is high metal translocation (Garba et al., 2012). Hussain et al.
(2017) explained the four-degree scale where BF < 0.01 indicates no accumulation, 0.01-0.1 low accumulation (0.01-0.1), medium accumulation (0.1-1.0) and > 1.0 high bioaccumulation. Salix species have been widely-studied regarding their potential to translocate metals from roots to aerial parts (Tőzsér et al., 2017). Salix growing on the polluted soils have different metals accumulation capacity as well as high genetic variability in growth rates (Salam et al., 2016; Yang et al., 2014;
Zhivotovsky et al., 2011).
The translocation factor and BF values increased significantly with an increase in N10O doses in all Cu, Ni, and Zn polluted soils (Fig. 5 AB). In all metal treatments, medium translocation values were observed for Cu (0.30), Ni (0.45) and a high value for Zn (4.40). On the other hand, in the amended metal treatments (with N10O), high translocation values (TF > 1) were found for Cu, Ni and Zn (Fig. 5A). Moreover, when S. schwerinii were grown in unamended metal treatments (without N10O), medium BF values were found for Cu (0.60) and Ni (0.80) and a high value for Zn (4.0). In contrast, high BF values (BF > 1) were found in the amended metal treatments (Fig. 5B).
The average uptake of the metals in the polluted soil treatments were as follows: Ni (90%), Cu (75%), and Zn (64%) (Fig. 6). The highest metal uptake of S. schwerinii was found in the Ni treatment, followed by Cu and Zn treatments. Correspondingly, when N10O was added to the Cu, Ni, and Zn polluted soils, about 6 –12%, 3 – 4% and 16 – 23% improvement was observed in the metal uptake in S. schwerinii, respectively. It has been observed by Vigliotta et al. (2016) that the addition of chelates resulted in an increase of Cu, Ni, and Zn uptake in many plant species, which is in line with our findings. Consequently, N10O could be a novel approach to enhance the phytoextraction of Cu, Ni, and Zn by S. schwerinii. These findings reveal that S. schwerinii has the capability to grow on Cu, Ni, and Zn polluted soils as well as produce high biomass which could be used for bioenergy purposes.
4. Conclusions
This study demonstrates the utilization of chelate N10O for the environmentally-friendly phytoextraction of Cu, Ni, and Zn contaminated soils and the chelate effect on Salix schwerinii growth parameters. The chelate N10O enhanced the translocation and accumulation of Cu, Ni, and Zn in S. schwerinii through alleviating the toxic effects of metals. Also, N10O showed fertilizing
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properties and provided some nutrients (i.e. NPK) that could effectively stimulate S. schwerinii growth and be seen in biometric and ecophysiological parameters. The application of N10O using S.
schwerinii could be a novel approach for the phytoextraction of Cu, Ni, and Zn contaminated soils.
However, further research is needed at larger scales to investigate the chelating efficiency of N10O using different Salix varieties.
Acknowledgements
This study was financially supported by the Kone foundation (Grant No. f5a0d4). We would like to acknowledge anonymous reviewers for their valuable comments. Authors also thanks company BioSO4 Oy to deliver chemical N10O.
Competing interests
The authors declare that they have no competing interests.
Author contributions
Muhammad Mohsin performed the experiment, collected data, and prepared the manuscript. Mir Md Abdus Salam contributed in data analysis and manuscript preparation. Suvi Kuittinen, Saila Laine and Erik Kaipiainen helped in conducting the experiment. Ari Pappinen supervised and designed the experiment. Pertti Pulkkinen, Sirpa Peräniemi, Jouko Vepsäläinen, and all authors improved the manuscript.
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