Populus nigra as a phytoremediator for Cd, Cu, and Pb in contaminated soil

Abstract

The contamination of agricultural soil with heavy metals is a complex phenomenon that causes negative consequences for various organisms. Poplars may have considerable phytoremediation potential, and this plant species can tolerate Cd, Cu, and Pb up to 15.6, 63.6, and 173.3 mg kg-1 soil, respectively, with 100% survival. The analyzed data revealed significant reduction in vegetative growth traits and leaf N, P, K, and carbohydrate (%) and leaf green color degree. However, a simultaneously significant increase in enzymatic activities and electrolyte leakage were recorded in comparison to control plants. A bioconcentration factor of plant organs was ˂ 1, and the translocation factors (TF) of Cd and Cu were ˂ 1 ( ˂100%) under various concentrations of each heavy metal, while TF of Pb was ˃ 1(>100%), except for the first level. More Cd, Cu, and Pb contents were localized in roots compared to leaves or stems. Thus, the risk of contamination through leaf can be minimized. Populus nigra has defense mechanisms against Cd, Cu, and Pb up to 7.8, 29.8, and 91.1 mg/kg soil, respectively because the tolerance index (TI) of either biomass or root was >0.8. Finally, it is a good candidate for research of phytoremediation and phytoextraction.

Full Article

Populus nigra as a Phytoremediator for Cd, Cu, and Pb in Contaminated Soil

El-Sayed Mohamed El-Mahrouk,^a Eman Abd El-Hakim Eisa,^b Hayssam M. Ali,^c,d,* Mahmoud Abd El-naby Hegazy,^a and Mohamed El-Sayed Abd El-Gayed ^b

The contamination of agricultural soil with heavy metals is a complex phenomenon that causes negative consequences for various organisms. Poplars may have considerable phytoremediation potential, and this plant species can tolerate Cd, Cu, and Pb up to 15.6, 63.6, and 173.3 mg kg^-1 soil, respectively, with 100% survival. The analyzed data revealed significant reduction in vegetative growth traits and leaf N, P, K, and carbohydrate (%) and leaf green color degree. However, a simultaneously significant increase in enzymatic activities and electrolyte leakage were recorded in comparison to control plants. A bioconcentration factor of plant organs was ˂ 1, and the translocation factors (TF) of Cd and Cu were ˂ 1 ( ˂100%) under various concentrations of each heavy metal, while TF of Pb was ˃ 1(>100%), except for the first level. More Cd, Cu, and Pb contents were localized in roots compared to leaves or stems. Thus, the risk of contamination through leaf can be minimized. Populus nigra has defense mechanisms against Cd, Cu, and Pb up to 7.8, 29.8, and 91.1 mg/kg soil, respectively because the tolerance index (TI) of either biomass or root was >0.8. Finally, it is a good candidate for research of phytoremediation and phytoextraction.

Keywords: Phytoremediation; Contamination; Heavy metals; Cadmium, copper; Lead; Populus nigra

Contact information: a: Hort. Dept.- Fac. Agric.- Kafrelsheikh Univ. 33516, Egypt; b: Sakha Horticulture Research Station 33717, Horticulture Research Institute, Agric. Res. Center, Giza, Egypt; c: Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; d: Timber Trees Research Department, Sabahia Horticulture Research Station, Horticulture Research Institute, Agriculture Research Center, Alexandria 21526, Egypt;

* Corresponding author: hayhassan@ksu.edu.sa

INTRODUCTION

Pollution of agricultural soils by heavy metals is a complicated and dangerous phenomenon (Puschenreiter et al. 2005). Heavy metals are the main components of inorganic contaminants, which pose a different problem than organic contaminants (Wu et al. 2018). Heavy metals are ubiquitous, highly persistent, and non-biodegradable (Gardea-Torresdey et al. 2005). Rocks in the soil are subjected to natural weathering, which is a source of metals ions. Also, the disposal of waste and use of fertilizers, pesticides, insecticides, and industrial effluent can contaminate the soil by increasing their concentrations of heavy metals (Abdullahi et al. 2009). Among the most common heavy metals are Cd and Pb, and Cu at a higher concentrations. Of these, Cd is one of the big HMs poisons and is not known for any essential biological process (Campbell 2007). Cd can cause harm to various organless entities including chloroplasts, nucleus, vacuole, mitochondria, and it inhibits activities of many enzymes, such as those rich in sulfurhydryl groups (Clemens 2006). Pb is one of the toxic HMs (Yang et al. 2005). Pb leads to inhibition of photosynthesis, oxidative stress, and geno toxicity including DNA damage and defects in mytosis (Küpper 2017). Cu becomes a toxic component when its concentration in the tissue of plants raises above optimal levels (Lombardi and Sebastiani 2005). Cu ions can catalyze the productin of highly toxic hydroxyl radicals, particularly through Fenton chemistry, causing the adverse impact to macromolecules and disturbance of metabolic pathways (Hänsch and Mendel 2009).

Many mechanically or physiochemically based remediation technologies have been conducted to treat contaminated soil (Marques et al. 2009). These technologies are expensive and disturb the soil, sometimes rendering the land useless for plant growth. Hence, phytoremediation is an alternative using plants to clean heavy metals from contaminated soils, sediments, and water. Phytoremediation is environmentally friendly and low cost, and it is effective in reducing ions in contaminated soils to very low levels (Rakhshaee et al. 2009). It is applicable for a wide range of toxic metals and radionuclides (Liu et al. 2000). Zárubová et al. (2015) mentioned that the bioavailability of metals in soil and different physiological processes in plants affect HM toxicity, adsorption, and distribution in the plant. These variables are dependent on plant growth stage, time of exposure and heavy metal concentrations in soil, soil pH, organic matter, added fertilizers, and temperature.

Heavy metals induce oxidative stress through the production of reactive oxygen species (ROS) that promote membrane lipid peroxidation, protein oxidation, enzyme inhibition, and damage to nucleic acid, resulting in cell death. To improve oxidative stress, plants employ the antioxidative machinery by alternations in antioxidative enzyme activities (e.g., superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione s-transferase) and the levels of low molecular weight antioxidants (ascorbic acid, reduces glutathione, carotenoids, phenolics (Hossain et al. 2012). Redovniković et al. (2017) supported this fact on P. nigra ‘ Italica’ cv. The balance between ROS and the antioxidative system is a key factor for plant adaptation and survival under stressful conditions such as heavy metals (Sharma et al. 2012)

Poplars are able to remove soil contaminants by phytoextraction, phyostabilization, phytovolatilization, and rhizofiltration. Advantageous characteristics of poplars for phytoremediation include quick stablishment, fast growth, large biomass accumulation, extensive and deep root systems, high transpiration rates, easy asexual propagation, exceptional growth on marginal lands, not being part of the food chain, long life (25 to 30 years), and poplars ability to be harvested and then regrown (Sebastiani et al. 2004; Zalesny et al. 2008). The plants can decrease the HMs concentration in the soil by phytoextraction in which the plants uptake heavy metals from soil by concentrating the metal in areal plant organs (Sebastiani et al. 2004). Phytostabilization is the use of plants to stabilize the soil surface via retaining the metals in the roots (Marques et al. 2009), and rhizolphiltration is where the plant roots absorb or adsorb metals from water and aqueous waste streams (Erakhrumen and Agbontalor 2007).

It is important to estimate whether a tree under any phytoremediation strategy will contaminate the wider environment through leaf fall. The heavy metals content in the green and fallen leaves is dependent on plant species and metal concentration and metal type in the soil. Rafati et al. (2011) who reported that the fallen leaves of Populus alba had more Cd content than the green leaves, while Morus alba had more Cd content in the green leaves than the fallen leaves. The same authors added that Cr was high in the fallen leaves and Ni was high in the green leaves.

Populus nigra L. (Salicaceae) is native to Europe, southwest and central Asia, and northwest Africa. It is widely distributed in Egypt and used for many purposes. Populus nigra produces economically valuable non-food biomas that is exploitable for energy production. Most previous studies have been conducted in acidic soil or a hydrobonic system for a short period (less than one year). Data of the effect Cd, Cu, and Pb on leaf chemical and biochemical traits and electrolyte leakage in P. nigra are limited in the previous studies. Moreover, there are no available data about the using of P. nigra as phytoremediator for Cd, Cu, and Pb in Egypt. Furthermore, large areas in Egypt have a high amount heavy metals, particularly Cd, Cu, and Pb owing to the irrigation with sewage effluent, industrial effluent or drainage water, and also usage of sewage sludge as organic fertilizer for fertilization the soil. This study investigated the effect of heavy metals such as Cd, Cu, and Pb on vegetative growth traits, chemical compositions, enzymatic activity, electrolyte leakage, and efficiency of metal phytoextraction of P. nigra. Its use as a phytoremediator for heavy metal-contaminated slightly alkaline soils was examined.

EXPERIMENTAL

Plant Material

Black poplar is a medium to large deciduous tree, reaching 20 to 30 m and rarely 40 m tall, and 1.5 m in diameter. Shoot cuttings (one-year-old wood) that were 15 cm in length and 0.5 cm in diameter were taken from a 10 year-old mother tree of P. nigra L. grown in the Experimental Farm of the Fac. Agric. Kafrelsheikh Univ. Egypt, on the first of February. Cuttings were cultured in the plastic bags of 10 cm diameter (one cutting per bag) filled with clay soil, which was used for experiments. The cultured bags with cuttings were placed in an air-condition in plastic greenhouse adjusted 25 ± 2 °C, 40 to 50% relative humanity, and a photoperiod 16 h light/8 h dark, with a light intensity of 300 µmol m^-2s^-1. The cuttings were watered manually every 10 days using 10-L watering cans; the same amount of water was applied to each bag.

Heavy Metals Treatments

The heavy metals were applied as follows: cadmium chloride [Cd Cl₂.H₂O] at rates of 20, 40, 60, and 80 mg/kg soil, equal to 3.9, 7.8, 11.9, and 15.6 mg Cd/kg soil, respectively; copper chloride [Cu Cl_2.2H₂O] at rates of 50, 100, 150, and 200 mg/kg soil, equal to 14.4, 29.8, 47.7, and 63.6 mg Cu/kg soil, consecutively; and lead acetate trihydrate [(CH₃COO)₂ Pb.3H₂O] at rates of 250, 450, 650 and 850 mg/kg soil, equal to 50, 91.1, 132.1, and 173.3 mg Pb /kg soil, each in turn. The soil was placed in the plastic pots of 40 cm in diameter with 9 kg air-dried weight soil per pot, spiked with solutions of the aforementioned concentrations of heavy metals, and equilibrated for 60 days outdoor under a waterproof tarpaulin. Each metal was added separately to prevent interaction effects. World soil average of Cd, Cu, and Pb are 0.41, 27, and 39.9 mg/kg soil and maximum allowable concentrations of such metals are 1 to 5, 60 to 150, and 20 to 300 mg/kg soil, respectively (Kabata-Pendias 2011).

Planting Date

Identical growth three month-old transplants (average 35 cm height and 0.9 cm stem diameter at the soil surface) were transplanted into the pots (one transplant/ pot) on May 1, 2015. The transplants were placed in the replicated plots after the planting. The plants were irrigated with tap water to reach the field capacity when needed.

Design of the Experiment

The experiment was designed using a complete randomized design (Snedecor and Cochran 1980). The experiment replicated 3 times, each replicate consisted of 13 treatments (3 heavy metals × 4 concentrations, and the control). Each replicate was represented by three plants; thus, each treatment was represented by nine transplants.

Recorded Measurements

At the end of the experiment on August 1, 2017, six plants (30 months old) for each treatment (two plant of each replicate) were chosen randomly to determine the following parameters described below.

Vegetative growth traits

Plant height (from the soil surface to shoot apex, cm), the number of branches per plant, the stem diameter (measured 5 cm from the soil surface, cm), the area per leaf (cm²) using a C1-202 laser area meter (Cid Bio-Science, Camas, WA, USA), and length of the longest root (cm) were determined. To determine the dry weight, harvested plants were separated into roots, stems, and leaves, which were washed twice (first with tap water to remove soil, then with deionized water) and then oven-dried at 80 °C for 24 h (Rautio et al. 2010). The degree of greenness was measured for the fifth leaf from the apex meristem using a portable leaf chlorophyll meter (SPAD-501, Minolta, Osaka, Japan), as described by Markwell et al. (1995).

Leaf chemical composition and Cd, Cu, and Pb contents and uptake in the plant parts (leaves, stems, and roots (

Dry samples were ground to obtain a homogenous powder in a metal-free mill (IKa-Werke, M 20, Staufen, Germany). Dried samples of 0.2 g were wet digestion to produce a clear solution according to Evenhuis and de Waard (1980) as follows: concentrated sulfuric acid (95%, 5 mL) was added to the sample (0.2 g), and the mixture was heated for 10 min. on a sand hot plate.Then, 0.5 perchloric acid was added, and heating was continued until a clear solution was obtained, The solution was left to cool bfore it was filtered and diluted to 50 mL with distilled water.

The digested samples were prepared for measuring nitrogen (N%) using a modified micro Kjeldahl methods as described by Chemists and Horwitz (1990). Phosphorus (P%) was extracted according to Murphy and Riley (1962) and determined calorimetrically using spectrophotometer (Beijing Purkinje instrument, T 80⁺, London, UK). Potassium (K%) was extracted as described by Cotteine et al. (1982) and determined using an atomic adsorption spectrophotometer (GPC Avanta E, Victoria, Australia). The total carbohydrate percentage in leaves was determined according to Herbert et al. (1971). The cadmium, copper, and lead concentrations (mg/kg D.W.) in different plant parts were determined according to Page et al. (1982) using an atomic absorption spectrophotometer (GPC Avanta E, Victoria, Australia). The uptake of heavy metal (mg/organ d.w.) in different plant parts and total uptake /plant(mg/plant) were calculated as follows,

 (1)

where MU is metal uptake, MC is metal concentration (mg/kg d.w), and ODW is organ dry weight (g).

TPU =LU +SU+RU (2)

In Eq. 2, TPU is total plant uptake (mg/plant), LU is leaves uptake (mg), SU is steam uptake (mg) and RU is root uptake (mg).

Biochemical assays of antioxidant enzymes and electrolyte leakage in the leaves

To determine antioxidant enzyme activities, 0.5 g of fully expanded young leaves were homogenized in liquid nitrogen with 3 mL of extraction buffer [50 mM TRIS buffer (pH 7.8) containing 1 mM EDTA-Na2 and 7.5% polyvinylpyrrolidone)] using a prechilled mortar and pestle. The homogenate was filtered through four layers of cheesecloth and centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant, which was re-centrifuged at 12,000 rpm for 20 min at 4 °C, was used for the total soluble enzyme activity assay using an ultraviolet-160A spectrophotometer (Shimadzu, Japan).

Catalase assay. Catalase (CAT; EC 1.11.1.6) activity was measured by following the consumption of H₂O₂ at 240 nm (Aebi 1984). A total of 1 mL of the reaction mixture contained 20 mg total protein, 50 mM sodium phosphate buffer (pH 7.0), and 10 mM H₂O₂. The reaction was initiated by adding the protein extract. For each measurement, the blank corresponded to the absorbance of the mixture at time zero, and the actual reading corresponded to the absorbance after 1 min. One unit of CAT activity was defined as a 0.01 decrease in absorbance at 240 nm/mg of protein/min.

Polyphenol oxidase assay. Polyphenol oxidase (PPO; EC 1.10.3.1) activity was determined according to the method described by Malik and Singh (1980). The reaction mixture contained 3.0 mL of buffered catechol solution (0.01 M) freshly prepared in 0.1 M phosphate buffer (pH 6.0). The reaction was initiated by adding 100 mL of the crude enzyme extract. Changes in the absorbance at 495 nm were recorded at 30 s for 3 min. Enzyme activity was expressed as an increase in the absorbance min–1·g–1 fresh weight.

Peroxidase assay. Peroxidase (POD; EC 1.11.1.7) activity was determined according to the procedure proposed by Hammerschmidt et al. (1982). The reaction mixture consisted of 2.9 mL of a 100-mM sodium phosphate buffer [pH 6.0 containing 0.25% (v/v) guaiacol (2-methoxy phenol) and 100 mM H₂O₂]. The reaction was started by adding 100 mL of crude enzyme extract. Changes in absorbance at 470 nm were recorded at 30^-s intervals for 3 min. Enzyme activity was expressed as an increase in the absorbance min^–1·g^–1 fresh weight.

Electrolyte leakage. Electrolyte leakage (EL) measurements were performed as described by Szalai et al. (1996), with some modifications. Twenty leaf discs (1 cm²) were placed individually into flasks containing 25 mL of deionized water (Milli-Q 50, Millipore, Bedford, MA). Flasks were shaken for 20 h at an ambient temperature to facilitate electrolyte leakage from injured tissues. Initial EC measurements were recorded for each vial using an Acromet AR20 EC meter (Fisher Scientific, Chicago, IL). Flasks were then immersed in a hot water bath (Fisher Isotemp, Indiana, PA) at 80 °C (176 °F) for 1 h to induce cell rupture. The vials were again placed on the Innova 2100 platform shaker for 20 h at 21 °C (70 °F). Final conductivity was measured for each flask. The percentage of electrolyte leakage for each bud was calculated as the initial conductivity/final conductivity %100.

Evaluation of Cd, Cu, and Pb phytoextraction potential of Populus nigra

The phytoextarction potential of heavy metals was determined by calcatating bioconcentration factor (BCF), translocation factor percentage (TF%), tolerance index biomas (TI_b), and root tolerance index (TI_r) as follows,

 (3)

where CO is the metal concentration in the plant organ (mg/kg D.W.), and CS is the metal concentration in the soil (mg/kg soil D.W.)

Depending on BCF values, accumulation efficiency was estimated using one of four groups: BCF< 1 [intensive], 1 to 0.1 [medium], 0.1 to 0.01 [weak], and 0.01 to 0.001 [no accumulation] (Kabata-Pendias and Pendias 1999),

 (4)

where CH is metal content in the shoots (mg/kg D.W.), and CR is the metal content in the roots (mg/kg D.W.)

TF % was calculated to estimate the metal efficiency ion transport from roots to aerial plant organs (Maiti and Jaiswal 2008) whereas, shoots=leaves and stems.

 (5)

In Eq. 5, MP is D.W. of metal- treated plant (g/plant), CP is D.W. of control plant (g/plant).

TI_b was calculated according to Wilking (1978) to estimate the heavy metal resistance through there values as follow TI_b < 1 (a net decrease in biomass and a stressed condition of plants), TI_b = 1 (no difference relative to control treatment) and TI_b > (a net increase in biomass and correct plant development).

TI_r was calculated according to Wilkins (1978),

 (6)

where RM is root length of the metal- treated plant (cm), RC is root length of control plant (cm).

Statistical Analysis

Data were subjected to analysis using the SAS program (Version 6.12; SAS Institute, Cary, NC), and the mean separation was performed using Duncan’s Multiple Range Test method. The significance was determined at p ≤ 0.05.

Soil Analysis

The used soil (virgin soil without contamination) was analyzed prior to and after plantation (one sample of each replicate were mixed carefully as one sample, then it was analyzed). The particle size distribution was analysed by the hydrometer method (Gee and Bauder 1986) before planting only (Table 1). Samples of three replicates (one sample of each replicate) of each treatment were taken from the soil around roots at the end of the experiment and mixed carefully in one sample for the analysis according to the procedure of Cools and De Vos (2011). The soil samples were prepared to determine the following parameters. The EC was measured in a 1:5 ratio (soil: deionized water) using an EC-Meter (MI 170, Szeged, Hungary). The pH was measured in a 1:1 ratio (soil: deionized water suspension) using a calibrated pH-meter (JENEWAY3510, Staffordeshire, UK). Soluble ions in saturated extracted samples were measured according to Jackson (1973). Total carbonate was determined by volumetric calcimeter (Nelson and Sommers 1996). Organic matter content was determined by the dichromate oxidation method (Nelson and Sommers 1996). The available N (%) was determined using the micro Kjeldahl method (Bremner and Mulvaney 1982), and available P (%) was determined according to Olsen and Sommers (1982). Both Ca and Mg contents were measured according to Jackson (1973). The concentrations of Cd, Cu, and Pb were quantified through atomic absorption spectrophotometer (AAS; Page et al. 1982). The Na and K contents were examined according the methods described by Black et al. (1965) and determined using a flame photometer PSP7 (JENEWY, Staffordeshire, UK). Chloride content was determined by titration with a standard solution of silver nitrate (Jackson 1973).

RESULTS AND DISCUSSION

Soil Analysis

Table 1 shows the soil characteristics before and after contamination. The texture of soil was clayey, with a pH of 7.84, EC of 3.30 dsm^-1, and organic matter (OM) of 1.31%. The cations, anions, and EC increased after plantation compared with before, except for the control treatment and Na under 250 and 450 mg Pb acetate kg^-1 soil. The available N, P, and K values and, Cd, Cu, and Pb concentrations decreased at the end of the experiment relative to their concentrations prior to the planting. Where Cd after plantation in soil was 3.47, 6.47, 7.04, and 9.03 mg/kg soil against the added concentration of 3.9, 7.8, 11.9, and 15.6 mg/kg soil , repectively. The Cu in soil was 1.65, 5.38, 10.19, 16.88, and 29.51 mg/kg soil against the added concentration of 3.6, 14.4, 29.8, 47.7, and 63.6 mg/kg soil, respectively. The Pb in soil was 13.14, 16.20, 19.95, and 23.61mg/kg after plantation against 50.00, 19.11, 132.10, and 173.30 mg/kg before planting, respectively.

At the end of experimentation, there were negligible changes in pH values. Vyslouzilová et al. (2006) reported that soil pH did not differ among rhizobax compartments Cd, Pb, and Zn polluted soil after plantation of two willow clones (Salix × rubens). Soil pH, OM, and cations exchange capacity (CEC) positively correlated with Cu retention (King 1988) and Cd and Pb retention (Jopony and Young 1994). A rise in EC values might have taken place due to the addition of the tested metals. The presence of a large amount of ionic substances and soluble salts have resulted in raising the value of EC in industrial effluents treated soil sample (Sharma and Raju 2013). The reduction in available NPK and Cd, Cu, and Pb concentrations after plantation of poplar might be attributed to uptake by plant roots and the loss fractionally with water irrigation.

Vegetative Growth and Leaf Chemical Composition

In general, the tested levels of Cd, Cu, and Pb significantly decreased the values of the growth traits (Table 2) in comparison to the control (p ≤ 0.05). The highest significant values of plant height, stem diameter, branches number per plant, area per leaf, root length, dry weights of leaves, stems and roots, and leaf green color degree were the outcomes of control plants. The negative impact was higher with increasing heavy metal concentrations (e.g., more than 50% reduction of leaves dry weight, 23.40, 21.55, and 22.44% in stem dry weight and 34.62, 32.96, and 33.17 % in root dry weight at the high level of each heavy metal, respectively). Meanwhile, the reduction in root length was 27.62, 30.73 and 31.03% at the higher level of each heavy metal.

Table 1. Soil Analysis Before and After Plantation

Note: Each value represents the average of three replicates after plantation.

The obtained results are in agreement with those of Borghi et al. (2008), who found a general reduction of P.× canadensis (Adda clone) biomass and growth variable at Cu equals or higher than 100 µM. Too total leaf area in Yillow clones was affected by 38.5 mg/L Cd sulphate (Zacchini et al. 2009) and by 0.19 mg/L Cu in Salix viminalis (Ga̧secka et al. 2012). Also, Houda et al. (2016) reported that there were inhibition effects on the growth prameters on Populus nigra and P. alba irrigated by treated wastewater that contained nickel, zinc, cadmium, and lead at rates of 0.5, 0.4, 0.2, and 0.2 mgL^-1, respectively. Additionally, Redovniković et al. (2017) concluded that there was a reduction in dry root biomass of P. nigra ‘Italica’ treated with 50 mg Cd and 1200 mg Pb kg^-1 soil. In spite of the fact that the heavy metals tested exerted adverse effects on the vegetative growth, the survival was 100%. This agrees with the study of Polle et al. (2013), who found that P. eurphratica and P. canescens could grow in treated soil with Cd. Similer results were obtained by Kubátová et al. (2016), who reported that P. maximowiczi × P. nigra could grow in 7.3 mg Cd, 1368 mg Pb, and 218 mg Zn /kg soil in spite of the inhibition effects on the growth parameters that correlated with higher levels of Cd, Pb, and Cu in the soil.

At the same time, the impact of HMs used on the percentages of N, P, K (Fig. 1, 2, and 3), and total carbohydrates (Fig. 4) showed a similar downward tendency in the presence of increasing HMs concentations. Moreover, the highest and least significant percentages of such chemical contents were the results from the control plants and the highest levels of heavy metals, respectively. Meanwhile, non-contaminated plants (control) had 2.35, 0.20, 2.14, and 14.98% for N, P, K, and total carbohydrates, consecutively. On the other side, the contaminated plants with 80 mg CdCl₂/kg soil resulted in lower significant N and P% of 0.83 and 0.03% each it turn. Meanwhile, the plants received 850 mg Pb acetate produced 1.28 and 9.10 % for K and total carbohydrates, respectively. Furthermore the treatments of 80 mg CdCl₂ and 850 mg Pb acetate gave non-significant values of P, K, and total carbohydrates % .The adverse effects of Cd, Cu, and Pb on N, P, and K% may be owing to the competition between them and essential nutrients. HMs exert toxicities in plants through: (i) similarities with the nutrient cations, which result into a competition for absorption at root surface; for example, As and Cd compete with P and Zn, respectively, for there absorption, (ii) displacement of essential cations from specific binding sites that lead to a collapse of function (Sharma and Dietz 2009; Dalcorso et al. 2013). Further, Cd affects N metabolism by inhibiting nitrate uptake and transportation and nitrate reductase (Lea and Miflin 2003). Cu can also substitute for Mg in the chlorophyll present in both antenna and reaction centres (Küpper et al. 1996). This adversely affects the structure and function of chlorophyll, leading to a reduction in carbohydrate accumulation. In addition, Pb accumulated in plant tissue reacts with the phosphate group of ADP or ATP and replaces essential ions, thus impaired essential elements uptake such as Mg and Fe and inducing deficiency of CO₂ resulting from stomatal closure (Pourrut et al. 2011).

Likewise, Pieterini et al. (2010) found that CdSO₄ at 50 mM interferes with the uptake, transport and use of different elements (e.g., Fe, Zn, and Mg) in poplar clones. In addition, Ga̧secka et al. (2012) pointed out that the disturbance in starch hydrolysis and inhibition of soluble carbohydrate transport to different organs of S. viminalis under Cu stress (at 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM) probably leads to a decrease in leaf length, root biomass, and root length. This is because carbohydrates formed during photosynthesis are the source of carbon and energy necessary for growth, lignification, and biomass formation. Overall, foliage elements differ according to their concentrations in soil or soil chemistry and due to the plant species or clones of Salix (Mosseler and Major 2017). According to El-Mahrouk et al. (2019), the decrement in N, P, and K % in leaves of Salix mucronata was increased with raising the concentrations of Cd Cl₂ (20 to 80 mg/kg soil ), CuCl₂ (50 to 200 mg/kg soil), and Pb acetate (250 to 850 mg/kg soil) in the soil.

Table 2. Effect of Different Levels of Cd, Cu, and Pb on the Vegetative Traits of Populus nigra L.

Means followed by a similar letter within a column are not significantly different at 0.05 level probability by Duncan’s Multiple Range Test. P≤0.05.

Fig. 1. Effect of different levels of Cd, Cu and Pb in soil on the leaf N% of Populus nigra L.

Means followed by a similar letter within a figure are not significantly different at 0.05 level probability by Duncan’s Multiple Range Test (P ≤ 0.05)

Fig. 2. Effect of different levels of Cd, Cu and Pb in soil on the leaf P %of Populus nigra L.

Means followed by a similar letter within a figure are not significantly different at 0.05 level probability by Duncan’s Multiple Range Test (P ≤ 0.05).

Fig. 3. Effect of different levels of Cd, Cu and Pb in soil on the leaf K %of Populus nigra L.

Means followed by a similar letter within a figure are not significantly different at 0.05 level probability by Duncan’s Multiple Range Test (P ≤ 0.05).

Fig. 4. Effect of different levels of Cd, Cu and Pb in soil on total carbohydrate% of Populus nigra L.

Means followed by a similar letter within a figure are not significantly different at 0.05 level probability by Duncan’s Multiple Range Test (P ≤ 0.05).

Cd, Cu, and Pb Contents and Uptake in the Plant Parts and Plant Total Uptake

The obtained results showed that all tested treatments of Cd, Cu, and Pb caused a significant (p ≤ 0.05) effect on their contents and uptakes in the different plant parts, as well as plant total uptake (Table 3). Differences among treatments of each heavy metal reached a significant level. In the same time period, Cd content reached 3.53, 2.57, and 6.65 mg/kg D.W., and its uptake reached 0.017, 0.107, and 0.180 mg, Cu content reached 30.67, 20.38, and 61.78 mg/kg D.W., and its uptake reached 0.15, 0.87, and 1.74 mg, and Pb content reached 47.63, 35.15, and 76.20 mg/kg D.W., and its uptake reached 0.25, 1.48, and 2.13 mg in leaves, stems, and roots, respectively, when 80 mg Cd Cl₂, 200 mg Cu Cl₂, and 850 mg Pb acetate/kg soil were applied, respectively. Meanwhile, total plant uptake of Cd, Cu, and Pb were 0.31, 2.76, and 3.85 mg/plant at 80 mg Cd Cl₂ , 200mg Cu Cl_{2 ,} and 850 mg Pb (CH₃COO)₂ / kg soil, respectively. The content of Cd, Cu, and Pb in the plant parts depended on their concentration in the soil, and their contents were found to be in the order of roots > leaves > stem, except for CdCl₂ at 20 and 40 mg/kg soil, where the content of Cd in the stem was more than leaves. At the same time, the uptake of the used HMs in the different plant parts was in order of roots >stems>leaves.

The present results showed that the roots had Cd, Cu, and Pb content and uptake more than aerial parts. This finding was supported by Zhivotovsky et al. (2011) in various tree species, where Cd and Pb mainly accumulated in the roots rather than leaves and stem. The results matched those of Samuilov et al. (2016) on Populus tremula × P. alba, where the concentration of Pb in the different plant parts depends on concentrations in soil. The plant roots can accumulate Pb more than the above-ground parts. Redovniković et al. (2017) exposed P. nigra ‘Italica’ cv. to Cd (10, 25, and 50 mg kg^-1 soil) and Pb (400, 800, and 1200 mg kg^-1 soil) and found that in the majority of cases Cd and Pb accumulated.

Thus the roots of Populus nigra had the highest accumulation of Cd, Cu, and Pb, when compared to the stems and leaves; in this case, the contamination risk of the wider environment through leaf fall can be considered minimal. This was supported by Baker (1981), who reported that many deciduous plant species are considered to translocate accumulated heavy metals to their above-ground tissues before senescence. Also, Vervaeke et al. (2003) mentioned that willow colonies, which do not accumulate metals in their leaves, can be used to minimize the contamination risk of the wider environmental through leaf fall. Additionally, Rafati et al. (2011) reported that the green leaves of P. alba had Ni less than the fallen leaves under 120 mg Ni nitrate/kg soil. Therefore, these results suggest that P. nigra is a suitable alternative to deciduous hyperaccumulators. To keep the soil at low heavy metals levels, short rotation forestry systems can be used to remove heavy metals contamination from the soil through repeated harvest of the biomass for energy purposes (Punshon and Dickinson 1997).

Table 3. Effect of Different Levels of Cd, Cu, and Pb on Their Content and Uptake in the Different Plant Parts as well as Plant Total Uptake

Means followed by a similar letter within a column are not significantly different at 0.05 level probability by Duncan’s Multiple Range Test. P≤0.05.

Enzymatic Activities and Electrolyte Leakage (EL) of the Leaves

There were significant increases in PPO, POD, and CAT activities under the tested independent variables levels in comparison to the control treatment (Fig. 5, 6, and 7). The activities of PPO, POD, and CAT enzymes were increased up to the second concentration then decreased gradually from the third concentration of each tested heavy metal, but the enzymatic activities were higher in control plant leaves. The differences among all tested treatments reached the significance level for most treatments. The significantly maximum activities of PPO, POD, and CAT were observed at 100 ppm Cu Cl₂/kg soil in comparison to the other tested heavy metal levels.

The visual toxicity symptoms of leaves as yellowish, chlorosis, and necrotic spots were noticed on the adult leaves, and browning of roots of poplar under study may take place due to inducing ROS under heavy metals stress (Wang et al. 2008). The same authors added that elevated Cd increased the enzymatic activities rapidly in metal-accumulator plants, especially SOD and CAT. ROS decreases with increased exposure time to heavy metals stress (Jakovljević et al. 2014). This finding was supported by Redovniković et al. (2017), who found that P. nigra ‘Italica’ cv., subjected to Cd (50 mg/kg) and Pb (1200 mg/kg) heavy metals, induced increasing oxidative stress during short-term exposure (one month), compared to long-term HM exposure (4 months). Increasing enzymatic activities with increasing heavy metal concentrations then decreased at high levels of such metal; this was explained by Nagajyoti et al. (2010), who reported that the activity of antioxidative enzymes under heavy metal stress (e.g., Cd, Cu, Pb) is concentration-dependent and can be retarded or stimulated. This was also reported by Stobrawa and Lorenc-Plucińska (2007) in the fine roots of P. nigra grown in Cu (849.7 and 1174.75 mg/kg) and Pb (255.30 and 411.13 mg kg^-1) polluted soil. Tauqeer et al. (2016) on Alternanthera bettzickiana concluded that under lower levels of both Cd and Pb (0.5 and 1.0 mM), both POD and CAT activities increased, while under higher Cd and Pb levels (2.0 mM) the activeties of such enzymes were decreased. Also, Zou et al. (2017) mentioned that the enzymatic acitvity in Salix matsudana was increased under stress of Cd (10, 50, and 100 μM). Emamverdian et al. (2018) reported increasing enzymatic activities in Indocalamus latifolius due to Cu, Pb, and Zn at rates of 0, 500, 1000, and 2000 mg kg^-1 soil. Bankaji et al. (2019) studied Atriplex halimus, finding that raising the Pb level from 0 to 600 µM in the soil increased CAT activity.

Concerning electrolyte leakage (EL), Fig. 8 shows that with increasing heavy metal levels in the soil, the EL gradually increased. The maximum significant electrolyte leakage value reached 95.74 µS/cm² owing to the application of 80 mg CdCl₂/kg soil, followed by Pb acetate and CuCl₂ at 850 and 200 mg/kg soil. Each in turn resulted in 88.95 and 87.81 µS/cm², respectively but without significant difference between themselves against 37.17 µS/cm² for the negative heavy metals treated plants. EL is ubiquitous among various species, tissues, and cell types and can be triggered by all stress factors including heavy metals (Demidchik et al. 2003). The membrane damage that resulted from Cu can be assessed by EL determintion (Liu et al.2004). Elevated Cd levels (200 mgkg^-1) and exposure time increase the EL in Brasica juncea (Ahmad et al. 2016). Furthermore, EL in Alternanthera bettzckiana increases with increasing Cd and Pb from 0.5 to 1.0 mM (Tauqeer et al.2016). Thus, reduced vegetative growth traits, leaf chemical composition, and TI may be due to the increase in EL with the increasing heavy metal levels. Thus, PPO, POD, and CAT activities and EL can be used as environmental biomarkers of heavy metal concentrations.

Fig. 5. Effect of different levels of Cd, Cu, and Pb in soil on peroxidase (PPO) activities in the leaves of Populus nigra L. Means followed by a similar letter within a figure are not significantly different at 0.05 level probability according to Duncan’s Multiple Range Test (P≤0.05).

Fig. 6. Effect of different levels of Cd, Cu, and Pb in soil on peroxidase (POD) activities in the leaves of Populus nigra L. Means followed by a similar letter within a figure are not significantly different at 0.05 level probability according to Duncan’s Multiple Range Test (P≤0.05).

Fig. 7. Effect of different levels of Cd, Cu, and Pb in soil on catalase (CAT) activities in the leaves of Populus nigra L. Means followed by a similar letter within a figure are not significantly different at 0.05 level probability according to Duncan’s Multiple Range Test (P≤0.05).

Fig. 8. Effect of different levels of Cd, Cu, and Pb in soil on electrolyte leakage activitiy in the leaves of Populus nigra L. Means followed by a similar letter within a figure are not significantly different at 0.05 level probability according to Duncan’s Multiple Range Test (P≤0.05).

Bioconcentration Factor (BCF), Translocation Factor (TF, %), and Tolerance Index (TI)

To evaluate the Cd, Cu, and Pb phytoextraction ability in poplar heavy metals accumulation, BCF and TF were calculated (Table 4). The BCF was calculated to estimate ion transport from soil to the plant organs. Higher significant BCF Cd of leaves, stem, and root were found at 80, 80, and 20 mg Cd Cl₂ kg^-1 soil, respectively. Also, higher significant BCF Cu of leaves and root were recorded for the treatments of 50 and 200 mg CuCl₂/kg soil, without a significant difference between themselves. Higher significant BCF Cu of stem was found at 50 mg Cu Cl₂/kg soil. On the other side, higher significant BCF Pb of leaves and root were achieved on 250 and 450 mg Pb acetate/kg soil, and the difference between such treatments did not reach the significant level (P≤ 0.05). Meanwhile, higher significant BCF Pb of stem resulted from 450 and 850 mg Pb acetate/kg soil, without a significant difference between such treatments. Soil properties such as pH, CEC, and Ca concentration have a significant (p ≤ 0.05) effect on the bioavailability of Pb in soil (Zhang et al. 2019). BCF and TF are key values that evaluate a plant’s potential for phytoextraction or phytoestabilization (Rafati et al. 2011). In this study, the BCF of plant organs at concentrations of each metal was estimated as medium (BCF = 1 to 0.1), which is in accordance to Kabata-Pendias and Pendias (1999). Also, shoots BCF of Cd, Cu, and Pb at the various concentrations of each metal was less than the unity (<1). This is incompatible with the previous study of Redovniković et al. (2017) on poplar ‘Italica’cv., where they mentioned that shoots BCF of Cd or Pb < 1 under Cd at 10, 25, and 50 mg/kg soil and Pb at 400, 800, and 1200 mg/kg soil. The results indicated that root BCF at the levels of Cd, Cu, or Pb was < 1. Rafati et al. (2011) concluded that root BCF of P. alba was <1 under 40, 80, and 160 mg Cd/kg soil. Additionally, at levels of each heavy metal, roots BCF Cd, Cu, and Pb was higher than of leaves or stems. Thus, P. nigra roots were more effective in storing these metals than other organs.

Concerning TF, there was a significant (p ≤ 0.05) increase in TF of Cd and Pb values with increasing their concentrations in soil. While TF of Cu did not show a clear trend. It is obvious from data of tested heavy metal levels that TF Cd or TF Cu < 1 (less than 100%), with an exception of 50 mg CuCl₂, while TF Pb is > 1 (over than 100%), with an exception of 250 mg Pb acetate. Higher TF of Cd, Cu, and Pb is 91.82, 141.01 and 108.79% for 80 mg CdCl₂, 50 mg CuCl₂ and 850 mg Pb acetate/kg soil, respectively.

The movement of heavy metals from roots to shoots probably occurs within the xylem. The free Cd, Cu or Pb levels can be influenced by cellular sequestration of these metals, and therefore, it can affect their movement through the plant (Niu et al. 2007; Fulekar et al. 2009; Pourrut et al. 2011). The TF Cd values at various levels were not higher because it is often restricted due to its ability to create Cd-phytoextraction complex by sequestration in the vacuole (Lux et al. 2010). Also, the present results indicated that TF Cd or Cu is <1 (˂ 100%) at levels of each, with an exception of TF Cu, which was 50 mg CuCl₂ kg^-1 >1 (˃ 100%). This is in agreement with the result of Redovniković et al. (2017) on P.nigra ‘Italica’cv, who found that TF Cd is <1 under 10 and 50 mg Cd kg^-1 soil. Meanwhile, TF Pb was >1(˃ 100%) under its concentrations, with the exception of TF Pb at 250 mg Pb acetate kg^-1 <1 (˂ 100%). This was contrary to TF Pb of P.nigra ‘Italica’cv, which was <1 under 400, 800, and 1200 mg Pb kg^-1 soil (Redovniković et al. 2017). In contrast, TF Cd >1 of P. alba grown in 40 to 160 mg Cd kg^-1 soil (Rafati et al. 2011). Whereas, Saraswat and Rai (2009) and Zacchini et al. (2009) concluded that plants recording a shoot BCF>1 are suitable for phytoextraction and those with a root BCF>1 and TF<1 are suitable for phytostabilization. From the results either shoot BCF or TF Cd or Cu <1, P. nigra has a potential for phytostabilization of these ions. At the same time BCF Pb of Populus nigra root was <1 and TF Pb was >1 (>100%) under the used levels of Pb in this study, with the exception of TF Pb at 250 mg Pb acetae /kg soil, which was <1 (<100%). This means that Populus nigra can be used as a phytoextractor of Pb contaminated soil at 450 to 850 Pb acetate /kg soil. The results of Rafati et al. (2011) indicated that BCF Cd of P. alba and Morus alba roots were <1 and TF Cd of such spesies were >1 under Cd at levels of 40, 80, and 160 mg/kg soil, and they added that the two species were suitable for phytoextraction of Cd contaminated soil at such levels.

To estimate the black poplar resistance for Cd, Cu, and Pb phytoextraction and to determine the plant ability to grow in the presence of tested heavy metals, TI_b and TI_r were calculated, as shown in Table 4. A significant reduction in TI_b and TI_r values was recorded with increasing heavy metal concentrations in the soil. Likewise, the values of either TI_b or TI_r of Cd, Cu, and Pb were less than the unity at different heavy metal levels. The TI_b reached 69, 71, and 70% and TI_r reached 72, 69, and 69% relative to control treatment (100%) at higher levels of Cd, Cu, and Pb, respectively. The result indicated that the values of TI_b under all tested levels of Cd, Cu, or Pb were lower than 1; the plants were under stress and the biomass production was limited or a net decrease in biomass and stressed condition of plants (Wilkins 1978). Upon TI_r < 1, there was a limited length of the root. The results in this study showed that TI_b of Populus nigra was 0.80, 0.83, and 0.87 and TI_r of Populus nigra was 0.85, 0.83, and 0.84 at 40 mg CdCl₂, 100 mg Cu Cl₂, and 450 mg Pb acetate /kg soil. This means that P. nigra has better tolerance against 40 mg CdCl_2, 100 mg CuCl₂, and 450 mg Pb acetate. Metal tolerance varied greatly among 20 different clones of willow and poplar spesies treated with Cd and Zn (Dos Santos Utmazian et al. 2007). The present results were supported by the result of Wu et al. (2010), who revealed that TI of P. deltoides x P. nigra was significantly decreased at 4.35g Cd/kg purble soil. Melczek et al. (2013) showed that tolerance index of biomas or root of S. viminalis was <1 under 3 mM Cu (NO3)₂ and at ratios of Ca/Mg (20:1 and 1:10). According to El-Mahrouk et al. (2019), the decrement in TI_b or TI_r of Salix macrunata was increased with increasing the levels of Cd, Cu, and Pb. In general, BCF, TF, and TI_b or TI_r factors could be used to define Cd, Cu, and Pb hyperaccumulators. The suitable plant for phytoremediation should have much more metal tolerances and high accumulation capacity in its tissues (especially in harvestable parts) (Shi et al. 2009). Thus, the results imply that P. nigra is a good candidate plant for phytoremediation applications in Cd, Cu, and Pb contaminated soil.

Table 4. BCF, TF%, TI_b and TI_r of Populus nigra as Affected by Cd, Cu, and Pb Concentrations in the Soil

Means followed by a similar letter within a column are not significantly different at 0.05 level probability by Duncan’s Multiple Range Test (P≤0.05).

CONCLUSIONS

1. Populus nigra had high survival rate (100%) under Cd, Cu, and Pb treatments up to 15.6, 63.6, and 173.3 mg kg^-1 soil, respectively.
2. The used levels of Cd, Cu, and Pb had an adverse effect on both vegetative traits and leaf N, P, K, and carbohydrates (%).
3. The stimulating effect occurred for the activities of PPO, POD, and CAT enzymes. Moreover, EL increased compared to the control after applications of the different levels of used HMs.
4. There was greater content and uptake of Cd, Cu, and Pb in roots than in the aerial parts.
5. The risk of contamination of the wider environment through leaf fall can thus be considered as minimal.
6. According to BCF, TF, TI_b, and TIr, Populus nigra can use as a phytostabilizator for Cd and Cu and a phytoextractor for Pb contaminated slightly alkaline soil at the used levels in this study.
7. Populus nigra can be regarded as a suitable alternative plant to hyperaccumulator plants for Cd, Cu and Pb contaminated slightly alkaline soil.

ACKNOWLEDGMENTS

Researchers Supporting Project number (RSP-2019/123) King Saud University, Riyadh, Saudi Arabia.

The authors would also like to thank Kafrelsheikh University for facilitating this work.

Funding

Researchers Supporting Project number (RSP-2019/123) King Saud University, Riyadh, Saudi Arabia

REFERENCES CITED

Abdullahi, M. S., Uzairu, A., and Okunola, O. J. (2009). “Quantitative determination of heavy metal concentrations in onion leaves,ˮ Int. J. Environ. Res. 3, 271-274. DOI: 10.22059/IJER.2009.54

Aebi, H. (1984). “Catalase in vitro,ˮ Methods in Enzymology 105, pp. 121-126.

Ahmad, P., Allah, E. F. A., Hashem, A., Sarwat, M., and Gucel, S. (2016). “Exogenous application of selenium mitigates cadmium toxicity in Brassica juncea L. (Czern & Cross) by up-regulating antioxidative system and secondary metabolites,ˮ J. Plant Growth Regul. 35, 936-950. DOI: 10.1007/s00344-016-9592-3

Baker, A. J. (1981). “Accumulators and excluders‐strategies in the response of plants to heavy metals,” J. Plant Nutrition 3, 643-654.‏ DOI: 10.1080/01904168109362867

Bankaji, I. Pérez-Clemente, R. M., Caçador, I., and Sleim, N. I. (2019). “Accumulation potential of Atriplex halimus to zinc and lead combined with NaCl: Effects on physiological parameters and antioxidant enzymes activities,” S.A.J.B. 123, 51-61. DOI: 10.1016/j.sajb.2019.02.011

Black, C. A., Evans, D. D., Whils, J. L., Ensminger, L. E., and Clerk, F. E. (1965). Methods of Chemical Analysis of Ecological Materials, Madison, Wisconsin, USA: Am Soc of Agronomy Inc.

Borghi, M. Tognetti, R. Monteforti, G., and Sebastiani, L. (2008). “Responses of two poplar species (Populus alba and Populus x canadensis) to high copper concentrations,ˮ Environ. Exp. Bot. 62, 290-299. DOI: 10.1016/j.envexpbot.2007.10.001

Bremner, J. M., and Mulvaney, C. S. (1982). “Nitrogen—Total 1. Methods of soil analysis. Part 2. Chemical and microbiological properties,ˮ (methodsofsoilan2) 595-624.‏

Campbell, P. G. C. (2007). “Cadmium-a priority pollutant,ˮ Environ. Chem. 3, 387-388. DOI: 10.1071/EN06075

Clemens, S. (2006). “Toxic metal accumulation, responses to exposure and mechanisms oftolerance in plants,ˮ Biochimie. 88, 1707-1719. DOI: 10.1016/j.biochi.2006.07. 003

Chemists, A. A., and Horwitz, W. (1990). Official Methods of Analysis, Vol. I. 15^th Ed. AOAC, Arlington, VA

Cools, N., and De Vos, B. (2011). “Availability and evaluation of European forest soil monitoring data in the study on the effects of air pollution on forests,ˮ iForest-Biogeosciences and Forestry 4(5), 205.‏ DOI: 10.3832/ifor0588-004

Cotteine, A., Verloo, M., Velghe, M., and Camerlynck, R. (1982). Chemical Analysis of Plant and Soil, Laboratory of Analytical and Agrochemistry, State Univ. Ghent-Belgium 100-192.

DalCorso, G., Manara, A., and Furini, A. (2013). “An overview of heavy metal challenge in plants: From roots to shoots,ˮ Metallomics 5, 1117-1132. DOI: 10.1039/C3MT00038A

Demidchik, V., Shabala, S. N., Coutts, K. B., Tester, M. A., and Davies, J. M. (2003). “Free oxygen radicals regulate plasma membrane Ca²⁺-and K⁺-permeable channels in plant root cells,ˮ J. Cell Sci. 116, 81-88. DOI: 10.1242/jcs.00201

Dos Santos Utmazian, M. N., Wieshammer, G.,Vega, R., and Wenzel, W. W. (2007). “Hydroponic screening for metal resistance and accumulation of cadmium and zinc in twenty clones of willows and poplars,ˮ Environ Pollut. 148, 155-165. DOI:10.1016/j.envpol. 2006.10.045

El-Mahrouk, E. S. M., Eisa, E. A. H., Hegazi, M. A., Abdel-Gayed, M. E. S., Dewir, Y. H., El-Mahrouk, M. E., and Naidoo, Y. (2019). “ Phytoremediation of cadmium-, copper-, and lead-contaminated soil by Salix mucronata (Synonym Salix safsaf),” HortScience 54(7), 1249-1257.‏

Emamverdian, A., Ding, Y., Mokhberdoran, F., and Xie, Y. (2018). “Antioxidant response of bamboo (Indocalamus latifolius) as affected by heavy metal stress,ˮ J. Elem. 23, 341-352. DOI: 10.5601/jelem.2017.22.2.1410

Erakhrumen, A., and Agbontalor, A. (2007). “Review. Phytoremediation: An environmentally sound technology for pollution prevention, control and remediation in developing countries,” Educational Res. Rev. 2, 151-156.

Evenhuis, B., and de Waard, P. W. F. (1980). Principles and Practices in Plant Analysis, Rome (Italy) FAO.

Fulekar, M. H., Singh, A., and Bhaduri, A. M. (2009). “Genetic engineering strategies for enhancing phytoremediation of heavy metals,ˮ African J. Biotechnol. 8, 529-535.

Gardea-Torresdey, J. L., Peralta-Videa, J. R., De La Rosa, G., and Parsons, J. G. (2005). “Phytoremediation of heavy metals and study of the metal coordination by X-ray absorption spectroscopy,ˮ Coord Chem. Rev. 249 (17-18), 1797-1810. DOI: 10.1016/j.ccr.2005.01.001

Ga̧secka, M., Mleczek, M., Drzewiceka, K., Magdziak, Z., Rissmann, I., Chadzinikolau, T., and Golinski, P. (2012). “Physiological and morphological changes in Salix viminalis L. as a result of plant exposure to copper,ˮ Journal of Environmental Science and Health – Part A Toxic/Hazardous Substances and Environmental Engineering 47, 548-557. DOI. 10.1080/10934529.2012.650557

Gee, G. W., and Bauder, J. W. (1986). “Particle-size analysis 1. Methods of soil analysis: Part 1—Physical and mineralogical methods,ˮ (methodsofsoilan1), 383-411.‏

Hammerschmidt, R., Nuckles, E.M., and Kuć, J. (1982). “Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium,ˮ Physiol. Plant Pathol. 20, 73-82. DOI: 10.1016/0048-4059(82)90025-X

Hänsch, R. and Mendel, R. R., (2009). “Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl),ˮ Curr. Opin. Plant Biol. 12, 259-266.
DOI:10.1016/j.pbi.2009.05.006

Herbert, D., Phipps, P. J., and Strange, R. E. (1971). “Determination of total carbohydrates,ˮ Methods Microbiol. 5, 290-344.

Hossain, M. A., Piyatida, P., da Silva, J. A. T., and Fujita, M. (2012). “Molecular mechanism of heavy metal toxicity and tolerance in plants: Central role of glutathione in detoxification of reactive oxygen species and methylglyoxal and in heavy metal chelation,ˮ J. Bot. 1-38. DOI: 10.1155/2012/872875

Houda, Z., Bejaoui, Z., Albouchi, A., Gupta, D. K., and Corpas, F. J. (2016). “Comparative study of plant growth of two poplar tree species irrigated with treated wastewater, with particular reference to accumulation of heavy metals (Cd, Pb, As, and Ni),ˮ Environ. Monit. Assess. 188(2), 99. DOI: 10.1007/s10661-016-5102-0

Jackson, M. L. (1973). Soil Chemical Analysis, Prentice Halla of India Private Limited, New Delhi, Indian.

Jakovljević, T., Bubalo, M. C., Orlović, S., Sedak, M., Bilandžić, N., Brozinčević, I., and Redovniković, I. R. (2014). “Adaptive response of poplar (Populus nigra L.) after prolonged Cd exposure period,ˮ Environ. Sci. Pollut. Res. 21(5), 3792-3802. DOI: 10.1007/s11356-013-2292-7

Jopony, M., and Young, S. D. (1994). “The solid solution equilibria of lead and cadmium in polluted soils,ˮ Eur. J. Soil. Sci. 45(1), 59-70. DOI: 10.1111/j.1365-2389.1994.tb00487.x

Kabata-Pendias, A., and Pendias, H. (1999). “Biogeochemia pierwiastków śladowych [Biogeochemistry of trace elements]–PWN–Polish Scientific Publishers,ˮ Warszawa,400.

Kabata-Pendias, A. (2011). “Trace elements in soils and plants,ˮ Force.Taylor and Francis Group,LLC.

King, L. D. (1988). “Retention of metals by several soils of the southeastern United States,ˮ J. Environ. Qual. 17, 239-246. DOI: 10.2134/jeq1988.00472425001700020014x

Kubátová, P., Hejcman, M., Száková, J., Vondráčková, S., and Tlustoš, P. (2016). “Effects of sewage sludge application on biomass production and concentrations of Cd, Pb and Zn in shoots of Salix and Populus clones: Improvement of phytoremediation efficiency in contaminated soils,ˮ BioEnergy Research  9(3), 809-819.‏DOI: 10.1007/s12155-016-9727-1

Küpper, H., Küpper, F., and Spiller, M. (1996). “Environmental relevance of heavy metal-substituted chlorophylls using the example of water plants,ˮ J. Exp. Bot. 47, 259-266.‏ DOI:10.1093/jxb/47.2.259

Küpper, H. (2017). “Lead toxicity in plants,ˮ Metal Ions in Life Sciences, 17.‏ DOI: 10.1515/9783110434330-015

Lombardi, L., and Sebastiani, L. (2005). “Copper toxicity in Prunuscerasifera: Growth and antioxidant enzymes responses of in vitro grown plants,ˮ Plant Sci. 168, 797-802.‏ DOI: 10.1016/j.plantsci.2004.10.012

Lea, P. J., and Miflin, B. J. (2003). “Glutamate synthase and the synthesis of glutamate in plants,ˮ Plant Physiol. Biochem.. 41, 555-564.‏ DOI:10.1016/S0981-9428(03)00060-3

Liu, D., Jiang, W., Liu, C., Xin, C., and Hou, W. (2000). “Uptake and accumulation of lead by roots, hypocotyls and shoots of Indian mustard [Brassica juncea (L.)],ˮ Bioresource Technol. 71 (3), 273-277. DOI: 10.1016/S0960-8524(99)00082-6

Liu, J., Xiong, Z., Li, T., and Huang, H. (2004). “Bioaccumulation and ecophysiological responses to copper stress in two populations of Rumex dentatus L. from Cu contaminated and non-contaminated sites,ˮ Environ. Exp. Bot. 52(1), 43-51. DOI: 10.1016/j.envexpbot.2004.01.005

Lux, A., Martinka, M., Vaculik, M., and White, P. J. (2010). “Root responses to cadmium in the rhizosphere: A review,ˮ J. Exp. Bot. 62(1), 21-37. DOI: 10.1093/jxb/erq281

Maiti, S. K., and Jaiswal, S. (2008). “Bioaccumulation and translocation of metals in the natural vegetation growing on fly ash lagoons: A field study from Santaldih thermal power plant, West Bengal, India,ˮ Environ. Monit. Assess. 136(1-3), 355-370. DOI: 10.1007/s10661-007-9691-5

Malik, C. P., and Singh, M. B. (1980). “Plant enzymology and histo-enzymology,ˮ Indian Print. Navin.Shanndra.Delhi, pp. 54-56.

Markwell, J., Osterman, J. C., and Mitchell, J. L. (1995). “Calibration of the Minolta SPADA-502 leaf chlorophyll meter,ˮ Phtosyn. Res. 46, 467-472.

Marques, A. P. G. C., Moreira, H., Rangel, A. O. S. S., and Castro, P. M. L. (1995). “Arsenic, lead and nickel accumulation in Rubus ulmifolius growing in contaminated soil in Portugal,ˮ J. Hazard Mater 165, 174-179. DOI: 10.1016/j.jhazmat.2008.09.102

Marques, A. P., Moreira, H., Rangel, A. O., and Castro, P. M. (2009). “Arsenic, lead and nickel accumulation in Rubus ulmifolius growing in contaminated soil in Portugal,ˮ J. Hazard. Mater., 165, 174-179.‏ DOI:10.1016/j.jhazmat.2008.09.102

Mleczek, M., Gąsecka, M., Drzewiecka, K., Goliński, P., Magdziak, Z., and Chadzinikolau, T. (2013). “Copper phytoextraction with willow (Salix viminalis L.) under various Ca/Mg ratios. Part 1. Copper accumulation and plant morphology changes,” Acta physiologiae Plantarum 35(11), 3251-3259.‏ DOI: 10.1007/s11738-013-1360-4

Mosseler, A., and Major, J. (2017). “Phytoremediation efficacy of Salix discolor and S. eriocephela on adjacent acidic clay and shale overburden on a former mine site: Growth, soil, and foliage traits,ˮ Forests 8(12), 475.‏ DOI: 10.3390/F8120475

Murphy, J., and Riley, J. P. (1962). “A modified single solution method for the determination of phosphate in natural waters,ˮ Anal. Chim. Acta. 27, 31-36.

Nagajyoti, P. C., Lee, K. D., and Sreekanth, T. V. M. (2010). “Heavy metals, occurrence and toxicity for plants: A review,ˮ Environ. Chem. Lett. 8(3), 199-216. DOI: 10.1007/s10311-010-0297-8

Nelson, D. W., and Sommers, L. E. (1996). “Total carbon, organic carbon, and organic matter,ˮ Methods Soil Anal. Part III. Chemical Methods, (methodsofsoilan3), pp. 961-1010.

Olsen, S. R., and Sommers, L. E. (1982). “Phosphorus,” pp. 403-430. in: A. L. Page (ed.), Methods of Soil Analysis,ˮ Agron. No. 9, Part 2; Chemical and Microbiological Properties, 2^nd ed., Am. Soc. Agron., Madison, WI, USA.

Niu, Z-x., Sun, L-n., Sun, T-h., Li, Y-s., and Wang, H. (2007). “Evaluation of phytoextracting cadmium and lead by sunflower, ricinus, alfalfa and mustard in hydroponic culture,ˮ J. Environ. Sci. 19(8), 961-967. DOI: 10.1016/S1001-0742(07)60158-2

Page, A. L., Miller, R. H., and Keeney, D. R. (1982). Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties, 2.

Polle, A., Klein, T., and Kettner, C. (2013). “Impact of cadmium on young plants of Populus euphratica and P.× canescens, two poplar species that differ in stress tolerance,ˮ New Forests 44(1), 13-22.  DOI: 10.1007/s11056-011-9301-9

Pourrut, B., Shahid, M., Dumat, C., Winterton, P., and Pinelli, E. (2011). “Lead uptake, toxicity, and detoxification in plants,ˮ in: Reviews of Environmental Contamination and Toxicology 213, Springer, 113-136. DOI: 10.1007/978-1-4419-9860-6_4

Pietrini, F., Zacchini, M., Iori,V., Pietrosani, L., Bianconi, D., and Massacci, A. (2010). “Screening of popolar clonesfor cadmium phytoremediation using phothensis, biomass and Cd content analysis,” Inter. J. Phytoremediation 12, 105-120. DOI: 10.1080/15226510902767163

Punshon, T., and Dickinson, N. M. (1997). “Acclimation of Salix to metal stress,” New Phytol. 137, 303-314. DOI:10.1046/j.1469-8137.1997.00802.x

Puschenreiter, M., Horak, O., Friesl, W., and Hartl, W. (2005). “Low-cost agricultural measures to reduce heavy metal transfer into the food chain – A review,” Plant Soil Environ. 51(1), 1-11.‏

Rafati, M., Khorasani, N., Moattar, F., Shirvany, A., Moraghebi, F., and Hosseinzadeh, S. (2011). “Phytoremediation potential of Populus alba and Morus alba for cadmium, chromuim and nickel absorption from polluted soil,ˮ Int. J. Environ. Res. 5, 961-970. DOI: 10.22059/IJER.2011.453

Rakhshaee, R., Giahi, M., and Pourahmad, A. (2009). “Studying effect of cell wall’s carboxyl–carboxylate ratio change of Lemna minor to remove heavy metals from aqueous solution,ˮ J. Hazard Mater 163, 165-173. DOI: 10.1016/j.jhazmat.2008.06.074

Rautio, P., Fürst, A., Stefan, K., Raitio, H., and Bartels, U. (2010). “Sampling and analysis of needles and leaves,ˮ Manual Part XII. Man. methods criteria Harmon. sampling, assessment, Monit. Anal. Eff. air Pollut. For. UNECE, ICP For. Program. Co-ord. Centre, Hamburg. ISBN, pp. 973-978.

Redovniković, I. R., De Marco, A., Proietti, C., Hanousek, K., Sedak, M., Bilandžić, N., and Jakovljević, T. (2017). “Poplar response to cadmium and lead soil contamination. Ecotoxicol,ˮ Environ. Saf. 144,482-489. DOI: 10.1016/j.ecoenv.2017.06.011

Samuilov, S., Lang, F., Djukic, M., Djunisijevic-Bojovic, D., and Rennenberg, H. (2016). “Lead uptake increases drought tolerance of wild type and transgenic poplar (Populus tremula x P. alba) overexpressing gsh 1,ˮ Environ. Pollut. 216, 773-785. DOI: 10.1016/j.envpol.2016.06.047

Saraswat, S., and Rai, J. P. N. (2009). “Phytoextraction potential of six plant species grown in multimetal contaminated soil,ˮ Chem. Ecol. 25, 1-11. DOI: 10.1080/02757540802657185

Sebastiani, L., Scebba, F., and Tognetti, R. (2004). “Heavy metal accumulation and growth responses in poplar clones Eridano (Populus deltoides× maximowiczii) and I-214 (P.× euramericana) exposed to industrial waste,” Environmental and Experimental Botany 52(1), 79-88.‏ DOI.ORG/10.1016/J.ENVEXPBOT.2004.01.003

Sharma, S. S. and Dietz, K. J. (2009). “The relationship between metal toxicity and cellular redox imbalance,” Trends Plant Sci. 14, 43-50. DOI: 10.1016/j.tplants.2008.10.007.

Sharma, M. S. R., and Raju, N.S. (2013). “Effect of heavy metal pollution on fungal diversity in soil around industrial areas of Mysore, Karnataka,ˮ Int. J. Pharma. Bio. Sci. 4, 267-273.

Sharma, P., Jha, A. B., Dubey, R. S., and Pessarakli, M. (2012). “Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions,ˮ J. Bot. 1-26. DOI: 10.1155/2012/217037

Shi, G. R., Cai, Q. S., Liu, Q. Q., and Wu, L. (2009). “Salicylic acid-mediated alleviation of cadmium toxicity in hemp plants in relation to cadmium uptake, photosynthesis, and antioxidant enzymes,ˮ Acta Physiol. Plant 31, 969-977. DOI: 10.1007/s11738-009-0312-5

Snedecor, G. W., and Cochran, W. G. (1980). “Statistical methods of analysis,ˮ Stat. Methods 7^th Ed., Iowa State Univ. Press Ames, pp. 117-120.

Stobrawa, K., and Lorenc-Plucińska, G. (2007). “Changes in carbohydrate metabolism in fine roots of the native European black poplar (Populus nigra L.) in a heavy-metal-polluted environment,ˮ Sci. Total Environ. 373, 157-165. DOI: 10.1016/j.scitotenv.2006.11.019

Szalai, G., Janda, T., Páldi, E., and Szigeti, Z. (1996). “Role of light in the development of post-chilling symptoms in maize,ˮ J. Plant Physiol. 148, 378-383. DOI: 10.1016/S0176-1617(96)80269-0

Tauqeer, H. M., Ali, S., Rizwan, M., Ali, Q., Saeed, R., Iftikhar, U., Ahmad, R., Farid, M., and Abbasi, G. H. (2016). “Phytoremediation of heavy metals by Alternanthera bettzickiana: Growth and physiological response,ˮ Ecotoxicol. Environ. Saf. 126, 138-146. DOI: 10.1016/j.ecoenv.2015.12.031

Vervaeke, P., Luyssaert, S., Mertens, J., Meers, E., Tack, F. M. G., and Lust, N. (2003). “Phytoremediation prospects of willow stands on contaminated sediment: A field trial,ˮ Environ. Pollu. 126, 275-282. DOI:10.1016/S0269-7491(03) 00189-1‏

Vyslouzilová, M., Puschenreiter, M., Wieshammer, G., and Wenzel, W. W. (2006). “Rhizosphere characteristics, heavy metal accumulation and growth performance of two willow (Salix x rubens) clones,ˮ Plant soil Environ. 52, 353-361.

Wang, Z., Zhang, Y., Huang, Z., and Huang, L. (2008). “Antioxidative response of metal-accumulator and non-accumulator plants under cadmium stress,ˮ Plant Soil 310, 137. DOI: 10.1007/s11104-008-9641-1

Wilkins, B. D. A. (1978). “The measurement of tolerance to edaphic factors by means of root growth,ˮ New Phytologist 80(3), 623-633. DOI: 10.1111/j.1469-8137.1978.tb01595.x

Wu, F., Yang, W., Zhang, J., and Zhou, L. (2010). “Cadmium accumulation and growth responses of a poplar (Populus deltoids×Populus nigra) in cadmium contaminated purple soil and alluvial soil,ˮ J. Hazard Mater 177(1-3), 268-273. DOI: 10.1016/j.jhazmat.2009.12.028

Wu, W., Wu, P., Yang, F., Sun, D. L., Zhang, D. X., and Zhou, Y. K. (2018). “Assessment of heavy metal pollution and human health risks in urban soils around an electronics manufacturing facility,ˮ Sci. the Total Environ.  630, 53-61.‏ DOI: 10.1016/j.aoas.2018.05.007

Yang, X., Feng, Y., He, Z. and Stoffell, P. J. (2005). “Molecular mechanisms of heavy metal hyperaccumulation and phytoremediation,” J. Trace Elem. Med. Biol. 18, 339-353. DOI:10.1016/j. jtemb.2005.02.007

Zacchini, M., Pietrini, F., Mugnozza, G. S., Iori, V., Pietrosanti, L., and Massacci, A. (2009). “Metal tolerance, accumulation and translocation in poplar and willow clones treated with cadmium in hydroponics,ˮ Water Air 197, 23-34. DOI: 10.1007/s11270-008- 9788-7

Zalesny, J. A., Zalesny Jr, R. S., Wiese, A. H., Sexton, B., and Hall, R. B. (2008). “Sodium and chloride accumulation in leaf, woody, and root tissue of Populus after irrigation with landfill leachate,” Environmental pollution 155(1), 72-80.‏ DOI.org/10.1016/j.envpol.2007.10.032

Zárubová, P., Hejcman, M., Vondráčková, S., Mrnka, L., Száková, J., and Tlustoš, P. (2015). “Distribution of P, K, Ca, Mg, Cd, Cu, Fe, Mn, Pb and Zn in wood and bark age classes of willows and poplars used for phytoextraction on soils contaminated by risk elements,ˮ Environ. Sci. Pollut. 22, 18801-18813. DOI: 10.1007/s11356-015-5043-0

Zhang, L., Verweij, R. A., and Van Gestel, C. A. M. (2019). “Effect of soil properties on Pb bioavailability and toxicity to the soil invertebrate Enchytraeus crypticus,ˮ Chemospher 217, 9-17. DOI: 10.1016/j.chemosphere.2018.10.146

Zhivotovsky, O. P., Kuzovkina, Y. A., Schulthess, C. P., Morris, T., and Pettinelli, D. (2011). “Lead uptake and translocation by willows in pot and field experiments,ˮ Int. J. Phytoremediation 13(8), 731-749. DOI: 10.1080/15226514.2010.525555

Zou, J., Wang, G., Ji, J., Wang, J., Wu, H., Ou, Y., and Li, B. (2017). “Transcriptional, physiological and cytological analysis validated the roles of some key genes linked Cd stress in Salix matsudana Koidz,” Environ. Exp. Bot. 134, 116-129. DOI: 10.1016/j.envexpbot.2016.11.005

Article submitted: July 11, 2019; Peer review completed: October 10, 2019; Revised version received: December 10, 2019; Accepted; December 12, 2019; Published: December 16, 2019.

DOI: 10.15376/biores.15.1.869-893