PHYTOEXTRACTION OF CADMIUM AND LEAD FROM A CONTAMINATED SOIL USING EUCALYPTUS SEEDLINGS

This study aims to estimate critical concentrations of cadmium (Cd) and lead (Pb) in the soil which negatively affect growth of Eucalyptus camaldulensis Dehnh. seedlings, and to estimate some phytoextraction parameters for heavy metals (HM) from the soil to evaluate efficiency of seedlings in their potential use in phytotechnology to improve the environment with phytoremediation. Eucalyptus seedlings were treated with Cd concentrations 0, 25, 55, 85, and 110 mg kg -1 dry soil as CdCl2, and Pb concentrations 0, 125, 250, 450, and 550 mg kg -1 dry soil as PbCl2, and the experiment was designed using the completely randomized design (CRD) as a two-factor factorial experiments and the data were analyzed using SAS system. Results showed that the highest percentage decrease in dry weight of stems, leaves and roots were 55, 68.6, and 67.2%, respectively, at the interaction (110 Cd and 550 Pb) mg kg -1 dry soil compared with control, and Cd concentrations in stems, leaves and roots ranged between (0.375-372.167), (0.417-128.167) and (0.583-162.083) mg kg -1 , respectively and Pb concentrations in stems, leaves and roots ranged between (9.583-62.375), (10.042-20.417) and (2.87573.500) mg kg -1 . It was found that values of translocation factor (TF), biological accumulation coefficient (BAC), bioconcentration factor (BCF) and concentration index (CI) for Cd ranged between (0.611-4.239), (1.333-28.790), (0.38316.840) and (1-490.812) respectively, and values of TF, BAC, BCF, and CI of Pb ranged between (0.275-5.702), (0.769-4.246), (0.295-7.539) and (1-3.833) respectively, and tolerance index (TI) values ranged between (0.370-1). We concluded that Eucalyptus seedlings are suitable for phytoextraction applications within phytoremediation processes of soils contaminated with Cd and Pb.


INTRODUCTION
functions in plant tissues, its high concentrations cause cellular stress that leads to plant death (6,17). In order to get rid of heavy metals, plants and micro-organisms were used to remove them from the environment, as they are the most preserving the environment and living organisms and for the economic and social benefits they generate, phytoremediation has become known as a modern innovation technology that is reflected in the absorption of heavy metals from soil, water and air are then transported and concentrated in their cellular tissues, and phytoextraction, which involves the use of plants to absorb excessively accumulated heavy metals from the surrounding environment, transport and concentrate them in their biomass (3,5,16), it is the most common in phytoremediation operations, as it is a very appropriate technique in terms of material cost and as it improves the quality of soil and water satisfactorily within an appropriate time limit, good for extraction, by analyzing and reserving the hazardous contents in the media in which the plants grow, as well as making plans a long-term strategy for making genetic modifications on good plants in the phytoextraction process in order to increase their relatively rapid growth rate as well as to obtain high-yielding biomass and to make them adaptable to different environmental conditions and to demonstrate the characteristics of the different genetic characteristics of the species (45). The accumulation of toxic heavy metals in agricultural areas leads to great concerns about food safety and its impact on the productivity of food crops and thus its negative impact on the food security of human societies (50), and there are types of plants called hyperaccumulators that can absorb and transport large quantities of the heavy metals, and storage in their cellular tissues, and despite their high storage capacity, in cases of soil contamination with large quantities of the heavy metals, plants do not fully perform their function due to their small biomass compared to the size of pollution, so it is preferable to choose plant species with large and fast-growing biomass, that can absorb greater amounts of inorganic pollutants in general and heavy metals in particular (10,40), therefore, recent studies have encouraged the treatment of polluted soils with heavy metals by selecting forest trees that are distinguished by their lack of contribution to the food chain, and high yield of biomass, and among the forests that are characterized by these characteristics are Eucalyptus, Willow, Poplar and Castor (2,21). Where a study was conducted on Poplar hybrids in Serbia for Populus deltoides for clone (B-81) and Populus × euramericana for clone (Pannonia) on the possibility of phytoremediation of soils contaminated with Cd using, which were treated with a concentration of Cd 8.14 mg kg -1 dry soil, the study showed that the decrease in biomass production was significantly greater in Pannonia than in B-81 (42). And in a study conducted by Lamb et al. (29) in Australia to investigate the effect of Cu, Zn, Cd and Pb on the root elongation for four types of native trees and other natural herbs which treated with concentrations of Cd and Pb 0, 0.1, 1, 10, 100, 500 and 1000 µM, as well as with concentrations of Cu and Zn 0, 0.1, 1, 10, 100, 1000 and 2000 µM, and the results showed that high concentrations of heavy metals reduced the growth rate to 50%, as Cd concentrations of root ranged from 27 µM in Lactuca sativa to 940 µM in Acacia species, and Pb concentrations ranged between 180 µM in L. sativa to 1000 µM in Acacia species, and Cd was found to be more toxic than Pb. As for the study by Ashraf et al. (8) showed an increase in stalk Cd concentrations from (4.20-9.70) and (3.30-8.50) mg kg -1 in the seedlings of Conocarpus erectus and Eucalyptus camaldulensis, respectively, when the Cd concentration increased from 5 to 5.15 mg kg -1 , and Cd concentrations in the roots increased from (8.20-24) and from (6.10-17.40) mg kg -1 in seedlings of C. erectus and E. camaldulensis, respectively, with increasing the soil Cd concentration from (5-5.15) mg kg -1 , while the BCF values were greater than one for the two species and for all Cd coefficients, and the BCF values for the two species were higher in C. erectus seedlings than E. camaldulensis, and the TF values were less than one for both species and for all Cd levels, it was higher in E. camaldulensis than C. erectus. Also studied Alkhatib et al. (4) the effects of Pb on growth rate, physiological and biochemical characteristics in Leucaena leucocephala seedlings, as the results showed that the effect of Pb led to a decrease in growth rate of seedlings treated with concentrations 300, 500 and 700 µM, whereas Pb concentrations of 25, 50 and 100 µM did not show any toxic effect on seedlings.

MATERIALS AND METHODS
Field work: On March 14, 2019, ten-monthold were transferred Eucalyptus camaldulensis Dehnh. seedlings from central nursery in the city of Mosul to the wooden shelter in nursery for College of Agriculture and Forestry at University of Mosul at a longitude (43° 09ˈ) north and latitude (36° 19ˈ) east, and seedlings were planted in pots its internal diameter and depth is 28 cm, then the soil was added to it to bring its dry weight to 13.125 kg pot -1 approximately, and seedlings were distributed randomly, and after two weeks of transferring the seedlings to the pots, they were treated with concentrations of Cd and Pb, and the service operations continued, such as watering and weeding, until November 21, 2019, three seedlings from each treatment, randomly selected, and then field measurements were made for them after extracting them from the pots, cleaning them from the soil, separating roots, stems and leaves separately, taking soil samples for each seedling from the area near the roots, and then the plant samples and soil samples were placed in cardboard bags with the number of treatment, refined, and sample type recorded, and (Picture 1) showed the seedlings in the wooden shelter at the start and end of the experiment. Soil analyzes: The analyzes of the soil sample taken at the start of study (before adding heavy metals) (Table 1), were conducted as indicated by El-Mahrouk et al. (17), which it included measuring the electrical conductivity (EC) according to the ratio (1 soil: 5 distilled water) and was measured using a device EC meter, and the pH was measured using a pH meter and according to the ratio (1 soil: 1 distilled water), and the soil texture was estimated using hydrometer method, and concentrations of Pb and Cd in the soil were estimated at the start of study according to Elmer (18), as well as when removing seedlings from pots (at the end of the experiment). Measuring dry weight of shoot and root: Stems, leaves and roots were dried at a temperature of 65° C for about three days until the stability of the weight, and dry weights were recorded immediately after they were removed from electric oven using a sensitivity balance of (1) mg.   (18), the dried plant samples were ground separately and 2 g were taken from each sample and placed in an conical flask, then 10 ml of concentrated nitric acid (HNO3) were added to it, and the samples were left until the next day and then heated on an electric heater until the red fumes from nitrogen dioxide (NO 2 ) stopped and the samples were left to cool down, and then (2-4) ml of perchloric acid (HClO 4 ) were added to them its concentration is 70%, and the samples were heated again until the end of the white vapors resulting from the perchloric acid, noting that a small volume of the sample remained bright as a result of the digestion process, then the samples were left to cool down and then transferred to a 50 ml beaker and the volume was supplemented with distilled water to the mark became the final volume of the plant samples 50 ml, then the samples were filtered using filter paper, and then the Cd and Pb concentrations of the dried plant samples were estimated by flame method, using an atomic absorption spectrophotometer, SHIMADZU AA7000, Japanese origin, in the Environment  (58) and as in the equation (TI = DW HM / DW control ), as DW HM = total dry weight of the plant treated with heavy metals, and DW control = total dry weight of the control plant. Statistical Analysis: Used the completely randomized design (CRD) as a two-factor factorial experiments, each factor had five levels with three replicates (5×5×3), and the results were analyzed using SAS system version 9.0 by applying CARDS orders for statistical analyzes required, the Duncan's multiple-range test was used to compare the means values at a probability level 5%.

RESULTS AND DISCUSSION Effect of Cd and Pb on dry weight of stems:
The results of Duncan's multiple range test (Table 2) showed a decrease in dry weight of stems with an increase in concentration of Cd and Pb in the soil, as for effect of interaction between Cd and Pb, it appears that the highest value of dry weight of the stems was 71.067 g at the control which exceeded significantly the smallest value of dry weight of stems was 31.967 g at the interaction (110 Cd and 550 Pb) mg kg -1 and dry weight of stems of 40.033 g at the interaction (110 Cd and zero Pb) mg kg -1 , and no significant difference was observed between the control and the value 62.933 g, at the interaction (550 Pb and zero Cd) mg kg -1 , the results of this study were lower than the results of Shah et al. (52) in their study on Eucalyptus camaldulensis seedlings, which showed that the highest dry weight value of stems reached 166 g at the control and significantly exceeded the smallest value 97 g at the interaction (0.40 Cd and 8 Cr) mg L -1 , and the results of this study are close to the values of Bajwa (9) in his study on seedlings of Eucalyptus tereticornis, Leucaena leucocephala, Melia azedarach and Dalbergia sissoo which Malted with Cd concentrations 0, 10, 20, 40, 80 and 120 and Pb concentrations 0, 30, 60, 120, 180 and 240 mg kg -1 dry soil, which showed in Eucalyptus seedlings that the highest dry weight of stems reached 91.4 g at the control and dry weight of stems was 55.9 g at the level of 120 Cd, the minimum dry weight of the stems was 45.9 g at a Pb level of 240 mg kg -1 .
The reason for the decrease in dry weight of stems at high concentrations of Cd and Pb may be attributed to cellular stress that affected the reduction in growth in tissue cells, reduced cell expansion and inhibition, prolongation by inhibiting heavy metals to the action of the proton pump responsible for gradually accumulating protein across the biofilm and thus reducing plant growth (12,20).

Effect of Cd and Pb on dry weight of leaves:
The results of Duncan's multiple range test (Table 3) showed a decrease in dry weight of leaves with an increase in the concentrations of Cd and Pb in the soil, while the effect of the interaction of Cd and Pb shows that dry weight of leaves reached 51.033 g at the control which significantly exceeded the smallest value for leaves dry weight of 16.033 g at the interaction (110 Cd and 550 Pb) mg kg -1 , and for a dry weight of leaves of 29.367 g at the interaction (110 Cd and zero Pb) mg kg -1 , while no significant difference was observed with the value 50.267 g at the interaction (550 Pb and zero Cd) mg kg -1 , and the results of this study are close to the results of Bajwa (9), which showed in Eucalyptus seedlings that the highest value for leaves dry weight was 92.8 g at a concentration of Cd 10 mg kg -1 dry soil, and the smallest value was for dry weight for leaves it was 55.9 g at the level of Cd 120 mg kg -1 , the smallest value for dry weight of leaves was 37.7 g at the level of Pb 240 mg kg -1 , and the highest value for dry weight of leaves was 55.5 g at the control. The decrease in the biomass of plant leaves exposed to heavy metals may be due to poor absorption and transfer of nutrients and water from root to shoot system and between the cells of plant themselves (55).

Table 3. Duncan's multiple-range test results for effect of Cd and Pb and their interactions in dry weight of leaves (g). Interactions means in dry weight of leaves (g)
Pb rates Cd concentrations (mg kg -1 dry soil)

Effect of Cd and Pb on dry weight of roots:
The results of Duncan's multiple range test (

Effect of Cd and Pb on Cd concentration in stems:
The results of Duncan's multiple range test (Table 5) showed an increase in Cd content in stems with an increase in the concentration of Cd and Pb in the soil, and the effect of the interaction between Cd and Pb shows that the highest concentration of Cd in stems was 372.167 mg kg -1 at the interaction (110 Cd and 550 Pb) mg kg -1 , it was significantly superior to all concentrations of other treatments, and the smallest content of Cd was 0.375 mg kg -1 at the control which did not notice a significant difference between it and the concentration of Cd 0.417 mg kg -1 at the interaction (550 Pb and zero Cd) mg kg -1 , while the concentration of Cd was significantly higher than the concentration of Cd 178.625 mg kg -1 at the interaction (110 Cd and zero Pb) mg kg -1 at the control, and the study indicated that 19 interactions in which the concentration exceeded the critical limit for Cd (5-10) µg g -1 dry weight (44,51), the values of this study were superior to what was reported by Khamis et al. (27) in their study of Melia azedarach and Populus alba seedlings, which were treated with Cd concentrations 0, 10, 20 and 40 and Pb concentrations 0, 200, 400 and 800 ppm, as they found that the highest concentration of Cd in stems of P. alba seedlings was 11.5 mg kg -1 and in stems of M. azedarach seedlings it reached 5.9 mg kg -1 in the concentration of Cd 40 mg kg -1 dry soil, which was superior to the smallest concentration for Cd whose value approached zero mg kg -1 at the control. The reason for the increase in Cd concentration in stems of seedlings at high concentrations of Cd and Pb in the soil may be due to the fact that with the increase in the time of exposure of plant to heavy metals, the activity of the antioxidant system as well as other defenses in plant decreases, which facilitates transfer of heavy metals into the shoot to collect in the cell walls, this is one of the most important sites devoted to heavy metals (22).

Effect of Cd and Pb on Cd concentration in roots:
The results of Duncan's multiple range test (Table 7) showed an increase in the Cd concentration in the roots with an increase in the concentration of Cd and Pb in the soil, and the effect of the interaction between Cd and Pb showed that the highest concentration of Cd in the roots was 162.083 mg kg -1 at the interaction (110 Cd and 550 Pb) mg kg -1 , and it was significantly superior to all concentrations of other treatments, also the smallest concentration of Cd was 0.583 mg kg -1 at the control, whereas the concentration of Cd was 30 mg kg -1 at the interaction of (110 Cd and zero Pb) mg kg -1 was significantly superior to the control, and the study showed that 15 interventions in which the concentration in seedlings exceeded the critical limit of Cd (5-10) µg g -1 dry weight, and the results of this study outperformed the results reported by Khamis et al. (27) in their study of Melia azedarach and Populus alba seedlings, who observed that the highest concentration of Cd in roots was found in P. alba and M. azedarach 17.5 mg kg -1 at a concentration of Cd 40 mg kg -1 dry soil. The reason for the increase in the concentration of Cd in the roots with the increase in the concentration of Cd and Pb in the soil is due to the fact that the roots of plants work to release all factors that help in the chelation and binding of minerals in the area of the soil close to them (28), and that most of the absorption of Cd occurs in The epidermis of the root cap (30), as the root cap lacks a casparian strip that controls the transport of water and salts through the cell wall cavity from the phloem to the xylem, so that Cd is easily transported through cell walls directly to the xylem (27). Table 7

. Duncan's multiple-range test results for effect of Cd and Pb and their interactions in Cd concentration in roots (mg kg -1 ). Interactions means in Cd concentration in roots (mg kg -1 )
Pb rates Cd concentrations (mg kg -1 dry soil)

Effect of Cd and Pb on Pb concentration in stems:
The results of Duncan's multiple range test (Table 8) showed an increase in the concentration of Pb in stems with an increase in the concentrations of Cd and Pb in the soil, while the effect of the interaction between Cd and Pb showed that the highest concentration of Pb in the stems was 62.375 mg kg -1 at the interaction (110 Cd and 550 Pb) mg kg -1 and this value was significantly superior to all the concentrations of the treatments, and the Pb concentration at the control treatment was 12.417 mg kg -1 , and it was found from the results of this study that three values exceeded the critical toxic limit (30 µg g -1 dry weight) (47), the results of this study outperformed the results of researchers Mleczek et al. (38) in their study on seedlings of Acer platanoides L., A. pseudoplatanus L., Betula pendula Roth, Quercus robur L., Tilia cordata Miller and Ulmus laevis Pall., which treated with mining sludge containing arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), thallium (TI) and zinc (Zn) at concentrations of 18022, 1030, 4511, 3865, 669 and 1565 mg kg -1 , respectively, results showed that the significant superiority of the Pb concentration that approaching 50 mg kg -1 in U. laevis seedlings grown in mining sludge on all seedlings. The increase in the concentration of Pb in the stems at high concentrations of Cd and Pb may be due to the possibility of plant accumulation of heavy metals in the shoot due to the productivity of the vegetative biomass that supports the extraction of minerals and their confinement in cell wall vacuoles (7). Table 8

Effect of Cd and Pb on Pb concentration in leaves:
The results of Duncan's multiple range test (Table 9) showed that there are no significant differences with increasing the concentration of Cd and Pb in the soil between the concentration of Pb in the leaves at the control and the interactions between Cd and Pb except for the two interactions (85 Cd and zero Pb) and (85 Cd and 250 Pb) mg kg -1 when the concentration of Pb in leaves decreased significantly compared to the control, while the effect of the interaction between Cd and Pb showed that the highest concentration of Pb in the leaves was 20.417 mg kg -1 at the interaction (110 Cd and 550 Pb) mg kg -1 and did not differ significantly from the control value (18.375 mg kg -1 ), and the results of this study were lower than the results of researchers Mleczek et al. (38) whose findings showed a clear significant superiority over all types of seedlings for the concentration of Pb whose value approached 50 mg kg -1 in the leaves of Ulmus laevis Pall. seedlings growing in mining sludge. The low movement of Pb from soil to leaves was the reason for the absence of significant differences between most of the interactions.

Effect of Cd and Pb on Pb concentration in roots:
The results of Duncan's multiple range test (Table 10) showed that the concentration of Pb increases with the increasing the concentration of Cd and Pb in the soil, as for the effect of the interaction between Cd and Pb, it appears that the Pb concentration in the roots was 24.958 mg kg -1 at the interaction of (110 Cd and 550 Pb) mg kg -1 , which It significantly exceeded the smallest value of the Pb concentration (2.875 mg kg -1 ) at the control, also Pb concentration was 34.417 mg kg -1 at the interaction (550 Pb and zero Cd) mg kg -1 was significantly superior to the control, concentrations of Pb exceeded 30 µg g -1 dry weight, which is the critical toxic limit for most plants, the results of this study were lower than the results of researchers Mleczek et al. (38), whose values showed that the highest concentration of Pb in the roots approached 600 mg kg -1 in Acer platanoides seedlings developing in mining sludge, which was not observed significant difference with Pb concentration in roots of Quercus robur and Ulmus laevis grown in mining sludge while it outperformed all other concentrations in seedlings. The reason for the increase in Pb concentration in the roots of seedlings with the increase in the concentration of Cd and Pb in the soil is due to the plant's resistance to the stress of heavy metals through the formation of complexes that work to encapsulate and isolate heavy metals in the root cell vacuoles that have been identified as a detoxification mechanism, as the inner dermis is one of the most important sites for concentrating heavy metals in the roots (53,33).

Effect of Cd and Pb on TF of Cd:
The results of Duncan's multiple range test (

Effect of Cd and Pb on BAC of Cd:
The results of Duncan's multiple range test (Table  12) showed that most of the BAC values of Cd increased in seedlings with increasing the concentration of Cd and Pb in the soil, while the effect of the interaction between Cd and Pb showed that the highest value of BAC was 28.790 at the interaction (110 Cd and zero Pb) mg kg -1 was superior significantly for all BAC values of Cd, and that BAC value of 20.597 at the interaction (110 Cd and 550 Pb) mg kg -1 significantly exceeded value 2.106 at the control, while no significant difference was observed between value of 1.333 at the interaction (550 Pb and zero Cd) mg kg -1 at the control, and the values of this study outperformed the results of Ng et al. (41) in their study on the vegetative accumulation of heavy metals in the soil, as they found in Imperata cylindrica plant that BAC value of Cd reached 1.519 at the control did not differ significantly from value 1.343 at a level of Cd 15 mg kg -1 soil, while it was found in Pennisetum purpureum that BAC value of Cd was 2.163 at a level of Cd 15 mg kg -1 dry soil, and it significantly exceeded value 1.055 at the control. The transport of heavy metals from the soil to the shoot through the roots depends on the environment of the plant and the soil that determines the availability of heavy metals, and the plants with a value (>1) for BAC were considered to be an hyperaccumulator of heavy metals (31), and plant extraction efficiency is related to mineral concentration in plant and dry matter productivity, in order for the plant to be ideal in phytoremediation of a polluted site, it must have high biomass productivity and the ability to withstand and accumulate mineral pollutants (56).

Effect of Cd and Pb on BCF of Cd:
The results of Duncan's multiple range test (

Effect of Cd and Pb on TI:
The results of Duncan's multiple range test (Table 19) showed that the TI values decreased in seedlings with an increase in the concentration of Cd and Pb in the soil, as for the effect of the interaction between Cd and Pb, it appears that the smallest value of TI was 0.370 at the interaction (110 Cd and 550 Pb) mg kg -1 , and TI value was 0.536 at the interaction (110 Cd, zero Pb) mg kg -1 , and the results of this study relatively outperformed the results reported by Kabir et al. (26) in their study on Leucaena leucocephala seedlings treated with concentrations of Pb and Cd 0, 25, 50, 75 and 100 ppm each single and mixed, as they found that the TI value was 0.623 when the interaction (100 Cd and 100 Pb) mg kg -1 . The reason for the decrease in TI values with increasing concentrations of Cd and Pb in the soil is due to the stress of heavy metals effect, which leads to inhibit the growth of the plant, which causes a decrease in the biomass, according to the type of plant and type and amount of toxins absorbed in the cellular tissues (13,15). CONCLUSIONS: Eucalyptus seedlings have good ability to accumulate and concentrate Cd in their shoot, based on TF values greater than one, as well as for BAC, BCF, and CI which confirmed the ability of seedlings to accumulate and transfer elements from soil to root and shoot, and that seedlings have a good ability to absorb and accumulate Pb in their roots, when increasing the concentration of Pb in the soil, whether alone or in the presence of Cd, as BCF values for Pb increased than one, as well as for TF, BAC, CI and TI, which confirms the seedlings ability to resist high concentrations of Pb. And despite the increase in Cd and Pb concentrations at critical limits in plant parts, seedlings resisted these concentrations even though their growth was negatively affected.
And TF and BAC value greater than one showed that Eucalyptus camaldulensis Dehnh. seedlings were suitable for use in phytoextraction applications of soils contaminated with Cd and Pb within the phytoremediation processes.