3.1. Chemical Analysis of Irrigation Water and Soil
The concentrations of nutrients and heavy metals in TWW used for irrigation of various tree and shrub species are shown in
Table 3. Treated wastewater contained nitrate, phosphate, nitrogen, and potassium, with mean values for each nutrient of 42.3 mg L
−1, 16.2 mg L
−1, 14.7 mg L
−1, and 36.5 mg L
−1, respectively (
Table 3). The analysis of various nutrient elements found levels to be lower than the standard limits for irrigation water described previously (JISM, 2013; WHO, 2006) [
31,
32]. Significant quantities of essential elements were reported in treated wastewater when compared to fresh water in the Wadi Musa region, according to previous studies [
33,
41,
42,
43]. Furthermore, higher quantities of Na
+ and Cl
– ions were also found in the TWW with concentrations of 93.0 mg L
−1 and 134.5 mg L
−1, respectively.
Significant levels of HMs were also detected in TWW used for irrigation purposes, with higher concentrations of Pb and Cr detected compared to Cd and Ni (
Table 3). On the other hand, no significant concentrations of Cu, Zn, or Mn were observed in the irrigation water samples (
Table 3). High levels of Pb, Cr, Cd, and Ni were found to exceed the acceptable Jordanian (JISM) and WHO standard limits for irrigation water, which agrees with Al-Habahba et al. [
33]. Variability in the elemental composition of TWW was reported in Jordan depending on the source of municipal or industrial water effluents. Maximum values of Fe and Cu were recorded, with values reaching 200 and 85 mg L
−1, respectively [
44,
45]. Manasreh and Alzaydien [
46] found similar values of Fe and Cu, in addition to high concentrations of Zn, Pb, Cd, and Cr, with values of 90, 21, 2.1, and 2 mg L
−1, respectively.
Soil collected from the vicinity of woody trees and shrubs was found to contain considerably high levels of nitrate, phosphate, total nitrogen, potassium, sodium, and chlorine (
Table 3). The buildup of elements in the soil samples could be attributed to the long-term use of TWW for irrigation. Estimated amounts of N, P, and K added by continuous irrigation with TWW in this study might reach 105, 30, and 67 Kg ha
−1 yr
−1 for these elements, respectively. Treated wastewater is well-known for its high content of ions, particularly nitrate and phosphate [
47]. Furthermore, the higher contents of organic matter that exist in TWW could also be responsible for increasing ion availability, especially N, P, and K [
48,
49]. Such an increase in the concentrations of essential nutrients usually has positive effects on the growth of various plant species [
10,
28]. However, although high levels of Na (374 mg Kg
−1) and Cl (784 mg Kg
−1) were detected in TWW-irrigated soil, no toxicity symptoms were observed on the leaves of the studied trees and shrub species. It is known that the use of TWW for long periods leads to the accumulation of significant quantities of toxic metals in the soil [
42]. In this study, the results of the soil heavy metal analysis showed comparatively low heavy metal levels in the soil around the trees and shrubs (
Table 3), which might suggest the possibility of higher uptake of these elements by plants. Higher Fe and Cr levels were detected, with values of 2.03 mg L
−1 and 0.20 mg L
−1, respectively (
Table 3).
3.2. Effects of Irrigation with TWW on HM Content in Different Tissues of Woody Trees
The average concentrations of HMs in roots, bark, and leaves of six woody trees irrigated with TWW are summarized in
Table 4. The concentrations of Zn, Fe, Mn, and Cu in roots differed considerably among tree species, with the highest content of Zn detected in
F. nitida roots at a mean value of 54.05 mg Kg
−1, while the lowest mean value (11.1 mg Kg
−1) was found in
P. nigra roots. For root Fe concentrations,
R. pseudoacacia was significantly the highest (625.5 mg Kg
−1) among all tree species, while
C. sempervirens contained the lowest Fe concentrations (120.5 mg Kg
−1). Mn concentrations in
N. oleander, R. pseudoacacia, and
F. nitida roots were also significantly higher than the rest of the tree species, with mean values of 31.9 mg Kg
−1, 26.4 mg Kg
−1, and 27.3 mg Kg
−1, respectively, while the lowest concentration of Mn (8.99 mg Kg
−1) was found in the roots of
P. nigra. From
Table 4, it is clear that
F. nitida and
M. viminalis contained, significantly, the highest concentrations of Cu in their roots, with mean values of 41.25 mg Kg
−1 and 34.86 mg Kg
−1, respectively, whereas Cu concentrations in the roots of
C. sempervirens were, significantly, the lowest, with a mean value of 7.13 mg Kg
−1. The Cr and Ni root concentrations reached 4.47 and 4.95 mg Kg
−1, respectively, with no significant differences between different tree species, except for
P. nigra, which had the lowest concentrations of 1.82 mg Kg
−1 and 2.06 mg Kg
−1 for Cr and Ni, respectively. Cd root concentrations were as high as 0.71 mg Kg
−1, except for
F. nitida and
C. sempervirens, where they were significantly lower, with values of 0.16 mg Kg
−1 and 0.18 mg Kg
−1, respectively. It was clear that all roots of all tree species accumulated high concentrations of Pb, with a maximum content of 45.64 mg Kg
−1 in
M. viminalis (
Table 4). The trends in mean concentrations for each metal in the roots of different trees were in the order of Fe > Pb > Zn > Mn > Cu > Ni > Cr > Cd, with average values, in mg Kg
−1, of 355.63, 29.00, 23.24, 20.20, 17.72, 3.82, 3.81, 0.40, respectively. High levels of Pb and Fe correlates with their high levels in TWW (Pb > Fe > Ni > Cd > Zn > Cr > Mn > Cu) and in the soil content (Mn > Pb > Fe > Zn > Cu > Cr > Ni > Cd).
The concentrations of various measured heavy metals in the bark tissues significantly varied among the tree species in response to TWW irrigation (
Table 4).
Robinia pseudoacacia bark was found to have the highest Zn (45.31 mg Kg
−1) and Mn (68.89 mg Kg
−1) concentrations among the studied tree species. On the contrary,
P. nigra concentrations of Zn and Mn were the lowest, with mean values of 8.64 mg Kg
−1 and 7.03 mg Kg
−1, respectively. The highest concentrations of Fe, Cu, Cr, Ni, and Cd were found in
C. sempervirens bark, with mean concentrations of 227.95, 20.68, 4.08, 4.14, 1.84 mg Kg
−1, respectively. The lowest Fe and Ni contents were found in the bark of
P. nigra trees, with mean values of 91.03 mg Kg
−1 and 2.10 mg Kg
−1, respectively, whereas the bark of
M. viminalis trees contained the lowest Cu and Cr contents, with mean values of 2.50 mg Kg
−1 and 0.83 mg Kg
−1, respectively. On the other hand, Cd content was the lowest in the bark of
N. oleander trees (0.35 mg Kg
−1), while Pb content was the highest in
M. viminalis (26.43 mg Kg
−1), and the lowest was in
P. nigra (3.19 mg Kg
−1). The trends in the mean concentrations of each metal in the bark of different genotypes were in the order of Fe > Mn > Zn > Pb > Cu > Ni> Cr > Cd, with average values, in mg Kg
−1, of 159.17, 24.56, 18.22, 14.72, 8.74, 3.15, 2.20, 1.20, respectively. Mn and Cd concentrations in bark ranged from 122% to 301% of those in roots, respectively.
The leaves of the various tree species also exhibited variable HM contents, as shown in
Table 4. High concentrations of Zn, Mn, Cu, and Cd were detected in
R. pseudoacacia leaves, with mean values of 17.50, 142.19, 22.01, and 0.79 mg Kg
−1, respectively, whereas
N. oleander leaves were found to have the highest concentrations of Cr and Ni, with mean values of 2.26 mg Kg
−1 and 5.56 mg Kg
−1, respectively. Fe and Pb were the highest in
C. sempervirens leaves, with mean values of 326.70 mg Kg
−1 and 22.13 mg Kg
−1, when compared to other tree species leaves.
Populus nigra leaf contents of Zn, Fe, Mn, Cu, Cr, and Pb were the lowest among all tree species (
Table 4). The lowest mean value of leaf Ni content (3.73 mg Kg
−1) was found in
M. viminalis, whereas the lowest Cd content (0.26 mg Kg
−1) was found in
N. oleander leaves. The trends in mean concentrations of each metal in the leaves of different trees were in the order of Fe > Mn > Pb > Zn > Cu > Ni > Cr > Cd, with average values of 242.39, 69.71, 16.56, 14.15, 13.95, 4.34, 1.54, 0.61 mg Kg
−1, respectively. Mn, Ni, and Cd concentrations of leaves were higher than those for roots, with values of 345%, 113%, and 152% compared to roots, respectively. The results also show that Fe accumulation was the highest in roots, as compared to leaves and bark, with the exception of
C. sempervirens (
Table 4).
Absorption of heavy metals by plants depends primarily on their concentration and bioavailability in soil, and the rate at which elements transfer from solid to liquid phases and to plant roots [
50]. Plants can potentially accumulate metal ions in their roots at magnitudes greater than the surrounding medium [
51]. This can be explained by the fact that one of the normal functions of the roots is to absorb ions from the aqueous solution of the rhizosphere as compared to the aerial parts [
52]. Soil pH and organic matter content are major factors that affect the solubility and availability of metal ions in contaminated areas, and therefore, their absorption [
53]. Phytoaccumulator plants have developed efficient mechanisms to absorb metal ions from their environment, such as the ability of their roots to induce soil pH changes and redox reactions that increase the solubility of these metals, and therefore, increase their absorption rates [
54]. Furthermore, soil microorganisms (bacteria and fungi) that live in the vicinity of the rhizosphere may contribute to metal solubility and bioavailability in the soil [
55]. This occurs primarily due to the altering of soil pH and the production of chelators, such as siderophores and organic acids [
56].
Metal contents of most plant species are quite variable. Essential metalloid micronutrient concentrations have been averaged to be in the range of 2–20 μg g
−1 for Cu, 70–700 μg g
−1 for Fe, 20–70 μg g
−1 for Mn, and 20–40 μg g
−1 for Zn for many plant species [
57,
58]. These elements have a significant role in plant growth and development [
59], including ionic balance, membrane stability, coenzymes, protein synthesis, and participation in major cellular redox reactions [
58,
59]. In comparison with previous studies, the accumulation of nutrient elements in tree species irrigated with WWT was in the range of normal levels, except for zinc concentrations in the roots of
C. sempervirens and
F. nitida, where the values exceeded the normal range [
58,
60]. Furthermore, Cu concentrations exceeded the normal values for
F. nitida and
M. viminalis in their roots. Upper critical concentrations of Zn and Cu were reported for plant leaves with values up to 300 μg g
−1 and 100 μg g
−1 of dry weight, respectively [
60,
61]. These levels are much higher than those found in all studied species in our analysis. Moreover, no toxicity symptoms were evident in any of the studied tree species (data not shown).
On the other hand, non-essential heavy metals levels in crop plants occur in the range of 0.2–2.4 μg g
−1 for Cd, 1–5 μg g
−1 for Ni, 1–13 μg g
−1 for Pb, and 1–10 μg g
−1 for Cr [
62,
63]. Ni, Cr, and Cd concentrations in all parts of the six tree species were found to be within the normal range, whereas the concentrations of Pb were in excess of the normal limits, but still below phytotoxic levels (50 μg g
−1) [
64].
3.3. Effects of Irrigation with TWW on HM Bioconcentration and Translocation Factors in Different Parts of Woody Trees
The uptake of heavy metals and their translocation and partitioning into various plant parts is summarized by the measurements of their BCF and TF values. As shown in
Table 5, BCF mean values of Zn, Fe, Mn, Cu, Cr, Ni, Cd, and Pb were 23, 233.7, 3.4, 54.3, 22, 55, 18.5, 12.6 for roots, whereas for bark they were 15.8, 108.9, 3.6, 30.6, 13.5, 45.8, 55.7, 6.1, and 13.5, 162.2, 11.8, 45.7, 9.1, 64.2, 30.2, 7.2 for leaves, respectively. Roots showed higher accumulation ability for Fe, Cu, Cr, and Pb, whereas bark accumulation ability was the highest for Cd and Zn; in contrast, for leaves, higher accumulation ratios were observed for Ni and Mn. According to the calculated values of BCF, the capability of tree species to accumulate metals was in the order of Fe > Ni > Cu > Cd > Zn > Cr > Pb > Mn (
Table 5).
Ficus nitida,
R. pseudoacacia, and
P. nigra were found to have the highest Zn BCF in the roots (54.87), bark (25.69), and leaves (23.21), respectively.
Robinia pseudoacacia also exhibited the highest Fe BCF (347) in its roots, whereas for
C. sempervirens, maximum Fe BCF occurred in the bark (163) and leaves (233).
Nerium oleander,
R. pseudoacacia, and
C. sempervirens were also found to have the highest BCF values of Mn in the roots (5.68), bark (7.49), and leaves (23.74), respectively. Maximum Cu BCF was found in the roots of
F. nitida (97.66), while the highest values in bark (90.09) and leaves (85.19) were found in
C. sempervirens. On the other hand,
F. nitida showed the maximum BCF of Cr for both roots (38.02) and leaves (17.34), as compared to
C. sempervirens bark (29.21). The highest Ni BCF values were also found in the roots (94.7) and bark (82.75) of
C. sempervirens, as compared to the high BFC values in the leaves of
P. nigra and
M. viminalis. Maximum Cd BCF values in roots and bark were found in
R. pseudoacacia and
M. viminalis, while the maximum BCF in leaves was found in
R. pseudoacacia (
Table 5). High Pb BCF was also detected in the roots of
P. nigra (17.85), and in the bark and leaves of
C. sempervirens (10.27 and 12.1, respectively). In summary, and according to the obtained averages of the BCF for overall plant parts,
C. sempervirens and
F. nitida showed an active accumulation of different heavy metals from the surrounding soil.
In addition to BCF, the TFs of various HMs in the bark and leaves of the woody trees were also measured (
Table 6). According to Anderson [
65], plants are considered to be accumulators and hyperaccumulators once their TF values exceed one (TF > 1).
Robinia pseudoacacia appeared to be an accumulator for Zn in their bark, as compared to other species that had TF values > 1. Similarly,
R. pseudoacacia, C. sempervirens, and M. viminalis were also considered to be accumulators for Mn. Furthermore,
C. sempervirens was found as an accumulator for Fe, Cu, Cd, Pb, and Cr, whereas
P. nigra had the highest TF values for Cr in their bark.
Cupressus sempervirens was considered a hyperaccumulator plant for metalloids in bark tissue, except for Zn and Ni; on the contrary,
N. oleander was clearly an excluder plant.
Cupressus sempervirens had the highest TF values for leaves, and was evidently a hyperaccumulator for Fe, Mn, Cu, Cd, and Pb, with TF mean values of 2.76, 6.73, 2.72, 10.40, and 1.24, respectively (
Table 6). On the contrary, all TF values of Cr for all tree species leaves were < 1, with no significant differences among them.
Populus nigra and
N. oleander leaves possessed the highest TF values for Ni, at 1.96 and 1.65, respectively. Consequently, it is evident that
C. sempervirens can be considered a hyperaccumulator plant for the different heavy metals in its bark and leaves, and it can be recommended as a good choice for soil remediation in the long term.
The translocation of heavy metals into plants occurs either through energy-dependent active transport using special membrane-bound transporters, or passively through the transpiration stream [
66]. Increased transpiration losses in arid environments may be responsible for the increased uptake of metal ions and their accumulation in various plant parts [
66]. Furthermore, decreased temperature and metabolic rates also result in the decreased accumulation of metals, such as Cd in the leaves of tobacco plants. Heavy metal accumulation in hyperaccumulator genotypes have been associated with high translocation rates to areal parts [
67].
Previously, BCF and TF were utilized by researchers to select plant species based on their phytoremediation capabilities [
28,
68]. The selection of species with high translocation rates of harmful metalloids to aerial parts, primarily to stem tissues that can be harvested later and utilized for various purposes, is crucial for phytoremediation studies. Peuke and Rennenberg [
69] suggested that an effective economical phytoremediation process requires the use of plants that have metal BCF values of 10–20. The majority of forest species in this study have shown a prevalent pattern of metal accumulation and compartmentalization in the roots, and low translocation to areal parts. As illustrated in
Table 6,
C. sempervirens had higher TF values for several heavy metals in their areal parts than the other tree species. In addition to exhibiting a TF > 1 for Fe, Mn, Cu, Cr, Cd, and Pb in their leaves and bark,
C. sempervirens was also found to have the ability to transfer the high molecular weight Pb to its bark and leaves. This is in agreement with the results of several reports indicating that
C. sempervirens are phytoaccumulator trees and showed high translocation potentials for Pb, Zn, Mn, Cu, and Cd [
70,
71,
72].
The role of different Ficus species (
Ficus stranglers,
Ficus infectoria Roxb,
Ficus palmata Forsk,
Ficus religiosa L.) in phytoremediation studies has indicated marginal potential to phytoextract soil contaminants [
73,
74]. Calculated TF values < 1 for Cd, Cr, Co, Cu, Fe, Mn, Pb, and Zn were reported [
73,
74]. On the contrary, this study indicates that
F. nitida has the potential to be hyperaccumulators for Cd in their bark, since the TF value exceeds 12.
Although TF values were < 1 for Zn, Mn, Cu, and Ni in oleander tree bark, higher values (>1) were found in their leaves, suggesting the potential of oleander shrubs for removing such contaminants from the soil, and for use in the phytoextraction process. These results are in agreement with previous studies indicating that oleander trees had BCF and TF < 1 for metals such as Pb, and TF > 1 for Cd and Zn, in their areal parts [
75,
76,
77]. The deciduous trees, Poplar and Robinia, were not considered hyperaccumulators, nevertheless, they were recommended as an effective species for soil remediation due to their fast-growing behavior and deep root systems [
69,
78]. The results of this study indicate that Poplar and Robinia have a high TF for Ni and Cd toward their areal parts, whereas a high TF for Cr was observed in Poplar bark only (1.21). These results agree with those of Bhargava et al. [
79], who also concluded that poplar trees preferentially accumulated high Ni and Cd in their leaves and bark, respectively.