Phytoremediation potential and vegetation assessment of plant species growing on multi-metals contaminated coal mining site

This study aims to evaluate the plant species potential to accumulate, concentrate and translocate the heavy metals around the coal mining contaminated site with heavy metals at Harnoi, Abbottabad. The phytosociological surveys involve the systematic study of plant communities within the particular area to show their composition, structure and distribution showed that the contaminated coal mining-associated area was poor in vegetation. Among these, 11 plant species with higher important values (IV) are collected with associated soil and analyzed for the total concentrations of Cadmium (Cd), Copper (Cu), Chromium (Cr), Lead (Pb) and Nickel (Ni) using Atomic Spectrophotometer. The phytoremediation indices (BAF, BCF, TF and TI) were used to evaluate the multi-metals hyperaccumulator and stabilizer plant species. Dodonaea viscosa was evaluated as multi-metals (Cd, Cu and Ni) stabilizer. While the Ajuga bracteosa and Sonchus espera, Sisybrium officinale and Platango ovata stabilize Cd and Cr respectively. The other plant species that can stabilize as single heavy metal are Ajuga bracteosa and Sonchus espera (Cd), Sisybrium officinale and Platango ovata (Cr) and Amaranthus spinosus (Ni) respectively. While the multi-metals accumulator plant species are Bidens pilosa (Cu, Pb and Ni), Chenopodium ambrosioides (Cd, Cu and Ni), Amaranthus spinosus (Cd, Cu and Cr), Ajuga bracteosa (Pb and Ni) and Rumex hastatus (Cd and Ni). However, the single heavy metal accumulator plant species are Sonchus espera (Pb), Conyzea Canadensis (Ni), Platango ovata and Malvastrum coromandelianum (Cu) respectively. These plants could find valuable applications in practical phytoremediation for the remediation near mining tailings at Abbottabad. Moreover, the use of local plants is a promising approach not only for in situ accumulation and stabilization of heavy metals but also for tolerance and environmental adaptations in the contaminated area.


Introduction
Mining is an important economic operation in numerous nations of the world.The industry ranks as the second-oldest on a global scale and holds paramount significance [1].However, the discharge of massive volume of heavy metals near the vicinity of abandoned mining sites poses an extensive environmental danger to the ecosystem [2] and constitutes the most significant environmental hazard [3].Heavy metals are naturally occurring metals having atomic number and density more than 20 and 5 g cm −3 [4] .Fundamentally, literature showed that the heavy metals are transition and post transition metals such as Lead (Pb), Vandium (V), Cobalt (Co), Mercury (Hg), Chromium (Cr), Copper (Cu), Iron (Fe), Arsenic (As), Nickel (Ni), Manganese (Mn), Tin (Sn) and Zinc (Zn).The availability and accessibility of these metals and metalloids by natural and anthropogenic routes remain a major global concern in the ecosystem [5,6].Based on the availability in living organisms, heavy metals are grouped into essential and non-essential.The essential heavy metals (Cu, Fe, Mn, Ni and Zn) have role in the biochemical and physiological activities in plant species [7] but excess amount may cause toxicity [8].While non-essential heavy metals (Pb, Cd, As and Hg) are highly toxic and no known functions in the plant species [9].Heavy metal contamination has received considerable focus in recent past because of its persistence, bioaccumulation, and possibly harmful impacts on the environment [10].Therefore, it is prerequisite to undertake the intervention of isolating and treating heavy metals regions that have been afflicted by heavy metal pollutants and to ameliorate their deleterious consequences on the atmospheric ecosystem [11].
Heavy metals are processed using variety of techniques but in comparison [12] phytoremediation represents an economically efficient and universally acknowledged bioremediation technique that aligns with ecological principles and offers sustainability.This method utilizes plants to purify heavy metals from polluted soil [13,14].The plants with phytoremediation potentials can be employed in soil management and decontamination of heavy metals [15] It is an ecologically friendly way of extracting low-grade metal bio-ore from the environment because it can extract metal and recover biomass energy [16,17].The plants accumulating and stabilizing capacity provides important data into their potential targeted deployment at contaminated sites for successful phytoremediation [14].
Phytoremediation indices include various mechanisms, notably Bioaccumulation Factor (BAF), Bioconcentration Factor (BCF), and Translocation Factor (TF).BAF gauges the concentration of contaminants gathered by plants from soil, revealing their uptake efficiency.Similarly, BCF assesses ability of plants to absorb contaminants from water.These metrics highlight plants' pivotal role in removing pollutants from the environment.Moreover, TF clarifies how contaminants move within plants, especially from roots to aerial parts, affecting their distribution.The BF and TF calculate the heavy metals hyper-accumulation capability in plants [14].In a study [18], hyperaccumulator plant species were identified within copper mine tailings situated in the Antofagasta region of Chile and revealed that Gazania rigens and Pelargonium hortorum exhibited properties of metal excluders for specific elements Fe, Mn, Pb, Al, and Zn.Furthermore, these plants displayed characteristics suggesting their potential to accumulate Cu indicating their significance in the context of phytoremediation efforts in contaminated environments.
The plants must be identified and employed for successful and effective phytoremediation (phytomining) programs.So, the investigations that screen local plants are desired to explore in situ accumulation potential in the mining areas [14,19].Several studies have been reported that screened hyperaccumulating and phytostabilizing plant species [2,15,[20][21][22][23][24][25].The plants response varies for heavy metals depend upon their total bioavailability, total concentration, physical and chemical parameters of soil, and climatic factors [14,19,22,[26][27][28].The local plant species that have acclimated to local habitats and are thriving at heavy metal contaminated sites.These plants could be utilized instead of viable non-native heavy metal accumulator species [29,30].Several local plant species Sporobolus pyramidalis, Leucaena leucocephala, Melinis repens, Lantana camara, Arthraxon hispidus, Blepharis maderaspatensis, Boehmeria nivea, Polygonum capitatum, Miscanthus floridulus, Chrysanthemum indicum, Conyza canadensis, Rubus setchuenensis, Senecio scandens, Scirpus validus, Typha orientalis, Phragmites australis, Euphorbia heterophylla, Juncus acutus, Typha angustifolia,and Cyperus haspan were evaluated for phytoremediation of polluted soil [11,13,14,16,31,32].Atriplex nummularia has a potential for Cd phytostabilization in treated tailings, offering a viable remediation option to remediate copper mine tailings in northern Chile [33].These findings inform the selection of plant species and amendments, fostering sustainable approaches for copper mine tailings restoration.The study analyzed the concentration of chromium in the soil, roots, and shoots of at Dir Lower, Khyber Pakhtunkhwa, Pakistan and showed several plant species feasibility for the phytostabilization and phytoextraction of chromium.While six species were identified as chromium hyperaccumulators [34].
The mine tailings and waste is a usual occurrence in Pakistan [35] caused contamination in the surrounding environment.The objectives of this study are twofold: first, to evaluate the overall vegetation composition and diversity in these challenging environment, and second, to identify plant species capable of effectively accumulating multi-metals from the contaminated coal mining soil.Therefore, this study through a combination of field surveys and laboratory analyses explore the vegetation analysis and heavy metal contents in the native dominant plant species to screen out the candidate plant species (hyperaccumulators and phytostablizors) for remediation purpose.It also provide valuable insights for sustainable land management practices in coal mining affected areas.

Study area
The present study was study out near coal mining site in Harnoi (Kali-Mitti) approximately 11 km from the Abbottabad city.It is positioned at a 33°− 50°and 34°− 23°North and 73°− 35°and 73°− 31°East and elevation 1256 meters in a hilly area along the Murree road in district Abbottabad, Khyber Pakhtunkhwa of the Pakistan (figure 1).It has experienced rapid mining activities and urban expansion in recent years.Consequently, soil contamination has become a pressing issue, threatening both environmental sustainability.The hottest month is June with an average high of 88 °F, while the coldest month is January with an average low of 31 °F.The area average annual temperature is 60.6°F and rainfall is 1532 mm.This is a major tourist destination due to its scenic location and diverse biodiversity.Futhermore, the coal mining site is surrounded by the Nathiagali hills and Thandiani hills.

Vegetation analyses
The vegetation of the area was evaluated by the quadrat method.The sampling plots were 10 × 10 meters for trees, 05 × 05 meters for shrubs and 01 × 01 meter for herbaceous plants.The phytosociological attributes are computed by the following formulae [36,37]

Data collection, sample prepartion and analyses
The sampling of the soil and plant species was conducted thrice (April, June and September).The plant species possessing the I.V greater than 16.5 are collected for phytoremediation potentials to investigate the heavy metals accumulation, concentration and translocation abilities.This methodology is helpful to check the extinction of the plants from the area.The plant species are collected based on their important values (I.V).The plant species are identified, collected, labelled and persevered in a paper bags and oven dried.While, the samples of soil were acquired at depth of 30 centimeters in air-tight bag and sealed, precisely at points where the respective plants were gathered to facilitate subsequent analysis of heavy metal content.For the tolerance index (T.I) the plant species are also collected from un-polluted sites (forest).If the BAF, BCF, and TF indices are > 1 then the plant is worthy of phytoremediation [15,41].

The tolerance index of plants
It is calculated by the biomass of the plant species [42].

Statistical analyses
The analysis within three replicate samples was conducted, focusing on determining the mean and standard error of plant species.The computation of means and standard error were carried out utilizing Statistix 8.1 and MS Excel 365 software.Furthermore, Principal Component Analysis (PCA) was employed by using PAST 4.03 software.

Species composition
Vegetation can serve to reduce the impact of heavy metals in soils and their deleterious effects by mining activities on soil can also be evaluated by plant composition and vegetative analyses.The vegetation is leaded by herbaceous plants at Harnoi coal mining area (table 1).A floristic composition revealed that there are 23 plant species belonging to 09 families including herbs (14) and shrubs (07) and trees (02).The plant species with I.V less than 16.5 are not selected for phytoremediation (table 2) due to their low numbers at the mining site.These results of low I.V indicate the ecological impact of coal mining on vegetation is severe, with limited plant colonization and establishment in the affected coal mining area.

Ajuga bracteosa
It is commonly known as Persian Bugle.It is a perennial herbaceous plant, rosette leaves, tubular purple flowers, upright stem.It is used for medicinal and ornamental purposes [43].

Rumex hastatus
It is commonly known as Heartwing Sorrel.It is a perennial herb, arrow-shaped leaves, produce tall slender flower typically grown in disturbed areas.It is used for culinary and medicinal purpose [44].

Sisybrium officinale
It is commonly known as Hedge mustard.It is a biennial or perennial herb, erect stem, pinnately lobed leaves, cluster of small yellow leaves typically grows along roads, waste places and disturbed areas.It is used for Culinary and medicinal purposes [45].

Platango ovata
It is commonly known as Psyllium.It has lanceolate, small inconspicuous flowers at the end of stem and rich in mucilage.It has dietry fiber supplement and medicinal value [46].

Amaranthus spinosus
It is commonly known as Amaranth.It is found at road sides, waste places and disturbed areas, erect stem, alternate leaves, small greenish flowers.It is a weed used by animal as fodder [47].

Sonchus esper
It is commonly known as Prickly Sowthistle.It is a biennial or perennial herb.It is characterized by erect stem, covered with spiny prickles.The leaves are alternate, bright yellow flowers arranged at tips of stem.It is found at along roadsides and disturbed areas.It is used as culinary, medicinal and animal feed [48].

Malvastrum coromandelianum
It is an Indian mallow.It is a perennial herbaceous plant grows along waste places, roadsides and disturbed areas.
It is characterized by upright stem and palmate lobed leaves, small pale yellow flowers.It has ornamental and medicinal uses [49].

Bidens pilosa
It is commonly known as Spanish Needle.It is an annual herb typically grows in disturbed areas, roadsides and waste places.It is characterized by slender stems, lobes leaves arranged oppositely along the stem, leaves are usually green and serrated, small yellow flowers.It has medicinal and culinary uses.It improve soil fertility and structure [50].

Conyza canadensis
It is commonly known as Canadian Horseweed.It is an annual herb typically found at waste places, roadsides and disturbed areas.It is characterized by erect stem, serrate lanceolate leaves, small white or pinkish flowers.It has medicinal and culinary uses.It help to control soil erosion and improve soil health [51].

Dodonaea viscosa
It is commonly known as Hopbush.It is a perennial shrub.It is characterized by its dense foliage.The leaves are usually green or buish-green, alternate and serrated edges.It produces inconspicuous flowers greenish-yellow in colour and arranged in clusters along the stem.It is known for its ornamental qualities and drought tolerance [52].The area exhibits poor plant species diversity due to the disturbance caused by mining activities.However, a variety of plant species persist in the area.Both native and non-native plant species have been identified in the area.The dominant plant species are Malvastrum coromandelianum, Amaranthus spinosus, Bidens pilosa and Dodonaea viscosa.These plant species have adaptations that allow them to thrive in the disturbed coal mining site of Harnoi and tolerate harsh environmental conditions.The plant species Malvastrum coromandelianum (I.V = 20.9),Amaranthus spinosus (I.V = 20.0),Dodonaea viscosa (I.V = 19.6),Bidens pilosa (I.V = 19.5),Platango ovata (I.V = 18.4),Sonchus espera (I.V = 18.2),Conyzea Canadensis (I.V = 18.0),Ajuga bracteosa (I.V = 17.8),Rumex hastatus (I.V = 16.8),Sisybrium officinale (I.V = 16.5), and Chenopodium ambrosioides (I.V = 16.5) are selected for the screening of phytoremediation potentials (table 2).These results provide ecological potentials and challenges associated with vegetation dynamics in response to mining activities.
To investigate the inter-species multi-metals concentration, the cadmium surrounding the soil was found maximum in Ajuga bracteosa ( 0.08 mg kg −1 ), copper in Ajuga bracteosa (0.64 mg kg −1 ), chromium in Rumex hestatus (10.15 mg/kg), lead in Dodonaea viscosa (2.41 mg kg −1 ) and nickel in Malvastrum coromandelianum (0.63 mg kg −1 ) respectively.The results of the biplot of PCA (figure 3. A) of plant soil indicated a strong positive correlation of Cd with the soil of Rumex hastatus, a weak positive correlation of Cd with the soil of Sonchus espera, Conyzea Canadensis, Dodonaea viscosa and Cu weak positive correlation with the soil surrounding Bidens pilosa and Dodonaea viscosa (figure 3(A).

Heavy metals concentration in plant shoot
The plant shoots showed chromium (7.95 to 5.23 mg kg −1 ) > lead (2.41 to 0.07 mg kg −1 ) > nickel (0.7 to 0.19 mg kg −1 ) > cadmium (0.13 to 0.01 mg kg −1 ) > and copper (0.09 to 0.05 mg kg −1 ) concentrations illustrated non-significant differences (p > 0.05) (table 2).The heavy metals accumulated from plants relate to heavy metals present in the soil [25].The cadmium present in shoot was found higher in Ajuga bracteosa (0.136 mg kg −1 ), copper in Conyzea Canadensis and Chenopodium ambrosioides (0.09 mg kg −1 ), chromium in Chenopodium ambrosioides (7.95 mg/kg), lead in Ajuga bracteosa (2.41 mg kg −1 ) and nickel in Ajuga bracteosa (0.7 mg/kg) as shown in table 3. The F1 axis of the biplot of the PCA (figure 3(B) explains that cadmium has a strong positive correlation with Chenopodium ambrosioides, Sonchus espera and Platango ovata.Copper also shows a strong positive correlation only with Ajuga bracteosa while the other metals do not have a strong correlation with plant species.A weak positive correlation was observed in Amaranthus spinosus and Dodonaea viscosa for Cd and Conyzea Canadensis for Cu respectively.

Heavy metal concentration in plant roots
The plant roots show marginally lower concentrations than shoots with chromium ranging (7.73 to 5.52 mg kg −1 ) > lead (1.73 to 0.28 mg kg −1 ) > nickel (1.36 to 0.04 mg kg −1 ) > copper (0.19 to 0.06 mg kg −1 ) > and cadmium (0.12 to 0.01 mg/ kg).These metal concentrations are non-significant (p > 0.05) in the roots of plants (table 2).Moreover, the roots of plants showed that cadmium is concentrated highest in Dodonaea viscosa (0.12 mg kg −1 ), copper in Dodonaea viscosa, Chenopodium ambrosioides and Sisybrium officinale (0.19 mg kg −1 ), chromium in Dodonaea viscosa (7.73 mg/kg), lead in Dodonaea viscosa (1.73 mg kg −1 ), and nickel in Dodonaea viscosa (1.3 mg/kg) respectively.The F1 axis of the biplot of the metal concentrations showed that cadmium has a strong positive correlation with the roots of Dodonaea viscosa.A weak positive correlation was observed in Malvastrum coromandelianum and Conyzea Canadensis for Cd and Platango ovata and Dodonaea viscosa for Cu respectively (figure 3(C).

Bioconcentration factor
Similarly, the BCF determine the link of metal concentration in roots and soil anchoring the plants.The 3).Moreover, the plant species reported to stabilize heavy metals in the roots are Dodonaea viscosa (Cd, Cu and Ni), Ajuga bracteosa and Sochus espera (Cd), Sisybrium officinale and Platango ovata (Cr) and Amaranthus spinosus (Ni) respectively.

Tolerance index
The tolerance index (TI) based on the biomass of plant species from the Harnoi mining site revealed significant variations among the studied plant species.It ranged from 1.43 to 0.59 (p > 0.005) including a wide spectrum of tolerance levels to mining-related stressors by heavy metals.The plant species with higher TI such as Ajuga bracteosa (1.43), Chenopodium ambrosioides (1.17), Dodonaea viscosa (1.02) and Amaranthus spinosus (1.02) exhibited gresater resilience to the adverse conditions prevailing in mining sites, as evidenced (figure 2(D).These plants with higher TI species could serve as potential candidates for reclamation and restoration efforts in degraded mining sites.Conversely, the plant species with lower TI such as Rumex hastatus (0.88), Platango ovata (0.83), Bidens pilosa (0.75), Sisybrium officinale (0.75), Conyzea Canadensis (0.67) and Sonchus espera (0.59) demonstrated lower biomass at Harnoi mining site, suggesting their susceptibility to the harsh environmental conditions prevailing in mining sites.It provides valuable insights into the ecological resilience of plant species to mining-related disturbances and provides ecosystem restoration initiatives in mining landscapes.

Species composition
Variations in soil composition cause changes in vegetation composition [54].The use of suitable local plants holds great potential in repairing heavy metals tainted soil because it enhances the environmental quality within the regional ecology [13].Similar findings [55] that mining activities are correlated with removing vegetation and diminution of nutrients in plants.The assessment of native hyper-accumulator plants must also be considered.Plants should be area-specific, and restriction compliance should be highlighted prohibiting invasive and competitive alien species [55].Amaranthus retroflexus was identified for the heavy metals analysis based on the highest frequencies and densities at the polluted area.The knowledge about more tolerant or resistant plant species to certain levels of metals could provide guidance for land management decisions in reclamation efforts [11].The restoration of the mining areas by plantation can be performed by identifying the native plants with the prospect to thrive in these disturbed environment.

Concentration of heavy metals in plant species
The metal concentrations were found to be affiliated to the distance from mine tailings while vegetation coverage and richness enhanced with increasing distances [11].The concentrations of Cd (0.06 mg kg −1 ), Cr (2.19 mg kg −1 ), Pb (0.52 mg kg −1 ) and Ni (0.73 mg/kg) were observed in Ajuga bracteosa [10].Similarly, Amaranthus spinosus was not regarded as a hyperaccumulator since it accumulated metals in proportion below 1,000 mg kg −1 .However, it demonstrated phytoaccumulation efficiency, accumulating Cu, Pb and Cd substantial proportions in the roots > stem and > leaves [56].Bidens pilosa shoots and roots have a positive correlation with Pb accumulation accompanied by multi-accumulation of other heavy metals [57].The BCF show heavy metals concentrations in plants and soil indicating the transfer of heavy metals from soil into plants [64].The plants reported to stabilize heavy metals in the roots are Dodonaea viscosa (Cd, Cu and Ni), Ajuga bracteosa and Sochus espera (Cd), Sisybrium officinale and Platango ovata (Cr) and Amaranthus spinosus (Ni) respectively.
The TF determined the translocation ability of heavy metals from soil into the shoot of plants [13].Plants with translocation and bioaccumulation values below one are deemed unacceptable candidates and are explicitly excluded from extracting heavy metals from soil.The excluder plant species for the cadmium are Sisybrium officinale, Platango ovata, Malvastrum coromandelianum, Sonchus espera and Conyzea canadensis.The excluders for copper are Ajuga bracteosa, Rumex hastatus, Sisybrium officinale, Sonchus espera and Conyzea Canadensis.The excluders for chromium are Ajuga bracteosa, Rumex hastatus, Sonchus espera, Malvastrum coromandelianum, Bidens pilosa, Conyzea Canadensis, Dodonaea viscosa and Chenopodium ambrosioides.The excluders for lead are Rumex hastatus, Sisybrium officinale, Platango ovata, Amaranthus spinosus, Malvastrum coromandelianum, Conyzea canadensis, Dodonaea viscosa and Chenopodium ambrosioides.The excluders for Nickel are Sisybrium officinale, Platango ovata, Sonchus espera and Malvastrum coromandelianum.The accumulated heavy metals are less concentrated in shoots and roots than in the associated soil demonstrating the tolerance ability of plants [21].

Conclusion
The research highlights the significant phytoremediation potentials of various plant species thriving in multimetal contaminated coal mining sites.The vegetation assessment, metals uptake and tolerance in the plant species tailored to specific site conditions warranted to optimize their utilization in environmental restoration efforts.The harnessing of indigenous vegetation offers a sustainable and cost-effective approach towards mitigating the environmental impacts of mining activities.The plant species Ajuga bracteosa, Rumex hastatus, Amaranthus spinosus, Bidens pilosa and Chenopodium ambrosioides showed multi-metals hyperaccumulator potentials while Dodonaea viscosa stabilize multi-metals.The plantation of these plants reduces heavy metals significantly and can act for effective restoration of the contaminated mining sites.Further, an immediate necessity exists to focus on the use of heavy metals accumulated in plants in industries to block their entry into the food chain and the environment.

Figure 2 .
Figure 2. Phytoremediation Indices (A) Bioaccumulation Factor (B) Bioconcentration Factor (c) Translocation Factor (D) Tolerance Index of plant species collected from Harnoi Coal Mining Site (Columns shows Means; with Standard Errors and letters present Significant difference).

Figure 3 .
Figure 3. Biplot of Principal Component Analyses in (A) Soil (B) Shoots (C) Roots of plant species collected from Harnoi Coal Mining Sites.
The collected soil samples are crushed and sieved through the sieve (2 mm).The Plant samples are carefully separated into shoot and root and are oven-dried at 65 °C for 2 days.Afterwards, samples are grinded, acid digested in a 1HClO 4 :3HNO 3 and analyzed by atomic absorption spectrophotometer.

Table 1 .
General description of plant species collected from harnoi coal mining site.
It is commonly known as Mexican Tea.It is an annual herb.It is widespread including waste places, disturbed areas and roadsides.It has alternate, lanceolate to ovate leaves.The flowers are arranges at the tip of stem.The

Table 2 .
[53]osociology of Plant species present at harnoi coal mining site.planthasability to colonize at the disturbed areas quickly aggressively.It has culinary, medicinal and Insect repellent uses[53].

Table 3 .
Heavy accumulation in plant species collected from harnoi mining site (means values of heavy metals; ± represents Standard errors & letters (a)-(k) shows significant difference).

Table 4 .
List of hyperaccumulators, phytostabilizers and Excluder Plant Species collected from harnoi coal mining site.