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Assessment of Lead (Pb) Remediation Potential of Senna obtusifolia in Dareta Village, Zamfara, Nigeria

Background. Environmental contamination by lead (Pb) and other toxic metals is of significant environmental and human health concern. Heavy metals are not readily eliminated by degradation, and thus remediation of contaminated media (soil, sediment and water/sludge) requires the outright removal or cleanup of these metals. Evaluation of the performance and cost efficiency of various remediation methods has led to the development of bioremediation as an inexpensive, innovative and environmentally friendly cleanup strategy. Objectives. The present study was designed to assess the Pb remediation potential of wild Senna obtusifolia (Sicklepod), in Dareta Village, Zamfara, Nigeria. Methods. Soil and Senna obtusifolia samples were collected from established plots and Pb content was determined using a Shimadzu atomic absorption spectrophotometer (model AA-6800, Japan) after wet digestion. Results. The mean concentrations of Pb (mg/kg) in soil, roots, stems and leaves, respectively, were 130.68±5.2, 61.33±17.86, 66.64±18.10 and 173.39±13.73 for plot 1, 287.84±6.5, 69.42±11.62, 123.4±3.67 and 294.28±4.38 for plot 2, 315.73±4.13, 68.42±10.22, 86.89±6.08 and 290.61±7.47 for plot 3, 396.86±5.48, 91.64±2.87, 150.58±2.21 and 282.53±5.69 for plot 4 and 264.23±8.02, 72.71±2.18, 124.60±2.27 and 282.40±3.79 for plot 5. Average values for the translocation factor, bioaccumulation factor and bioconcentration factor were 3.65±0.66, 1.01±0.23 and 0.29±0.10, respectively. Discussion. Soil Pb levels in the present study were found to be within the United States Environmental Protection Agency (USEPA) standards and the Dutch Intervention Values for Pb in soil. Lead content of Senna obtusifolia leaves was found to be higher than the Pb content of the stem and root, indicating relatively low restriction and the efficiency of internal transport of the toxic metal from the roots towards the aerial parts. High translocation and bioaccumulation factors indicate that the plant has vital characteristics for phytoextraction of Pb. The mean Pb concentration of Senna obtusifolia leaves was found to be far above Codex general standards and the European Union (EU) maximum levels for Pb in leafy vegetables. Conclusions. The study concludes that wild Senna obtusifolia has significant characteristics for phytoextraction of Pb and that consumption of Senna obtusifolia leaves from the study area would pose a serious risk of Pb intoxication. Competing Interests. The authors declare no competing financial interests.


Introduction
Environmental contamination by heavy metals such as lead (Pb) or cadmium (Cd) is of significant concern. These metals cannot be destroyed by degradation, and thus remediation of contaminated medium requires outright removal or cleanup. 1 A number of technologies are available to clean up metalcontaminated environments. However, a majority of these technologies are costly to implement and may cause further disturbance to the already damaged environment. Evaluation of the performance, cost implication and public acceptability of various methods applied to clean up different types of pollutants from the environment has led to the development of bioremediation as an evolving, cost-effective, innovative and environmentally friendly cleanup strategy. Bioremediation entails the use of living organisms for the recovery or cleanup of a contaminated medium (soil, sediment, sludge or water) and is defined by Phillips as any process that uses living organisms (microorganisms, fungi, green plant or enzymes) to return a medium altered by a contaminant to its original condition. 2 Despite this broad definition, bioremediation usually refers specifically to the use of microorganisms for the cleanup of a contaminated medium. 3 The use of green plants to clean up contaminated sites is referred to as phytoremediation and is considered a form of bioremediation. The process Research of phytoremediation is an emerging green technology for the cleanup of toxic chemicals in soil, sediment, ground water, surface water, and air. 4 The use of metal-accumulating plants to remove heavy metals and other compounds was first introduced in 1983, but the concept has been implemented for the past 300 years on wastewater discharges. 3, 1 The technique relies heavily on the use of plant interactions in the contaminated site to mitigate the toxic effects of pollutants. 5 The complex chemical, physical and biological interactions that take place within the medium adjacent to plant roots allow for cleanup of contaminated sites through a number of phytoremediation mechanisms. 4,6 The phytoremediation mechanism most commonly employed for the treatment of heavy metalcontaminated soil involves the use of green plants to absorb, concentrate and precipitate contaminants from the soil into the above ground parts of the plant (phytoextraction). One of the major advantages of this remediation approach is that some precious metals can bioaccumulate in plants and be recovered after remediation, a process known as phytomining. 5 With ever increasing global metal contamination, plant remediation provides efficient, cost effective and ecologically sound approaches for sequestration and removal through leaves, stem and roots. 7 The success of the phytoextraction process, whereby pollutants are effectively removed from soil, is dependent on an adequate yield of plants and/or the efficient transfer of contaminants from the roots of the plants into their aerial parts. 8, 9 The discovery of plant species capable of accumulating 100 times more metals (hyperaccumulators) than other non-accumulating plants in the same medium demonstrates that plants have significant potential to remove metals from contaminated soil and thus bioremediation is considered a green alternative to the problem of heavy metal pollution.
Plants with exceptional metal accumulating capacity (hyperaccumulators) occur throughout the plant kingdom. 9 Globally, the discovery of hyperaccumulating plants has been slow-footed due to a lack of systematic screening of plant species in several regions of the world. 10 In 2017, a global data base for plants that hyperaccumulate metal and metalloids shows that about 721 hyperaccumulator species representing over 45 families have been documented. 10 Currently, they are only a few known hyperaccumulators for Pb; eight plant species belonging to six families. 10 Melastoma melabathricum for instance has been shown to accumulate up to 13 800 mg/kg in the root of the plant, but only a small amount of the Pb could be translocated from the root to the above ground part (translocation factor (TF) <1), indicating that it is a good bioaccumulator (bioavailability factor >1) of the toxic metal and that phytostabilization is the mechanism at work in the uptake of Pb. 11 However, certain species of the Noccaea genus and other plants have been identified to have the potential to uptake Pb and transport it from the roots to the shoot. Noccaea praecox has been reported to accumulate over 4000 mg/ kg Pb in the shoot, while Noccaea rotundifolia and Noccaea caerulescens have recorded Pb uptake greater than 28 700 mg/kg and 65 631 mg/kg, respectively. 9 Once introduced into the soil, Pb is difficult to remove. The heavy metal is strongly bounded to soil and all interactions within the soil matrix are pH dependent. Under acidic conditions (pH < 5.5) Pb is more mobile and readily available to plants. Lead is one of the constituents that makes up the earth's crust in nature. The metal is commonly found in water, soil and plants at barely detectable levels. 12 In nature, the occurrence of metallic Pb is rare. The principal ores of Pb are cerussite (PbC0 3 ) and galena (PbS). Other ore minerals such as pyromorphite (Pb 5 (PO 4 ) 3 Cl) and anglesite (PbSO 4 ) also occur frequently, but are less important. 12 Lead is often present as a constituent of ores that contain gold, silver, as well as copper, and is usually obtained "as a co-product of these metals". 13 Due to anthropogenic activities, Pb has come to be known as the most widely scattered toxic metal in the world. 14 The accumulation of Pb in plants depends upon the species, plant cultivar, plant organ, the exogenous concentration of Pb and the presence of other ions in the environment. The accumulated Pb content generally increases with the increase in the metal in the environment. 15 Mass acute Pb pollution and poisoning was reported in Zamfara state in 2010. The source of the widespread poisoning was traced to artisanal gold mining and ore processing in the villages. Lead concentration exceeding 100 000 mg/kg, far above 400 mg/kg considered acceptable for residential Over 735 children were reported to have died and thousands sickened by the toxic metal in what is believed to be the worst Pb poisoning worldwide in the last forty years. 16 Mineral ore processing activities involving crushing, washing and gold recovery were carried out at many sites within the residential areas in the affected villages. An immediate medical response protocol was developed to provide oral chelation therapy to children between the ages of 0-5 years, pregnant women and breast-feeding mothers. In order not to compromise the efficacy of the chelation therapy, immediate remediation of the affected villages was carried out. 17 The remediation of Dareta village (perhaps the most affected village) took place from June to July 2010. The remediation was a simple process involving the removal of 5 cm of contaminated topsoil in areas with soil Pb level greater than 1000 mg/kg and replacing with uncontaminated soil. The excavated contaminated topsoil was then buried in landfills. Areas with soil Pb levels between 400-1000 mg/ kg were simply covered with about 8 cm of clean soil and compacted. Immediately after the remediation exercise, soil Pb levels were reported in the village in the range of 81.65-684.27 mg/kg, indicating over 95% reduction in soil Pb levels. 18, 19 Three years after the remediation exercise, Udiba et al. recorded soil Pb levels ranging from 1029.42±98.50 mg/kg to 6724.68±184.00 mg/kg in the area and observed that the old grinding mill and other areas where ore processing took place were eventually covered with wild Senna obtusifolia growing aggressively and stifling the growth of preexisting plants. 20 The present study was designed to assess the Pb remediation potentials of wild Senna obtusifolia, in Dareta Village, Zamfara State, Nigeria. Despite the high soil Pb concentrations recorded, areas covered with wild Senna obtusifolia were found to be within permissible limits, hence the need to investigate the phytoremediation potentials of the plant. A fast growth rate, high above ground biomass, tolerance to Pb pollution, survival and adaptability to prevailing environmental conditions exhibited by wild Senna obtusifolia in Dareta village are some of the important characteristics to consider when choosing a plant to phytoremediate Pb in soil. 21, 22 The ability of this plant to cleanup Pb-contaminated sites depends on the amount of metals that can be accumulated by it, soil Pb concentration, rate of uptake, and translocation and accumulation in harvestable tissues. These are important properties for phytoextraction of toxic metals, which this study was designed to investigate. The success of any phytoremediation approach is anchored primarily on optimizing the remediation potentials of native plants growing in polluted sites. The findings of the present study may present the need for optimizing the remediation potentials of Senna obtusifolia. Putshaka

-Map of Anka Local Government Area of Zamfara State, Nigeria, showing Dareta Village with sampling points
Research an international boundary with Niger Republic (Figure 1). 24 The Soil samples from each plot were thoroughly mixed to obtain a representative sample, air dried, crushed and sieved with 2 mm mesh before wet digestion. One (1) g of a well mixed sample from each sampling point was taken into a 250 ml glass beaker and digested with 10 ml of concentrated nitric acid, perchloric acid and hydrofluoric acid in the ratio 3:1:1 on a hot plate. After evaporating to near dryness, 10 ml of 2% nitric acid was added, filtered into a 50 mlvolumetric flask and then made up to mark with distilled deionized water. 18

Sample analysis
Lead concentrations in the digests were determined using a Shimadzu atomic absorption spectrophotometer (model AA-6800, Japan) equipped with Zeaman background correction and graphite furnace at the National Research Institute for Chemical Technology, Zaria, Nigeria. The instrument was then set to 0 by running the respective reagent blanks and Pb concentration was determined at a wavelength of 283. 3 nm. An average value of three replicates was taken for each determination. Data obtained were subjected to statistical analysis.

Analytical quality assurance
Appropriate quality assurance procedures and precautions were taken to ensure the authenticity of the results. Samples were carefully handled to avoid cross-contamination. Glassware was properly cleaned and deionized water was used throughout the study. All of the reagents, including nitric acid (Riedelde Haen, Germany), hydrofluoric acid (Sigma-Aldrich, Germany) and perchloric acid (British Drug House Chemicals Limited, England) were of analytical grade. In order to check the reliability of the analytical method employed for Pb determination, one blank and combine standards were run with every batch of 12 samples to detect background contamination and monitor consistency between batches. The result of the analysis was validated by digesting and analyzing standard reference materials (Lichen coded IAEA-336) following the same procedure. The analyzed values and the certified reference values of the elements determined were compared to determine the reliability of the analytical method employed.

Statistical analysis
Test for normality was carried out using the Shapiro-Wilks test, and the Z-score test was used to check for outliers.

Analysis of variance test
Having passed the test for normality Research and outliers, data collected were subjected to statistical test of significance using the analysis of variance (ANOVA) test to assess significant variation in Pb concentration in the soil, Senna obtusifolia roots, stem and leaves across the five different plots under study. Probabilities less than 5% (p < 0.05) were considered to be statistically significant. Independent t-test was used to compare Pb content of Senna obtusifolia from the study area and the Pb content of Senna obtusifolia from Zaria (control). Probabilities less than 5% (p < 0.05) were considered to be statistically significant.

Pearson product-moment correlation coefficient
Pearson product-moment correlation coefficient was used to determine the association between Pb levels in the soil, stem, roots and leaves of Senna obtusifolia at α = 0.05.
All the above-mentioned statistical analyses were performed by SPSS software 17.00 for Windows.

Bioavailability factor
The bioavailability factor of heavy metals in plants, also known as the bioavailability index, was calculated according to Malik et al., and expressed in Equation 1. 28

Equation 1
Bioavailability factor = HML / (HMS) × 100 where, HM L is mg of heavy metal per kg of plant leaves and HM S is total content of heavy metal per kg of soil.

Biological concentration factor, translocation factor and bioaccumulation factor
The biological concentration factor (BCF) was calculated as a metal concentration ratio of plant roots to soil, and is given in Equation 2. 28

BCF = [metals] root / [metals] soil
The TF was described as the ratio of heavy metals in plant shoots to that in the roots, as given in Equation 3. 28

TF = [metals] shoot / [metals] root
The bioaccumulation factor (BAC) was calculated as the ratio of a heavy metal in plant shoots to that in soil, as shown in Equation 4. 28

Results
To evaluate the accuracy and precision of the employed analytical procedure, standard reference materials of Lichen coded IAEA-336 was analyzed in a like manner to the samples in the present analysis. The analyzed values were found to be within the ranges of the certified reference values for the elements in the present study, suggesting the reliability of the employed methods (Table 1).
Lead content of soil and Senna obtusifolia tissues across sampling plots in Dareta village are presented in Table 2. Correlations between Pb concentration in soil and Senna obtusifolia tissues are presented in Table 3. Translocation factor, BCF and BAC are presented in Table 4. Average Pb levels of soil and Senna obtusifolia tissues are shown in Figure 2.   Statistical analysis revealed a significant (ANOVA, p < 0.05) difference in overall Pb concentration between the soil, Senna obtusifolia roots, Senna obtusifolia stem and Senna obtusifolia leaves, and Pb concentrations in soil were significantly higher than the Pb concentration in roots and stem. The Pb concentration in Senna obtusifolia leaves was also found to be significantly (p < 0.05) higher than in Senna obtusifolia stem and Senna obtusifolia roots. The Pb concentration in Senna obtusifolia stems was found to be significantly (p < 0.05) higher than in the root. The difference in overall Pb concentration between in soil and Senna obtusifolia leaves was not statistically significant (p > 0.05). The differences in Pb concentrations between soil and Senna obtusifolia tissues across the plots were found to be statistically significant (ANOVA, p < 0.05) ( Table 2). Table 3    The observed high Pb levels in Senna obtusifolia leaves, stem and roots, (Table 2, Figure 2) despite the fact that soil Pb levels were within the USEPA acceptable limit and Dutch soil remediation intervention values, may suggest that a good proportion of Pb in Dareta soils is present in the mobile phase, thus readily available for uptake by plants. Plants growing in a Pb-enriched environment are able to accumulate large quantities of the metal in their tissues depending on the percentage bioavailabilty/mobility of the toxic metal. Susceptible plant species that are not resistant to the metal usually die off, allowing the resistant ones to thrive. This could possibly explain the fact that the old grinding mill and other areas where Pb processing had taken place in