The research was conducted at the nursery site of the Department of Forestry, Aliko Dangote University of Science and Technology, Wudil, located in Wudil Local Government Area of Kano State, Nigeria (11°48′N, 8°50′E; elevation ~457 m above sea level). The area lies within the Sudan Savannah ecological zone, characterized by distinct dry and wet seasons. Mean annual rainfall ranges between 800–1000 mm (May–September), mean annual temperature is approximately 27°C, and relative humidity averages 30–40% in the dry season and up to 80% during the wet season (NIMET, 2023). Soils are predominantly alfisols and entisols, sandy loam to loamy in texture, with low organic matter, nitrogen, and cation exchange capacity [31-32].

Soil Collection and Preparation

Disturbed topsoil samples were collected from the 0–20 cm depth within the university premises using a soil auger. A total of 20 auger samples were taken randomly across the collection area and combined to form a composite sample. The composite soil was air-dried for seven days, crushed gently, and passed through a 2 mm mesh sieve to ensure uniformity and remove debris, stones, and plant residues. The sieved soil was thoroughly homogenized and divided into 17 experimental units, each receiving 12 kg of soil. The soil type was predominantly sandy loam, characteristic of the Sudan Savannah zone.

Petroleum Hydrocarbon

Fresh petroleum (premium motor spirit) was sourced from Gano Investment petrol station, Wudil. It was applied at two contamination levels: 1000 mL and 1500 mL per 12 kg of soil. Based on an average crude oil density of 0.85 g/mL, these volumes correspond to approximately 70,833 mg/kg and 106,250 mg/kg, respectively, simulating realistic oil pollution scenarios [33-34].

Planting Materials

Seeds of Jatropha curcas were sourced from healthy, disease-free mother plants in Gaya Local Government Area, Kano State. Stem cuttings consisting of semi-hardwood sections with an average height of 22.27 cm and girth of approximately 4.0 cm were harvested from healthy, mature Jatropha curcas plants, as recommended for optimal root initiation [35-36].

Experimental Containers

Standard plastic buckets weighing 0.40 kg with dimensions of 32 cm height, 106 cm upper diameter, and 69 cm lower diameter (7 L capacity) were perforated at the base to allow for drainage. Each bucket was filled with 12 kg of prepared soil.

The experiment was laid out in a 3 × 3 factorial arrangement using a Completely Randomized Design (CRD). Factor A was propagation method (unplanted control [J0], Jatropha from cutting [JC], Jatropha from seed [JS]). Factor B was petroleum contamination level (0 mL [P0], 1000 mL [P1000], 1500 mL [P1500]). The nine treatment combinations are described in Table 1.

Table 1: Treatment Combinations

Treatments F–I were replicated three times, while controls (A–E) were not replicated, resulting in a total of 17 experimental units. Measured volumes of petroleum were thoroughly mixed with the soil in each bucket and allowed to equilibrate for 7 days prior to planting to ensure uniform hydrocarbon dispersion [37]. After equilibration, seeds and cuttings were planted directly into the treated soil. The experiment was maintained for 12 weeks with regular irrigation using borehole water to maintain near-field capacity moisture.

Soil Sample Collection and Preparation

At the end of the 12-week experimental period, soil samples were collected from each experimental unit at the root zone. Samples were air-dried, gently crushed, sieved through a 2 mm mesh for physical and chemical analysis, and through a 0.5 mm mesh for TPH and heavy metal analyses. Samples were stored in labeled, airtight polythene bags at 4°C until analysis [38-39].

Total Petroleum Hydrocarbon (TPH) Determination

TPH concentration was determined using Gas Chromatography-Flame Ionization Detector (GC-FID) following extraction with n-hexane, according to USEPA Method 8015B [39]. Results were expressed in mg/kg dry soil. Remediation efficiency (%) was calculated as:

Remediation Efficiency (%) = C0−Ct/C0×100

Where C0 = initial TPH concentration and Ct = TPH concentration after phytoremediation.

Heavy Metal Analysis

Concentrations of lead (Pb), cadmium (Cd), zinc (Zn), and copper (Cu) were analyzed using Atomic Absorption Spectrophotometry (AAS) after wet digestion with a mixture of nitric acid (HNO₃) and perchloric acid (HClO₄), following AOAC methods [40]. Results were expressed in mg/kg dry weight.

Soil Physicochemical Properties

The following soil physicochemical properties were assessed using standard methods: soil pH potentiometrically in a 1:2.5 soil-to-deionized water suspension; electrical conductivity (EC) using a conductivity meter in a 1:2.5 soil-to-water extract [41]; organic carbon (OC) by Walkley-Black wet oxidation [43]; total nitrogen (N) by Macro-Kjeldahl digestion method [43]; available phosphorus (P) by Bray-1 method [44]; cation exchange capacity (CEC) by ammonium acetate (1N NH₄OAc) method at pH 7.0 [45]; bulk density (BD) by core method [43]; porosity (PR) calculated as [1−(BD/PD)]×100 assuming particle density (PD) of 2.65 g/cm³; and mean weight diameter (MWD) by wet-sieving method [40].

Data were subjected to two-way Analysis of Variance (ANOVA) for a Completely Randomized Design using SAS software. Where significant differences were observed (p < 0.05), means were separated using Tukey's Studentized Range (HSD) test at 5% probability [46]. Results are presented as mean ± standard error.

The effects of Jatropha curcas propagation method and petroleum contamination level on TPH concentration and remediation efficiency are presented in Table 2. ANOVA revealed that propagation method significantly (p < 0.05) influenced final TPH concentration, while contamination level and its interaction with propagation method were not significant. The unplanted control (J0) recorded the highest TPH concentrations: 65,217.1 ± 6483.43 mg/kg at P1000 and 98,490.4 ± 10,030.39 mg/kg at P1500, compared to 23.1 ± 14,460.14 mg/kg in the uncontaminated control (P0). This indicates negligible natural degradation in the absence of vegetation. Cutting-propagated Jatropha (JC) reduced TPH concentrations to 28,430.4 ± 421.98 mg/kg (P1000) and 40,104.0 ± 485.75 mg/kg (P1500), representing remediation efficiencies of 56.4% and 59.3%, respectively. Seed-propagated Jatropha (JS) achieved the greatest reductions: 24,530.5 ± 356.14 mg/kg (P1000) and 36,683.9 ± 367.57 mg/kg (P1500), corresponding to efficiencies of 62.4% and 62.7%. These values were significantly different (p < 0.05) from both the unplanted control and the JC treatments. The general trend indicates that TPH concentration increased with contamination level but decreased markedly under vegetation, with seed-propagated plants consistently outperforming cutting-propagated plants.

Table 2: Mean TPH Concentration and Phytoremediation Efficiency of Jatropha curcas Under Different Treatments

Note: Trt. = Treatment; Pet. = Petroleum; TPH = Total Petroleum Hydrocarbon; Rem. Eff. = Remediation Efficiency; Phyto. Pot. = Phytoremediation Potential. J0 = Unplanted Control; JC = Jatropha from Cutting; JS = Jatropha from Seed

Caption: Figure 1: Mean TPH concentrations (mg/kg) in soils after 12 weeks of phytoremediation under different treatments. J0 = Unplanted Control, JC = Jatropha from Cutting, JS = Jatropha from Seed. Error bars represent standard error of the mean. Bars with different letters are significantly different at p < 0.05 (Tukey's HSD).

Post-remediation heavy metal concentrations varied significantly (p < 0.05) among treatments as shown in Table 3. Lead (Pb) concentrations in remediated soils ranged from 96.90 ± 1.04 mg/kg (JS×P1000) to 110.70 ± 0.72 mg/kg (JC×P1500), significantly lower than the unplanted controls (124.63–134.33 mg/kg). Cadmium (Cd) concentrations ranged from 3.20 ± 0.043 mg/kg (JS×P1000) to 3.79 ± 0.030 mg/kg (JC×P1500), representing significant reductions compared to unplanted controls (4.94–5.54 mg/kg). Zinc (Zn) concentrations ranged from 120.50 ± 1.25 mg/kg (JS×P1000) to 138.83 ± 0.78 mg/kg (JC×P1500), all significantly lower than unplanted controls (151.47–164.10 mg/kg). Copper (Cu) concentrations ranged from 83.93 ± 0.72 mg/kg (JS×P1000) to 94.20 ± 0.69 mg/kg (JC×P1500), compared to 104.37–114.83 mg/kg in unplanted controls. For all metals, seed-propagated treatments (JS) achieved lower residual concentrations than cutting-propagated treatments (JC), and both vegetated treatments were significantly lower than unplanted controls.

Table 3: Heavy Metal Concentrations (mg/kg) in Soil after Phytoremediation

Caption: Figure 3. Heatmap showing relative concentrations of heavy metals (Pb, Cd, Zn, Cu) across all treatment combinations. Darker colors indicate higher concentrations. Values are expressed in mg/kg

The influence of Jatropha curcas propagation method and petroleum contamination on soil physicochemical properties is presented in Table 4. Petroleum contamination significantly reduced soil pH from 7.02 (J0×P0) to 6.25 (J0×P1500). Vegetation improved pH, with JS×P1500 reaching 6.65 and JC×P1500 reaching 6.58, compared to 6.25 in the unplanted control. The highest pH among contaminated vegetated treatments was recorded in JS×P1000 (6.82). Electrical conductivity (EC) increased with contamination level in unplanted controls (0.160 dS/m at P0 to 1.250 dS/m at P1500), but vegetation reduced EC, with JS×P1000 recording the lowest EC (0.477 dS/m) among contaminated treatments. Organic carbon (OC) decreased sharply with contamination in unplanted controls (2.39% at P0 to 1.06% at P1500). Vegetated treatments showed higher OC, with JS×P1000 (1.88%) and JS×P1500 (1.66%) outperforming JC treatments (1.73% and 1.53%, respectively). Total nitrogen (N) followed a similar trend, with JS×P1000 (0.200%) and JS×P1500 (0.162%) showing higher values than JC treatments (0.182% and 0.162%) and unplanted controls (0.120% and 0.095%). Available phosphorus (P) was significantly higher in vegetated treatments, with JS×P1000 (18.8 mg/kg) and JS×P1500 (16.3 mg/kg) compared to JC treatments (17.6 and 15.5 mg/kg) and unplanted controls (14.2 and 12.5 mg/kg). Cation exchange capacity (CEC) improved under vegetation, with JS×P1000 (5.80 cmol/kg) and JS×P1500 (5.10 cmol/kg) exceeding JC treatments (5.40 and 4.80 cmol/kg) and unplanted controls (4.20 and 3.80 cmol/kg). Bulk density (BD) increased with contamination in unplanted controls (1.413 to 1.823 g/cm³), but vegetated treatments showed lower BD, with JS×P1000 (1.577 g/cm³) and JS×P1500 (1.650 g/cm³) outperforming JC treatments (1.620 and 1.700 g/cm³). Porosity followed the inverse pattern. Mean weight diameter (MWD), an indicator of soil structural stability, decreased with contamination (1.003 mm at P0 to 0.480 mm at P1500 in unplanted controls). Vegetated treatments improved MWD, with JS×P1000 (0.753 mm) and JS×P1500 (0.650 mm) exceeding JC treatments (0.700 and 0.600 mm).

Table 4: Selected Soil Physicochemical Properties after the Experiment

Note: Means within a column followed by the same letter are not significantly different at p < 0.05 according to Tukey’s HSD test. EC = Electrical Conductivity; OC = Organic Carbon; N = Nitrogen; P = Phosphorus; K = Potassium; BD = Bulk Density; MWD = Mean Weight Diameter; CEC = Cation Exchange Capacity

Caption: Figure 5. Principal Component Analysis biplot showing relationships among soil physicochemical properties and treatment groups. Vectors indicate direction and strength of variable influence on treatment separation.

Residual contaminant concentrations were compared with national and international regulatory standards (Table 5).

Table 5: Comparison of Residual Contaminants with Regulatory Standards

All TPH values in vegetated soils remained well above the DPR ecological intervention limit of 1,000 mg/kg. Pb concentrations slightly exceeded FAO and WHO limits [47-49]. Cd concentrations exceeded all permissible limits. Zn and Cu remained within acceptable limits.

Soil fertility parameters (pH, OC, N, P, CEC) in remediated soils fell within FAO medium fertility ranges, indicating partial restoration [48]. However, bulk density (1.59–1.68 g/cm³) exceeded the FAO optimum (<1.60 g/cm³), and porosity (37.0–42.5%) was lower than the uncontaminated control (48.5%), suggesting slight structural deterioration.

The significant reduction in TPH concentration in vegetated treatments compared to unplanted controls confirms the effectiveness of Jatropha curcas in enhancing hydrocarbon degradation. The higher remediation efficiency achieved by seed-propagated plants (62.4–62.7%) compared to cutting-propagated plants (56.4–59.3%) can be attributed to differences in root architecture and rhizosphere dynamics. Seed-propagated Jatropha develops a deeper taproot system, which enhances soil aeration, increases the volume of rhizosphere soil, and supports a more diverse and active microbial community capable of hydrocarbon degradation [50-51]. Root exudates from seed-propagated plants likely provided carbon sources that stimulated hydrocarbon-degrading microbes, accelerating rhizodegradation [24,52]. In contrast, cutting-propagated plants, with shallower fibrous roots, had a more limited rhizosphere volume, reducing contact with deeper contaminants and limiting microbial stimulation. These findings align with Al-Hadith, who reported that propagation method significantly influences root development and pollutant uptake in Jatropha curcas [26]. Rao similarly found that seed-propagated plants exhibited greater root biomass and deeper penetration, enhancing their remediation potential [35]. Despite achieving substantial reductions (up to 62.7%), residual TPH concentrations (24,530–40,104 mg/kg) remained far above the DPR intervention limit of 1,000 mg/kg. This indicates that a single 12-week remediation cycle is insufficient for complete restoration of heavily contaminated soils [47]. Similar observations have been reported by Pilon-Smits and Prasad, who emphasized that phytoremediation efficiency depends on contamination load and treatment duration, with heavily polluted sites requiring multiple cropping cycles or integrated approaches [17,53]. When compared with regulatory standards, the residual TPH values in all vegetated soils remained well above the DPR ecological intervention limit of 1,000 mg/kg, indicating that extended remediation periods are necessary [47].

The differential reduction patterns among heavy metals reflect the specific uptake and accumulation mechanisms of Jatropha curcas. The significant reductions in Zn and Cu concentrations indicate effective phytoextraction of these essential micronutrients, which are required for plant metabolic processes including enzyme activation and photosynthesis, driving their active uptake and accumulation in plant tissues [22,52]. In contrast, Pb and Cd reductions were more limited, suggesting that Jatropha curcas primarily stabilizes these non-essential toxic metals in the rhizosphere (phytostabilization) rather than translocating them to harvestable tissues [23]. Ali explained that non-essential metals are often sequestered in root vacuoles or bound to cell walls, limiting their upward movement [19]. The superior performance of seed-propagated plants in reducing all metal concentrations further supports the importance of root architecture in metal uptake and stabilization. Deeper root systems provide greater surface area for metal adsorption and access to contaminants throughout the soil profile [25]. When compared with international regulatory standards, Zn concentrations (120.5–138.8 mg/kg) fell within FAO and USEPA permissible limits, and Cu concentrations (83.9–94.2 mg/kg) also complied with FAO and WHO standards [47,48,53]. However, Pb concentrations (96.9–110.7 mg/kg) exceeded FAO limit of 100 mg/kg and WHO limit of 85 mg/kg, while Cd concentrations (3.20–3.79 mg/kg) exceeded both FAO limit of 3 mg/kg and WHO limit of 0.8 mg/kg [48,49]. These findings indicate that while Jatropha curcas effectively manages micronutrient contaminants within acceptable timeframes, remediation of toxic metals to regulatory standards requires extended periods or integrated approaches.

The improvement in soil physicochemical properties under Jatropha curcas vegetation demonstrates the plant's capacity to restore soil health beyond simple contaminant removal. The increase in soil pH from acidic levels (6.25) in contaminated controls to near-neutral values (6.65–6.82) in vegetated treatments can be attributed to the release of basic cations (Ca²⁺, Mg²⁺, K⁺) through root activity and microbial decomposition of organic residues [50-51]. This pH neutralization is critical for reducing metal bioavailability and creating favorable conditions for microbial activity and nutrient cycling. The increase in organic carbon (OC) in vegetated soils (1.53–1.88%) compared to unplanted contaminated controls (1.06–1.26%) reflects continuous addition of root exudates, senesced root tissues, and leaf litter, which serve as substrates for microbial growth and contribute to soil organic matter buildup [54-55]. Higher OC in seed-propagated treatments suggests greater root biomass and exudation compared to cuttings. The improvement in CEC (4.80–5.80 cmol/kg) correlates strongly with increased OC, as organic matter contributes significantly to the soil's exchange capacity [56-57]. This enhancement in nutrient retention capacity is essential for supporting subsequent vegetation and agricultural productivity on remediated sites. Increased available phosphorus (15.5–18.8 mg/kg) in vegetated treatments likely results from organic acid and phosphatase release by Jatropha roots and associated microbes, which solubilize bound phosphorus [58-59]. Higher values in seed-propagated treatments again indicate greater rhizosphere activity. The reduction in bulk density (1.59–1.68 g/cm³) and increase in porosity (37.0–42.5%) in vegetated soils compared to unplanted contaminated controls (1.75–1.82 g/cm³; 32.5–35.3%) reflect improved soil structure through root penetration, aggregate formation, and organic matter incorporation [29]. However, values had not fully recovered to uncontaminated control levels (1.39 g/cm³; 48.5%), indicating that structural restoration requires longer timeframes than chemical recovery. Soil fertility parameters in remediated soils fell within FAO (2020) medium fertility ranges, indicating partial restoration. However, bulk density (1.59–1.68 g/cm³) exceeded the FAO optimum (<1.60 g/cm³), and porosity (37.0–42.5%) was lower than the uncontaminated control (48.5%), suggesting slight structural deterioration that requires continued management.

The failure of residual TPH, Pb, and Cd concentrations to meet regulatory standards after 12 weeks has important implications for phytoremediation practice in the Sudan Savannah. While Jatropha curcas demonstrated significant remediation capacity, the high initial contamination levels (70,833–106,250 mg/kg TPH) exceeded the typical range for which phytoremediation alone is recommended. Prasad noted that phytoremediation is most effective for low-to-moderate contamination levels, with heavily polluted sites requiring integrated approaches. However the compliance of Zn and Cu with international standards suggests that Jatropha curcas can effectively manage micronutrient contaminants within acceptable timeframes. This differential performance based on metal type should inform site-specific remediation planning, with expectations adjusted for different contaminant classes. The partial restoration of soil fertility parameters to FAO (2020) medium ranges indicates that phytoremediation with Jatropha curcas can simultaneously achieve contaminant reduction and soil quality improvement. This dual benefit enhances the economic and environmental viability of the approach, particularly in resource-limited settings where land rehabilitation must support eventual agricultural use [29-30].