Lead and zinc chemical fraction alterations in multi-metal contaminated soil with pomelo peel biochar and biochar/apatite incubation

The issue of heavy metal soil contamination is widespread, and the negative effects of heavy metals on the ecosystem depend on their chemical speciation in contaminated soil. Pomelo peel-derived biochar produced at 300 °C (PPB300) and 500 °C (PPB500) and its combination with apatite ore (AP) was applied to immobilize lead (Pb) and zinc (Zn) in agricultural multi-metal polluted soil. Soil amendments including biochar (PPB300 and PPB500) at concentrations of 3%, 5%, and 10%, as well as a mixture of biochar and apatite (AP) at 3:3% and 5:5% weight ratios, were introduced into the soil matrix. The chemical forms of Pb and Zn in incubated soil samples and control soil (CS) were studied utilizing the Tessier’s sequential extraction procedure. The chemical partitioning of heavy metals was carried out using Tessier’s extraction protocol, yielding fractions representing exchangeable (F1), carbonate (F2), Fe/Mn oxide (F3), organic matter (F4), and residual (F5) forms. Quantification of Pb and Zn concentrations was accomplished via inductively coupled plasma mass spectrometry (ICP-MS). Observations revealed notable elevations in pH, organic carbon (OC), and electrical conductivity (EC) levels within the treated soil relative to the control soil (p < 0.05). After incubating PPB300, PPB500, and AP for 30 days, there was a reduction in the exchangeable fraction of Pb and Zn by approximately 64% and 58% respectively. This reduction was achieved by transforming heavy metals from mobile fractions to immobile fractions using five primary mechanisms: chemical and physical adsorption, electrostatic attraction, the formation of complexes via interactions with active groups, cation exchange processes, and precipitation events mediated by phosphate, carbonate ions, or hydroxyl ions within an alkaline environment. Therefore, pomelo peel-derived biochar and the blend of biochar/apatite show promise as materials for mitigating heavy metal pollution in soil.


Introduction
Heavy metal pollution has significantly expanded worldwide, inflicting severe detriment to the ecological environment due to the rapid development of industrial production and the acceleration of urbanization [1,2].Natural occurrences and anthropogenic activities, including mining, agricultural practices, industrial and municipal discharge, have rapidly intensified heavy metals (HMs) and metalloid contamination in soil [3,4].These activities might seriously endanger environmental ecology, public health, and food security issues [4].Because HMs are non-biodegradable, they may persist in the soil, get into the food chain from contaminated soil, water, and air through agricultural products, and even build up in humans through bioaccumulation.As a result, they pose a threat to human and animal health [5,6].The ability of soil to carry out some of its essential roles in the ecosystem can also be negatively impacted by soil contamination.Although soil is a living resource, it may become 'functionally dead' once contamination surpasses a certain permissible level.This can adversely affect plant quality and yield, as well as seriously impair the composition and activity of the soil microbial community [6].Thus, when soils are contaminated with HMs, remediation technologies are needed to enhance soil quality while reducing metal concentrations [6].
To address the problem of soil heavy metal pollution, numerous techniques, including physical, chemical, and phytoremediation, have been used [6].Within these methodologies, the immobilization of HMs using organic or inorganic substances is perceived as a rapid, effective, and environmentally benign approach.This strategy aims to reduce the mobility and bioavailability of HMs by transforming them into inert forms, thereby mitigating their impact on agricultural soil [7].Biochar, which is the product of pyrolyzing biomass (such as agricultural wastes, wood, and leaves) without or with limited air [2], is one of the most ubiquitous amendments applied for heavy metal remediation in contaminated soil [8].It is a carbon-rich material with numerous specific properties, such as an expansive surface area, a negatively charged surface, a neutral to alkaline pH, and abundant active organic functional groups and aromatic carbon structures [9].Numerous prior investigations have substantiated the efficacy of biochar in mitigating heavy metal contamination within polluted soil environments [10][11][12][13][14][15].Moreover, it has been combined with other amendments, such as fly ash [16,17] or apatite [18][19][20], to enhance biochar's effectiveness in ameliorating heavy metals in contaminated soil.Apatite, a phosphorous-rich mineral, is widely utilized to immobilize heavy metals in soil polluted by heavy metals [21][22][23][24].
The physical-chemical properties of biochar are typically crucial in determining how well it can immobilize heavy metals in soil.The properties of biochar such as surface area, porous pores, functional groups, and minerals were significantly dependent on the pyrolysis temperatures and feedstocks [25].The various feedstock kinds had a noticeable impact on the surface morphological characteristics of the biochars [26].The majority of the time, the various biomass waste and pyrolysis conditions determine the major characteristics of biochars, such as surface area, pore volume, and mineral distribution [26].Liao et al 2022 discovered that the amount of carbon and degree of graphitization in the biochars made from various materials varied [27].Therefore, it is imperative to conduct a systematic investigation into the remediation efficacy of biochar derived from diverse feedstocks for mitigating heavy metal contamination in soil.
In general, HMs' toxicity and mobility in the soil are influenced by their total concentration, chemical fractions, and environmental factors.The comprehensive assessment of metal concentration may not suffice to elucidate the biological accessibility and potential risks associated with hazardous metals.However, identifying the chemical speciation of HMs offers more comprehensive information regarding their availability, movement, and toxicity.It is vital to understand their chemical speciation to comprehend the bioavailability and chemical form of HMs, which significantly impact their fate and redistribution [7].Sequential extraction and fractionation procedures have been popularly used as valuable tools that may provide more comprehensive knowledge about the source, bioavailability, and toxicity of HMs [28].Tessier's sequential extraction procedure stands out as the predominant and extensively applied technique for evaluating the bioavailability, toxicity, and redistribution dynamics of metals across various chemical states.This method has been extensively used to investigate the chemical fractions of heavy metals in polluted soil [20,[29][30][31][32].According to Tessier, the chemical fractions of heavy metals are categorized as the exchangeable fraction (F1), carbonate fraction (F2), Fe/Mn oxide fraction (F3), organic substances fraction (F4), and residual fraction (F5) [33].
The Pb/Zn mining area in Hich village, in Dong Hy district, Vietnam, has been reported in preceding studies as highly heavy metal contamination in the soil caused by human activities [18][19][20]34].Hence, it becomes imperative to identify an appropriate method for remediating heavy metals in the contaminated soil of this region, particularly in agricultural areas.Previous research has scarcely explored the utilization of agricultural waste-derived biochar in conjunction with apatite to immobilize heavy metals in contaminated soil in this region [18][19][20]34].However, there is a scarcity of studies that comprehensively investigate the impacts of biochar derived from various biomass sources and its combination with apatite.Information regarding the effects of pomelo peel-derived biochar and its amalgamation with apatite on the chemical fractions of heavy metals remains limited.The objective of this study is to bridge this gap in knowledge.
The primary objective of this study is to assess the impact of pomelo peel-derived biochar, a readily available agricultural residue, and a composite of biochar/apatite ore on the chemical fractions of heavy metals in soil contaminated with multiple pollutants.This investigation aims to enhance the utilization of biochar/apatite blends in mitigating heavy metal contamination in soil.The hypothesis posits that biochar sourced from pomelo peel and the amalgamation of biochar/apatite can immobilize heavy metals in polluted soil by transitioning them from labile or exchangeable fractions to more stable chemical forms.Consequently, this study aims to: (i) analyze the physicochemical characteristics of biochar derived from pomelo peel at 300 °C and 500 °C; (ii) assess the influence of different types of biochar and their application rates on the chemical fractions of heavy metals in contaminated soil; and (iii) evaluate the effects of biochar on soil pH, soil organic carbon content, and soil electrical conductivity, as well as the interplay between these factors and the exchangeable fraction of heavy metals.

• Soil samples
The soil samples under analysis were obtained from the surface of a cornfield adjacent to a Pb/Zn mine in Thai Nguyen Province, situated in northern Vietnam (21°43′46.27′′N,105°51′2.75′′E).Each soil sample was collected at intervals of approximately five meters, with an average weight of about two kilograms and dimensions measuring approximately 30 cm in width, 30 cm in length, and ranging from 0 to 20 cm in depth.To ensure homogeneity and representativeness, five subsamples were collected and thoroughly mixed to form a composite soil sample [35].

• Biochar
Pomelo peel was cleaned using distilled water and then dried in the oven at 45 °C for 48 h.Pomelo peelderived biochar was produced using a drum pyrolyzer where pomelo peel was burned in limited air condition at 300 °C and 500 °C for 1 h.The biochar produced from pomelo peel at 300 °C and 500 °C were abbreviated as PPB300 and PPB500.

• Apatite
The apatite ore was procured from Vietnam Apatite Ltd in Lao Cai Province, Vietnam.Both the biochar and apatite were finely ground to particles smaller than 1 mm before being thoroughly mixed with the soil under investigation to ensure uniform distribution [19].

Design experiment
The studied soil was thoroughly homogenized and blended with various ratios of biochar and apatite.Apatite and biochar were added in three different ratios of 3%, 5%, and 10% to soil (w/w) in a plastic cup.There were twenty-one microcosm incubation plots in total.Three analyses of every microcosm plot were conducted.Therefore, the experimental analysis was carried out through all 63 runs.The setup of experiments is shown in table 1. Soil samples were incubated for 30 days at room temperature.Deion water was added to soil samples once every two days to maintain the proper moisture level at roughly 70% [18,19].After 30-day incubation, soil samples were dried in the oven at 45 °C for 48 h.After that, they were pulverized in a small size to pass through a 2 mm sieve and preserved for further analysis [19].

Methodologies and techniques for soil and amendment analysis 2.3.1. Physicochemical analysis
A comprehensive evaluation of the primary physicochemical properties of the soil samples, biochar, and apatite ore was conducted both pre-and post-incubation with amendments (biochar and apatite) over a 30-day period.Measurement of pH and electrical conductivity (EC) values for both the soil and amendments (PPB300, PPB500, and AP) was accomplished using a pH meter (Hanna HI 9124 , Rumani).Following a 1 : 10 (w/v) dilution with distilled water, the materials were agitated and allowed to stand for one hour prior to assessment [20].Soil texture analysis was performed via the pipette method [19,36], while the organic carbon (OC) content of the materials (biochar and apatite) was determined employing the C/N multi 3100 instrument (Analytik Jena, Germany) [37].

Heavy metal analysis
The heavy metal concentration in soil samples was analyzed by digesting the soil sample in the microwave oven according to the EPA 3051A [38].Briefly, 0.5000 g of the substance was weighed, and a mixture of 8 ml concentrated HNO 3 : HCl (v/v = 1 : 3) was employed for digestion using the Mars 6 microwave system.Operational settings for the microwave system are detailed in table S1 (refer to Supplementary Materials).Post-digestion, the resulting solution was cooled, filtered with a Whatman No. 42 filter, and diluted to a 100 ml volume with distilled water.Quantitative analysis of heavy metal levels was accomplished utilizing ICP-MS (Agilent 7900, Agilent Technologies, UK) (table S2, see in Supplementary Materials) provides an overview of the operational specifications for the Agilent 7900 ICP-MS instrument.The recovery assessment of heavy metals (Pb and Zn) entailed analyzing their concentrations in the sediment standard reference material (MESS-4).Previous investigations [19] indicated recovery rates of 109.27% for Pb and 103.22% for Zn (see table S2 in Supplementary Materials (SM)).Tessier's sequential extraction procedure was utilized for the analysis of heavy metal chemical fractions [33], which is described in depth in table S3 (see SM).

Apatite and biochar's surface properties
The investigation of functional groups present on the surfaces of apatite and biochar was conducted utilizing Fourier transform infrared spectroscopy (FTIR) with a JASCO FT/IR-4600 instrument (JASCO International Co. Ltd, Tokyo, Japan) [19].Surface morphology and chemical composition analyses of biochar and apatite were conducted using a field emission electron microscope (FE-SEM), namely the JSM-6700F model (JEOL, Akishima Tokyo, Japan), outfitted with an energy dispersive spectrometer (EDS) [20].Furthermore, dimensional pore characteristics and surface area of apatite and biochar were assessed employing a BET analyzer, specifically a TriStar II 3020 instrument (Micromeritics Instrument Corporation, Norcross, GA, United States) located at 4356 Communications Dr, Norcross, GA 30093, United States [19].

Data analysis and statistics
The data underwent analysis utilizing Origin Pro 2021 (OriginLab Corp., Northampton, MA, USA) and Microsoft Excel 2019 software.Excel 2019 was utilized to calculate the average values and standard deviations of the triple results.The results are displayed in tables and figures, presenting the data as mean ± standard deviation.The Tukey's honestly significant difference (HSD) test with a 95% level of confidence was used to identify differences between the mean values.Spearman correlation analysis was performed to determine how the factors under study correlated utilizing Origin Pro 2021.

Physicochemical properties of the studied soil and materials
The basic properties of soil, biochar (PPB300, PPB500) and apatite are shown in table S4 (See SM).pH values of PPB300 and PPB500 were 11.56 ± 0.01 and 11.65 ± 0.01, respectively, while the pH value of apatite was 9.06 ± 0.01.The observed pH values notably surpassed those of the investigated soil, suggesting a potential elevation in soil pH subsequent to incubation with biochar and apatite.The alkalinity observed in biochar specimens was linked to the degradation of acidic functional groups (-COOH and -OH), the formation of carbonate, and the release of alkali salts from organic molecules during the progressive escalation of pyrolysis temperatures [39][40][41].
Table S4 shows that the organic carbon (OC) values of biochar were 75.55 ± 0.34% and 72.21 ± 0.32%, whereas those of the studied soil and apatite were much less with 1.95 ± 0.31% and 3.34 ± 0.11%, respectively.The OC result indicates that biochar can build up the OC content of the incubated soil after the 30-day incubation.Moreover, the EC values of biochar were 2670 ± 2.5 μS cm −1 and 4620 ± 3.5 μS cm −1 , which were extremely high compared to that of apatite (380.4 ± 0.2 μS cm −1 ) and the studied soil (138.4 ± 0.2 μS cm −1 ), showing that biochar might raise the EC values of the incubated samples after being treated with biochar that might facilitate the exchange reaction of heavy metals in the soil solution.In addition, the concentration of Pb and Zn, in the studied soil, biochar, and apatite were also investigated.The amount of Pb and Zn in the soil sample was 3369.5 ± 55.6 and 2414.1 ± 51.6 mg kg −1 , respectively, but biochar and apatite had very few of these elements (table S4).This finding suggests these materials were suitable for heavy metal treatment in multi-contaminated soil [18][19][20]34].According to Vietnamese Regulation (2015) [42], the acceptable thresholds for Pb and Zn levels in agricultural soil are 70 and 200 mg kg −1 , respectively.Analysis revealed that the Pb and Zn content in the examined soil exceeded these limits by approximately 48 and 12 times, respectively, indicating severe contamination.Consequently, an appropriate strategy is imperative to mitigate heavy metal contamination in this multi-contaminated soil.Given the exceptionally elevated concentrations of lead and zinc, this study exclusively concentrated on the remediation of Pb and Zn.

Analysis of amendment properties 3.2.1. Characterization of amendments using FTIR analysis
• FT_IR of biochar The FTIR spectra of the raw pomelo peel (PP), biochar-derived from pomelo peel produced at 300 °C (PPB300) and 500 °C (PPB500) are illustrated in figure 1(A).All the spectra of PP, PPB300, and PPB500 have a distinct peak at ~3432 cm −1 , which can be assigned to hydroxy (O-H) [43,44].The intensity of the peak decreased in the order of PP > PPB300 > PPB500.When the temperature of the pyrolysis increased, the intensity of this peak was most apparent in PP, less noticeable in PPP300, and least in PPB500.This finding can be explained due to the decomposition of hydroxy in materials when burned at high temperatures.In addition, figure 1(A) showed a peak at around 2925 cm −1 , which was most evident in PP, and almost negligible in PPP300 and PPB500.This peak can be assigned to C-H [45].The peak at around 1741 cm −1 appeared obviously in the spectra of PP but was negligible in that of PPB300 and PPB500.This peak corresponded to the stretch vibration of the aromatic C=O functional group, carboxylic groups, or conjugated ketone [45,46].The peak at about 1627 cm −1 was attributed to the aromatic C=C vibrations [47].The intensity of this peak was reduced in the order of PP > PPB300 > PPB500.Whereas the peak at around 1383 cm −1 refers to the phenolic -OH [48] or -C-H bend [49].In addition, the peak at ~1048 cm −1 was attributed to C-O-H or C-O-C stretching or allopathic [43,48].This peak is noticeable in PP but almost negligible in PPB300 and PPB500.Similar prominent peaks of functional groups were reported in previous studies when they analyzed pomelo peel-derived biochar [49,50].

• FT-IR of apatite
The FT-IR of apatite was investigated in previous studies [19,20], which reported that the apatite sample was a fluor-hydroxide-carbonate-apatite with the prominent peaks representing the functional groups of PO 4 3-, -OH, or CO 3 2-(figure 1(B)).These main groups might facilitate heavy metal immobilization in contaminated soil via exchange and precipitate reactions [19].
To sum up, the PPB300 and PPB500 had oxygenic functional groups such as hydroxyl, carboxyl and aldehyde on the biochar's surface, which may facilitate the immobilization of heavy metals these groups were less in PPB500 than in PPB300.Whereas AP had active groups such as PO 4 3-, -OH, or CO 3 2-.These functional groups may be crucial in the removal of heavy metals from contaminated soil through reactions involving complexion and precipitation.

SEM-EDS investigation of amendments
• SEM analysis The surface morphology of PP, PPB300, PPB500, and apatite was conducted using a scanning electron microscope (SEM) instrument outfitted with an energy-dispersive x-ray fluorescence spectrometer to determine the elemental composition of the material's surface.The SEM results of PP, PPB300, PPB500, and apatite are illustrated in figure 2. The surface of raw pomelo peel (PP) was not flat but had no holes.The PPB300 and PPB500 had a porous surface with many holes on their surface, indicating that PPB300 and PPB500 might facilitate the absorption of heavy metals on their surface.In contrast to biochar, apatite had a pretty flat surface without holes; therefore, it might not enable the immobilization of heavy metals by physical absorption on the surface.The flat surface of apatite was documented in earlier research [19,20].

• EDS analysis
The elemental composition of the surface of Biochar and apatite was analyzed using EDS.The results are shown in figure 3. The finding showed that PPB300 (figure 3(A)) and PPB500 (figure 3(B)) had similar main components on the surface with C, O, Mg, P, K, and Ca, in which C and O were the most dominant elements.Whereas apatite's surface (figure 3(C)) had various elements: O, Mg, Si, P, Ca, K, C, F, N, S, and Na.These results were consistent with the FT-IR result and previous studies [19,20], which approved that the apatite was a fluorhydroxide-carbonate-apatite, a phosphorous-rich mineral.
In conclusion, the SEM-EDS results demonstrated that PPB300 and PPB500 were rich in organic-oxygencontaining groups and had a porous surface which might facilitate the heavy metal immobilization process.Although apatite's surface was not porous, it was rich in inorganic elements like phosphorus, which could help with the remediation of heavy metals through exchange and precipitate reactions, particularly regarding lead ions [1,7,19].

Changes in OC, pH, and EC of the studied soil after a one-month incubation
The critical soil quality factors are OC, EC, and pH, especially in the heavy metal ameliorating process.These factors of the amended soil samples were investigated after 30-day incubation.The results are illustrated in table 2.

• Organic carbon (OC)
The OC value of the control soil sample was 20,16 ± 0,46 g kg −1 .The OC values of the incubated soils were significantly higher than that of CS (p < 0.05) and increased when the amended ratios rose.The highest OC values were 89.06 ± 0.64 g kg −1 and 87.24 ± 0.51 g kg −1 in the PPB3:10 and PPB5:10 samples when the incubated biochar ratio was 10%.The rise in organic carbon (OC) levels was ascribed to the elevated OC content inherent in biochar types PPB300 and PPB500 (75.55 ± 0.34% and 72.21 ± 0.32%).The OC values' increase in the incubated soils was also documented in former studies [1,51] when they incubated agricultural waste-derived biochar in contaminated soil.

• Electrical conductivity (EC)
The control soil sample had an EC value of 119,1 ± 1,3 μS cm −1 , while all the incubated samples had significantly higher EC values than CS (p < 0.05).The more the amended ratios of amendments increased, the higher the EC values of amended samples were.This finding can be explained due to the high value of biochar and apatite, especially PPB300 and PPB500, which were 2670 ± 2.5 μS cm −1 and 4620 ± 3.5 μS cm −1 , respectively.The soil sample PPB5:10 had the highest EC value with 571,9 ± 1,5 μS cm −1 when the incubated ratio of PPB500 was 10%.This soil sample had an EC value much higher than that of PPB3:10 when the amended proportion was 10% as well.It was due to the high EC value of PPB500 (4620 ± 3.5 μS cm −1 ) compared to that of PPB300 (2670 ± 2.5 μS cm −1 ).The previous studies [19,20] also reported that the biochar produced at the higher temperature had a higher EC value than that of the biochar produced at the lower temperature.The present study's result was in accordance with previous studies, which informed that the incubated soil's EC values increased after being amended with agricultural waste-derived biochar [1,52].
• pH pH is an essential factor controlling the chemical fraction of heavy metals in soil [53], and it frequently has a strong correlation with the heavy metals' exchangeable fraction in the soil [19].The control soil sample (CS) had a pH value of 6,69 ± 0,01 and this value was significantly lower than that of incubated soil samples (p < 0.05).The pH values of the amended soil samples rose when the biochar's application rate increased.The highest pH value of the incubated soil was PPB5:10, with a pH value of 7,18 ± 0,01 when the biochar application rate was 10%.In general, the soil samples incubated with PPB500 had slightly higher pH values compared to those of soil samples incubated with PPB300.This finding might be attributed to a higher pH value of PPB500 (11.65 ± 0.01)  in comparison with PPB300 (11.56 ± 0.01).The elevated pH levels of the treated soil samples had a significant role in remediating heavy metals by immobilizing them via precipitation reactions [7,20].
To sum up, the properties of the treated soils dramatically altered when compared to the control soil after the 30-day incubation with amendments (PPB300 and PPB500, and AP) (p < 0.05).The significant increase in incubated soils' pH values may be crucial for immobilizing heavy metals through precipitate reactions.In addition, the rise of pH, OC, and EC values of incubated soil samples might assist in the remediation of heavy metals in contaminated soil.

• Exchangeable fraction (F1)
The results in table S4 illustrated that the concentration of lead in the exchangeable fraction (F1-Pb) in control soil (CS) was 440,6 ± 17,2 mg kg −1 , while the lead content in incubated soils increased significantly compared to that of CS (p < 0,05), apart from PPB3:3 (p > 0.05).The more biochar application rates increased, the less exchange fraction of Pb was.The most significant decrease of F1-Pb was in sample PPB3:10 when the contaminated soil was incubated with 10% of biochar PPB300.In this case, the F1-Pb in the PPB3:10 sample decreased by approximately 64% compared to CS.Overall, the sample incubated with PPB500 caused the reduction of F1-Pb better than PPB300 at the application rate of 3% and 5%, but at 10%, the biochar PPB500 had a slightly better effect on the diminish of F1-Pb in the soil (158.4 ± 6.1 and 179.7 ± 11.9 mg kg −1 ).Besides, the soil samples incubated with biochar/apatite at 3:3% and 5:5% had better results in diminishing F1-Pb than those with only 3% or 5% of biochar PBB300.However, when applied with PPB500, the rate of 5:5% (PPB5A5) had a better effect on reducing F1-Pb than only 5% (PPB5:5) biochar, while the rate of 3:3% (PPB5A3) had a worse impact on reducing F1-Pb than 3% biochar (PPB5:3).The decrease in the exchangeable fraction of lead in contaminated soil when the biochar application rates increases was reported in previous studies [1,7,18].

• Carbonate fraction (F2)
The control soil's carbonate fraction lead (F2-Pb) concentration was 1921.9 ± 75.4 mg kg −1 .All the incubated soil had a value of carbonate fraction content similar to that of control soil (CS) (p > 0.05), except PPB3:10, which had the F2-Pb slightly higher than that of CS (p < 0.05).Previous studies reported that contaminated soil samples incubated with biochar produced from rice straw [18] and peanut shell [19] had F2-Pb higher than that of the control soil.
• Fe/Mn-Oxide fraction (F3) The lead concentration in the control soil (CS) was 488.2 ± 12.9 mg kg −1 .Overall, the F3-Pb values of the incubated soils were less than that of CS (p < 0.05), aside from the PPB3A3 and PPB5:3 samples, which did not differ significantly from CS in the F3-Pb value (p > 0.05).Previous studies informed diverse results in the change of this fraction after the incubation compared to the control soil.Dang et al (2018) informed that the oxide-bound proportion had not significantly changed when applied with biochar derived from rice straw [18], while Awad et al (2021) documented that incubating with biochar made from garden waste resulted in no appreciable change [54] when paulownia biochar (PB) and bamboo biochar [1] applied at the rate of 4% and 6%.In addition, when they were amended at the rate of 2%, the F3-Pb was marginally lower than it was in the control soil [54].
• Organic carbon bound fraction (F4) The F4-Pb value of CS was 55.5 ± 1.9 mg kg −1 , whereas this figure of incubated soils increased significantly compared to that of CS (p < 0.05).The increase of F4-Pb in the incubated soil correlated with the increase in the biochar application rate.The highest increase percentage of F4-Pb was in PPB5:10 (74%) when the biochar application rate was 10% of PPB500.This outcome was consistent with earlier research [1,7,19], which reported that biochar incubation raised the F4-Pb in contaminated soil substantially as compared to control soil.

• Residual fraction (F5)
The lead concentration within the residual fraction of the control soil was quantified at 549.2 ± 13.3 mg kg −1 .Following the incubation with biochar and apatite, no statistically significant alteration in F5-bound lead (F5-Pb) levels was observed in samples PPB3:3, PPB3:5, PPB3A3, and PPB5:3 compared to the control soil (CS) (p > 0.05); however, there was a significant increase in F5-Pb in the incubated soils when the application rates were 5% and 10%.Previous studies reported different changes in this fraction after incubation with biochar.Some showed no significant change [7,18], while others showed there was a decrease of F5-Pb in the incubated soil compared to CS [1], and there was a significant increase in this fraction after the incubation [19].

Zn speciation
The chemical fractions of zinc are illustrated in figure 5(B).The result showed various changes in fractions after incubating with biochar PPB300, PPB500 and apatite.

• Exchangeable fraction (F1)
In the control soil (CS), the exchangeable fraction of zinc (F1-Zn) was measured at 320.6 ± 16.0 mg kg −1 .Subsequently, in the biochar-treated soils, this parameter exhibited a significant decrease compared to the control soil (p < 0.05), except for the PPB3A3 sample, where no significant difference in F1-Zn concentration was observed relative to the control soil.(p > 0.05).Overall, the F1-Zn decreased with the increase of amendments application rates, and the most substantial reduction of this fraction (about 58%) happened in sample PPB3:10 (133.2 ± 16.0 mg kg −1 ) and PPB5:10 (148.5 ± 13.9 mg kg −1) when the soil incubated with 10% of PPB300.In general, the F1-Zn in the incubated soil had a slightly better impact on the diminish of F1-Zn in the contaminated soil in some cases when incubated with PPB500 compared to that of PPB300 (PPB3:5 and PPB5:5; PPB3A3 and PPB5A3; PPB3A5 and PPB5A5).In addition, soil samples incubated with only biochar had less effect on the reduction of PPB3A5 in comparison with soil samples incubated with PPB300.The significant difference between biochar and biochar/apatite were seen in PPB3:5 and PPB3A5, PPB5:3 and PPB5A3; PPB5:5 and PPB5A5 (p < 0.05), but there was no significant difference between PPB3:3 and PPB3A3 (p > 0.05).The significant decrease of F1-Zn after the incubation with amendments was also reported in previous studies [7,18].
• Oxide bound fraction (F3) The Fe/Mn oxide-bound fraction of Zn in the control soil (CS) was 695.1 ± 41.5 mg kg −1 and this figure almost had no significant change in the incubated soil (p > 0.05).This finding did not agree with the reported results of previous studies, which showed no significant change in the biochar application rate of 3%, but at a higher application ratio of 10%, this fraction in the incubated soil was significantly less than that of [19].while previous studies reported a significant reduction of F3-Zn after the incubation [1,22] or there was an increase in this fraction [10].The different results were attributed to the difference in biochar type and the studied soil properties.
• Organic compound fraction (F4) The control soil (CS) had a zinc content of 42.7 1.2 mg kg −1 in the organic fraction (F4-Zn), and after 30 days of incubation, this value dramatically rose in all of the treated soils.The content of zinc in the organic substance fraction (F4-Zn) of the control soil (CS) was 42.7 ± 1.2 mg kg −1, and this figure in all incubated soils increased significantly after a 30-day incubation (p < 0.05), apart from in sample PPB3:3 when the soil was amended with 3% of PPB300 biochar.The highest values of F4-Pb in incubated soils were in samples PPB3:10, PPB5:10 and PPB5A5.The increase of F4-Zn in the present study agreed with previous studies [18,20,56], which have reported significant changes in F4-Zn levels following soil incubation with biochar.

• Residual fraction (F5)
The control soil (CS) had a zinc content of 569.8 ± 25.3 mg kg −1 in the residual fraction (F5-Zn).Almost the incubated soil had an F5-Zn value more significant than that of CS (p < 0.05), except for samples PPB3:3, PPB3A3, and PPB5:3.These samples had no notable divergence in F5-Zn compared to CS.Previous studies reported that adding biochar to contaminated soil had almost no noticeable effect on the incubated soils [1,7,18].The contrast findings could be assigned to the disparity in biochar types and the application ratio.For example, the studies of Awad et al (2021) [7], the studies investigated 2, 4 and 6% of biochar, and the study of Dang et al (2018) investigated the application rate of 1, 3 ad 5%, while the present study studied the higher ratio of 5 and 10% [18].
Overall, after one month of incubation with biochar produced from pomelo peels (PPB300 and PPB500) and apatite, the soil was considerably different from the control soil in terms of the chemical fractions of Pb and Zn.The most obvious difference between incubated soil and control soil was seen in the exchangeable fraction of Pb and Zn.The decrease in F1-Pb and F1-Zn reached levels of 64% and 58% respectively, indicative of substantial reductions in both metals.This demonstrated that biochar and apatite, particularly when applied at rates of 5% and 10%, can immobilize Pb and Zn in soil.Additionally, the amendment of biochar (PPB300 and PPB500) and apatite increased the proportions of the stable fractions F4 and F5 for lead and zinc, proving that biochar and apatite assisted in the transformation of heavy metal from the mobilizing fraction into immobilizing fractions.Compared to the exchangeable fraction, which is very labile, these stable fractions, such as F4 and F5, had a less detrimental effect on the environment since they are stable under natural conditions.

Mechanism for rendering heavy metals immobile
It has been documented in numerous studies that biochar could remediate heavy metals from contaminated soil through a variety of mechanisms, including chemical and physical adsorption [57,58], electrostatic attraction [53], the formation of complexes via interactions with active groups [2,59], cation exchange processes [2], and precipitation events mediated by phosphate, carbonate ions, or hydroxyl ions within an alkaline environment [60,61].
In this study, FT-IR and EDS results of biochar before and after incubation in soil were analyzed (figures S1 and S2, see SM).Besides, the FT-IR results of PBB300 and PPB500 before and after the incubation are demonstrated in figures 5(A), (B).The FT-IR of PPB300 before and after 30-day incubation illustrated that there were significant changes in the intensity of the primary peaks of PPB300 before and after amendment in the contaminated soil.Especially, it had more new peaks in the area around 465 cm −1 , indicating the presence of PO 4  3-[58].The shift and the decrease of intensity of peaks at 3441, 2927, 1604, 1389, and 1039 cm −1 show that the possibility of Pb 2+ and Zn 2+ combined with OH-or phenolic OH via complexation reaction and with PO 4 3and OH -through the precipitation reaction confirming that the biochar absorbed the Pb 2+ and Zn 2+ [58,62].In the meantime, the FT-IR of PPB500 showed that there were significant changes and shifts of peaks at 2922, 1973, 871, 663 and around 500 cm −1 as well.The peak at 871 cm −1 was not present in the IR spectra of biochar PPB500 before incubation but appeared in the IR spectra of biochar PPB500 after incubation.This peak might be assigned to the presence of CO 3  2-[58].Additionally, the new presence of a peak at 663 cm −1 in the IR spectra of PPB500 after incubation was attributed to the precipitation form of Pb 2+ with PO 4  3-[58, 63].To sum up, the IR spectra of biochar before and after incubation showed that the shift and the change of peak intensity demonstrated the adsorption of Pb and Zn by biochar via exchange or complexation reactions, and the presence of the peak appeared only in the IR spectra of biochar after incubation indicated the precipitation form of heavy metals with carbonate or phosphate.EDS results showed that after being incubated in the contaminated soil (figure S2, see SM).Before the incubation, the PPB300 had no Pb and Zn on its surface (figure S2(A)); however, after the incubation, it had more elements, such as Pb and Zn presented on its surface (figure S2(B)).
In addition, when PPB300 was mixed with apatite in the contaminated soil, Pb and Zn also appeared on its surface (figure S2(C)).Therefore, the EDS results approved that Pb and Zn accumulated on the surface of biochar PPB300 after the 30-day incubation.The changes of EDS and FT_IR results of biochar before and after incubation in the studied soil could be clarified that when biochar and apatite were incubated in the contaminated soil, they were moistured and broken into small pieces because of redox reactions during the incubated time.As a result, soluble ions (Ca 2+ , Mg 2+ , Al 3+ , Na + , Cl -, SO ) and organic substances would be released from amendments [18,20].Consequently, the pH and EC of the soils surrounding these tiny particles of amendments rose, and they might combine with the heavy metals via diverse processes such as ion exchange, complexation, and precipitation reactions [53,64].According to previous studies, the key factor causing the significant decrease in the exchangeable fraction of heavy metals was the rise in pH and EC of the soil solution when incubated with the high pH amendments [65,66].The pH and EC of the soil solution increased as the application rates increased.In this study, PPB300 had a higher content of organic functional groups and a larger surface area than PPB500.However, there were minimal differences in lead and zinc immobilizing results in the incubated soils.This finding might be speculated by the high pH value of both PPB300 (11.56 ± 0.01) and PPB500 (11.65 ± 0.01), which facilitated the hydrolysis precipitation reaction that occurred.
In addition, the FTIR and EDS data revealed that apatite was an ore that is abundant in phosphorus and other inorganic elements including Mg, Si, Ca, K, N, F, and Na, which might facilitate the remediation of Pb 2+ by precipitating with phosphate to form Pb 3 (CO 3 ) 2 (OH) 2 or Pb 5 (PO 4 ) 3 OH [66], Pb 5 (PO 4 ) 3 X( X: Cl, F, Br, I) [58].Numerous studies informed that the main mechanism of the immobilization of lead when incubating contaminated soil with apatite was the formation of pyromorphite Pb 5 (PO 4 ) 3 X (X = F, Cl, Br, OH) [67][68][69][70].Pyromorphite Pb 5 (PO 4 ) 3 X are the most stable ambient Pb compounds, developed under a wide range of pH and EC natural circumstances, increasing the amount of Pb in the residual or insoluble fraction [69,71,72].The fact that these processes made heavy metals in the soil less mobile and converted them into more stable fractions suggests that biochar and apatite could be utilized to remediate heavy metals like lead and zinc in the polluted soil [20].The conversion of mobilizing fractions of Pb and Zn into stable fractions could be attributed to the formation of the organo-mineral layer.The development of an organo-mineral layer, which consists of microagglomerates comprised of inorganic and mineral nanoparticles that are held together by organic substances [73], on the surface of the biochar after one-month incubation can be used to explain the formation of stable fractions [18].When the biochar is broken up, these micro-agglomerates create micro aggregates that might be integrated into the stable micro-aggregate component of the soil [18,74].
To sum up, we concluded by speculating that a combination of mechanisms, including adsorption, complexation reactions, exchange, and precipitation, made up the primary mechanism of the immobilization of lead and zinc in the studied soil.
3.6.Relationships between exchangeable lead and zinc fractions and key soil parameters (pH, organic carbon, electrical conductivity) following a 30-day incubation The correlation of the soil's key factors, such as pH, EC, and OC, with the exchangeable fraction of lead (F1-Pb) and zinc (F1-Zn) was investigated using Spearman correlation.The results are displayed in figures 5(A) and (B). Figure 5(A) demonstrates that F1-Pb exhibited a strongly negative connection with OC (r = −0.78)and EC (r = −0.71)and pH (r = −0.80).Similar results were seen for the exchangeable fraction of Zn (F1-Zn), which showed very strong negative correlations with pH (r = −0.84),OC (r = −0.82),and EC (r = −0.84).While pH had a very strong positive correlation with both OC (r = +0.86)and EC (r = +0.84).In addition, OC and EC had a strongly positive association (r = +0.86)(figures 5(A), (B)).These findings were consistent with previous studies, which informed that the lead and zinc exchangeable fractions had a strong negative correlation with the pH and OC of soil samples [19,20].Because of the high pH, EC, and OC values of amendments, particularly biochar, these factors have strong positive correlations.Compared to the control soil, PPB300 and PPB500 were much richer in OC and had higher values of EC and pH.The pH, EC, and OC values increased with more amendments.Thus, the incubated soils showed a high positive correlation between the pH, EC, and OC.
In general, elevated application rates of the amendments correlated with increases in pH, organic carbon (OC), and electrical conductivity (EC) values, while concurrently reducing the exchangeable fractions of heavy metals.Consequently, these parameters may play a pivotal role in the immobilization of heavy metals in contaminated soil, as indicated by previous studies [19,20].

Conclusions
(i) The physicochemical attributes of biochar and apatite, encompassing parameters such as pH, concentrations of lead (Pb) and zinc (Zn), electrical conductivity (EC), and surface characteristics, underwent comprehensive analysis.
(ii) The amendments of biochar and biochar/apatite led to an augmentation in the proportions of F4 and F5 for lead and zinc, resulting in their conversion into more stable fractions under ambient conditions.However, the exact mechanisms underlying these transformations remain unresolved.Nonetheless, they are hypothesized to involve exchange, precipitation, and complexation reactions facilitated by the functional groups and minerals present in PPB300 and AP.Additionally, physical adsorption onto the extensive, porous surface of PPB300 and the hydroxide precipitation reaction attributable to the high pH values of materials such as PPB300 and PPB500 may contribute to these processes.The proportion of Pb and Zn's exchangeable fraction in the incubated soil could be altered when the contaminated soil was applied with biochar and apatite at rates of 5%, 10%, and 5:5%, however, the 3% ratio had little to no impact on this fraction.PPB300 and PPB500 had almost the same effects on the alteration of the Pb and Zn's exchangeable fraction when applied in the contaminated soil at the same ratios of 3, 5, and 10%, even though they had different properties such as surface area, the quantity of active functional groups, but the same high pH values.This finding suggests the concurrent operation of multiple mechanisms, including cation exchange, physical adsorption, precipitation, and complexation, within contaminated soil when subjected to incubation with PPB300, PPB500, and their combination with PPB300/AP and PPB500/AP.
(iii) After one month of incubation, the amendments had favorable effects on the pH, OC, and EC of the incubated soils.When compared to the control soil, these values of the treated soil samples increased dramatically.The pH, OC, and EC values increased with the amendment application rates, facilitating the application of pomelo peel-derived biochar and the mixture of Biochar/apatite in immobilizing Pb and Zn in the studied soil.
The results of this study underscore the potential of PPB300, PPB500, and apatite as viable materials for immobilizing heavy metals in multi-contaminated soil, particularly when applied at appropriate rates.

3. 4 .
Changes in chemical fractions of heavy metals after 30-day incubation with amendments 3.4.1.Pb speciation The lead's chemical fractions in the treated soils and control soil (CS) are illustrated in tables S5, S6 (see SM) and figure 4.

Figure 4 .
Figure 4.Chemical partitioning of lead (A) and Zinc (B) in soil specimens.

Table 1 .
Designation of incubation experiments.

Table 2 .
Pb and Zn's exchangeable fraction, OC, EC and pH values after 30-day incubation with amendments.
As per the t-test results, identical letters denote a lack of statistically significant difference between the two groups (p < 0.05).