Aça í Seed Biochar-Based Phosphate Fertilizers for Improving Soil Fertility and Mitigating Arsenic-Related Impacts from Gold Mining Tailings: Synthesis, Characterization, and Lettuce Growth Assessment

: Biochar represents a promising alternative for enhancing substrates and remediating contaminants in mining areas. Given that arsenic (As) and phosphorus (P) share similar chemical forms, the combination of biochar and P fertilizers may reduce As uptake, thereby mitigating As-related impacts. This study aimed to evaluate the potential of biochar-based P fertilizers in improving soil fertility and mitigating human health risks from gold mining tailings in the eastern Brazilian Amazon. Biochar from aça í palm ( Euterpe oleracea Mart.) seeds was produced through enrichment with single and triple superphosphate at a ratio of 1:4, at 400 ◦ C, and applied to mining tailings at 0.5%, 1%, and 2%. After one year of incubation, lettuce plants were grown for 70 days. Biochar reduced As absorption by lettuce and improved biomass and nutrient accumulation, resulting in improved vegetation indices. Biochar was effective in reducing non-carcinogenic As risks via ingestion of soil and plants to acceptable levels. Regression equations explained the As absorption behavior as affected by the biochar and the importance of biochar-related nutrients in reducing As stress. This study demonstrates the potential of P-enriched biochar as an amendment for As-contaminated soils, reducing As absorption, increasing P availability, and improving plant growth.


Introduction
Artisanal mining may cause several negative impacts on exploration sites, especially with the generation of large amounts of residues that are inappropriately deposited in the ecosystem [1,2].These residues commonly have low fertility and release potentially toxic elements (PTEs) that cause several adverse effects.Arsenic (As) is considered the most dangerous PTE for human health [3], with various damages already evidenced, including skin diseases, diabetes, cardiovascular diseases, cognitive problems, liver and kidney dysfunctions, reproductive complications, and cancer [4].Therefore, it is essential to reduce the entry of As into the human body, including reducing the levels absorbed by plants normally consumed by humans.
Concentrations of As in edible parts of plants depend on the mobility and bioavailability of this element in the soil, in addition to the potential for accumulation and translocation [5].It is an element chemically similar to phosphorus (P) and acts as a substitute in all biochemical and physiological processes related to P in plants [6], which contributes to increased absorption of As by plants and represents a threat to the ecosystem and hu-man health [5].Therefore, the development and application of techniques to reduce the bioaccumulation of As in plant tissue is essential for ensuring food security [7].
Ecological and human health risks have been associated with As in gold (Au) mining areas in the eastern Amazon [8,9].Remediation techniques must be designed not only to reduce the bioavailability, bioaccessibility, and bioaccumulation of As, but also to improve the quality of residues deposited in mining areas, which generally have low fertility, limited biological activity, and adverse physical conditions [10].In this context, biochar is a material that may be easily produced via pyrolysis in muffle furnaces under controlled time and temperature conditions, representing a promising alternative to improve soil fertility and promote As immobilization, due to its highly porous structure, large surface area, and diverse functional groups [11][12][13].In addition, the association of biochar with P fertilizers can improve As remediation capacity [14,15].
Several plant species generate biomass that has been used for the production of biochar, aiming to improve soil quality and remediate PTEs, such as sugarcane, rice, and wheat, which have already shown potential for the remediation of As [16], cadmium (Cd) [17], and lead (Pb) [18], respectively.Among palm tree species, the date palm, oil palm, and coconut deserve special mention for this purpose [19][20][21][22].In the Amazon, the açaí palm (Euterpe oleracea Mart.) production chain generates significant quantities of seeds that are randomly deposited in large urban centers, which suggests the need for studies to reuse these residues [23].Previous studies have indicated biochar from açaí seeds as a valuable tool for improving fertility and remediating contaminants in substrates [24][25][26][27].Mainly, this biochar was suggested as an easily obtainable product that is promising for the remediation of mining tailings rich in As [28].
The conversion of açaí seeds into biochar and the enrichment with P fertilizers may constitute a viable option for treating As-impacted areas, favoring plant growth, and mitigating the impacts of this metalloid.Thus, the objectives of this study were to (i) evaluate the effects of açaí biochar and P fertilizers on the chemical attributes, with emphasis on P availability; (ii) determine the bioavailability and bioaccessibility of As; and (iii) assess the potential risk associated with ingesting lettuce (as an indicator plant) in Au mining residues in the eastern Amazon.This study is a pioneer in the combination of biochar and P fertilizers to improve substrate fertility and mitigate As risks in Au mining areas in the Brazilian Amazon, with results that could directly contribute to the protection of the environment and health of the affected population in the region.

Sampling of Mining Tailings
Mining tailings were collected from underground Au mining areas in the municipality of Cachoeira do Piriá (01 • 45 ′ 35 ′′ S, 46 • 32 ′ 42 ′′ W).The municipality belongs to the Guamá microregion, located in the northeastern mesoregion of Pará, in the eastern Amazon, with a territorial extension of approximately 2419 km 2 and climate classified as tropical monsoon, with an annual mean temperature of 26 • C and total annual precipitation of 2300 mm [8].These tailings were collected from the superficial layer of piles (0.0-0.2 m), air-dried, homogenized, and analyzed in terms of mineralogy and total concentrations of aluminum (Al), As, calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), P, sulfur (S), and zinc (Zn), according to the methodology described by Ref. [8].

Production and Characterization of Biochars
Açaí seeds were collected at urban fairs in the metropolitan region of Belém, state of Pará, and dried for 24 h in an oven at 60 • C. Afterward, the açaí seeds were crushed and mixed with single superphosphate (SSP) and triple superphosphate (TSP) at a ratio of 4:1 (biomass:phosphate) [29].Such mixtures were placed in closed porcelain crucibles (to ensure low oxygenation) and subjected to pyrolysis at 400 • C in a muffle-type furnace (model Q318M24, Quimis Instruments, Diadema, Brazil) with a heating rate of 3.3 • C min −1 [27,28].The final pyrolysis temperature was maintained for one hour (slow pyrolysis).The resulting biochars were crushed, sieved, and identified as BA (biochar without enrichment), BS (biochar enriched with SSP), and BT (biochar enriched with TSP).

Experiment Conduction
The experiment was conducted in a greenhouse (randomized block experimental design), with ten treatments and five replicates, totaling 50 experimental units (1 kg each).The treatments were represented by the application of three different rates (0.5%, 1.0%, and 2%) of the three produced biochars (BA, BS, and BT) to the mining tailings (T), in addition to T applied alone (Table 1).The treatments were incubated for one year to ensure better interaction of biochars and residues, with moisture maintained at 70% of the total pore volume.The cultivated plant was lettuce due to its efficiency in the uptake and accumulation of contaminants and for being widely used in toxicity tests and evaluations of risks to human health [32].The experiment was conducted for 70 days, and then several substrate and plant analyses were carried out.

Fertility Analyses
After the experiment, fertility analyses were carried out according to Ref. [33].Thus, pH was obtained in water (1:2.5)using potentiometer (model HI9811-51, Hanna Instruments, Barueri, Brazil); available P and potassium (K + ) concentrations were extracted using Mehlich 1 solution, which is composed of 0.05 M HCl and 0.0125 M sulfuric acid (H 2 SO 4 ), and determined by colorimetry and flame photometry, respectively.Calcium (Ca 2+ ), magnesium (Mg 2+ ), and aluminum (Al 3+ ) concentrations were extracted using 1 M potassium chloride (KCl) and determined by titration.Potential acidity (H + Al) was extracted with 0.5 M calcium acetate (Ca(C 2 H 3 O 2 ) 2 ) and quantified by titration with 0.1 M sodium hydroxide (NaOH) solution in the presence of 1% phenolphthalein (C 20 H 14 O 4 ) as an indicator.Cation exchange capacity (CEC) and anion exchange capacity (AEC) were found using the compulsive exchange method, with ammonium nitrate (NH 4 NO 3 ) solu-tion and subsequent washing with KCl, followed by quantification via colorimetry [30,31].Organic carbon (OC) and inorganic carbon (IC) were estimated by loss of mass in a muffle furnace (model Q318M24, Quimis, Diadema, Brazil) at 450 • C and 950 • C, respectively, and total carbon (TC) was obtained through the sum of OC and IC [34].

Inorganic P Fractionation
Inorganic P fractionation was carried out according to the methodology in Ref. [35], evaluating the following P fractions: soluble P (P-F1) extracted with ultrapure water, labile P (P-F2) extracted with 0.5 M sodium bicarbonate (NaHCO 3 ), adsorbed P (P-F3) extracted with 0.1 M NaOH, and mineral-associated P (P-F4) extracted with 1 M HCl.The remaining solid material was digested using the EPA 3051A method and considered as residual P. All P fractions were quantified via colorimetry [36].

Nutrients and Arsenic in Plants
Plant material was collected and separated (roots and leaves), washed, and dried in an oven at 50 • C until constant weight was achieved for dry mass evaluation.Nutrient and As content in roots and leaves were obtained by digestion using nitric acid (HNO 3 ) and hydrogen peroxide (H 2 O 2 ) [9] in a microwave oven (model Mars Xpress 6, CEM Corporation, Matthews, NC, USA).All analyses were carried out in triplicate and analytical quality was assessed using the reference material ERM-CD281 (rye grass) and blank samples.Concentrations were obtained using flame atomic absorption spectrometry (FAAS) (model iCE3000, Thermo Fisher Scientific, Cambridge, UK) with a hydride generator for As quantification (model VP100, Thermo Fisher Scientific, Cambridge, UK).
The contents of As and biomass values were used to calculate the bioconcentration factor (BCF), translocation factor (TRF), and tolerance index (TI), following Equations ( 1)-( 3), respectively.

BCF =
As in plant mg kg −1   As in substrate mg kg −1 (1)

TRF = (
As in leaves mg kg −1   As in roots mg kg −1

Arsenic Fractionation and Bioaccessibility
The fractionation of As was performed using the method proposed in Ref. [37]: soluble As (As-F1) was extracted with ultrapure water; adsorbed As (As-F2) was extracted with ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ); As bound to amorphous minerals (As-F3) was extracted with ammonium oxalate (NH 4 ) 2 C 2 O 4 ); As bound to crystalline minerals (As-F4) was extracted with (NH 4 ) 2 C 2 O 4 (80 • C); residual As (As-F5) was extracted with KCl + HCl + HNO 3 solution.The concentrations of As in each fraction were quantified using FAAS (model series iCE 3000, Thermo Fisher Scientific, Cambridge, UK) with a hydride generator (model VP100, Thermo Fisher Scientific, Cambridge, UK).
Oral bioaccessibility was determined with the simple bioaccessibility extraction test (SBET), simulating human gastrointestinal absorption using glycine (C 2 H 5 NO 2 ) solution (0.4 M, pH 1.5), followed by centrifugation, filtration, and quantification using FAAS (model series iCE 3000, Thermo Fisher Scientific, Cambridge, UK) with a hydride generator (model VP100, Thermo Fisher Scientific, Cambridge, UK).The bioaccessible percentage was determined using Equation ( 4): where BFAs is the bioaccessible fraction of As (%), BAs is the bioaccessible concentration of As (mg kg −1 ), and TAs is the total concentration of As (mg kg −1 ).

Evaluation of Risk to Human Health
The potential adverse effects of As on human health were evaluated using the risk assessment model developed in Ref. [38].The hazard quotient was calculated for ingestion of contaminated soil (HQ s ) (Equation ( 5)) and plant (HQ p ) (Equation ( 6)) from contaminated sites.The exposure risk (HI) was obtained from the sum of HQ s and HQ p for children and adults [39].
where C s and C p represent the total concentrations of As in soil and plant (mg kg −1 ); BFAs is the bioaccessible fraction of As (%); IR s is the soil ingestion rate (100 mg d −1 for adults and 200 mg d −1 for children) [38] and IR p is the plant ingestion rate-200 mg for adults and 100 mg for children [38]; EF is the exposure frequency, 279 d [40]; ED is the exposure duration-24 years for adults and 4 years for children [40]; BW is the body weight-70 kg for adults and 16 kg for children [40]; AT is the average time without carcinogenic effects (ED × 365 d); CF is the conversion factor-10 −6 kg mg −1 [38]; Rfd is the reference dose-0.0003for As (mg kg d −1 ) [38]; and SF is the slope factor-1.51[39].

Statistical Analysis
The results were submitted to Shapiro-Wilk's normality test (p < 0.05).Given normality, analysis of variance (ANOVA) was performed and means were compared using Tukey's test (p < 0.05).Multiple linear regression analysis was carried out to generate a robust model for estimating As accumulation by the plant.The variables for modeling were selected through the variance inflation factor (VIF), and the equation of multiple linear regression was obtained with the Stepwise method, eliminating variables without statistical significance for the model and with better Akaike Information Criterion (AIC) values.The precision and accuracy of the selected model were evaluated through the significance of the model (p-value), adjusted determination coefficient (adjusted R 2 ), and normalized root-mean-square error [41].All analyses were performed using R, version 4.4.1 [42].

Characterization of Tailings
The chemical elements presented concentrations in the order of Fe > Al > As > Ca > Mg > Mn > K > P > Cu > S > Zn > Co in the mining tailings (Table 2).Among the PTEs determined, Cu, Co, and mainly As had concentrations above the prevention values (PV) established by Brazilian legislation [43], suggesting potential risks to soil functions.The concentrations of Cu and Co were also above investigation values (IV) for agricultural areas.Arsenic is the element in the most worrying scenario, with concentrations (3000 mg kg −1 ) above IV defined by national legislation for agricultural, residential, and industrial areas [43], indicating possible risks to public health.

Biochar Characterization
Biochar exhibited different characteristics with the treatments (Table 3).The addition of fertilizers changed the amount of charges on the biochar and decreased the pH value.The excessive anionic charge on the biochar surface may be due to the presence of Al and Fe oxide and hydroxides [44] since these elements are commonly found in the phosphate rocks that originate in SSP and TSP [45].In addition, the use of acid in P fertilizer production may have contributed to the acidification of the biochar.At the same time, the higher contents of chemical elements in BT and BS may be due to the greater richness of elements in P fertilizers, mainly Ca and P in SSP (21% P and 16% Ca) and TSP (46% P and 12% Ca).The enrichment of biochar with fertilizers may add nutritional value to the carbonaceous material [46].The increase in ash content in P-rich biochars is due to the possible increase in inorganic compounds that tend to accumulate in the form of oxides, carbonates, hydroxides, and phosphates, which are not broken down and volatilized in the pyrolysis process [27].Similar results (higher ash content) were found by Ref. [47], who studied the enrichment of bamboo biochar with P and K under different temperatures.

Effect of Biochars on Mining Tailings
The addition of biochar altered all chemical properties of the mining tailings (p < 0.05) (Table 4).The pH value became more alkaline with the addition of BA and BS, while the highest dose of BT acidified the residue.Biochar normally has an alkaline pH due to the formation of ash during the pyrolysis of organic matter, which is rich in carbonates and hydroxides and tends to solubilize and alkalize the environment [48,49].At the same time, the exogenous addition of ions can protect organic compounds that tend to decompose during maturation, releasing compounds that acidify the environment [50,51].On the other hand, the higher amount of P in BT is probably the cause of the higher acidity due to the more acidic characteristics of TSP [29,52].Potential acidity (H + Al) increased with the addition of BT and BS (Table 4).In addition, it was proportional to the total Al content of the biochar (BT > BS > BA) and the exchangeable Al content (Table 4).Under acidic conditions, Al availability is higher and leads to increased Al uptake by plants, with negative effects in the root environment [49,53].In a study carried out by Ref. [54], different results were observed, with increased liming potential in biochar enriched with potassium phosphate associated with sources of Ca and Mg.
Nutrients (Ca, Mg, K, and P) varied with increasing levels and were proportional to the ash content of the biochar (Table 4).The addition of BT and BS to biochar increased the availability of P and Ca in mining tailings, respectively.Nutrient-rich biomass tends to produce and supply nutrient-rich biochar [52].The effectiveness of nutrients depends directly on the properties of biomass and the pyrolysis temperature [55].In addition, the amount of ash produced is fundamental because it is a direct nutrient source to the soil [56,57].
Biochar increased the CEC/AEC charge and OC content of the tailings as a function of the amount applied (Table 4).Carbon not mineralized in the pyrolysis process is responsible for the increase in soil OC [58].Organic residues that are still present after biochar production tend to solubilize and increase soil carbon and nutrients [24,25,59].The increase in OC content increases the number of negative charges since it is rich in organic functional groups that, when oxidized, increase the number of active sites available for the adsorption of cations and anions [60,61].

Inorganic P and As Fractionation
The addition of biochars altered the P fractions in the mining tailings (Table 5).There was a decrease or stabilization in P levels after the addition of BA, while treatments with P-enriched biochars presented elevated P levels in all fractions.The addition of P biochar was effective in increasing the availability of P in a form easily absorbed by plants (Table 5).The soluble form of P is highly mobile and absorbable by plant roots and has a high potential for adsorption by soil colloids/minerals [62,63].The increase in adsorption/mineral fraction with the addition of biochar is an alternative to elevated P pools and is very important for sustained release [64], supplying the P needs under stress conditions.
The addition of biochar altered the As fractions in the mining tailings (Figure 1).All biochars increased As concentrations in fraction F1 (soluble) and decreased in fraction F2 (adsorbed).These more mobile fractions were inversely correlated with each other and with all fractions of P, OC, CEC, and pH (Table S1).Due to chemical and structural similarities, the form and availability of P affect the solubility of As [65], which favors the greater availability of As in conditions of excess P [66].Changes in OC, CEC, and pH have a direct effect on the mobilization of As in the soil, and the electrostatic repulsion caused by the increase in the effective charge of the organic compounds (from the addition of biochar) promotes the solubilization of As [67,68].Similar results were observed in Ref. [69], who verified greater mobility of As in the soil with the application of sugarcane biochar rich in P, originating from pyrolysis at a high temperature.
The addition of P-enriched biochar decreased the F5 fraction and increased the As content of the F3 and F4 fractions in residues (Figure 1).Both fractions were related to each other and to F5, with F3 related to P-F2, P-F3, IC, and AEC, and F4 related to P-F1 (Table S1).The stabilization of As in the F3 and F4 fractions is due to the addition of biochar rich in Ca, Al, and Fe (Table 3) in the inorganic carbon fraction of biochar, which helps form insoluble/stable As complexes [70,71].Solubilization of As from the most stable fraction occurred due to excess P addition, which promoted ion exchange or solubilization of As from minerals in the residues [65], consequently increasing environmental risks arising from possible leaching of more mobile fractions in the soil.The OC content is also important in the solubilization of As due to the proportional increase in low molecular weight organic compounds [72], which play an important role in the reductive dissolution of As-containing minerals present in the residual fraction [73].

Arsenic-Plant Interaction
Biochar additions showed different results, with BA and BS increasing biomass and nutrient contents in leaves and roots (Table 6).The nutrients (Ca, K, and P) accumulated the most in the roots and leaves, as they are found in greater levels in the biochars used (Table 3).The increase of these nutrients in the plant helps with development in environments with high As levels.The TF and BCF indices decreased with the addition of biochar (except for the treatment with BT), indicating that the greater translocation observed in the treatment without biochar was reflected in the greater amount of As absorbed, resulting in a lower increase in plant biomass (Table 7).TI values greater than 1 for most biochar treatments suggest that the amendments improved plant tolerance to excess As in mining residues, making more nutrients available.The reduction in root-to-leaf translocation has the direct effect of reducing physiological problems associated with As accumulation [74,75].The lower content of toxic elements in leaves reflects a better photosynthetic capacity, resulting in greater accumulation of nutrients and biomass production [76].Increasing nutrient absorption is fundamental for resistance to As stress [77] and directly reflects better plant development [78].Differently, in a study conducted by Ref. [79], Ipomoea asarifolia plants tended to increase the translocation of As from the roots to the leaves with the application of Fe enriched açaí biochar, with TRF > 1.
Nutrients may improve plant development and inhibit As absorption [65,80].For example, accumulation of P in roots occurs through competition with As for soil adsorption sites or root uptake, as well as the antagonism between these elements.Greater P uptake suppresses As absorption, which in turn reduces accumulation in leaves [81].Accumulation of Ca and Mg in roots and leaves stabilizes cell integrity and physiological activity and improves plant growth under As stress [78].Also, the accumulation of Fe in plant roots affects As chelation and serves as a defense mechanism to reduce absorption and translocation [82].
Multiple regression analysis indicates that biochar enrichment with P fertilizers had distinct effects on As accumulation in the root (R-As) and leaves (L-As) (Table 8).For the BA models, the characteristics of the tailings (As and OC fractions) and plant (Ca and P) influenced foliar As, while root As was related to Ca and the F2 fraction of P in tailings.The low nutrient content found in biochars without enrichment provided greater absorption of As, which had a negative effect on plant development.In the BT models, the F5 fraction of residue As and leaf nutrients (K and P) influenced foliar As, while root As was related to residue characteristics (Al, Ca, pH, and P fractions) and leaf Ca and Mg.For the BS models, the characteristics of the residue (F1 fraction of As and OC) and plant (Ca, Mg, and P) influenced L-As, while only the characteristics of the residue (F4 fraction of P, Ca, and K) influenced R-As.The number of nutrients (especially Ca) and P found in BS improved biomass and nutrient accumulation, which consequently resulted in lower TF values and higher TI values.

Human Health Risk Assessment
The application of all biochars statistically reduced carcinogenic and non-carcinogenic HI values from plant consumption, both for adults and children (Figure 2).All treatments reduced the non-carcinogenic risk for adults to levels below the limit (1.0) established by Ref. [38].Furthermore, the application of T + 1.0BS also reduced the non-carcinogenic risk for children to a value below the aforementioned limit.Risk mitigation is primarily due to the reduction of As translocation to the edible part of the plant and the increase in As bound to amorphous and crystalline minerals (less bioavailable).The authors in Ref. [70] proposed the use of mitigating agents (limestone, organic material, and iron oxide) to minimize As mobility and bioavailability, as such products may reduce As concentration in the plant's aerial part.On the contrary, the biochars used by Ref. [80] increased As availability but did not result in greater translocation by Brassica juncea.The authors justified this low ratio by the possible nutritional improvement of plants with the addition of biochars.The reduction of TF values is essential for mitigating potential health risks related to the consumption of plants from As contaminated areas [83].

Conclusions
The addition of P fertilizer to biochar improved the chemical properties of mining tailings and enhanced biomass and nutrient accumulation in lettuce plants, mainly with the addition of BS.The toxic effects of As were mitigated mainly through reducing element translocation.Regression equations indicated the importance of biochar-related nutrients in reducing As stress.
Considering that açaí seeds are easily obtained in the Amazon, the preparation of this P-enriched biochar can significantly contribute to the remediation of As-contaminated Au mining areas in the region, enhancing soil quality improvement, revegetation, and resumption of ecosystem services.Attention should be directed to the costs of obtaining P fertilizers, which can make the amendment manufacturing process expensive.Thus, alternative sources can be tested, including co-products such as rock dust.
The results of this study may also support the use of P biochar in improving the substrate for the development of As phytoremediation plants.This is of special interest in the Amazon, which has several plant species that may present phytoremediation potential and should be tested due to their high biomass production, rapid growth, and easy propagation.Such studies could contribute to the mitigation of As impacts in Au mining areas in the Amazon.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/min14070732/s1, Figure S1: Main minerals found in the studied mine tailings; Table S1: Pearson correlation between arsenic fractions and chemical characteristics of treatments.

Figure 2 .
Figure 2. Carcinogenic and non-carcinogenic risk index for soil and plant ingestion.Different letters indicate a significant difference between treatments using Tukey's test (p < 0.05).Red line represents the levels of non-carcinogenic risk (1) and carcinogenic risk (0.0001).

Table 1 .
Description of the tested treatments.

Table 2 .
Characterization of the studied mining tailings.

Table 3 .
Chemical properties of the produced biochars.

Table 4 .
Chemical properties of mining residues after biochar application.
Different letters indicate a significant difference between treatments using Tukey's test (p < 0.05).

Table 5 .
Fractionation of phosphorus after biochar application.

Table 6 .
Biomass and chemical elements in the roots and leaves of lettuce plants.

Table 7 .
Bioconcentration factor for roots (BCF r ) and shoots (BCF s ), translocation factor (TRF), and tolerance index (TI) of As in lettuce plants.

Table 8 .
Multiple regression equations related to arsenic (As) accumulation in leaves (L-As) and roots (R-As).