Influence of calcination temperature on equine bone hydroxyapatite structure and lead adsorption efficiency

Abstract

Lead (Pb²⁺) contamination in aquatic environments represents a serious global threat due to its toxicity, persistence, and non-biodegradable nature. In this study, hydroxyapatite (HA) was synthesized from equine scapula bone and investigated as a low-cost, sustainable adsorbent for Pb²⁺ removal from aqueous media. HA was thermally treated at 100 °C, 500 °C, and 900 °C to assess the effect of calcination on physicochemical and adsorption properties. Characterization techniques including XRD, FTIR, SEM/EDX, TGA and XRF confirmed structural and compositional evolution with temperature. Among the samples, HA-500 exhibited the highest adsorption performance, achieving 99% Pb²⁺ removal and a maximum capacity of 50 mg/g under optimal conditions (0.25 g dose, 20 mg/L initial Pb²⁺ concentration). Kinetic studies followed a pseudo-second-order model (R² > 0.999), and equilibrium data were best described by the Freundlich isotherm (R² = 0.9839), indicating multilayer adsorption on heterogeneous surfaces. Thermodynamic analysis revealed that Pb²⁺ adsorption is spontaneous and exothermic, with negative Gibbs free energy values and positive entropy changes, confirming increased randomness at the solid–liquid interface. Furthermore, regeneration studies showed that HA-500 retained 73% of its efficiency after three cycles. These findings demonstrate the potential of equine bone as a novel bio-based source of hydroxyapatite for efficient and sustainable heavy metal remediation.

Introduction

Water pollution caused by heavy metals poses a significant threat to both environmental and human health¹. Among these contaminants, lead (Pb²⁺) is of particular concern due to its toxicity, persistence, and bioaccumulative nature². Even at low concentrations, lead can cause serious damage to the nervous system, kidneys, and other organs³. Its widespread presence in industrial wastewater resulting from activities such as battery manufacturing, mining, and metal plating necessitates the development of efficient and cost-effective remediation strategies⁴.

One of the most promising approaches for the removal of heavy metals from aqueous solutions is adsorption, owing to its simplicity, high efficiency, and low cost⁵. In recent years, natural and bio-derived materials have attracted growing interest as environmentally friendly and sustainable adsorbents. Among them, hydroxyapatite (HA) a calcium phosphate compound with the general formula Ca₁₀(PO₄)₆(OH)₂ has shown excellent potential for metal ion sequestration due to its ion-exchange capacity, high surface reactivity, and biocompatibility⁶. Hydroxyapatite can be synthesized through various chemical routes, but the use of natural sources such as animal bones offers a low-cost and sustainable alternative, particularly relevant in the context of circular economy and waste valorization⁷.

Animal bones especially those rich in calcium phosphate are excellent candidates for the production of bio-hydroxyapatite⁸. Previous studies have focused on bovine, ovine, and porcine bones; however, little to no attention has been given to equine bones, despite their abundant availability and rich mineral content⁹. This oversight represents a gap in current research, especially considering the unique mechanical and structural properties of equine bone, which result from the high mechanical loads it supports¹⁰. These properties could potentially enhance the performance of the derived apatite in adsorption applications¹¹.

This study presents, the extraction and characterization of hydroxyapatite from equine bone, specifically the scapula bones, and its application as an adsorbent for Pb²⁺ ions in aqueous solution. The originality of this work lies not only in the unexplored use of equine bone as a raw material but also in the systematic investigation of the effect of calcination temperature (100 °C, 500 °C, and 900 °C) on the structural, chemical, and adsorption properties of the resulting material.

Despite extensive studies on hydroxyapatite derived from bovine, porcine, and ovine bones, the utilization of equine bone remains underexplored in the field of bio-hydroxyapatite synthesis and heavy metal adsorption¹². This represents a significant research gap, as equine bones possess unique structural and mechanical properties due to the high mechanical loads they support, potentially offering enhanced physicochemical characteristics compared to more commonly studied sources. Moreover, equine scapula bones are abundant and often considered waste in many regions, presenting an opportunity for sustainable valorization aligned with circular economy principles. Exploring equine bone as a raw material not only diversifies the sources of bio-hydroxyapatite but also addresses environmental and economic challenges by converting waste into a value-added product for efficient lead removal from aqueous solutions¹³. Through a comprehensive suite of characterization techniques (XRD, FTIR, SEM/EDX, TGA XRF), adsorption isotherms (Langmuir and Freundlich), and kinetic models (pseudo-first and second-order), we demonstrate the structure property relationship that governs Pb²⁺ removal efficiency.

Furthermore, the regeneration and reusability of the adsorbents are evaluated, providing insight into their long-term applicability and stability. The findings not only contribute to the body of knowledge on bio-based adsorbents but also open new possibilities for valorizing equine waste materials in environmental remediation. In sum, this work bridges a critical knowledge gap and offers an innovative approach to lead removal, supporting the broader goals of green chemistry, waste reuse, and sustainable water treatment technologies.

Materials and methods

Availability of equine bone material

Equine bones are a readily available by-product of the global horse industry, which spans breeding, sports, meat production, and veterinary sectors. With an estimated worldwide horse population exceeding 60 million, equine bone waste is generated in significant quantities, particularly in regions such as North America, Europe, Asia, and parts of Africa¹⁴. This makes equine bone a sustainable and accessible raw material for hydroxyapatite synthesis in many countries. While availability may vary regionally due to differences in equine industry scale and regulatory frameworks, the material’s global presence supports its feasibility for large-scale environmental and biomedical applications.

Preparation of apatite from equine bone

Bone is a natural source of apatite. This study represents the first exploration of equine bone (specifically scapula) as a matrix for apatite extraction using our research methodology, and was purchased from Orchard Equestrian, Limerick, Ireland.

Material selection

The choice of equine scapula bone for apatite preparation is driven by several key factors. First, these bones are rich in calcium phosphate, making them particularly suitable for apatite extraction¹⁵. Their large size and availability enable the collection of substantial quantities of raw material, offering clear economic advantages. Furthermore, due to the high mechanical stress they endure, scapula bones possess a dense and solid structure, promoting the production of powders with excellent mechanical properties¹⁶. Additionally, scapula bone allows for the generation of materials with a high specific surface area, which is beneficial for adsorption and interactions with other substances particularly in applications related to heavy metal adsorption, such as lead. Thus, apatite derived from equine scapula bone proves to be a valuable biomaterial, suitable for effective use in various environmental and biomedical applications¹⁷.

Bone treatment

The scapula bones were degreased by boiling them twice consecutively, with each boiling lasting 2 h. The bones were then immersed for 18 h in a 0.1% NaOH solution in 500 mL beakers, followed by draining and rinsing with deionized water. Subsequently, the bones were soaked in acetone for 2 h, drained again, rinsed with deionized water, and dried for 8 h.

Preparation of hydroxyapatite

The raw equine bone was first cleaned, degreased, and subjected to a drying process at 100 °C for 8 h. This sample, labeled HA-100, represents the non-calcined form of hydroxyapatite and was used as a baseline to compare the effects of thermal treatment on adsorption behavior. Additional samples were then calcined at 500 °C (HA-500) and 900 °C (HA-900) in a Nabertherm furnace for 6 h, with a heating rate of 10 °C/min to evaluate the influence of crystallinity and thermal stability. After calcination, the samples were slowly cooled inside the furnace, then ground with a mortar and sieved to obtain particles smaller than 160 μm.

The selected temperatures (100 °C, 500 °C, and 900 °C) were chosen to cover the full spectrum of thermal transformation of equine bone material. At 100 °C, minimal thermal treatment preserves organic matter, allowing insight into the initial matrix composition. The intermediate temperature of 500 °C allows for substantial decomposition of organic content while maintaining surface functionality and porosity, which is often favorable for adsorption processes. Finally, 900 °C ensures complete organic removal and full crystallization, enabling evaluation of the effect of excessive thermal treatment on hydroxyapatite performance. This range was specifically designed to assess how the progressive structural evolution of the material influences its physicochemical and adsorption properties.

Calculation of mass loss percentage

To determine the percentage of mass loss (Y), the bone sample is weighed before and after calcination at room temperature. This percentage reflects the amount of material lost during the calcination process, primarily due to the elimination of organic matter and other volatile components. The mass loss percentage (Y) is calculated using the following equation:

$Y = \frac{{W_{2} - W_{1} }}{{W_{1} }} \times 100$

Where: W₁ is the weight of the raw bone before calcination. W₂ is the weight of the bone after calcination.

This measurement provides insight into the thermal stability of the material and the proportion of organic content removed during heat treatment. A higher mass loss typically indicates a greater presence of organic matter in the original bone structure.

Characterization of the treated bone

X-ray diffraction (XRD)

X-ray diffraction (XRD) is a technique used to identify the crystalline phases present in materials and to obtain information regarding their purity, crystallinity, and crystallographic parameters. In this study, XRD was employed to characterize the various crystalline phases within the apatite synthesized from natural phosphate. The analysis was carried out on powder samples under ambient temperature and pressure conditions. A Philips PW131 X-ray diffractometer was used, equipped with a copper anode (λ = 1.5406 Å) and computer-controlled for data acquisition and processing.

Fourier transform infrared spectroscopy (FTIR)

FTIR spectroscopy was used to investigate the functional groups and confirm the presence of characteristic chemical bonds in the synthesized apatite. The spectra were recorded under standard ambient conditions, providing complementary information to XRD regarding the material’s structural and chemical composition.

Scanning electron microscopy (SEM)/energy dispersive X-ray spectroscopy (EDX)

Scanning Electron Microscopy (SEM) coupled with Energy Dispersive X-ray Spectroscopy (EDX) was carried out using a JEOL JCM-6000 Plus microscope operated at an accelerating voltage of 5 kV. The SEM detector captures a combination of secondary and backscattered electrons, the proportions of which vary depending on parameters such as beam energy, working distance, incident angle, and the composition of the sample. Secondary electrons provide high-resolution images of the surface topography, allowing detailed visualization of morphological features, while backscattered electrons enhance contrast based on variations in atomic number, enabling differentiation of material phases. In addition, EDX analysis was employed to determine the elemental composition of the samples before and after Pb²⁺ adsorption, allowing the confirmation of lead ion uptake and the identification of associated surface changes.

X-ray fluorescence (XRF)

X-ray fluorescence spectroscopy was performed using a benchtop spectrometer (PANalytical Epsilon 3X), equipped with a 37 W X-ray tube with a molybdenum anode, a multilayer monochromator (energy resolution < 150 eV at the MnKα line), and a 30 mm² silicon drift detector. Measurements were conducted for 500 s at 50 kV and 0.75 mA. Quartz holders were used as reflectors. Spectrum processing was carried out using Spectra 7 software in the normal fitting mode, with background correction based on 1000 stripping cycles.

Thermgravimetric analysis (TGA)

The thermal stability of the prepated samples was evaluated using thermogravimetric analysis (TGA) performed on a Versatherme HM system. The measurements were conducted up to 500 °C under controlled conditions, with a constant heating rate of 20 °C/min.

Leaching test

Each adsorbent (HA-100, HA-500, and HA-900) was immersed in deionized water at neutral pH (pH ≈ 7) under agitation at room temperature. Aliquots were withdrawn at regular intervals (1, 2, 3, 5, 7 and 10 days) and filtered before analysis. The concentration of calcium (Ca²⁺) and phosphorus (PO₄³⁻) ions in the supernatant was measured using ICP-OES, which allowed us to monitor any structural degradation or dissolution of the hydroxyapatite matrix.

Lead (Pb²⁺) adsorption on equine bone treated at various calcination temperatures

We investigated the use of apatite extracted from horse bone as an adsorbent for the removal of heavy metals from aqueous solutions. The study focused on evaluating the effects of various parameters including contact time, metal ion concentration, and adsorbent massn in order to optimize the conditions influencing the adsorption process of heavy metals. Based on the characterization results, apatite derived from horse bone thermally treated at three different temperatures (100 °C, 500 °C, and 900 °C) was used in this study to assess the impact of calcination temperature on adsorption performance.

Adsorption experiments were conducted in a volume of 100 mL. The aqueous solutions of Pb²⁺ ions, prepared at various concentrations from lead nitrate (Pb(NO₃)₂), were used as the model contaminant. The solutions were agitated at 160 rpm for 30 min to reach equilibrium. After agitation, the suspensions were filtered to separate the adsorbent, and the remaining metal ion concentrations in the filtrates were analyzed using atomic absorption spectroscopy (AAS).

Determination of the point of zero charge (pHpzc)

The point of zero charge (pHpzc) is the pH at which the net surface charge of a material is zero. At this pH, the surface carries equal amounts of positive and negative charges rather than being entirely uncharged. This parameter is essential for understanding adsorption behavior and surface interactions, as it helps determine whether a material exhibits acidic, neutral, or basic characteristics. The pHpzc also provides valuable insight into the chemical and electronic properties of the functional groups present on the material’s surface.

In this study, the pHpzc of the apatite used as a bioadsorbent was determined using the solid addition method. Six distilled water solutions with initial pH values ranging from 2 to 12 were prepared by adding 0.1 M HCl or NaOH. To each 100 mL solution, 0.1 g of the bioadsorbent was added. The resulting suspensions were stirred for 24 h at room temperature (28 ± 2 °C), after which the final pH of each solution was measured. The pHpzc corresponds to the point where ΔpH = 0, which is determined by plotting the final pH against the initial pH and identifying the intersection point.

Theoretical equations for pollutant removal process

The amount of pollutant adsorbed from aqueous solution was calculated using the following equation :

$$q_{e} = \frac{{c_{{0 - }} c_{e} }}{m} \times V$$

Where: C₀: Initial concentration of the pollutant (mg/L). C_e: Equilibrium concentration of the pollutant (mg/L). V: Volume of the solution (L), m: Mass of the adsorbent (g).

Adsorption kinetics modeling

To understand the adsorption mechanism, various controlling factors were examined, including chemical reaction, diffusion control, and mass transfer. Two commonly used kinetic models were applied to fit the experimental data:

• Pseudo-First-Order Kinetic Model.

$$\frac{{dq_{t} }}{{dt}} \times = K_{1} (q_{{e,1}} - q_{t} )^{2}$$

• Pseudo-Second-Order Kinetic Model.

$$\frac{{dq_{t} }}{{dt}} \times = K_{2} (q_{{e,2}} - q_{t} )^{2}$$

Where:

q_e,1 and q_e,2: Amount of pollutant adsorbed at equilibrium (mg/g).

q_t: Amount of pollutant adsorbed at time t (mg/g).

k₁: Rate constant of the pseudo-first-order model (1/min).

k₂: Rate constant of the pseudo-second-order model (g/mg·min).

Adsorption isotherm models

Equilibrium data were analyzed using two widely recognized adsorption isotherm models: Langmuir and Freundlich.

In addition to Langmuir and Freundlich models, three additional isotherm models Dubinin–Radushkevich (D–R), Temkin, and Redlich–Peterson (R–P) were applied to further explore the adsorption mechanism and potential multi-pathway behavior. These models provide insights into adsorption energy (D–R), adsorbate–adsorbent interactions (Temkin), and hybrid behavior between Langmuir and Freundlich (R–P), thus offering a more comprehensive understanding of the adsorption process.

• Langmuir Isotherm Model.

$$q_{e} = q_{{e,\max }} \frac{{\beta C_{e} }}{{1 + ~\beta C_{e} }}$$

Where:

q_e, max: Maximum adsorption capacity (mg/g).

β: Langmuir constant related to the energy of adsorption (L/mg).

• Freundlich Isotherm Model.

$$q_{e} = K_{{f~}} C_{e} ^{{\frac{1}{n}}}$$

Where:

K_f : Freundlich constant related to adsorption capacity.

n: Adsorption intensity parameter.

• Dubinin–Radushkevich (D–R) Isotherm:

$$q_{e} = q_{{\max }} \exp ( - \beta \varepsilon ^{2} )$$

$\varepsilon = RT~\ln ~\left( {1 + \frac{1}{{C_{e} }}} \right)$

Where:

q_e: amount of adsorbate at equilibrium (mg/g).

C_e: equilibrium concentration (mg/L).

q_max: theoretical saturation capacity (mg/g).

β: activity coefficient related to mean adsorption energy.

ε: Polanyi potential.

Mean free energy (E) of adsorption is calculated as:

$$E = \frac{1}{{\sqrt {2\beta } }}$$

If E < 8 kJ/mol → physisorption.

If 8 < E < 16 kJ/mol → ion exchange or chemisorption.

• Temkin Isotherm.

$$q_{e} = B~\ln A_{T} + B~\ln C_{e}$$

Where:

$B = \frac{{RT}}{{b_{T} }}$.

b_T: Temkin constant related to heat of sorption (J/mol).

A_T: Temkin isotherm equilibrium binding constant (L/g).

• Redlich–Peterson (R–P) Isotherm.

$\frac{{C_{e} }}{{q_{e} }} = \frac{1}{{a_{{RP}} }} + \frac{{b_{{RP}} }}{{a_{{RP}} }}C_{e}$

Where:

a_RP: the Redlich–Peterson constant related to adsorption capacity (L/g).

b_RP: the Redlich–Peterson constant related to the adsorption affinity (1/mg).

The efficiency of the adsorbent can be evaluated using the parameter K_RP, calculated as:

$K_{{RP}} = ~\frac{{a_{{RP}} }}{{b_{{RP}} }}$.

This parameter K_RP, provides a global indication of the system’s adsorption performance, with higher values suggesting more effective adsorption.

Thermodynamic studies

Adsorption experiments were conducted at four different temperatures (298 K, 308 K, 318 K and 328 K), and the corresponding equilibrium data were used to calculate the standard Gibbs free energy change (ΔG°), enthalpy change (ΔH°), and entropy change (ΔS°) based on the Van’t Hoff equation:

$\ln K_{c} = ~ - \frac{{\Delta H^{^\circ } }}{{RT}} + \frac{{\Delta S^{^\circ } }}{R}$.

Where:

K_c: the equilibrium constant (calculated from qe/Ce).

R: the universal gas constant (8.314 J/mol·K).

T: the absolute temperature (K).

From the slope and intercept of the Van’t Hoff plot (ln Kc vs. 1/T), values of ΔH° and ΔS° were determined. The Gibbs free energy change ΔG° was then calculated using the relation:

$$\Delta G^{^\circ } = \Delta H^{^\circ } - T\Delta S^{^\circ }$$

The activation energy for adsorption can be estimated using the Arrhenius equation, which relates the adsorption rate constant to temperature:

$$\ln K = ~\ln A - \frac{{E_{a} }}{{RT}}$$

Where:

K: adsorption rate constant.

A: pre-exponential factor.

E_a: activation energy (J/mol).

R = 8.314 J/mol·K (gas constant ).

T: absolute temperature (K).

Surface coverage θ is a measure of how much of the adsorbent surface is covered by the adsorbate. It can be estimated using:

$\theta = \frac{{q_{e} }}{{q_{{\max }} }}$

Where:

q_e= amount adsorbed at equilibrium (mg/g).

q_max= maximum adsorption capacity, typically obtained from the Langmuir model.

Desorption and reuse study

To evaluate the regeneration and reusability of the adsorbents, a desorption–reuse study was carried out over three consecutive adsorption–desorption cycles. After each Pb²⁺ adsorption experiment, the used hydroxyapatite samples (HA-100, HA-500, and HA-900) were separated by filtration, washed thoroughly with deionized water, and subjected to desorption treatments under various conditions. Two different eluting agents hydrochloric acid (HCl, 0.1 M) and sodium hydroxide (NaOH, 0.1 M) were employed to investigate the effect of both acidic and basic media on Pb²⁺ desorption behavior.

For each condition, 0.3 g of used adsorbent was dispersed in 100 mL of the chosen eluting solution and stirred at 150 rpm for 2 h. The desorption tests were conducted at three different temperatures: 25 °C (room temperature), 40 °C, and 60 °C, in order to assess the influence of thermal activation on desorption efficiency and material stability. Following desorption, the samples were filtered, rinsed repeatedly with deionized water until a neutral pH was reached, and then dried at 60 °C for 12 h. The regenerated materials were reused under the same experimental conditions (Pb²⁺ concentration: 20 mg/L, adsorbent dose: 0.3 g, contact time: 180 min, stirring speed: 160 rpm). The remaining Pb²⁺ concentration in solution was determined using atomic absorption spectroscopy (AAS), and the adsorption efficiency of each regenerated material was calculated for every cycle.

The adsorption efficiency (%) for each cycle was determined using the following formula:

$$\text{Re} moval~Efficiency~\left( \% \right) = \frac{{c_{{0 - }} c_{e} }}{{c_{0} }} \times 100$$

Where:

C₀: is the initial concentration of Pb²⁺ (mg/L).

Ce: is the equilibrium concentration of Pb²⁺ after adsorption (mg/L).

Results and discussion

Mass loss percentage

Equine bone was subjected to calcination at three different temperatures: 100 °C, 500 °C, and 900 °C. The corresponding mass loss percentages were approximately 93% at 100 °C, 60% at 500 °C, and 51% at 900 °C. These values were determined by measuring the initial mass of the untreated bone and comparing it to the final mass after thermal treatment at each temperature.

Fig. 1

Effect of calcination temperature on mass loss of equine bone.

As illustrated in Fig. 1, a noticeable decrease in mass loss percentage was observed with increasing calcination temperature. This inverse relationship is primarily due to the progressive decomposition and volatilization of organic components such as collagen and lipids that are present in raw bone tissue¹⁷. At lower temperatures (e.g., 100 °C), the bone still contains a high amount of moisture and organic matter, most of which is rapidly lost during initial heating, resulting in a higher mass loss. As the temperature increases to 500 °C, a significant portion of the organic content continues to degrade, but at a slower rate. At 900 °C, the majority of the organic matter has already been removed, and the remaining material is predominantly inorganic, leading to a smaller mass loss.

These results clearly demonstrate the impact of calcination temperature on the thermal decomposition behavior of equine bone and the yield of the resulting mineral material primarily hydroxyapatite. Understanding this behavior is essential for optimizing the preparation process of bio-derived apatite, particularly in applications requiring high purity and thermal stability, such as in environmental remediation and biomedical engineering.

Diffraction des rayons X (XRD)

X-ray diffraction (XRD) analysis was performed on powder samples prepared under ambient temperature and pressure conditions. The XRD patterns for the samples calcined at 100 °C (HA-100), 500 °C (HA-500), and 900 °C (HA-900) are presented in Fig. 2.

Fig. 2

X-ray diffraction patterns of equine bone-derived apatite samples (HA-100, HA-500, and HA-900).

The XRD analysis of equine bone samples calcined at 100 °C (HA-100), 500 °C (HA-500), and 900 °C (HA-900) reveals significant changes in the crystallinity and structural organization of the apatite as a function of calcination temperature.

At 100 °C (HA-100), the diffraction peaks are broad and of low intensity, notably at 2θ angles around 25.9° (002), 31.8° (211), 32.2° (112), and 32.9° (300). These broad and weak peaks indicate poor crystallinity and suggest an amorphous or poorly crystallized structure. The calcination temperature of 100 °C is insufficient to induce full crystallization of the apatite, which explains the low definition and intensity of the observed peaks.

At 500 °C (HA-500), a notable improvement in crystallinity is observed. The diffraction peaks become sharper and more intense, reflecting better crystalline organization. Key 2θ angles include 25.9° (002), 31.8° (211), 32.2° (112), 32.9° (300), 39.9° (310), 46.7° (222), and 49.5° (213). The enhanced peak sharpness and intensity suggest that the apatite structure is becoming more ordered and crystalline at this temperature. This improvement is indicative of atomic rearrangement promoted by the increased thermal energy at 500 °C.

At 900 °C (HA-900), the apatite exhibits a highly crystalline structure. The diffraction peaks are extremely sharp, narrow, and intense, with well-defined peaks at 2θ angles such as 25.9° (002), 31.8° (211), 32.2° (112), 32.9° (300), 39.9° (310), 46.7° (222), 49.5° (213), 53.2° (004), and 56.5° (116). These results indicate the formation of a well-ordered and highly crystalline apatite phase. The calcination temperature of 900 °C is sufficient to induce complete crystallization, allowing atoms to arrange optimally within the apatite’s crystal lattice.

In conclusion, the XRD analysis demonstrates that the crystallinity of the apatite increases significantly with the calcination temperature. At 100 °C, the material remains largely amorphous or poorly crystalline; at 500 °C, an intermediate crystalline structure emerges; and at 900 °C, a highly crystalline and well-organized structure is obtained¹⁸.

Fourier transform infrared spectroscopy (FTIR)

Figure 3 displays the infrared absorption spectra of apatite samples subjected to calcination at different temperatures: 100 °C, 500 °C, and 900 °C.

Fig. 3

FTIR absorption spectra of equine bone-derived apatite samples (HA-100, HA-500, and HA-900).

The FTIR spectrum of the sample dried at 100 °C (HA-100) reveals several characteristic bands that help identify specific organic and mineral components in the bone matrix. A broad absorption at 3266 cm⁻¹ corresponds to the stretching vibrations of OH groups, indicating residual water in the sample despite the drying process. Bands observed at 2921 cm⁻¹ and 2852 cm⁻¹ are attributed to C-H and N-H stretching vibrations from collagen amide groups, confirming the presence of organic matter¹⁸. Further, absorption bands at 1647 cm⁻¹ and 1535 cm⁻¹ correspond to amide II vibrations, indicative of protein content primarily collagen, the dominant structural protein in bone¹⁹.

Characteristic carbonate bands appear at 1454 cm⁻¹ and 1413 cm⁻¹, along with bending vibrations at 871 cm⁻¹ and 720 cm⁻¹, suggesting the presence of carbonate species, likely in the form of calcium carbonate within the mineral matrix. Phosphate (PO₄³⁻) ions are identified through strong absorption bands at 1021 cm⁻¹ and 961 cm⁻¹, with additional bending vibrations at 600 cm⁻¹, 560 cm⁻¹, and 480 cm⁻¹, representing phosphate groups in hydroxyapatite the primary mineral component of bone. This spectrum indicates that the sample retains a complex composition of both organic (collagen) and inorganic (hydroxyapatite, carbonates) constituents at 100 °C²⁰.

At 500 °C (HA-500), a noticeable reduction in the intensity of organic bands is observed. The C-H and N-H stretching vibrations at 2921 cm⁻¹ and 2852 cm⁻¹, as well as the amide II bands at 1647 cm⁻¹ and 1535 cm⁻¹, are significantly diminished, reflecting partial decomposition of the organic matrix. In contrast, the carbonate bands at 1454 cm⁻¹, 1413 cm⁻¹, 871 cm⁻¹, and 720 cm⁻¹ remain visible, indicating that the inorganic components are relatively stable at this intermediate temperature. The phosphate bands at 1021 cm⁻¹ and 961 cm⁻¹, along with the bending bands at 600 cm⁻¹, 560 cm⁻¹, and 480 cm⁻¹, become more distinct. This reflects an increase in the relative concentration of mineral phases as organic matter volatilizes²¹.

At 900 °C (HA-900), the spectrum shows the near-complete disappearance of all bands associated with organic compounds, confirming full decomposition of the organic matrix. The OH stretching band at 3266 cm⁻¹ is no longer present, indicating the removal of residual moisture. While carbonate bands at 1454 cm⁻¹, 1413 cm⁻¹, 871 cm⁻¹, and 720 cm⁻¹ are still detected, they may exhibit changes due to structural rearrangement at high temperature. The phosphate bands at 1021 cm⁻¹ and 961 cm⁻¹, together with the well-defined bending bands at 600 cm⁻¹, 560 cm⁻¹, and 480 cm⁻¹, are highly intense and dominant. This suggests the formation of a nearly pure mineral phase, primarily composed of crystalline apatite²².

In conclusion, the FTIR analysis of equine bone samples calcined at various temperatures reveals a progressive transition from an organic and hydrated matrix to a predominantly mineral composition. At 100 °C, the sample retains a complex structure of collagen and minerals; at 500 °C, partial decomposition of organic matter occurs; and at 900 °C, the transformation is complete, resulting in a structure mainly consisting of hydroxyapatite and carbonates²³. These observations underscore the significant influence of calcination temperature on the chemical and structural evolution of bone-derived materials.

SEM-EDX

The surface morphology and elemental composition of the hydroxyapatite samples (HA-100, HA-500, and HA-900) were examined using scanning electron microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDX). (Fig. 4).

Fig. 4

SEM images of treated equine bones at different calcination temperatures.

The SEM micrographs reveal that the calcination temperature significantly influences the structural features of the synthesized materials. At 100 °C (HA-100), the surface appears irregular and porous, with disordered and agglomerated particles. This morphology indicates the presence of residual organic matter and a low degree of crystallinity, which is consistent with minimal thermal treatment. The rough and heterogeneous surface may provide numerous active sites, favoring heavy metal adsorption. However, the material lacks structural compactness²⁴.

With calcination at 500 °C (HA-500), the sample displays more defined and homogeneous crystalline particles. The surface becomes denser and more organized, suggesting a transition toward partial crystallization of the apatite structure. This morphological evolution enhances the material’s stability while retaining sufficient porosity to support effective adsorption. HA-500 thus shows a good compromise between crystallinity and surface reactivity²⁵.

Although not shown in this specific image, HA-900 typically presents a more compact and sintered morphology, characterized by smoother, larger particles and lower porosity. Such dense packing results from complete crystallization at high temperatures, which, while improving structural integrity, may reduce surface area and thus limit the number of accessible adsorption sites²⁶.

The EDX spectra of HA-100 and HA-500 confirm the presence of calcium (Ca), phosphorus (P), and oxygen (O), the primary elements in hydroxyapatite. The Ca/P atomic ratios derived from the EDX data provide further insight into the chemical structure. For HA-100, the Ca/P ratio is approximately 1.47, indicating a calcium-deficient apatite, potentially due to the retention of organic components or the presence of phosphate-rich phases. In contrast, HA-500 exhibits a Ca/P ratio of 1.60, approaching the theoretical value of 1.67 for stoichiometric hydroxyapatite. This suggests a more stable and mature mineral phase, consistent with the SEM observations. These results collectively demonstrate that the calcination process plays a critical role in tailoring the microstructure and composition of equine bone-derived hydroxyapatite, directly influencing its performance in adsorption applications²⁷.

X-ray fluorescence (XRF) spectroscopy

The chemical composition of the calcined equine bone samples, expressed in terms of oxide forms, was analyzed using X-ray fluorescence (XRF) spectroscopy. The results are presented in Table 1.

Table 1 Oxide composition (mass %) of equine bone samples calcined at different temperatures.

The XRF analysis of equine bone samples calcined at 100 °C, 500 °C, and 900 °C reveals significant changes in their oxide compositions with increasing temperature. At 100 °C (HA-100), the samples exhibit a high concentration of calcium oxide (CaO) at 84.409% and phosphorus pentoxide (P₂O₅) at 10.550%, consistent with the presence of hydroxyapatite, along with minor quantities of magnesium oxide (MgO), strontium oxide (SrO), and zinc oxide (ZnO). At this stage, the material still contains residual moisture and organic matter, which influences the overall elemental profile.

When the samples are calcined at 500 °C (HA-500), the P₂O₅ content increases significantly to 20.864%, while CaO slightly decreases to 77.564%. This shift suggests partial decomposition of organic matter, exposing more phosphate groups. The slight increase in MgO content may indicate the onset of mineral stabilization, while the noticeable decrease in SrO and ZnO concentrations may be attributed to partial volatilization or redistribution of these trace elements at elevated temperatures.

At 900 °C (HA-900), the CaO content rises sharply to 86.861%, and P₂O₅ decreases to 11.443%, suggesting the formation of a nearly pure mineral structure, predominantly composed of thermally stable hydroxyapatite. The increase in MgO to 0.799% reflects a greater incorporation of magnesium into the crystalline matrix, while the further decline in SrO (0.113%) and ZnO (0.023%) likely results from thermal volatilization or structural reorganization at high temperature.

In conclusion, these findings illustrate how progressive calcination enhances the mineral composition of equine bone by eliminating water and organic matter. The result is a more purified and thermally stable hydroxyapatite, particularly evident at 900 °C, which makes it suitable for high-performance biomedical and environmental applications.

Thermogravimetric analysis (TGA)

The thermogravimetric analysis (TGA) curves of the hydroxyapatite samples HA-100, HA-500, and HA-900, derived from equine bone and treated at increasing calcination temperatures, are shown in Fig. 5. The thermal behavior of each sample reflects its composition and degree of thermal treatment, and provides insight into the decomposition of organic and inorganic phases.

Fig. 5

Thermogravimetric analysis (TGA) curves of equine bone-derived hydroxyapatite samples: HA-100 (untreated), HA-500 (calcined at 500 °C), and HA-900 (calcined at 900 °C).

The TGA curve of HA-100, which represents the untreated or raw bone-derived sample, reveals three distinct stages of weight loss. The first stage, occurring approximately between 30 °C and 150 °C, corresponds to the loss of physisorbed water and moisture. A more pronounced weight loss is observed between 150 °C and 250 °C, which is attributed to the thermal degradation of organic matter, primarily collagen and other proteins present in the bone matrix. A final weight loss phase, extending up to around 450 °C, is linked to the oxidation of residual carbonaceous material and potential decarbonation of poorly crystalline carbonate phases. These results confirm that HA-100 retains a significant amount of organic content, which aligns with the FTIR analysis showing strong amide bands and with SEM images showing an irregular, porous surface morphology. Although HA-100 demonstrates a reactive surface favorable for ion exchange, its thermal instability and residual organics limit its long-term performance, particularly during regeneration cycles.

In contrast, the TGA profile of HA-500 shows a smoother and more gradual mass loss, with a total weight reduction of only about 5% over the entire temperature range. This indicates that the sample had already undergone substantial decomposition during the initial calcination at 500 °C, and only minor residues of volatile or weakly bound carbonate species remained. This result confirms the partial removal of organic material and a transition toward a more stable structure. The thermal behavior of HA-500 supports the SEM observations of more defined and homogeneous surface morphology, and the XRD data indicating increased crystallinity. This intermediate treatment condition appears optimal, as HA-500 showed the highest lead (Pb²⁺) adsorption performance, with 99% removal efficiency, balancing sufficient surface activity with structural stability.

The sample HA-900, which was subjected to high-temperature calcination, exhibits minimal weight loss restricted to temperatures below 200 °C. This negligible loss is most likely due to the elimination of structurally bound water or hydroxyl groups. The stability of the sample throughout the rest of the thermal range confirms the complete elimination of organic material and the formation of a highly crystalline and thermally stable hydroxyapatite phase. These findings are consistent with the XRD patterns showing sharp diffraction peaks and with SEM images depicting a compact, sintered morphology. Although HA-900 exhibits excellent thermal and chemical stability, its adsorption capacity is lower than HA-500, likely due to reduced porosity and fewer accessible active sites for metal ion interaction.

Overall, the TGA results demonstrate how calcination temperature influences the composition, structure, and functional properties of equine bone-derived hydroxyapatite. HA-100 retains a high organic load and exhibits pronounced thermal degradation, while HA-500 achieves a balance between surface reactivity and thermal stability. HA-900, meanwhile, reflects a highly pure and crystalline structure with excellent stability but reduced adsorption performance. These trends correlate closely with the results obtained from FTIR, XRD, and adsorption experiments, reinforcing the conclusion that the thermal treatment process plays a key role in tuning the properties of hydroxyapatite for environmental remediation applications.

Leaching test

To assess the chemical stability of the synthesized hydroxyapatite materials (HA-100, HA-500, and HA-900), a leaching study was performed. Each adsorbent was immersed in deionized water (neutral pH, room temperature) for time intervals ranging from 1 day to 10 days. The release of calcium (Ca²⁺) and phosphate (PO₄³⁻) ions into the solution was measured by ICP-OES to evaluate the potential dissolution or degradation of the apatite structure (Fig. 6).

Fig. 6

Leaching behavior (Ca²⁺ and PO₄^3-).

The plotted leaching curves clearly demonstrate the influence of calcination temperature on the aqueous stability of hydroxyapatite samples derived from equine bone. The HA-100 sample, which was either untreated or calcined at a low temperature, exhibits the highest ion release over the 10-day period. The concentration of Ca²⁺ increases progressively from 2.5 mg/L on Day 1 to over 5.1 mg/L on Day 10, while PO₄³⁻ levels rise from 1.2 to 2.3 mg/L. This trend indicates significant structural degradation and confirms the poor water resistance of HA-100.

In contrast, HA-500 displays moderate leaching behavior, with Ca²⁺ and PO₄³⁻ concentrations stabilizing quickly after Day 3, plateauing around 1.5 mg/L and 0.9 mg/L, respectively. This suggests a partially crystalline structure that maintains some aqueous stability, supporting its intermediate performance in adsorption and regeneration tests. Remarkably, HA-900 shows minimal ion release, with Ca²⁺ levels remaining below 0.4 mg/L and PO₄³⁻ around 0.3 mg/L throughout the 10-day period. This confirms its excellent crystallinity and resistance to aqueous dissolution, making it the most chemically stable material among the three.

These results collectively validate that thermal treatment significantly enhances the physicochemical robustness of hydroxyapatite, with HA-900 emerging as the most promising candidate for long-term environmental applications in aqueous media.

Application of natural apatite for the removal of pb²⁺ ions through adsorption

Point of zero charge (pHpzc)

The point of zero charge (pHpzc) is a key parameter used to characterize the surface properties of an adsorbent material (Fig. 7). At this specific pH, the surface of the material is electrically neutral, with a balance between positive and negative surface charges. When the pH of the surrounding solution exceeds the pHpzc, the surface becomes negatively charged, thereby enhancing the adsorption of positively charged heavy metal ions.

Fig. 7

The zero point of charge (pHZPC) of naturally derived apatite.

Figure 7 illustrates the variation of ΔpH (pH_final – pH_initial) as a function of the initial pH (pHi) for the natural apatite samples calcined at different temperatures: HA-100, HA-500, and HA-900. The point at which ΔpH = 0 corresponds to the point of zero charge (pH_ZPC) the pH at which the surface of the material is electrically neutral.

From the curves, the pH_ZPC values can be identified as follows:

HA-100: pH_ZPC ≈ 7.6

HA-500: pH_ZPC ≈ 8.4

HA-900: pH_ZPC ≈ 8.8

These results indicate a clear influence of calcination temperature on the surface chemistry of the apatite. As the temperature increases, the pH_ZPC also increases, reflecting changes in surface functional groups and the overall acid-base behavior of the material.

At lower temperatures (HA-100), the surface remains more acidic, likely due to the presence of residual organic matter and hydroxyl groups. With increasing calcination temperature, these surface groups are progressively eliminated or transformed, leading to a surface that is less protonated and thus more basic reflected in the higher pH_ZPC observed for HA-900.

This shift in pH_ZPC has important implications for the material’s adsorption behavior. At pH values above the point of zero charge (pHpzc ≈ 8.4 for HA-900), the HA-900 surface becomes negatively charged, enhancing the electrostatic attraction of positively charged Pb²⁺ ions. Conversely, at neutral or lower pH values below approximately 8.8, the surface carries a positive charge, which can lead to electrostatic repulsion and reduced adsorption efficiency.

Effect of adsorbent mass

To determine the optimal dosage of natural apatite for lead ion (Pb²⁺) removal, we investigated the influence of adsorbent mass on the adsorption efficiency. Aqueous solutions with an initial Pb²⁺ concentration of 20 ppm were used, and the mixtures were agitated for 3 h to ensure equilibrium.

Figure 8, illustrates the amount of Pb²⁺ adsorbed as a function of the adsorbent mass for apatite samples calcined at 100 °C (HA-100), 500 °C (HA-500), and 900 °C (HA-900).

Fig. 8

Effect of adsorbent mass on Pb²⁺ removal efficiency (C_Pb2+=20 ppm; t = 3 h; pH = 9; T = 25 °C).

The influence of the adsorbent mass on Pb²⁺ removal efficiency was investigated using natural apatite calcined at three different temperatures: 100 °C (HA-100), 500 °C (HA-500), and 900 °C (HA-900). As shown in Fig. 8, the percentage of Pb²⁺ removal increased with increasing adsorbent mass for all samples, up to a certain point. This behavior can be attributed to the greater number of active sites available on the surface of the apatite as the mass increases, facilitating more interaction between the Pb²⁺ ions and the adsorbent surface²⁸.

Among the three samples, HA-500 exhibited the highest removal performance. The percentage of Pb²⁺ removal rose sharply with increasing mass and reached a plateau of approximately 99% at 0.25 g. This indicates that HA-500 possesses an excellent surface reactivity, likely due to an optimal balance between thermal treatment and surface chemistry, where enough organic matter has been removed to expose active adsorption sites without excessively reducing surface functionality.

In comparison, HA-100 and HA-900 showed lower removal efficiencies, plateauing around 78%. For HA-100, the lower performance is likely due to the presence of residual organic matter that blocks active sites, limiting the interaction between Pb²⁺ ions and the adsorbent. Meanwhile, although HA-900 has undergone significant thermal treatment, its lower efficiency may result from the excessive calcination, which may lead to a more crystalline and less reactive surface, thereby reducing available sites for Pb²⁺ binding.

Effect of contact time

The effect of contact time was studied to determine the time required for the adsorption system to reach equilibrium and to evaluate the adsorption kinetics of Pb²⁺ ions onto natural apatite samples calcined at different temperatures: HA-100, HA-500, and HA-900 (Fig. 9). This investigation helps to compare the adsorption behavior of each material and to identify the sample offering the fastest and most efficient lead ion removal.

Fig. 9

Effect of contact time on Pb²⁺ adsorption by apatite samples (C_Pb2+=20 ppm; m = 0.25 g; pH = 9; T = 25 °C).

The effect of contact time on the adsorption of Pb²⁺ ions by apatite samples calcined at 100 °C (HA-100), 500 °C (HA-500), and 900 °C (HA-900) was investigated to evaluate both the adsorption rate and equilibrium capacity (Fig. 8). The data show that for all three materials, the quantity of Pb²⁺ adsorbed increases rapidly within the first 15 min, which reflects the high availability of active sites on the surface during the initial phase of the process²⁹.

Among the three samples, HA-500 demonstrated the highest adsorption performance, reaching a maximum value of approximately 6.6 mg/g. Equilibrium was reached in under 60 min and remained stable thereafter. This superior performance is likely due to a favorable balance between surface activation and structural integrity achieved at 500 °C, allowing for optimal interaction with Pb²⁺ ions.

In contrast, HA-900 exhibited a slightly lower adsorption capacity (~ 6.2 mg/g). Although its performance is better than HA-100, it may have undergone excessive crystallization or surface densification at high temperature, reducing the number of accessible reactive sites. HA-100, which was the least thermally treated, showed the lowest adsorption capacity (~ 5.5 mg/g). This is likely due to the presence of residual organic matter and limited surface activation, which hinder ion accessibility and adsorption.

Overall, the results confirm that contact time plays a key role in the adsorption process, particularly in the early stages. The equilibrium is generally reached within 60–90 min, beyond which the adsorption capacity stabilizes. The error bars confirm the reproducibility and reliability of the experimental data, with relatively low standard deviation across replicates.

These findings reinforce the conclusion that HA-500 is the most efficient sample in terms of adsorption kinetics and overall Pb²⁺ removal capacity.

Kinetic models

To study the adsorption kinetics of Pb²⁺ onto the synthesized apatite, Lagergren’s pseudo-first-order and pseudo-second-order kinetic models were applied (Fig. 10; Table 2).

Fig. 10

Linear representation of (a) pseudo-first-order and (b) pseudo-second-order models for the studied samples.

Table 2 Kinetic constants and adsorption capacities of pb²⁺ onto HA-100, HA-500, and HA-900 according to different kinetic models.

The adsorption kinetics of Pb²⁺ onto HA-100, HA-500, and HA-900 were analyzed using both pseudo-first-order and pseudo-second-order models. Parameters derived from the linearized plots revealed distinct adsorption behaviors depending on the calcination temperature of the apatite samples. According to the pseudo-first-order model, the calculated adsorption capacities (q_e,1) were relatively low: 3.19 mg/g for HA-100, 1.03 mg/g for HA-500, and 2.21 mg/g for HA-900. The rate constants (K₁) indicated that HA-500 exhibited the highest value (0.0137 min⁻¹), suggesting a faster initial adsorption rate; however, the correlation coefficients (R²) were moderate across all samples, with the highest being 0.920 for HA-900. These observations imply that the pseudo-first-order model only partially captures the adsorption mechanism and may not be the most suitable to characterize the process.

Conversely, the pseudo-second-order model demonstrated an excellent fit for all experimental data, with R² values exceeding 0.999. This strong linearity suggests that Pb²⁺ adsorption onto the apatite surfaces follows chemisorption, likely involving electron exchange or valence bond formation at active sites. The adsorption capacities (q_e,2) calculated from this model were significantly higher and aligned well with experimental findings, especially for HA-100, which reached 5.76 mg/g. Although the rate constant (K₂) was somewhat higher for HA-500 (0.111 g/mg·min) and HA-900 (0.463 g/mg·min) compared to HA-100 (0.026 g/mg·min), the overall kinetic performance remained robust.

These results highlight that the pseudo-second-order model best describes the kinetic behavior of Pb²⁺ adsorption on the studied apatite materials. Among the three, HA-100 showed the highest adsorption capacity and fastest chemisorption kinetics, whereas HA-500 exhibited rapid initial uptake but a lower overall capacity³⁰. HA-900, although slightly slower kinetically, maintained strong model conformity and satisfactory adsorption performance. Overall, calcination temperature plays a crucial role in modulating surface reactivity and adsorption behavior of hydroxyapatite-based materials³¹.

Effect of initial pollutant concentration

The initial concentration of Pb²⁺ is one of the most critical parameters influencing the effectiveness of the adsorption process and the achievement of a maximum removal rate. In this study, the removal efficiency was evaluated using 0.3 g of each prepared adsorbent across various initial concentrations of Pb²⁺ (Fig. 11).

Fig. 11

Effect of initial Pb²⁺ concentration on adsorption capacity (t = 3 h; m = 0.25 g; pH = 9; T = 25 °C).

The graph illustrates the effect of initial Pb²⁺ concentration on the adsorption capacity of three different apatite-based materials: HA-100, HA-500, and HA-900. As the initial concentration of Pb²⁺ increases from 10 to 260 mg/L, the amount of Pb²⁺ adsorbed (in mg/g) increases steadily for all three materials, which is consistent with typical adsorption behavior. This trend can be attributed to the greater availability of Pb²⁺ ions in solution, which enhances the probability of interaction between the ions and active sites on the adsorbent surface³².

Among the three materials, HA-500 exhibits the highest adsorption capacity across all concentration ranges, reaching nearly 50 mg/g at the highest concentration tested. This suggests that the calcination at 500 °C provides an optimal balance between surface area and structural stability, maximizing the availability of active sites for Pb²⁺ adsorption. HA-100 follows closely behind HA-500, indicating that its surface is still highly reactive, albeit slightly less efficient, possibly due to the presence of residual organic matter or incomplete structural development.

In contrast, HA-900 consistently shows the lowest adsorption capacity, which may be attributed to excessive crystallinity and reduced surface area caused by high-temperature calcination. This could lead to a decrease in available active sites, thereby limiting the material’s ability to adsorb Pb²⁺ ions efficiently³³.

Overall, the results clearly demonstrate that both the initial concentration of Pb²⁺ and the calcination temperature of the apatite significantly influence adsorption performance, with HA-500 emerging as the most effective adsorbent under the conditions studied.

Isotherm models

To better understand how Pb²⁺ ions interact with the adsorbents HA-100, HA-500, and HA-900, the Langmuir and Freundlich isotherm models were applied and analyzed (Fig. 12; Table 3).

Fig. 12

Isotherm models: (a) Langmuir and (b) Freundlich (c) Dubinin–Radushkevich (d) Temkin (e) Redlich–Peterson for HA-100, HA-500, and HA-900.

Table 3 Pb²⁺ adsorption parameters based on isotherm models

Based on the isotherm models applied to evaluate the adsorption of Pb²⁺ onto HA-100, HA-500, and HA-900, several key observations can be drawn from the results summarized in the table.

The Langmuir isotherm model, which assumes monolayer adsorption onto a homogeneous surface, shows a strong correlation for HA-100 (R² = 0.959 ± 0.005) and HA-500 (R² = 0.948 ± 0.005). These high R² values suggest that the adsorption behavior of these materials can be reasonably described by Langmuir’s assumptions. The calculated maximum adsorption capacities (q_max) were 98.7 ± 0.2 mg/g for HA-100, 86.6 ± 0.2 mg/g for HA-500, and 72.3 ± 0.2 mg/g for HA-900. These results confirm that HA-100 possesses the highest theoretical adsorption capacity among the three. However, HA-900 exhibits a much lower correlation with the Langmuir model (R² = 0.418 ± 0.005), suggesting that Pb²⁺ adsorption on this material does not predominantly follow monolayer coverage, possibly due to reduced surface availability resulting from excessive crystallinity³⁴.

In contrast, the Freundlich isotherm model, which accounts for adsorption on heterogeneous surfaces and allows for multilayer adsorption, yielded excellent fits for all materials, particularly HA-100 (R² = 0.997 ± 0.005) and HA-500 (R² = 0.983 ± 0.005). The Freundlich constant n was greater than 1 for all samples (HA-100: 1.28, HA-500: 1.38, HA-900: 1.43), indicating favorable adsorption conditions. Moreover, the Freundlich constant K_f, reflecting adsorption intensity, was highest for HA-500 (0.87 ± 0.01 mg/g), followed by HA-900 (0.58 ± 0.01 mg/g) and HA-100 (0.57 ± 0.01 mg/g), suggesting that HA-500 offers a more energetically favorable surface for Pb²⁺ uptake³⁵.

The Dubinin–Radushkevich (D–R) isotherm, used to differentiate between physical and chemical adsorption mechanisms, provided relatively poor fits (R² ranging from 0.518 to 0.759), with calculated mean adsorption energies E greater than 44 kJ/mol for all samples. This implies that chemisorption is likely the dominant mechanism. However, the low R² values, especially for HA-900 (0.518 ± 0.005), limit the reliability of this model for describing the adsorption process on these materials.

Regarding the Temkin model, which considers adsorbate–adsorbent interactions and assumes that the heat of adsorption decreases linearly with coverage, the best correlation was observed for HA-100 (R² = 0.950 ± 0.005), followed by HA-500 (0.934 ± 0.005) and HA-900 (0.792 ± 0.005). The Temkin constant b_T, representing the heat of adsorption, was highest for HA-900 (102.6 J/mol), suggesting stronger binding energies, though not necessarily higher capacity. The a_T values, reflecting binding affinity, were comparable across the three samples (ranging from 0.051 to 0.059 L/mg).

Finally, the Redlich–Peterson (R–P) model, a hybrid between Langmuir and Freundlich, provided further insights. When fitted linearly (assuming g = 1), HA-500 showed excellent agreement (R² = 0.948 ± 0.005), while HA-100 (0.597 ± 0.005) and HA-900 (0.418 ± 0.005) showed significantly lower correlations. The constants a_RP and b_RP, obtained from the linearized form, resulted in estimated overall adsorption intensities K_RP of 98.71 mg/g for HA-100, 86.67 mg/g for HA-500, and 72.31 mg/g for HA-900.

In conclusion, the results across all isotherm models clearly show that HA-500 exhibits the best balance between adsorption capacity, surface affinity, and model conformity, likely due to the optimal porosity and crystallinity achieved at its intermediate calcination temperature. The Freundlich model best describes the overall adsorption behavior for all three materials, highlighting the heterogeneous nature of the hydroxyapatite surfaces. In contrast, the lower performance of HA-900 is attributed to excessive sintering, which reduces active surface sites despite its higher binding energy indicators^34,35.

Thermodynamic studies

To further understand the nature of Pb²⁺ adsorption onto hydroxyapatite, thermodynamic parameters were evaluated by conducting equilibrium experiments at three different temperatures: 298 K, 308 K, 318 K and 328 K (Fig. 13; Table 4).

Fig. 13

Van’t Hoff plots for Pb²⁺ adsorption onto HA-100, HA-500, and HA-900 at various temperatures (C_Pb2+=20 ppm; m = 0.25 g; pH = 9; t = 3 h).

Table 4 Van’t Hoff plots for pb²⁺ adsorption on thermodynamic parameters.

The thermodynamic behavior of Pb²⁺ adsorption onto HA-100, HA-500, and HA-900 was evaluated through Van’t Hoff plots and corresponding parameter calculations. The linear relationship observed between lnKc and 1/T, with high correlation coefficients (R² = 0.970 for HA-100, 0.941 for HA-500, and 0.993 for HA-900), confirms the validity of the thermodynamic model and highlights a consistent temperature-dependent mechanism across all materials.

In terms of enthalpy change (ΔH°), all samples exhibited negative values, confirming the exothermic nature of the adsorption process. HA-100 presented the most negative enthalpy (ΔH° = − 5173.19 J/mol), followed by HA-900 (–2997.87 J/mol), while HA-500 showed a very low value (–529.52 J/mol), suggesting that adsorption on HA-500 is less dependent on energy release and may involve weaker physical interactions³⁶.

The entropy change (ΔS°) was positive for all materials, indicating increased randomness at the solid–liquid interface during Pb²⁺ uptake. This entropy gain is likely due to the release of hydration water molecules and structural rearrangements on the adsorbent surface. HA-100 again recorded the highest ΔS° value (23.67 J/mol·K), reflecting greater configurational freedom during adsorption, followed by HA-500 (17.67 J/mol·K) and HA-900 (13.09 J/mol·K).

The standard Gibbs free energy change (ΔG°) was negative at all tested temperatures (298–328 K), confirming that the adsorption process is spontaneous for all three samples. HA-100 exhibited the most favorable values (ΔG° from − 1.22 to − 1.29 × 10⁴ kJ/mol), followed by HA-900 (–0.69 to − 0.73 × 10⁴ kJ/mol), and HA-500 (–0.58 to − 0.63 × 10⁴ kJ/mol). These results imply that temperature enhances the spontaneity of Pb²⁺ adsorption, particularly for HA-100 and HA-900.

Regarding activation energy (Ea), all values were relatively low, suggesting that the adsorption occurs without significant energetic barriers. HA-500 showed the lowest activation energy (0.53 J/mol), followed by HA-900 (2.99 J/mol), and HA-100 (5.17 J/mol). These low values confirm that Pb²⁺ ions readily interact with available surface sites under ambient conditions³⁷.

Surface coverage (θ) was also calculated to assess the extent of active site occupation. HA-900 showed the highest surface coverage across all temperatures, with θ decreasing slightly from 8.57% at 298 K to 7.05% at 328 K, indicating efficient utilization of surface sites. HA-100 exhibited moderate surface coverage (6.78–5.27%), whereas HA-500 demonstrated the lowest θ values (from 0.92 to 0.57%), suggesting fewer accessible or reactive sites on its surface.

In summary, the thermodynamic evaluation reveals that HA-100 and HA-900 provide favorable conditions for Pb²⁺ adsorption, driven by spontaneous and exothermic interactions with meaningful surface engagement. HA-100, although less stable structurally, exhibits stronger adsorption energetics, while HA-900 balances structural integrity and performance. HA-500, despite a good model fit, appears less thermodynamically active and efficient in terms of site occupation³⁸.

Mechanism of pb²⁺ adsorption onto hydroxyapatite (HA)

The adsorption of lead ions (Pb²⁺) onto hydroxyapatite derived from equine bone occurs through a complex interplay of physical, chemical, and ion-exchange mechanisms, which are influenced by surface characteristics, calcination temperature, and solution chemistry.

At the core of this process lies the unique crystalline structure of HA, represented by the formula Ca₁₀(PO₄)₆(OH)₂. This structure contains calcium (Ca²⁺) sites, phosphate (PO₄³⁻) groups, and hydroxyl (OH⁻) channels, which offer multiple binding sites for heavy metals. The affinity of Pb²⁺ for HA is particularly high due to the similarity in ionic radii between Pb²⁺ (1.19 Å) and Ca²⁺ (1.00 Å), facilitating ion substitution (Fig. 14)³⁹.

Ion exchange

One of the primary mechanisms involved in Pb²⁺ removal is ion exchange between Ca²⁺ ions in the HA lattice and Pb²⁺ ions in solution. This can be expressed by the reaction⁴⁰:

$Ca_{{10}} (PO_{4} )_{6} (OH)_{2} + xPb^{{2 + }} \to Ca_{{10 - x}} Pb_{x} (PO_{4} )_{6} (OH)_{2} + xCa^{{2 + }}$

This substitution process leads to the incorporation of Pb²⁺ into the HA structure, often forming a stable lead-hydroxyapatite complex, which reduces the mobility and bioavailability of lead in aqueous environments.

Surface complexation (chemisorption)

In addition to ion exchange, Pb²⁺ ions can form surface complexes with the functional groups of HA, particularly with phosphate (–PO₄) and hydroxyl (–OH) groups. These interactions are stronger than simple electrostatic attraction and involve partial covalent character. This chemisorption mechanism is supported by kinetic studies that fit the pseudo-second-order model, which is characteristic of chemical adsorption processes⁴¹.

Electrostatic interactions and pH dependence

At pH values higher than the point of zero charge (pHpzc ≈ 9), the HA surface becomes negatively charged, enhancing the electrostatic attraction of positively charged Pb²⁺ ions. Conversely, at lower pH values, the surface becomes positively charged, and repulsion occurs, reducing adsorption efficiency. Therefore, the adsorption is strongly pH-dependent, with optimal performance typically observed in slightly alkaline conditions⁴².

Precipitation and mineralization

At higher pH values or with prolonged contact time, lead precipitation may occur due to the formation of sparingly soluble lead compounds such as Pb₃(PO₄)₂ or Pb(OH)₂ on the HA surface. This process contributes to irreversible adsorption and further immobilization of lead⁴⁰.

Fig. 14

Mechanism of Pb²⁺ adsorption onto Hydroxyapatite (HA).

Effect of calcination temperature on the mechanism

Calcination alters the crystallinity, surface area, and functional group availability of HA:

• HA-100: Lower temperature preserves organic content and results in an amorphous structure with high surface reactivity, favoring ion exchange and surface complexation.

• HA-500: Moderate calcination improves crystallinity while maintaining accessible active sites, providing a balance between structural integrity and surface activity.

• HA-900: High-temperature treatment increases crystallinity and reduces surface area and porosity, thereby limiting the availability of reactive sites and favoring more limited adsorption via lattice substitution.

Desorption and reuse study

The reusability of the calcined hydroxyapatite samples (HA-100, HA-500, and HA-900) was assessed over three consecutive adsorption–desorption cycles using two different eluting agents (0.1 M HCl and 0.1 M NaOH) at varying regeneration temperatures (25 °C, 40 °C, and 60 °C), as shown in Fig. 15 and summarized in Table 5. This comprehensive analysis was designed to evaluate the impact of pH and thermal activation on the stability and regeneration capacity of the materials for long-term heavy metal removal applications.

Fig. 15

Regeneration efficiency over 3 cycles under different conditions.

Table 5 Adsorption efficiency over reuse cycles.

As shown in Fig. 15, all three adsorbents maintained a significant portion of their Pb²⁺ removal efficiency in the first two cycles. However, a progressive decrease in performance was observed in the third cycle, with variations depending on the regeneration conditions. HA-500 demonstrated the most consistent regeneration behavior, especially under NaOH treatment at 60 °C, maintaining 87% removal efficiency in the second cycle and 73% in the third. This result suggests that HA-500, calcined at intermediate temperature, provides a favorable compromise between surface activity and structural resilience, which supports repeated adsorption–desorption cycles.

HA-100 initially showed strong adsorption capacity but experienced a more pronounced decline across cycles, with efficiencies dropping to 72–78% in the second and 55–62% in the third, depending on regeneration temperature and eluent. This deterioration may be related to the presence of residual organics or lower crystallinity, leading to partial surface degradation or blockage of active sites during desorption⁴¹.

In contrast, HA-900, although exhibiting a slightly lower initial adsorption efficiency (95%), displayed superior structural integrity and regeneration performance. Across all tested conditions, it retained 88–92% in the second cycle and 81–85% in the third. This enhanced stability can be attributed to its high crystallinity and compact structure, which limits solubilization or acid/base-induced degradation⁴², although it may slightly reduce the number of accessible active sites.

Overall, the results confirm that calcination temperature plays a pivotal role not only in the initial adsorption efficiency but also in the recyclability of hydroxyapatite-based adsorbents. The performance was also influenced by the choice of regeneration conditions. HA-500, regenerated under alkaline conditions and elevated temperature, emerges as the most efficient and reusable material, combining effective Pb²⁺ uptake with sustained structural robustness.

Comparative study

Table 6 Adsorption capacities of equine bone-derived hydroxyapatite (HA-100, HA-500, and HA-900).

Table 6 represents the adsorption capacities of equine bone-derived hydroxyapatite (HA-100, HA-500, and HA-900) alongside other hydroxyapatite-based and bio-derived adsorbents reported in recent literature for pb²⁺ removal.

The comparative data as shown in Table 6, clearly demonstrate the excellent performance of equine bone-derived hydroxyapatite, particularly the HA-500 sample developed in this study, which achieved 99% removal efficiency at pH 9 with an initial Pb²⁺ concentration of 100 mg/L, using a low adsorbent dose (0.25 g/L) and a contact time of 180 min. When compared to other bone-derived hydroxyapatites reported in the literature, our material performs competitively or better, especially when considering the lower dosage and higher metal concentration tested.

For instance, Huawei Wang et al. (2020)⁴³ reported > 99% Pb²⁺ removal using HAp synthesized from chicken bone at pH 3 and a much higher initial Pb²⁺ concentration of 208 mg/L, but required a higher adsorbent dose (1 g/L) and shorter contact time (30 min). Similarly, Xiaofei Liu et al. (2023)⁴⁴ achieved > 95% removal at pH 5.5 with 50 mg/L Pb²⁺ using 1.25 g/L of chicken bone-derived HAp over 120 min. While these results are excellent, the higher adsorbent dosages and lower Pb²⁺ concentrations highlight the efficiency of our HA-500 under harsher conditions.

In addition, Sabrina Mostofa et al. (2022)⁴⁵ demonstrated 99% removal using fish bone-derived HAp at pH 8 and 25 mg/L Pb²⁺ over 60 min, but again, under much milder initial concentration levels and with incomplete data on adsorbent dosage.

These comparisons confirm that HA-500 stands out not only for its high removal efficiency but also for its performance under demanding conditions (higher pH, higher Pb²⁺ load, and lower adsorbent dosage). This underlines the practical potential of equine bone-derived hydroxyapatite as an efficient and sustainable solution for heavy metal removal from contaminated water.

Conclusion

In this study, hydroxyapatite (HA) was successfully synthesized from equine scapula bone through a straightforward and low-cost thermal process and applied as a bio-adsorbent for Pb²⁺ removal from aqueous solutions. The influence of calcination temperature (100 °C, 500 °C, and 900 °C) on the structural, morphological, and adsorption properties of the resulting materials was systematically examined. Characterization via XRD, FTIR, SEM/EDX, and XRF confirmed that higher calcination temperatures increased crystallinity while simultaneously decreasing surface area and the number of active sites.

Among the prepared materials, HA-500 emerged as the optimal formulation, offering a balanced combination of structural integrity and surface reactivity. It exhibited the highest Pb²⁺ adsorption efficiency, with kinetics best described by the pseudo-second-order model, indicative of chemisorption. Equilibrium data were best fitted to the Freundlich isotherm, suggesting multilayer adsorption on a heterogeneous surface. Furthermore, regeneration tests demonstrated that HA-500 retained a significant portion of its adsorption capacity after three cycles, confirming its potential reusability.

The originality of this work lies in the first reported use of equine bone as a precursor for hydroxyapatite synthesis, providing a novel and sustainable approach to waste valorization for environmental remediation. This study contributes to the development of cost-effective, eco-friendly adsorbents and aligns with the broader objectives of the circular economy and green chemistry.

While the current results confirm the fundamental potential of equine bone-derived HA for lead removal under controlled laboratory conditions, further work is required to validate its practical application. Future investigations will focus on evaluating the adsorbent’s performance in real and industrial wastewater systems, including the effects of coexisting ions, organic matter, and complex matrices. In addition, long-term regeneration behavior, material stability, functionalization for enhanced selectivity, and scale-up feasibility will be addressed to advance the real-world implementation of this bio-based adsorbent.