Biogenic fabrication of biochar-functionalized iron oxide nanoparticles using Miscanthus sinensis for oxytetracycline removal and toxicological assessment

J. Biodiv. & Environ. Sci. Sharmila et al. RESEARCH PAPER OPEN ACCESS Biogenic fabrication of biochar-functionalized iron oxide nanoparticles using Miscanthus sinensis for oxytetracycline removal and toxicological assessment Meenakshi Sundaram Sharmila, Gurusamy, Annadurai* Sri Paramakalyani Centre of Excellence in Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi, Tamil Nadu, India Key words: BC-Fe₃O₄ NPs, Miscanthus sinensis, (OTC) removal, Co-precipitation, Photocatalytic degradation DOI: https://dx.doi.org/10.12692/jbes/27.2.10-20 [ Published: August 07, 2025 ] ABSTRACT This study reports the green synthesis of biochar-coated iron oxide nanoparticles (BC-Fe₃O₄ NPs) using Miscanthus sinensis-derived biochar via a co-precipitation method. The resulting BC-Fe₃O₄ NPs were thoroughly characterized using X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy coupled with Energy-Dispersive X-ray Spectroscopy (SEM-EDX), Thermogravimetric Analysis/Differential Thermal Analysis (TGA/DTA), and Brunauer-Emmett-Teller (BET), which confirmed the successful incorporation of Fe₃O₄ onto the biochar matrix, along with enhanced surface functionality and mesoporosity. Photocatalytic degradation tests demonstrated efficient removal of oxytetracycline (OTC), achieving up to 90% degradation within 60 minutes under UV irradiation, with optimal activity observed at mention pH and dosage. Antibacterial assays revealed significant inhibition zones against Escherichia coli and Serratia marcescens, especially during the initial two hours of treatment. Reusability assessments showed moderate regeneration efficiency across three cycles, whereas zebrafish embryo toxicity tests demonstrated a dose-dependent rise in developmental abnormalities, decreased hatching rates, and increased mortality. Overall, the findings suggest that BCFe₃O₄ NPs hold promise as an eco-friendly, cost-effective material for water purification, though further investigation into their long-term environmental safety. *Corresponding Author: G. Annadurai  [email protected] Journal of Biodiversity and Environmental Sciences | JBES ISSN: 2220-6663 (Print); 2222-3045 (Online) Website: https://www.innspub.net E-mail contact: [email protected] Vol. 27, Issue: 2, p. 10-20, 2025 J. Biodiv. & Environ. Sci. Vol. 27, Issue: 2, p. 10-20, 2025 11 Sharmila et al. Journal of Biodiversity and Environmental Sciences | JBES Website: https://www.innspub.net INTRODUCTION Oxytetracycline, the most commonly used tetracycline antibiotic in animal husbandry, is poorly absorbed and largely excreted in faeces. Its stable molecular structure resists microbial degradation, allowing it to persist in the environment and contribute to pollution. This approach has emerged as a viable solution for wastewater management, particularly in the context of advancing green technologies (Omer et al., 2022). The urgency is further intensified by the rapid depletion of water reserves, driven by the exponential growth of the global population and the impacts of climate change (Eltaweil et al., 2022). In biochar/metal oxide composites, the biochar acts as a porous carbon framework that is surfacefunctionalized with metal oxide, thereby increasing the adsorbent's surface area (Dhila et al., 2025). The biochar (BC) was further modified with manganese and iron (Mn-Fe) to enhance its physicochemical properties and improve its adsorption efficiency for removing Pb²⁺ ions from synthetic wastewater (Ahmed et al., 2021). This study examines the influence of key operational parameterssuch as solution pH, initial metal ion concentration, and adsorbent dosageon the efficiency of Pb²⁺ removal (Shafiq et al., 2025). Nanobiochar can be synthesized from conventional biochar using various techniques, Mechanical grinding is commonly employed to reduce particle size to the nanoscale. In addition to mechanical approaches, flash heating has been used to directly produce graphitic nanosheets (Oleszczuk et al., 2016). utilized an ultrasonic vibrator to disperse biochar particles, followed by sonication to achieve nanoscale dimensions. Among the reported methods, ball milling has emerged as the most effective and widely preferred technique for nanobiochar production (Shui et al., 2016). The Fe oxide/biochar nanocomposite (FeBN) was synthesized using the co-precipitation method. In brief, brewery spent grain was pyrolyzed at 350 °C for 3 hours to produce biochar (Pap et al., 2023). This biochar was subsequently impregnated with FeCl₃·6H₂O through a co-precipitation process (Jin, Ying-Hui et al., 2020). This approach eliminates the need for high-temperature annealing and enhances the adsorption capacity of the magnetic biochar through the photocatalytic properties of ZnO (Zhang et al., 2020). In this study, it was hypothesized that a biochar/iron oxide composite could be successfully synthesized using a green method and would exhibit effective methylene blue (MB) removal capabilities. To test this, banana peel biochar was modified with banana peel extract and FeSO₄ under ultrasonic treatment at room temperature. The resulting material was characterized, and its adsorption behaviour was evaluated (Ahmaruzzaman et al., 2021). A Fe oxide/biochar nanocomposite (FeBN) made from brewery spent grain was tested to remove CLR from water. The highest CLR removal happened at pH 6, with a maximum adsorption capacity (qmax) of 7.91 mg/g at 30 °C (Li et al., 2021). This research presents a sustainable method for synthesizing biochar-coated iron oxide nanoparticles (BC-Fe₃O₄ NPs) using biochar derived from Miscanthus sinensis, targeting the removal of oxytetracycline (OTC) from aqueous environments (Shafiq et al., 2025). The Fe₃O₄ nanoparticles were fabricated via a co-precipitation process and subsequently combined with biochar. Characterization techniques such as XRD, FTIR, SEM, EDX, TGA/DTA, and BET confirmed the successful synthesis and integration of the nanomaterials, highlighting enhanced surface properties and a mesoporous structure. The BCFe₃O₄ NPs demonstrated notable photocatalytic activity, particularly under neutral pH and higher dosage conditions. These findings support the potential of BC-Fe₃O₄ NPs as a cost-effective and environmentally friendly material for wastewater treatment and environmental remediation. J. Biodiv. & Environ. Sci. Vol. 27, Issue: 2, p. 10-20, 2025 12 Sharmila et al. Journal of Biodiversity and Environmental Sciences | JBES Website: https://www.innspub.net MATERIALS AND METHODS Synthesis of biochar-coated iron oxide nanoparticles (BC-Fe₃O₄ NPs) Materials All chemicals used were of analytical grade and used without further purification. Ferric chloride hexahydrate (FeCl₃·6H₂O), ferrous sulfate heptahydrate (FeSO₄·7H₂O), and sodium hydroxide (NaOH) were obtained from Vishnu Priya Chemicals Pvt Ltd. Biochar was prepared from Miscanthus sinensis by pyrolysis at in a muffle furnace under 700°C a nitrogen atmosphere (Kumar et al.,2022). Preparation of biochar Miscanthus sinensis raw plant was washed, dried at 105°C for 24 h, and pyrolyzed at 700°C for 2 h in a nitrogen environment. The resulting biochar was ground and sieved to a particle size <100 µm. Biochar made from Miscanthus sinensis was passed through a sieve to get particles around 75 µm in size. Then, 5 g of this biochar was placed in a 500 mL conical flask with 2 M citric acid and shaken for 24 hours (Tang et al., 2013). After that, the mixture was spin in a centrifuge at 3000 rpm for 10 minutes. The biochar was washed two times with deionized water to remove any leftover acid, dried at 60 °C, and then stored in clean culture tubes for later use. Synthesis of Fe₃O₄ nanoparticles Iron oxide nanoparticles were synthesized the coprecipitation method Scheme 1. shows briefly, FeCl₃·6H₂O and FeSO₄·7H₂O were mixed in a 2:1 molar ratio in deionized water under nitrogen atmosphere with vigorous stirring at 80°C. Aqueous NaOH (1 M) was added dropwise until the pH reached 10, resulting in the formation of a black precipitate of Fe₃O₄ nanoparticles. The precipitate was magnetically separated, washed with deionized water and ethanol, and dried under vacuum at 60°C (El-Abid et al., 2023) (Fig. 1). Biochar coating iron oxide nanoparticles The dried Fe₃O₄ nanoparticles were dispersed in deionized water containing a known amount of biochar (typically 1:1 w/w ratio). The suspension was sonicated for 30 min and then stirred at 60°C for 4 h to allow effective adhesion of biochar onto the nanoparticle surfaces. The coated nanoparticles were magnetically separated, washed with water to remove unbound biochar, and dried under vacuum (Salim et al., 2023). Fig. 1. Schematic illustration of (BC-Fe₃O₄ NPs) synthesis Photocatalytic activity The efficiency of BC-Fe₃O₄ nanoparticles in removing (OTC) from an aqueous solution was evaluated. To ensure effective removal, 0.5 g of BC-Fe₃O₄ nanoparticles was added to 50 mL of a 10 ppm OTC solution in a 100mL vial. The mixture was stirred at room temperature for 25 minutes. At predetermined time intervals, samples were withdrawn from the vial to measure the remaining OTC concentration using a UV spectrophotometer (Akhtar et al., 2014). The percentage of OTC removal was calculated using the following equation: where C0 is the initial concentration of OTC and C is the OTC concentration after a specific time. Antibacterial activity The microorganisms used in this study were isolated from soil and included Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Bacillus species, and Serratia marcescens. All bacterial strains were obtained from 24-hour-old cultures. Two oxytetracycline (OTC) adsorption J. Biodiv. & Environ. Sci. Vol. 27, Issue: 2, p. 10-20, 2025 13 Sharmila et al. Journal of Biodiversity and Environmental Sciences | JBES Website: https://www.innspub.net samples, prepared in water, were analyzed at various time intervals: 15, 30, 45, and 60 minutes. Sterile discs were soaked in these samples and placed on agar plates inoculated with the test microorganisms. Discs containing untreated OTC served as control. The plates were incubated at 37 °C for 24 hours. Antibacterial activity was assessed by measuring the diameter of the inhibition zones around each disc, with results expressed in millimetres (mm) (Huang et al., 2014). Regeneration of BC-Fe3O4NPs To assess the recyclability of BC-Fe₃O₄ nanoparticles (NPs), a three-cycle regeneration test was conducted at room temperature. After each cycle, the spent adsorbent was separated from the antibiotic solution by centrifugation at 4000 rpm for 15 minutes. The collected BC-Fe₃O₄ NPs were then washed with 15 mL of ethanol, followed by 20 mL of distilled water, and shaken at 120 rpm for 45 minutes (Saraswathy, and Shaoqin et al., 2014). The regenerated BC-Fe₃O₄ NPs were dried in a hot air oven for 2 hours and subsequently reused for the removal of OTC. All regeneration experiments were carried out in triplicate. Zebrafish embryo toxicity study Zebrafish (Danio rerio) were maintained in water containing 18 g of ocean salt, 75 g of sodium bicarbonate (NaHCO₃), and 8.4 g of calcium sulfate (CaSO₄) per 1000 liters. Wild-type zebrafish were cultured under these conditions. Additional experiments were conducted in simulated marine environments. The night before spawning, adult zebrafish were placed in breeding tanks at a male-tofemale ratio of 2:1. They were kept under a 12-hour light and 9-hour dark photoperiod. Fertilization occurred within one hour after the lights were turned on the next morning. Viable embryos were collected and incubated in embryo medium. This medium contained 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄. The pH was adjusted to 7.2– 7.3. The embryos were maintained at a temperature of 28 ± 1°C with adequate dissolved oxygen (Chen et al., 2015). All experimental procedures were approved by the Institutional Animal Ethics Committee at the Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi. The study followed international guidelines for the care and use of laboratory animals. RESULTS AND DISCUSSION Scanning electron microscopy-energydispersive spectroscopy (SEM-EDX) analysis Fig. 2 (a), at lower magnification, displays layered, flake-like particles with rough surfaces. This morphology is typical of materials like clay minerals, graphene derivatives, or layered double hydroxides (LDHs). Fig. 2 (b), taken at higher magnification, reveals sharper edges and a lamellar (sheet-like) structure, confirming the flake-like morphology. Fig. 2. The SEM-EDX image of (a) &(b) BC-Fe₃O₄ NPs and (c) EDX analysis (BC-Fe₃O₄ NPs) Fig. 2 (c) presents the EDX spectrum. It shows a high-intensity carbon (C) peak, indicating a carbonrich structure, such as biochar. Oxygen (O) is also present, which is common in oxides or hydroxylated surfaces. Potassium (K), calcium (Ca), and magnesium (Mg) suggest the presence of mineral impurities. Iron (Fe) may be from iron oxides or natural contamination. Chlorine (Cl) might have been introduced during synthesis or sample preparation. Scandium (Sc) appears in trace amounts and may be a contaminant or analytical artifact, needing further verification. Overall, the J. Biodiv. & Environ. Sci. Vol. 27, Issue: 2, p. 10-20, 2025 14 Sharmila et al. Journal of Biodiversity and Environmental Sciences | JBES Website: https://www.innspub.net SEM and EDX results show that the material has a flake-like, layered structure. Its composition is mainly carbon and oxygen, with iron oxides and minor mineral components (Xie et al., 2022). X-Ray diffraction (XRD) analysis The biochar pattern displays a broad, intense peak centered around 2θ ≈ 23–30°, indicating an amorphous or poorly crystalline structure. This broad hump is typical of carbon-rich biochar due to disordered carbon stacking. In contrast, the Fe₃O₄– biochar composite exhibits several sharp peaks between 2θ ≈ 25–50°, indicating the presence of crystalline phases. These peaks suggest the successful incorporation of iron-based crystalline compounds into the biochar matrix. The XRD pattern of pure Fe₃O₄ nanoparticles shows sharp, distinct peaks, confirming their crystalline nature (Mou et al., 2016). Major diffraction peaks appear at 2θ ≈ 30.1°, 35.5°, 43.1°, 53.4°, 57.0°, and 62.6°, corresponding to the (220), (311), (400), (422), (511), and (440) crystal planes, respectively. Fig. 3. The XRD image of BC-Fe₃O₄ nanoparticles Thermogravimetric analysis (TGA) and DTA Thermogravimetric analysis (TGA) was conducted to assess the thermal stability of BC-Fe₃O₄ NPs at a heating rate of 10 °C/min under a gas mixture of 60% nitrogen and 40% air, as shown in Fig. 3. The TGA curve of the biochar displayed a two-step weight loss of approximately 11% upon heating from room temperature to 750 °C. An initial minor weight loss of about 0.343 mg at 66.0 °C corresponds to moisture evaporation. A more significant mass loss between 112.8 °C and 184.8 °C (8.573 mg and 8.229 mg) indicates the decomposition of volatile organic components. Further weight losses from 374.4 °C to 598.2 °C (7.769 mg, 6.937 mg, and 5.112 mg) suggest the progressive thermal degradation or combustion of the residual material. The DTA curve shows both endothermic and exothermic events. An endothermic peak appears at 177.7 °C (−25.43 μV), which may be due to dehydration or melting (Ntoutoume et al., 2016). After that, an exothermic peak is seen at 257.7 °C (61.237 μV), possibly caused by oxidation or the start of decomposition. Strong exothermic peaks are observed at 336.79 °C (130.244 μV), 468.47 °C (190.48 μV), and 606.33 °C (120.95 μV). These peaks suggest major decomposition or combustion processes. Moisture is lost below 100 °C. In Fig. 4 shows major decomposition happens between about 100 °C and 600 °C in several stages. The exothermic peaks confirm chemical changes like oxidation and decomposition. Fig. 4. Thermogravimetric analysis (TGA) Fourier transforms infrared spectroscopy (FTIR) FT-IR spectra of biochar and BC-Fe₃O₄ were recorded in the range 4000–400 cm⁻¹ to identify functional groups involved in nanoparticle capping and stabilization (Fig. 5). The spectrum of pristine biochar shows a broad band at 3400.56 cm⁻¹, corresponding to O–H stretching of hydroxyl groups, and a peak at 2906.17 cm⁻¹ due to aliphatic C–H stretching. The band J. Biodiv. & Environ. Sci. Vol. 27, Issue: 2, p. 10-20, 2025 15 Sharmila et al. Journal of Biodiversity and Environmental Sciences | JBES Website: https://www.innspub.net at 1400.58 cm⁻¹ is assigned to aromatic C=C stretching, while the peak at 1200.36 cm⁻¹ is attributed to C–O stretching from carboxylic acids, esters, or ethers. Following Fe₃O₄ modification, notable spectral changes occur. The O–H band shifts to 3407.23 cm⁻¹ with reduced intensity, suggesting interaction with Fe₃O₄. The C–H band shifts to 2923 cm⁻¹, and a new peak at 1583.34 cm⁻¹ appears, corresponding to C=O stretching of carboxyl groups stabilized by iron. A band at 1349.24 cm⁻¹ may indicate C–H bending or C–N stretching, reflecting structural reorganization (Abruzzo et al., 2016). The peak at 1074 cm⁻¹ is ascribed to C–O or Fe– O stretching, and a distinct signal at 473 cm⁻¹ confirms Fe₃O₄ deposition. Fig. 5. The FTIR image of BC-Fe₃O₄ Wavenumber (cm⁻¹) Functional group Sample 3407.23 O–H stretching (hydroxyl group) Fe coated biochar 2923.00 C–H stretching (alkyl group) Fe coated biochar 1583.34 C=C stretching (aromatic ring) Fe coated biochar 1349.24 C–N stretching (amine group) Fe coated biochar 1074.00 C–O stretching (alcohol/ether group) Fe coated biochar 473.20 Fe–O vibration (metal-oxygen bond) Fe coated biochar 3400.56 O–H stretching (hydroxyl group) Biochar 2906.17 C–H stretching (alkyl group) Biochar 1400.58 C–C stretching (aromatic ring) Biochar 1200.36 C–O stretching (alcohol/ether group) Biochar 470.36 Possible mineral/metal oxide vibration Biochar Brunauer-emmett-teller analysis (BET) The surface area, pore size, and pore volume of BCFe₃O₄ nanoparticles (NPs) were analyzed using nitrogen adsorption–desorption isotherms at 78.350 K while the Barrett–Joyner–Halenda (BJH) method was applied to measure the pore size. The BC-Fe₃O₄ NPs exhibited an average surface area of 3.9466 m²/g and an average pore diameter of 39.248 nm. These values are attributed to the presence of slit-like pores and a Type II isotherm, which indicates the formation of larger mesopores. In this Fig. 6 shows the suggests that the Fe₃O₄ coating altered the biochar's structure, influencing both its surface characteristics and pore architecture (Hazrati et al., 2021). Overall, these structural properties make BC-Fe₃O₄ NPs attractive for applications such as catalysis, environmental remediation, and other fields that benefit from high surface area and well-defined porosity. Fig. 6. (a) N2 adsorption-desorption isotherms graph and (b) Pore volume distribution of the BC-Fe₃O₄ nanoparticles (NPs) Photocatalytic activity Mechanism of photocatalytic activity The image illustrates the photocatalytic degradation process of pharmaceutical compounds using a biochar/Fe₃O₄ composite under UV light. Upon exposure to UV irradiation, electrons in the valence band of the Fe₃O₄ nanoparticles are excited to the conduction band, resulting in the formation of electron-hole pairs (Pathy et al., 2023). These charge carriers interact with oxygen (O₂) and water (H₂O) molecules present in the surrounding environment, leading to the generation of reactive oxygen species (ROS) such as superoxide radicals (O₂⁻) and hydroxyl radicals (•OH). These ROS play a crucial role in degrading pharmaceutical pollutants depicted in the image by a red-white capsule and a chemical structure by breaking down the complex molecules into simpler, non-toxic byproducts. This mechanism demonstrates the effectiveness of biochar/Fe₃O₄ J. Biodiv. & Environ. Sci. Vol. 27, Issue: 2, p. 10-20, 2025 16 Sharmila et al. Journal of Biodiversity and Environmental Sciences | JBES Website: https://www.innspub.net composites in environmental remediation through photocatalytic degradation (Fig. 7). Fig. 7. Reaction mechanisms for the degradation of OTC BC-Fe₃O₄ NPs + hν(UV) → BC-Fe₃O₄ NPs (e−(CB) + h+(VB)) H2O+ h+(VB) → OH˙ + H+ (O2−).O2 + e−(CB) → O2−˙ (OH˙).O2−˙+ H+ ⇄ HOO˙ 2HOO˙ → H2O2+ O2 H2O2→ 2OH˙ + OH˙ → CO2 + H2O (Antibiotics intermediates) OTC + h+(VB) → oxidation products OTC + e−(CB) → reduction products OTC + hν → OTC* OTC* + BC-Fe₃O₄ NPs → OTC+ BC-Fe₃O₄ NPs − OTC The main absorbance peaks of OTC appear between 200–400 nm, with distinct maxima around 275 nm and 360 nm. Over time, a reduction in absorbance reflects the progressive degradation of OTC. The control sample exhibits the highest absorbance, confirming that no degradation occurs in the absence of treatment. In contrast, after 60 minutes of exposure to BC-Fe₃O₄, a substantial decrease in absorbance is observed, indicating effective OTC removal likely driven by photocatalytic activity. The BC-Fe₃O₄ sample alone displays minimal absorbance, with a minor peak near 240 nm and an almost flat spectrum beyond 300 nm, suggesting it has negligible intrinsic absorbance and does not interfere with OTC detection. These UV-Vis results demonstrate that BC-Fe₃O₄ enables timedependent degradation of OTC, achieving approximately 90% degradation after 60 minutes (Peiris et al., 2017). In Fig. 8 relatively linear degradation trend further supports the effectiveness of BC-Fe₃O₄ as a photocatalyst for OTC removal. Fig. 8. UV-Visible spectra of BC-Fe₃O₄ nanoparticles (NPs) The dosage percentage increases over time for all three dosages. Among them, the 0.5 g dosage consistently exhibits Fig. 9 the highest percentage, reaching nearly 90% at 60 minutes. The 0.05 g dosage follows, achieving around 85%, while the 0.005 g dosage shows the lowest performance at approximately 80% after 60 minutes. These results indicate that the efficiency of BC-Fe₃O₄ improves with increasing dosage, with the 0.5 g dosage being the most effective in reaching a higher dosage percentage within the 60-minute time frame (Danner et al., 2019). Fig. 9. Maximum amount of antibiotics degradation of model antibiotics at different dosage with time J. Biodiv. & Environ. Sci. Vol. 27, Issue: 2, p. 10-20, 2025 17 Sharmila et al. Journal of Biodiversity and Environmental Sciences | JBES Website: https://www.innspub.net Fig. 10. Maximum amount of antibiotics degradation of model antibiotics at different pH with time pH with time Fig. 10 illustrates the percentage increase over time at different pH levels (pH 4, pH 7, and pH 8) for BCFe₃O₄, likely reflecting its adsorption efficiency or degradation rate. At pH 4, there is a steady increase, reaching approximately 83% at 60 minutes. At pH 7, performance slightly surpasses that of pH 4 after 40 minutes, achieving the highest overall efficiency of around 88% at 60 minutes. pH 8 shows the fastest initial increase, reaching about 70% within 30 minutes, but plateaus around 80% after 45 minutes. This suggests that pH 8 provides the quickest initial reaction or adsorption rate, while pH 7 offers the best overall performance. Although pH 4 starts off with the slowest rate, it nearly matches pH 8 by the end of the experiment (Felis et al., 2020). Overall, pH 7 is optimal for BC-Fe₃O₄, pH 8 yields the fastest initial response, and pH 4 is the least efficient, though its performance improves over time. Antibacterial activity BC-Fe₃O₄ exhibits the highest antibacterial activity within the first 15 minutes to 2 hours of exposure, showing inhibition zones between approximately 21% and 24%. Activity is moderate during this early period but significantly declines at 12 and 48 hours, indicating a time-sensitive release profile with peak efficacy shortly after administration. Among the tested bacteria, E. coli and Serratia marcescens are the most sensitive, showing the greatest inhibition, while Klebsiella pneumoniae and Bacillus subtilis exhibit lower sensitivity, particularly at extended exposure times. Staphylococcus aureus displays a consistent decline in response, highlighting reduced antibacterial efficacy over time. Overall, Fig. 11 the findings suggest that BC-Fe₃O₄ loaded with OTC provides time-dependent antibacterial activity, with rapid release and short-term exposure (within 2 hours) being critical for optimal performance (Wang et al., 2019). Future formulations may benefit from strategies to prolong drug release or enhance stability for sustained antibacterial effects. Fig. 11. BC-Fe₃O₄ -Oxytetracycline antibacterial activity Regeneration and reusability of BC-Fe₃O₄ BC-Fe₃O₄ illustrates the regeneration efficiency of BC-Fe₃O₄ over three cycles at two dosages: 0.5g/50ml and 0.05g/50ml.In the first cycle, both dosages demonstrate high regeneration efficiencies (above 80%), with the 0.05g/50ml dosage Fig. 12 showing slightly better performance. By the second cycle, regeneration efficiency declines for both dosages, falling to approximately 60–62%, indicating a reduction in the material’s regenerative capacity. In the third cycle, the 0.05g/50ml dosage continues to outperform the 0.5g/50ml dosage slightly, maintaining around 68% efficiency compared to 62%. Overall, the data suggests that BC-Fe₃O₄ retains moderate regeneration efficiency over three cycles, though performance diminishes with each use. The lower dosage (0.05g/50ml) consistently yields slightly better results, implying it may be J. Biodiv. & Environ. Sci. Vol. 27, Issue: 2, p. 10-20, 2025 18 Sharmila et al. Journal of Biodiversity and Environmental Sciences | JBES Website: https://www.innspub.net more effective or sustainable for repeated regeneration (Shakoor et al., 2020). Fig. 12. Recyclability study of BC-Fe₃O₄ CONCLUSION This study successfully demonstrated the green synthesis of biochar-coated iron oxide nanoparticles (BC-Fe₃O₄ NPs) using Miscanthus sinensis for the efficient removal of oxytetracycline (OTC) from water. The synthesized nanocomposites exhibited favorable physicochemical characteristicssuch as increased surface area, mesoporosity, and functional group modifications—confirmed through XRD, FTIR, SEMEDX, TGA-DTA, and BET analyses. Photocatalytic experiments showed a notable OTC degradation efficiency of up to 49% within 60 minutes under UV light, with optimal results observed at neutral pH and higher nanoparticle dosages. Furthermore, BC-Fe₃O₄ NPs displayed time-dependent antibacterial activity, particularly against E. coli and S. marcescens, and maintained moderate regeneration efficiency over three reuse cycles. In conclusion, BC-Fe₃O₄ NPs represent a promising and environmentally friendly material for wastewater treatment and environmental remediation. ACKNOWLEDGEMENTS Authors thank the Service of the instruments of the Manonmaniam Sundaranar University, Alwarkurichi and Periyar University for the SEMEDS, FTIR, TGA images. Sharmila M. Research Scholar (Register No: 22114012052014) acknowledges the Research centre, Sri Paramakalyani Centre of Excellence in Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi, for providing the support for this research work. REFERENCES Abruzzo A, Zuccheri G, Belluti F, Provenzano S, Verardi L, Bigucci F, Cerchiara T, Luppi B, Calonghi N. 2016. 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