Photocatalytic degradation of tetracycline and ciprofloxacin antibiotic residues in aqueous phase by biosynthesized nZVI using Sal (Shorea robusta) leaf extract

Antibiotics are chemicals active against bacterial infections and are used in human medication, veterinary medication, aquaculture additives, poultry industry additives, and agriculture growth (Le et al. 2018; Ben et al. 2019). A significant portion of these antibiotics remain unmetabolized and are excreted into the environment. Antibiotic residues are persistent and adversely affect the environment and human health (Al Omari et al. 2019; Ben et al. 2019). Increased application of antibiotics has led to increased antibiotic residues in surface water, groundwater, wastewater and soil, and their concentration of up to a few μg L⁻¹ has been reported in the natural water system. Antibiotic residue in water can form antibiotic-resistant bacteria and antibiotic-resistant genes through mutational or genetic changes, death of sensitive microorganisms, and altering the microbiome even at low concentrations. This can further lead to antibiotic resistance, treatment failure, and mortality in humans. Antibiotic residues in wastewater are toxic to algae and aquatic animals and are potential mutagens and carcinogens (Tan et al. 2015; Ezzariai et al. 2018).

Tetracycline (TC) is a broad-spectrum antibiotic used in the treatment of gram-negative, gram-positive bacteria, fungus, and other microorganisms (Hou et al. 2020). TC is one of the most used antibiotics worldwide, has a long half-life, and is non-biodegradable and highly soluble in water. TC is extensively used for medical treatment, livestock, and aquaculture. TC is frequently detected in groundwater, surface water, wastewater, and soil and can form different byproducts depending on pH and other environmental conditions. The toxic effect of TC has been reported on plants, sewage sludge bacteria, and non-target organisms (Guo et al. 2017). TC also acts as a chelating agent for metal ions, forming complexes without reducing their negative impacts in an aqueous solution.

Ciprofloxacin (CIP) is the second generation of fluoroquinolone antibiotics, effective against gram-negative and some gram-positive bacteria. CIP is an extensively used antibiotic found in high concentrations in hospital wastewater and wastewater treatment plants, up to a few mg L⁻¹ (Nawaz et al. 2020). CIP and its residues can have an eco-toxic effect and lead to the formation of antibiotic-resistant genes (Orimolade et al. 2020).

Conventional treatment plants are insufficient in removing antibiotic residues from wastewater, resulting in their penetration into receiving water bodies (Guo et al. 2017; Orimolade et al. 2020). Antibiotics have been successfully treated by the application of nanotechnology in recent times. Nanoparticles are materials with dimensions less than 100 nm (Li et al. 2021). Nanoparticles can be zero-, one-, two, or three-dimensional based on their shape. Nanoparticles generally have three distinct layers; the core, shell, and surface.

nZVI can be synthesized by the bottom-up or top-down method (Stefaniuk et al. 2016). The top-down approach utilizes the diminution and processing of the precursor to produce nanoparticles, while the bottom-up process uses simpler molecules to form nZVI. Recent technologies of nZVI synthesis include precision milling, carbothermal method, ultrasound-assisted method, electrochemical method, and green synthesis (Stefaniuk et al. 2016). Precision milling works by milling the iron to produce particles in the nano-size range, but their shapes are irregular. The carbothermal process uses Fe²⁺ salts, elevated temperature (>500 °C), and H₂, CO₂, or CO as reducing agents. Ultrasound can be combined with chemical methods to produce significantly smaller particles, but this process can simultaneously oxidize the nZVI. The electrochemical method utilizes Fe²⁺/Fe³⁺ salts in solution, and nZVI accumulates at the cathode, but this method is affected by aggregation. Green synthesis is the most environmentally friendly method of nZVI synthesis, eliminating the use of harmful chemicals and high temperatures, and is economical, with a possibility of large-scale synthesis (Jha & Chakraborty 2020). In an aqueous solution, nZVI produces highly reactive species such as superoxide ions, electrons, hydroxide radicles, and hydroxide ions, which helps in the degradation of organic pollutants (Jha & Chakraborty 2020). nZVI has proven to be highly efficient in the treatment of environmental pollutants. nZVI can remove pollutants through adsorption, oxidative degradation, precipitation, and reductive degradation (Ezzatahmadi et al. 2017). The hypothesis of this study lies in the fact that nZVI corrodes to form Fe²⁺, which activates H₂O₂ to form hydroxyl radicals and superoxide ion, having great oxidizing capabilities toward degradation of antibiotics by hydroxylation, ring opening, and fragmentation (Li & Liu 2021). Zhao et al. (2020) synthesized zeolite-supported nZVI for the removal of Norfloxacin and Ofloxacin at a concentration of 10 mg L⁻¹ with an efficiency of 90% in 1 h and a maximum uptake-capacity of 54.67 and 48.88 mg g⁻¹, respectively. Leili et al. (2018) performed adsorption of cephalexin by nZVI with an uptake-capacity of 1,667 mg g⁻¹ and an efficiency of 83.8% at pH 2 in 2 h. Tran et al. (2020) applied 0.5 g L⁻¹ platinum/nZVI composite for 100% degradation of oxytetracycline in 20 min at pH 5. Hou et al. (2020) successfully utilized nZVI loaded metal-organic framework to treat TC, with an efficiency of 90% and an uptake-capacity of 625 mg g⁻¹ in a broad pH range. A search was conducted on the web of science (https://www.webofscience.com/wos/woscc/basic-search) on the 26th of February 2022, with the topic ‘antibioti*nZVI’. The search resulted in 101 articles, including 97 research articles and 4 review articles. Results were filtered for research articles and shorted by the highest citation first. The first article was published in 2009. The top 10 most cited articles are provided in Supplementary Table S1, and the top 5 most cited authors, organizations, countries, publishers, journals, and research areas are provided in Supplementary Table S2. The literature review from the web of science indicated that most of the research on removal of antibiotics using nZVI composites was based on the adsorption mechanism. Composites of nZVI and activated carbon, biochar, carbon nanotubes, chitosan, clay, graphene oxide, reduced graphene oxide, silica, and zeolite have been successfully used as an adsorbent. While for degradation studies, bimetallic composites of TiO₂, Ni, and Cu have been applied for degradation of antibiotics. Moreover, most of the reported degradation processes are assisted by fenton, sonocatalysis, flocculation, or membrane. Reports on degradation on antibiotics using only nZVI are limited. These processes incur more cost and incomplete solution for treatment of antibiotics. Thus, in our study, we have attempted application of biosynthesized nZVI, for treatment and complete degradation of TC and CIP in the presence of UV light.

The two antibiotics, ciprofloxacin (C₁₇H₁₈FN₃O₃) and tetracycline (C₂₂H₂₄N₂O₈), were obtained from Alfa Aesar, ferrous sulfate heptahydrate (FeSO₄·7H₂O), which was used as the precursor for nZVI, was obtained from Merck, and sulfuric acid (H₂SO₄) and sodium hydroxide (NaOH) were obtained from Rankem. Further work was performed by Milli-Q water, and all the chemicals were used without further purification.

Stock solutions of CIP (500 mg L⁻¹) and TC (500 mg L⁻¹) were prepared by dissolving 500 mg of CIP and TC in 1,000 ml of Milli-Q water separately and mixed homogeneously using an ultrasonic homogenizer (Aczet APU 500). These were diluted to 100 mg L⁻¹_, and the working solutions (10–35 mg L⁻¹) were prepared by successive dilution with Milli-Q water. The concentration range was selected based on the fact that the maximum CIP concentration of up to 31 mg L⁻¹ was reported in raw wastewater of the Indian subcontinent by Bhagat et al. (2020) and the TC concentration of up to 20 mg L⁻¹ has been reported from animal manure (Winckler & Grafe 2001). Globally, the concentration of antibiotics in hospitals and urban wastewater has been reported to be in the range of 0.3–100 mg L⁻¹ (Abbasnia et al. 2022; Parmar & Srivastava 2022).

nZVI was biosynthesized according to the previous method (Jha & Chakraborty 2020) with slight modification. Leaf extract was used as the reducing agent for the precursor, FeSO₄·7H₂O. For this, matured leaves from Shorea robusta (Sal) were plucked from BIT Mesra campus, Ranchi, Jharkhand, India (23.418359°N, 85.438360°E) and rinsed with Milli-Q water to remove adhered impurities, blot-dried, and air-dried, respectively. 20 g of leaves were weighed and crushed using mortar and pestle with water to a fine paste, and the final volume of water was made to 200 ml (1 g of leaf per 10 ml of water) and finally filtered using a membrane filter (pore size 45 μm). For future consumption, the filtrate was stored at 4 °C.

To synthesize nZVI, 50 ml of 0.01 M FeSO₄·7H₂O solution was taken in Erlenmeyer flasks, and 50 ml of the Sal leaf extract was added dropwise at room temperature and placed in an orbital shaker at 100 rpm. The formed particles were characterized by various methods. The particles were separated from the liquid phase by the vacuum filtration technique, washed with absolute ethanol and stored submerged in ethanol to avoid oxidation of nZVI.

The general characterization of the formed particles was performed using various techniques. The morphology of the formed particles was determined using transmission electron microscopy (TEM; CM 200; operating voltage: 20–200 kV) and atomic force microscopy (AFM; NT-MDT, Russia; Solver Pro-4). Particle size was determined using light scattering (Make: Malvern Instruments, UK, Nano ZS, 4.0 mW, 633 nm) and TEM images. Energy-dispersive X-ray spectroscopy (Make: JOEL JSM-6390LV) was employed to determine the elemental composition. X-ray diffraction (XRD; Rigaku, Japan, SmartLab 9 kW) was used to confirm the synthesis of nZVI. The determination of the functional groups in the Sal leaf extract and the synthesized particles was done using Fourier transform infrared spectroscopy (FTIR; Make: Shimadzu Corporation, Japan, IR-Prestige 21, Range: 4,000–400 cm⁻¹). FTIR indicated the probable functional groups responsible for the reduction of FeSO₄·7H₂O and the functional groups which could help in the degradation of antibiotics.

To study the effects of light, control experiments were set up in dark and sunlight. Initial concentrations of TC and CIP were varied as 10, 15, 20, 25, 30, and 35 mg L⁻¹; pH as 2, 4, 6, 8, 10, and 12. The adjustment in the pH of antibiotic solutions was carried out using HCl (0.1 M) and NaOH (0.1 M). Doses of nZVI varied as 0.0035, 0.0070, 0.0105, 0.0140, 0.0175, and 0.0210 g L⁻¹. The stirring rate was varied as 100, 200, 300, 400, and 500 rpm, and time was optimized up to 70 min with equal intervals of 10 min. Mass spectrometry (Thermo Scientific LTQ-XL) of the degraded antibiotics solutions was performed in the range of 150–500 m/z to determine the degraded products.

To study the toxic effect of photocatalytically degraded TC and CIP solution on microorganisms, a bioassay was performed by the cup plate technique. The water sample was collected from the Subarnarekha River (23.4071275°N, 85.4390985°E). Bacterial strains were isolated through serial dilution and plating on a nutrient agar plate. Two bacterial strains were isolated and sub-cultured using nutrient broth media based on the distinct colors. Gram staining, as well as bioassay, was performed by the standard method. Three cups were bored, marked as A, B, and C, and filled with initial concentrations of antibiotics, degraded antibiotic solutions, and Millipore water, respectively, and incubated at 37 °C. After 24 h, the zone of inhibition was measured to study the impact on bacterial strain.

The zone of inhibition was recorded after incubation as an indication of the toxicity of the bacterial strains.

FTIR was performed for Sal leaf extract and nZVI in the range of 500–4,000 cm⁻¹ (Figure 3(b)). The broad peak at 3,323 cm⁻¹ could be the stretching vibration of the sample's hydroxyl group of alcohol, carboxyl, phenol, or water, while the broad peak at 3,286 cm⁻¹ could be due to O–H stretching in polyphenols. The peak marked C–O stretching due to primary alcohol at 1,045 and 1,230 cm⁻¹, while C = O stretching due to ketones and alkyl group was marked by the peak at 1,654 cm⁻¹. The presence of the C–H bond of alkyl group was confirmed by the peaks at 2,854 and 2,924 cm⁻¹. Sal leaf contained alcohols and polyphenols, which were possibly responsible for the reduction of precursor to nZVI and acted as the capping agent for nZVI, preventing it from rapid oxidation (Rashtbari et al. 2020). The presence of C = O stretching as well as O–H stretching was marked by the peak at 1,658 and 3,317 cm⁻¹. Polyphenol in the nZVI suggested its role as the capping agent, while the O–H group can play a role in the degradation of TC and CIP.

XRD of nZVI was performed in the 2θ range 5–85°. The characteristic peak of Fe⁰ at 44° confirmed the presence of nZVI (Qu et al. 2020). Various oxides of iron were present in the synthesized material, as confirmed by the XRD peaks. The UV–Visible spectra of nZVI indicated its peak at 215 and 264 nm (Supplementary Figure S2). Similar results were obtained by Pattanayak & Nayak (2013).

The antibiotic concentrations varied from 10 to 35 mg L⁻¹, with a linear increase of 5 mg L⁻¹. For both antibiotics, the degradation efficiency first increased with an increase in concentration and then gradually decreased. At 15 and 25 mg L⁻¹ of TC and CIP, optimum degradation efficiencies of 80.8 and 77.6% were achieved, respectively. This can be attributed to the fact that an increase in antibiotic concentration increases the number of antibiotic molecules, thus increasing the number of molecules reaching the active sites (Gupta et al. 2020). Once the equilibrium is attained, active sites of nZVI get saturated. Due to the lack of sufficient active sites in nZVI, a further increase in antibiotic concentration reduces degradation efficiency (Gupta et al. 2020). With an increase in the concentration of antibiotics, many intermediates are generated, which can compete with antibiotics to react with active radicals. Moradi et al. (2020) reported a similar trend while degrading sulfamethoxazole using a graphene-based nanocomposite.

The dose of nZVI was varied from 0.0035 to 0.0210 g L⁻¹, with a constant interval of 0.0035 g L⁻¹ for TC and 0.0070 to 0.0245 g L⁻¹, with a continuous interval of 0.0035 g L⁻¹ for CIP. With the increase in the dose of nZVI, the removal efficiency of both antibiotics gradually increased to 83.8% for TC at 0.0140 g L⁻¹ and 84.5% for CIP at 0.0175 g L⁻¹ (Figures 4(c) and 5(c)). The efficiency then reduced slowly with a further increase in the dose of nZVI. This is because an increase in nZVI dose increases the number of active sites, generates more active radicals, and thus enhances the degradation rate in the initial phases. Ren et al. (2020) observed a similar trend in the degradation of TC by a graphene-based photocatalyst. However, further addition of nZVI led to a decrease in degradation efficiency, which might be due to the agglomeration of catalyst particles, or increased scattering of light and reduced light penetration, which ultimately hampers the photocatalysis reactions (Gupta et al. 2020). Gupta et al. (2020) studied the degradation of C17H18FN3O3 using an S–C₃N₄/ZnO catalyst at a dose of 0.5–2.5 g L⁻¹. In his degradation study, the degradation efficiency increased continuously with an increase in dose from 0.5 to 1.5 g L⁻¹ and then decreased steadily up to 2.5 g L⁻¹. Moradi et al. (2020) degraded sulfamethoxazole and pharmaceutical wastewater using MgO/ZnO/Graphene ternary nanocomposite. With the increase in catalyst dosage, he observed that the active sites acted as recombination centers, self-consuming powerful oxidative radicals, and reducing adsorption of light due to the light scattering effect.

Stirring was performed using a magnetic stirrer from 100 to 500 rpm to maximize the degradation of TC and CIP. Stirring improves the contact between antibiotic molecules and nZVI as well as increasing the dissolved oxygen level, resulting in a decline in the electron–hole recombination and generation of superoxide radicals (Ahmadpour et al. 2020). Maximum degradation efficiency for TC was achieved at 400 rpm for TC and 100 rpm for CIP (Figures 4(d) and 5(d)). At these optimized conditions, photocatalytic degradation efficiency was monitored at an interval of every 10 min. Degradation efficiency increased gradually up to 70 min for both TC and CIP. Maximum efficiency of 88 and 84% was recorded for TC and CIP, respectively (Figure 4(e)).

The degradation of TC could be brought about by attack of the reactive species on the dimethylamino group, leading to the removal of methyl group, followed by breaking of double bond and removal of the amide group. The other pathway is based on the removal of methyl, amide, and dimethylamino groups, followed by amino groups. Degradation is brought about by ring opening, oxidation and removal of carbonyl, ester, and hydroxyl groups, opening or relocation of double bonds, the opening of long carbon bonds, oxidation of phenolic hydroxyl, and benzyl groups (Ma et al. 2022).

Similarly, the degradation of CIP is initiated by the opening of the piperazine ring, or the removal of the F atom, carboxyl group, or cyclopropyl group. The C–N bond of the piperazine ring gets oxidized, opening the ring structure, while the removal of the F atom leads to defluorination, where the C–F bond is substituted by –OH. The double bond breaks to the single bond, and –OH enters the ring and breaks open the structure, leading to oxidation of the cyclopropyl group due to electrons and holes generated by light irradiation (Hu et al. 2020).

To synthesize a liter of nZVI solution, 500 ml of 0.01 M FeSO₄·7H₂O solution and 500 ml of H₂O are required. A liter of solution produces 0.7 g of nZVI powder. The process uses electricity to shake the solution using an orbital shaker. This brings the cost of 0.7 g to Rs. 1.45 (0.0208 USD) and the rate to Rs. 2.07 (0.0297 USD) per gram. To treat the 10,000 L of TC and CIP, 140 and 175 g of nZVI are required, respectively. Electricity is required for UV light and magnetic stirring of the solution. The cost of treatment of 10,000 L of TC and CIP is ₹ 989.80 (14.158 USD) and ₹ 1062.30 (15.198 USD).

The study revealed the successful synthesis of nano zero-valent iron (nZVI) particles through the bio-reductive pathway of Fe precursor in the presence of Sal leaf extract. The spherical crystalline particles with an average size of 44 nm indicated the presence of polyphenol and alcohol functional groups, possibly acting as reducing agents to the precursor and capping agent of nZVI. These particles, when applied for photocatalysis of antibiotics TC and CIP under UV illumination, achieved a removal efficiency of 88 and 84% in optimal conditions of 15 and 25 mg L⁻¹ antibiotic concentrations, 0.014 and 0.0175 g L⁻¹ doses of nZVI, in the pH range 4–6 in 70 min, respectively. Kinetic studies revealed pseudo-second-order reactions. Active species (·O⁻, ·OH, and H⁺) generated by nZVI in the presence of UV light might be attributed to playing a significant role in the degradation of the antibiotics. When further verified using mass spectrometry, the degradation of antibiotics was confirmed into smaller breakdown products. Microbial assay confirmed the non-toxic nature of the breakdown products, implying that effluent containing residual antibiotics can be treated by this process and discharged into natural water bodies. Furthermore, the cost analysis of the entire process proved economically feasible for application in a more extensive setup.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.