Mycogenic TiO 2 nanoparticles for remedying toxic metals in industrial wastewater and their toxicity profiling

Heavy metal pollution, particularly cadmium and chromium, poses a global environmental challenge due to its detrimental effects. Nanobioremediation is an advanced approach to remediate heavy metal contamination. Bioactive molecules extracted from Aspergillus penicillioides VDRVYF serve as a stabilising and capping agent for producing TiO 2 nanoparticles. This study presents an eco-friendly approach using fungus-mediated TiO 2 nanoparticles to remove heavy metal ions from industrial effluents. The research adopted a comprehensive approach, employing batch adsorption studies, adsorption kinetics, and heavy metal analysis via atomic absorption spectroscopy (AAS). The collected effluent samples from the battery and automobile industries were treated with 50 μg/mL TiO 2 nanoparticles for 5 hours under sunlight. Physicochemical analysis was carried out, and the results were compared with the CPCB standard limit. Additionally, desorption and reuse studies were performed. Furthermore, toxicity assays were conducted to evaluate cytotoxic effects on living cells. AAS determines the optimal conditions for the adsorption of heavy metals employing Aspergillus penicillioides VDRVYF-mediated TiO 2 nanoparticles. The physicochemical parameters of untreated wastewater samples exceeded CPCB standards, rendering direct discharge infeasible. However, treated samples showed over 72% metal ion adsorption, with physicochemical parameters falling within permissible limits. Reusability studies revealed that the nanoparticles adsorption efficiency was maintained for upto six cycles, with a final rate of 65.2%. Furthermore, phytotoxicity and the MTT assay confirmed the biocompatibility of the nanoparticles. The findings demonstrate that bio-based TiO 2 nanoparticles are a promising agent for sustainable industrial wastewater treatment, effectively reducing heavy metal contamination.

The global water quality has declined substantially in recent times. Recently, one of the main causes of this condition is the amount of pollutants present in industrial effluents (Ma et al., 2020). The pollutants commonly present in industrial wastewater include volatile organic compounds, polycyclic aromatic hydrocarbons, endocrine-disrupting chemicals, heavy metals, and various other organic and inorganic materials. The detrimental impacts on aquatic life caused by industrial processes, such as mining, tanning, metal plating, pulp processing, smelting, and battery production, have garnered international attention and necessitated effective control of metal contamination (Yamini et al., 2023). Industrial effluents are frequently found to contain heavy metals among the contaminants, including iron, cadmium, zinc, lead, mercury, and nickel. In addition to posing long-term or harmful hazards to aquatic life, these compounds are known to endanger the overall biodiversity (Neal et al., 2005). Heavy metals and other contaminants present in wastewater can accumulate in growing crops, which can result in serious risks to the ecosystem. Toxic metals are known to have the ability to attach to bioactive compounds and cause toxicity in living things; some of these metals have also been established as mutagens and carcinogens. Crops and plant tissues are capable of accumulating heavy metals and other pollutants from industrial effluents, frequently at concentrations higher than those deemed safe for aquatic life and the ecosystem as a whole (Ma et al., 2020).

Cd is recognized as one of the most hazardous metal contaminants found in the environment. Despite its higher solubility in water, cadmium pollution negatively impacts wildlife and public health. As a result, there is greater interest in developing methods to reduce its hazardous effects in soil (Shankar, 2020). Cd poses serious threats to aquatic ecosystems by impairing the health of aquatic plants and animals, notably fish. Cd exposure has been shown to reduce reproductive success in fish and hinder plant development. In humans, prolonged exposure to Cd can cause severe health problems, such as kidney dysfunction, bone weakening (demineralization), pulmonary diseases, and various forms of cancer (Yamini et al., 2023).

Cr is another significant environmental contaminant that naturally occurs in mineral form rather than free elemental form. The hexavalent state of Cr, i.e., Cr(VI), is particularly toxic and disrupts the aquatic environment by inhibiting photosynthesis in aquatic plants, damaging the gill structures of fish, and negatively affecting their reproductive systems. In humans, exposure to Cr(VI) can lead to respiratory ailments, dermatological reactions, liver and kidney injuries, gastrointestinal disturbances, as well as the development of lung tumors upon prolonged exposure (Oliveira, 2012).

Given the growing concerns over the presence of heavy metal pollutants in industrial effluents, there is an urgent requirement for efficient, economical, and sustainable technologies to remediate contaminated water sources (Yamini et al., 2023). One such emerging and promising approach is nanobioremediation, which merges the principles of nanotechnology with bioremediation processes. This method enhances the degradation and removal of environmental pollutants through nanoparticles synthesized using a biological technique (Pete et al., 2021). Advancements in nanoscience have driven researchers to engineer nanoparticles of sizes less than 100 nm by capitalizing on their large surface areas, reactivities, and unique functional and structural attributes (Wu et al., 2011). Nanoparticles can be synthesized via chemical, physical, or biological routes. Biological synthesis is one of the most environmentally friendly remedies available, especially since it does not require hazardous chemicals that are frequently used in conventional chemical and physical processes.

Bioactive molecules from living organisms, such as bacteria, fungi, and plants, are used in this technique to generate nanoparticles; these compounds work as natural stabilizing and reducing agents (Subramanian and Tarafdar, 2011). Fungi are especially preferred for generating nanoparticles owing to their remarkable bioaccumulation capabilities, ability to build strong connections, and superior capacity for enduring metal stress compared to bacteria. The additional useful benefits of fungi include their ability to secrete a wide range of enzymes, cost-effective and scalable production procedures, and ease of biomass management (Zonooz et al., 2011). Because of their high biomass output and ability to withstand harsh climatic conditions, fungi are particularly advantageous for large-scale applications when used to synthesize nanoparticles, according to Kuppusamy et al. (2016). There is promising potential for developing efficient systems for heavy metal detoxification and environmental restoration by integrating fungal biotechnology with nanotechnology.

Titanium dioxide nanoparticles are extensively used in many applications, especially in sunscreens, cosmetics, and as photocatalysts, owing to their remarkable functional and structural characteristics (Rehman et al., 2021). TiO 2 is a key material in technological applications involving dielectrics and semiconductors and can be produced from metal ions through the actions of various microorganisms. One of the outstanding features of TiO 2 nanoparticles is their chemical stability; they remain stable in both acidic and alkaline environments and are highly durable in water. Modern methods for generating TiO 2 nanoparticles are not only cost-effective and sustainable but also recyclable and simpler than traditional chemical processes.

Using biological entities, especially microorganisms that produce proteins that act as natural reducing and capping agents, is a green alternative to conventional chemical synthesis that often depends on environmentally harmful substances. Biosynthetic methods not only simplify the process of producing nanoparticles but also show great potential for bioremediation, particularly in cleaning up wastewater tainted with heavy metals (Rehman et al., 2021). Among the different microbial species, the fungus Aspergillus penicillioides found in soil has shown impressive abilities to absorb toxic heavy metals like Cd and Cr (Paria et al., 2022). The natural prevalence, ease of cultivation, and environmental compatibility of this fungus make it a fantastic choice for large-scale bioremediation processes.

To ensure commercial success, bioremediation technologies must be affordable, easy to operate, and environmentally friendly. Accordingly, the main goal of the present work is to develop a straightforward yet effective method for synthesizing TiO 2 nanoparticles using the extracellular extract of the A. penicillioides VDRVYF strain (Yamini and Devi Rajeswari, 2024). The present study entails characterizing the nanoparticles generated and assessing their ability to adsorb heavy metals, specifically Cd and Cr ions, through atomic absorption spectroscopy (AAS). By optimizing various experimental conditions like the pH level, nanoparticle dosage, metal-ion concentration, and different irradiation sources over varying time frames, the efficiency of metal adsorption can be boosted significantly. Understanding the adsorption kinetics is a vital step as it reveals how quickly the solutes are taken up and determines how long the metal ions remain on the nanoparticle surface, both of which ultimately affect the effectiveness of the bioremediation strategy.

To ensure the reliability and stability of the synthesized TiO 2 nanoparticles, we performed a series of bioassays; these included tests for phytotoxicity, studies on algal growth inhibition, and MTT assays to evaluate the cytotoxic effects of the nanoparticles on living cells. These tests are crucial for confirming that the nanoparticles do not pose secondary environmental or health issues. The thorough methodological approach applied in this study includes nanoparticle characterization, adsorption studies, kinetic modeling, and bioassays to offer valuable insights into the potential of A.-penicillioides -VDRVYF-mediated TiO 2 nanoparticles as an effective solution for ecological remediation of industrial wastewater. Additionally, the present research is expected to contribute to the broader step toward sustainable nanotechnology aimed at addressing heavy metal contamination.

Synthesis and characterization of A.-penicillioides -mediated TiO 2 nanoparticles

Our earlier study provides a thorough description of the isolation, morphology, and molecular characterization of the fungus A. penicillioides (Yamini and Devi Rajeswari, 2024). The identified fungal sequence has been deposited in the NCBI gene sequence database. Using the extracellular biomolecules secreted by A. penicillioides, we also reported the biosynthesis and characterization of TiO 2 nanoparticles (Vinayagam and Vijayarangan, 2025). The adsorption efficiencies of Pb 2+ and Ni 2+ ions from aqueous media were determined through kinetic isotherm modeling and optimization studies for nanoparticle production. Furthermore, the photocatalytic activity of the synthesized TiO 2 nanoparticles was assessed under visible light to explore potential environmental applications, and the antimicrobial properties of the nanoparticles were evaluated against specific pathogenic strains to highlight their biomedical significance. To ensure the safety of the synthesized nanoparticles, hemolytic assays were performed to evaluate their cytocompatibility and eco-friendliness. Building on the foundational methodologies and findings from our earlier work (Yamini and Devi Rajeswari, 2024), the present study involved investigating the adsorption behaviors of Cd 2+ and Cr 6+ ions using TiO 2 nanoparticles mediated by A. penicillioides; we also performed advanced optimization of various parameters, including the pH, nanoparticle dosage, and metal-ion concentration, along with comprehensive kinetic and isotherm studies. Lastly, environmental toxicity assessments such as phytotoxicity and algal inhibition assays were incorporated to ensure the durability and effectiveness of the nanoparticles for wastewater remediation applications.

Optimization, kinetics, and isotherm studies

Batch adsorption studies for Cd and Cr

The efficacy of the TiO 2 nanoparticles for adsorptive photocatalytic reduction of pollutants in effluents was evaluated by analyzing the adsorption of Cd and Cr in simulated wastewater. To test the photocatalytic activity, approximately 2 µL of the TiO 2 nanoparticle solution (4 μg/mL) was added to wastewater containing chromium oxysulfate (100 μg/mL) and cadmium sulfate octahydrate. The mixture was agitated constantly at 150 rpm and maintained at 25 °C (room temperature or RT). We then explored various factors, such as the concentrations of chromium oxysulfate and cadmium sulfate octahydrate (ranging from 2 to 100 μg/mL), amount of TiO 2 nanoparticles needed (2–10 μg/mL), changes in pH level (from 5 to 9), and type of light used to achieve reactions (ultraviolet and sunlight) over 5 h to identify the optimized conditions for maximum adsorption. Samples were collected at regular intervals throughout the 5 h duration of the adsorption study. Once equilibrium was achieved, the nanoparticles (nanocatalysts) were eluted and analyzed using an ultraviolet spectrometer after spinning for 10 min at approximately 8,000 rpm. The optical density (OD) was determined, and the adsorption percentage was calculated using Equation 1:

Adsorption kinetics and isotherm studies for Cd and Cr

The adsorption kinetics and isotherms were analyzed to explore the processes, proportions, characteristics, and tendencies of the TiO 2 nanoparticles for Cd and Cr adsorption. To evaluate the scientific data gathered from the batch adsorption experiments, we examined the Langmuir and Freundlich adsorption isotherms along with the pseudo-first-order and pseudo-second-order kinetics models. The Langmuir isotherm (Equation 2) represents a single-layer configuration on a uniform surface with limited adhesion regions under the assumption of consistent adsorption energies and no interactions among the adsorbed molecules. In contrast, the Freundlich isotherm (Equation 3) accounts for adsorption on diverse surfaces with varying adsorption energies.

Here, q e denotes the amount adsorbed, q m indicates the highest efficacy, C e represents the equilibrium concentration, K L is the Langmuir coefficient, K F is the Freundlich coefficient, and n is the heterogeneity factor (where n > 1 signifies favorable adsorption).

The rate of adsorption is correlated with the total number of vacant regions as per pseudo-first-order kinetics (Equation 4); in contrast, the pseudo-second-order kinetics theory (Equation 5) proposes that the adsorption rate relies on the square of the number of vacant regions: where q e is the equilibrium adsorption, q t is the adsorption at time t, K 1 and K 2 are the respective coefficient rates for the pseudo-first-order and pseudo-second-order kinetic models.

AAS analysis of Cd and Cr

We identified the Cd and Cr pollutants in the sample wastewater using the Perkin Elmer Analyst 400 system in the AAS study. The radiation sources for Cd and Cr were hollow cathode lamps powered by acetylene and air. The standard absorbance wavelengths for Cd and Cr are 228 nm and 357 nm, respectively, with a slit width of 0.7. We evaluated the Cd and Cr levels in both untreated and treated wastewater test samples in triplicate. The blank and standard samples were then atomized, and the results were recorded. After generating the calibration graph for each compound, the test samples were atomized and analyzed. The calibration curves were then used to determine the quantities of the heavy metals in the test wastewater samples based on the absorbance observed for the standard sample.

Real sample study

Sample collection

Sterilized hard glass containers were used to gather industrial (battery and automobile) wastewater samples from five selected industries at their discharge points (S1–S5) in Chennai and Vellore, India. These glass containers were thoroughly sterilized with a disinfectant, rinsed with regular water, impregnated with 10% nitric acid (HNO 3), and subsequently washed with double-distilled H 2 O. Various samples were obtained for different assays to evaluate the pH, temperature, color, turbidity, and conductivity of the wastewater, particularly if the samples showed biodegradation. Each sample was carefully labeled, transferred to the laboratory, and stored in a freezer at approximately 4 °C until analysis. To preserve the dissolved oxygen (DO) level, a few drops of manganous sulfate was added to each wastewater sample and immediately placed in a dark DO bottle for DO and biological oxygen demand (BOD) assessments. The collected samples were maintained at RT and were treated with TiO 2 nanoparticles (50 μg/mL) under the optimized parameter conditions. The physicochemical parameters were analyzed for both untreated (S1–S5) and treated (T1–T5) industrial wastewater samples, and the observations were compared with the standards established by the Central Pollution Control Board (CPCB) of India.

Evaluation of physicochemical parameters by analytical method

The concentrations of various toxic metal ions, including Cr, Ni, Cd, and Pb, were measured according to the American Public Health Association (APHA) method along with various physicochemical parameters like pH, temperature, color, turbidity, BOD, chemical oxygen demand (COD), total dissolved solids (TDS), total organic carbon (TOC), conductivity, total suspended solids (TSS), total alkalinity, sulfates, nitrates, and phosphates from the industrial effluent samples before and after treatment with TiO 2 nanoparticles. The instruments used for these evaluations were within the required precision ranges, and all compounds used were of analytical reagent grade. We ensured all necessary efforts to avoid contamination during the collection process.

pH, color, temperature, conductivity, and TDS

The pH, temperature, and color were measured onsite, and the pH readings were obtained immediately after calibrating the equipment with existing buffer solutions (pH 4, 7, and 10). All equipment and field meters were inspected and validated according to the guidelines and standards of the suppliers. The conductivity was measured with a conductivity meter, and the TDS was measured using a TDS meter.

TSS

To calculate the TSS, we used Whatman filter paper that was treated with double-distilled water, dried at 105 °C for precisely 1 h, and cooled in an evaporator. An electronic weighing machine was employed to measure the residual weight (W 1). After filtering, a 100-mL wastewater sample was evaporated for an hour at 105 °C, and the weight of the filter paper with the residue (W 2) was noted. Then, the TSS was computed using Equation 6:

Total alkalinity

Titration was employed to evaluate the alkalinity level. In a sterile 150-mL flask, we introduced 50 mL of the wastewater sample along with three drops of phenolphthalein indicator. This mixture was titrated with 0.05 M H 2 SO 4 until the indicator color began fading. Before the titration, readings, and alkalinity calculation, we introduced methyl orange (3 drops) to the colorless solution and continued titration until the yellow color changed to an orange-red or consistent crimson hue.

BOD

Winkler’s method was adapted with azide for measuring the DO and BOD values. A 300-mL BOD bottle was filled with 200 mL of the wastewater sample, to which manganese sulfate (1 mL) and an alkaline alkali–iodide–azide solution (1 mL) were introduced. The mixture was titrated against 0.025 N sodium thiosulfate until a color change was observed; this titer reading was noted as the DO value. The BOD bottle with the sample was then placed in a dark place at 20 °C for 5 d; following this incubation, orthophosphoric acid (2 mL) was added to the bottle. Once the color changed, the mixture was gently shaken and titrated with sodium thiosulfate; this titer value indicates the DO on day five. The difference between the DO levels of days 1 and 5 was employed to compute the BOD.

COD

Standard procedures were used to determine the COD. In an inverted flask, the wastewater sample (50 mL) was mixed with potassium dichromate (K 2 Cr 2 O 7; 10 mL) and mercuric sulfate (1 g). Stirring beads were then introduced to facilitate steaming and mixing. Next, concentrated H 2 SO 4 containing silver sulfate (10 mL) was carefully introduced through the condenser’s wide opening and swirled. After approximately an hour of refluxing, the device was chilled. Ferroin (3 drops) was added as an indicator to the solution after the contents of the flask were diluted with distilled water (150 mL). Lastly, the sample was titrated with standard ferrous ammonium sulfate (FAS) until the blue-green hue turned reddish brown to ascertain the blank COD.

TOC

We followed standard procedures to assess the TOC. After finely grinding the evaporated sedimentary material, 0.2 g of each sample was placed in a 500-mL flask. Then, 0.5 M K 2 Cr 2 O 7 (10 mL) was introduced and stirred gently. Next, 20 mL of concentrated H 2 SO 4 was carefully introduced into the solution directly. After gentle stirring, the mixture was allowed to sit for 40 min; following this, 200 mL of distilled H 2 O was carefully added, and 10 mL of pure phosphoric acid (H 3 PO 4) was introduced. Once the mixture had cooled, ferroin (3 drops) was added as the indicator. Finally, 0.25 M FAS was used for titration, and the endpoint of this step was when the solution turned wine-red in color. Equation 7 was then used to calculate the TOC (%) of the water sample: where V b and V s are the volumes of FAS required for the blank and sample, respectively, and M is the molarity of FAS.

Sulfates, nitrates, and phosphates

To measure the sulfate level, the gravimetric/turbidimetric technique was employed using BaCl 2 as a precipitant. Accordingly, a 250-mL glass container was loaded with the sample (50 mL), which was then mixed with 150 mL of distilled water, four drops of methyl orange, and 1 mL of concentrated hydrochloric acid. After adding 10 mL of a 10% barium chloride solution, the mixture was heated for 5 min, allowed to sit overnight, and filtered through Whatman filter paper. Distilled water was used to wash the filter paper to remove the chloride ions, and the paper was dried in a silica beaker at 80 °C in an oven; the paper was then heated at 800 °C for an hour for calcination before being cooled in an evaporator and weighed. To ensure consistency, the combustion, cooling, and weighing processes were repeated. The sulfate content in the wastewater sample was then calculated from these repeated measurements. The phenoldisulfonic acid method was employed to measure the nitrate level, and a spectrometric approach was used to estimate the phosphate level.

Heavy metal ions

The quantities of toxic metal ions (Pb, Cd, Ni, and Cr) in the industrial wastewater (both untreated samples and those treated with TiO 2 nanoparticles) were assessed using AAS (APHA, 1926), and the obtained findings were compared with the limit specifications established by the CPCB.

Desorption and reuse efficiency

The adhesion of toxic metals onto the surfaces of nanoparticles is a reversible mechanism that permits reusing the nanoparticles after desorbing the metal ions. To elute the metal ions from the surfaces of the nanoparticles, four eluents were selected, namely, methanol and 1:1 combination of 0.1 mol/L sodium hydroxide (NaOH) with methanol, acetone, and propanol separately. The results showed that compared to the other eluents, 2.0 mL of the 1:1 mixture of 0.1 mol/L NaOH and methanol had the maximum desorption efficiency. The adsorbent was subjected to several adsorption–desorption cycles to evaluate its reusability. After each extraction cycle, the adsorbent was used in subsequent extractions after eluting with 2.0 mL of the 1:1 solution of methanol and NaOH (0.1 mol/L) for 10 cycles. After 5–10 adsorption–desorption cycles, we found that the regenerated adsorbent could remove more than 90% of the hazardous metal ions from the sample solution (Jethave et al., 2019). The adsorption–desorption experiment was repeated for 5–10 reuse cycles, and Equation 8 was employed to compute the adsorption efficiency:

Here, C o and C e are the initial and optimum concentrations of the dye (mg/L), respectively; V denotes the solution volume (L), and M refers to the mass of the TiO 2 nanoparticles (g).

Toxicity studies of the TiO 2 nanoparticles

Phytotoxicity assay

Seed origin

The seeds were gathered from native Pisum sativum plants grown in Vellore, Tamil Nadu, India.

Seed germination assay

This assay was conducted with P. sativum seeds and industrial effluents (both untreated and treated with TiO 2 nanoparticles). A 1.2% sodium hypochlorite solution was used to wipe the surfaces of the seeds to sterilize them and remove any fungal growth. All seeds were placed individually in shallow, round, clear dishes with flat lids and treated with TiO 2 nanoparticles at distinct concentrations (25–100 μg/mL). Normal water was used as the positive control. The plates were checked for germination regularly, and radicals emerging from the seeds were regarded as having germinated. After one week, the germination rate as well as lengths of the plumule (shoot) and radical (root) were observed.

Algal growth inhibition assay

Green algae species such as Chlorella sp. and Volvox sp. were utilized for the algal growth inhibition assays that were conducted according to the OECD 201 test guidelines. The algal isolates were cultivated in Bold’s basal medium (pH 7.5) for approximately 72 h at 25 °C in a shaking incubator using four different concentrations of the TiO 2 nanoparticles (25–100 μg/mL). The TiO 2 nanoparticles and algal species were both present in the test sample that was detected at a wavelength of 680 nm. The OD was measured every 24 h, and the control algal species were maintained. The obtained readings were plotted against time to produce the growth curve of the algal sample. The results enable assessment of the potential of the TiO 2 nanoparticles to suppress proliferation of the algal species.

MTT assay

Cell cultures

The 3T3-L1 normal fibroblast cell line was obtained from the National Centre for Cell Sciences (NCCS) in Pune, India. Next, 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin were added to Dulbecco’s modified Eagle medium (DMEM) and used to cultivate the cells. For logarithmic growth, the cultures were maintained at 37 °C in a humidified incubator with 5% CO 2 and 95% air.

Cytotoxic effects

Based on the method established by Mosmann (1983), the MTT assay was used to evaluate the cytotoxic potential of the TiO 2 nanoparticles. Following seeding at a density of 1 × 10 4 cells per well in 96-well plates, the cells were incubated for 48 h until they achieved roughly 70%–80% confluency. The cells were then exposed to different doses of TiO 2 nanoparticles (20, 40, 60, 80, and 100 μg/mL) and allowed to incubate for an additional 24 h. A digital inverted microscope set to ×20 magnification was employed to examine and record any morphological modifications of the treated cells and untreated controls. Next, 20 μL of the MTT solution (5 mg/mL in phosphate-buffered saline (PBS)) was introduced to each well after washing the cells with PBS (pH 7.4) at the end of incubation. Then, the plates were again incubated for 4 h at 37 °C under dark conditions. The formazan crystals formed at this step were dissolved in 100 μL of dimethyl sulfoxide (DMSO), and the absorbance at 570 nm was evaluated using a spectrophotometer for quantification. The positive control cells were treated with 1% Triton X-100, while the negative control cells were left untreated. For comparison, ampicillin was used as the standard reference compound (Vinayagam et al., 2025). Finally, the cell viability was calculated as a percentage based on the absorbance values (Equation 9):

Statistical analysis

All experimental procedures were conducted three times to validate the reproducibility and reliability of the results. The obtained results were analyzed using Microsoft Excel, Origin 2024, and Minitab software. Standard deviations were calculated to evaluate the variability within the datasets. The one-way analysis of variance was computed to evaluate the statistical variations between the experimental groups, and a significance threshold of p < 0.05 was applied to validate the results. Furthermore, post hoc tests were employed where necessary to identify specific differences between the treatment groups. Integrating multiple software platforms allowed us to comprehensively and precisely interpret the experimental data, thereby ensuring robust statistical validation of the findings.

Synthesis and characterization of A.-penicillioides -mediated TiO 2 nanoparticles

Our previous report (Yamini and Devi Rajeswari, 2024) describes the synthesis and characterization of A.-penicillioides -mediated TiO 2 nanoparticles. The optimization and kinetic studies were performed to enhance the rates of Pb and Ni adsorption. The identified fungus (A. penicillioides) was reported in the NCBI gene sequence repository as A. penicillioides VDRVYF with the accession number PQ570604.1 (https://www.ncbi.nlm.nih.gov/nuccore/2847402264; Vinayagam and Vijayarangan, 2025). The hemolytic assay confirmed the ecological sustainability of this strain for bioremediation. Figure 1 shows the significance of the A. penicillioides VDRVYF biomolecules, specifically the polysaccharides and proteins that influence the nucleation, growth, surface stabilization, and adsorption selectivity of nanoparticles.

Optimization, kinetics, and isotherm studies

Batch adsorption study

Using TiO 2 nanoparticles as the catalyst, we assessed how different factors like the type of light source, amount of adsorbent, pH level, and concentrations of Cd and Cr (toxic metal ions) affected the process over 1–5 h of stirring. The results from our batch adsorption tests revealed that the best conditions for adsorbing Cd were 60 μg/mL of Cd combined with 2 μg/mL of TiO 2 nanoparticles in a solution stirred for 5 h. Figure 2a shows that it is essential to adjust the solution pH to 6 along with exposure to sunlight. For Cr adsorption, the best conditions involve a reaction mixture with 60 μg/mL of Cr and 10 μg/mL of TiO 2 nanoparticles at a pH of 6 with exposure to sunlight (Figure 2b). After analyzing the parameters, the optimized values were determined for the adsorption study. Interestingly, we found that the adsorption of these toxic metal ions decreased as the stirring time and concentrations of Cd and Cr increased. When the Cd concentration was 60 μg/mL and the mixture was stirred for 5 h, the adsorption efficiency of Cd was 70%. At lower Cd concentrations, Cd compounds tended to stick to the active sites of the TiO 2 nanoparticles; this means that as more active sites are occupied, the adsorption of Cd slows down. Similarly, at the maximum concentration of 60 μg/mL of Cr, the adsorption rate of Cr was 75% after 5 h.

The process of adsorbing Cr and Cd is largely influenced by the amount of adsorption material available, which in turn increases the degree of absorption. As the concentration of the TiO 2 nanoparticles increases, there are more active sites available for the Cd and Cr ions to latch onto, leading to a higher rate of hazardous metal-ion adsorption. Over time, as the interactions between these metal ions and the nanoparticles continue, there is greater accumulation of Cr and Cd ions in the available spaces of the TiO 2 nanoparticles. Interestingly, the lowest pH value that effectively absorbs 90% of Cr and 97% of Cd within a 5-h timeframe is 6. At pH levels of 5, 7, 8, or 9, the adsorption of these metals is less effective. When the TiO 2 nanoparticles are exposed to a pH 6 environment, it triggers the protonation and adsorption of Cd and Cr ions. The evacuation of these ions was tested using 2 μg/mL and 10 μg/mL of TiO 2 nanoparticles as the photocatalyst under different light sources, including sunlight and ultraviolet light, which achieved maximum adsorption rates of 50% and 65%, respectively. We discovered that sunlight greatly accelerated the adsorption of Cd and Cr ions and that the maximum adsorption rates were 40% for Cd and 75% for Cr upon irradiation with sunlight. This demonstrates that TiO 2 nanoparticles can efficiently adsorb hazardous metal ions from industrial wastewater when exposed to direct sunlight (Kaliyamoorthy et al., 2023).

Adsorption isotherm and kinetics studies

The reaction mixture containing both Cr and Cd was subjected to isotherm and kinetics assessments to validate the accuracy of the test results from the batch adsorption experiments. Heterogeneous catalysis depends on different rate equations for the adsorption processes occurring on the reactant surfaces during these reactions (Rajabi et al., 2024). The C e vs. C e /q e values were used for the Langmuir adsorption isotherm analysis (Figures 3ai,bi. Similarly, Figures 3aii,bii show the Freundlich adsorption isotherms in terms of the log C e vs. log q e values. The pseudo-first-order (Figures 3aiii,biii and pseudo-second-order (Figures 3aiv,biv kinetics parameters for the Cd and Cr adsorption data were calculated from the plots of t vs. log (q e – q t) and t vs. t/q t, respectively. The kinetics and isotherm variables and corresponding R 2 values are shown in Table 1. Both the pseudo-second-order kinetic models and Freundlich adsorption isotherms demonstrate excellent correlations with the absorption of hazardous metal ions on the nanoparticle surfaces, with the R 2 values emphasizing the efficiencies of the TiO 2 nanoparticles in decomposing the Cd and Cr compounds.

According to the kinetics investigations, the TiO 2 nanoparticles show a linear fit with the Freundlich adsorption isotherm for toxic metal ions. We found that the adsorption capacity of the TiO 2 nanoparticles improved parallelly with the concentration of toxic metal ions and reaction time. This pseudo-second-order reaction shows that the time required is constant despite the number of variables (pH, concentrations of hazardous metals, concentration of TiO 2 nanoparticles, and irradiation source). The chosen model appropriately depicts the degradation process observed herein, and the high R 2 values are substantially correlated with the results of the experiments (Rajabi et al., 2024).

AAS evaluations of Cd and Cr

The results shown in Table 2 make it abundantly evident that the TiO 2 nanoparticles produced using A. penicillioides VDRVYF had effective adsorption rates for Cd and Cr. The exceptional adsorption abilities of the nanoparticles are shown by the discernible reductions in Cd and Cr concentrations after treatment. The high surface area, surface functional groups, and active binding sites of the biologically produced TiO 2 nanoparticles are responsible for this removal efficiency. Furthermore, the durability and repeatability of these biosynthesized nanoparticles were confirmed by the consistent removal tendencies observed across all trials. According to these findings, the nanoparticles show high affinities to heavy metal ions while providing a feasible and environmentally friendly method of treating industrial wastewater. Future studies could investigate the reusability cycles and field-level applications of the nanoparticles to validate their long-term effectiveness under diverse environmental conditions.

Real sample study

Physicochemical parameters

Temperature and color

The temperature significantly impacts the other characteristics of the wastewater. The temperature of the different samples obtained during wastewater collection from the industrial site was 27.5 °C. Following treatment of the wastewater with TiO 2 nanoparticles, all samples had a temperature of 25.5 °C. Some of the other characteristics were greatly impacted by the color of the industrial wastewater. The industrial wastewater samples (S1–S5) obtained for this study are shown in Figure 4a, and their colors vary from light green to dark brown, creamy white, turbid green, and orange yellow. All of these wastewater samples had different colors after treatment with TiO 2 nanoparticles. Samples T1, T3, and T5 were mostly colorless, whereas T2 and T4 were respectively bright green and brown after treatment (Figure 4a).

pH

A solution’s pH or hydrogen-ion activity is determined by calculating the logarithm of the reciprocal rate of hydrogen ions at a specific temperature. Sample S4 had a higher pH of 11, while S1 had a lower pH of 7.4. The pH values of the industrial wastewater samples collected from the release sites ranged from 7.4 to 11, and these values varied between 6.7 and 7.8 after treatment with the TiO 2 nanoparticles. Sample T5 had the lowest pH at 6.7, while T3 had the highest pH at 7.8 (Figure 4b). The average pH of each sample was higher than the values recommended by the WHO and local authorities. The government standard states that wastewater can have a maximum pH of 6–9. Variations in the carbonate, CO 2, and bicarbonate levels in the effluent samples can cause the pH values to fluctuate. Diseases like tuberculosis may be caused by lower pH levels, whereas corrosion, sedimentation, accumulation, and other issues are often caused by higher pH levels (Asaolu and Olaofe, 2004). The present study demonstrates that the pH of industrial wastewater can be reduced and brought within the acceptable range following treatment with the proposed TiO 2 nanoparticles.

Turbidity

The average turbidity values of the industrial effluent samples before treatment were 104 nephelometric turbidity units (NTU) for S1, 124 NTU for S2, 900 NTU for S3, 940 NTU for S4, and 1,042 NTU for S5 (Figure 5a). Meanwhile, after treatment with the nanoparticles, the mean values were 40 NTU for T1, 45 NTU for T2, 68 NTU for T3, 70 NTU for T4, and 71 NTU for T5 (Figure 5b). Here, the established permissible limit is 75 NTU; compared to the standard value established by the governing authority, the turbidity values of the industrial wastewater samples reduced upon treatment with the nanoparticles.

Conductivity

Electrical conductivity is a measure of the capacity of a material to carry an electric current. The electrical conductivity of water is known to be directly correlated with the amount of dispersed mineral particles in it. This conductivity is a result of the large amounts of dissolved salts released by various minerals, real salt in clean water, and salt that was added to food products. The conductivity values of the industrial wastewater samples S1–S5 (untreated samples) were found to be 3,685.7 μs cm −3, 11,400 μs cm −3, 9,028.6 μs cm −3, 3,828.6 μs cm −3, and 13,571.4 μs cm −3, respectively, as shown in Figure 5a. With regard to the industrial effluent samples, the water conductivity is a helpful measure of the salinity or total quantity of salt present irrespective of the value (high or otherwise). The average conductivity across all sample locations was higher than the 1,000 μs cm −3 limit established by the CPCB, and the local authorities recommend the release of such wastewater from industries into sewage systems. The conductivity values following treatment with the TiO 2 nanoparticles were 204.28 μs cm −3 for T1, 971.43 μs cm −3 for T2, 918.57 μs cm −3 for T3, 227.14 μs cm −3 for T4, and 1,122.85 μs cm −3 for T5 (Figure 5b); it is seen that most of these values are within the acceptable limit of the CPCB standard.

TDS

The mean TDS concentrations in the industrial wastewater samples before and after treatment are shown in Figures 4a,b. Accordingly, the TDS concentrations of the untreated samples are 2,580 mg/L for S1, 7,980 mg/L for S2, 6,320 mg/L for S3, 2,680 mg/L for S4, and 9,500 mg/L for S5 (Figure 5a). In the treated industrial wastewater samples, the TDS contents were found to be 143 mg/L for T1, 680 mg/L for T2, 643 mg/L for T3, 159 mg/L for T4, and 786 mg/L for T5 (Figure 5b). The TDS levels at all monitoring locations (treated samples) were below the 2,100 mg/L threshold established by the CPCB for sewage released into surface water.

TSS

An established quantity of the water sample was filtered through a pre-weighed filter paper. Then, the filter paper was evaporated at 103 °C–105 °C, and the TSS was calculated using Equation 8. The TSS concentrations of the untreated industrial wastewater samples were measured as 1,780 mg/L for S1, 2,678 mg/L for S2, 1,453 mg/L for S3, 1,568 mg/L for S4, and 3,010 mg/L for S5 (Figure 5a). According to the CPCB, the permissible limit for suspended solids in wastewater discharged into water bodies is 150 mg/L. Our study findings indicate that industrial wastewater cannot be released directly into the environment without treatment. Elevated TSS levels can affect the aquatic environment through physical impacts, such as obstructing the fish gills and respiratory pathways, smothering stream beds, disrupting spawning grounds, and blocking light essential for photosynthesis in aquatic plants (Phillips and Lipton, 1995). However, analysis of the treated samples (T1–T5) revealed significant reductions in the TSS levels, ranging between 72 and 150 mg/L (Figure 5b).

Total alkalinity

Excessively high alkalinity can lead to pipe corrosion by promoting mineral deposits and scaling within the pipes. Over time, this buildup may restrict water flow and potentially harm the plumbing system. However, in contrast to acidic water, highly alkaline water is generally less corrosive and more prone to causing scaling problems rather than directly corroding the metal surfaces. According to the Indian standard, the acceptable alkalinity level is 150 mg/L as CaCO 3. In this work, the alkalinity levels of the collected water samples varied between 130 and 550 mg/L (Figure 5a). Both alkalinity and pH play significant roles in the applicability of wastewater for biological treatment. The water samples treated with the synthesized nanoparticles exhibited total alkalinity levels ranging from 81 to 140 mg/L (Figure 5b), which are below the permissible limit.

BOD and COD

The amount of oxygen consumed by bacteria when breaking down organic materials is known as the BOD. The BOD values of the wastewater samples are imperative to this work as they are of primary concern to the outcome. BOD 5 was calculated by deducting the DO values assessed for an array of samples (in mg/L) from the corresponding DO values obtained after 5 d of incubation (Equation 10).

The untreated wastewater BOD values for S1 to S5 ranged from 358 to 709 mg/L (Figure 5a), while the COD values varied from 705 to 1,005 mg/L (Figure 5a), respectively. The BOD and COD values at each of the measurement locations are greater than the CPCB standard values of 30 and 250 mg/L for the release of industrial effluents into the aquatic environment, respectively. Inappropriate application of various substances may be the reason for the elevated values of the BOD and COD for the wastewater samples. The elemental concentration data for the industrial effluent samples from several sampling locations are displayed in Figures 5a,b. The COD of a sample is its oxygen requirement for the oxidation of both organic and inorganic materials; this value was obtained through blank titration by substituting distilled water for the sample. The COD values of the water samples were computed using Equation 11.

The average BOD and COD values of the treated industrial wastewater samples for T1–T5 were in the ranges of 20–30 mg/L and 160–210 mg/L, respectively (Figure 5b). These values are within the CPCB allowable limits, implying that the treated wastewater can be released into the ecosystem.

TOC

The TOC values for S1 and S4 (untreated) are within the CPCB permissible limit of 300 mg/L, whereas those of the remaining untreated industrial wastewater samples were much higher than the standard limit (above 400 mg/L) (Figure 5a). The treated samples T1 and T2 showed notable decreases in the TOC levels and were in the range of 100–143 mg/L (Figure 5b).

Sulfates

The sulfate concentrations at all the sampling points (S1–S5) varied from 6 to 17 mg/L (Figure 5a). Compared to the WHO guidelines and CPCB standards, the industrial wastewater samples contained high concentrations of sulfates. Water containing elevated levels of sulfates tastes unpleasant; moreover, children experience diarrhea-like symptoms from consuming excess levels of sulfates, including magnesium sulfate, especially in hotter areas. After treatment with the TiO 2 nanoparticles, the industrial wastewater samples showed reduced sulfate concentrations of 0.05–0.11 mg/L (Figure 5b), with T1 showing the lowest concentration (0.05 mg/L) and T5 showing the highest content (0.11 mg/L). The sulfate levels after treatment with the TiO 2 nanoparticles were within the WHO and CPCB permissible limits.

Nitrates

Nitrates are the most prevalent oxidized form of nitrogen in aquatic ecosystems and are capable of forming nitric acid. Nitrates act as growth-limiting nutrients in numerous instances but are important ingredients utilized by several photosynthetic autotrophs. Animals can use nitrates to form animal protein, while algae and various aquatic plants utilize them to produce plant proteins. Nitrates are important components of agricultural fertilizers and are vital for plant development and assimilation. Fish use nitrates as alternative food sources, which could lead to an increase in fish population. However, fish will perish if the oxygen levels are reduced by the excessive growth of algae. According to the WHO guidelines and CPCB standards, the permissible limit of nitrates is 10 mg/L. The nitrate concentrations at all the sampling points (S1–S5) varied between 7.4 and 9.8 mg/L (Figure 5a). Furthermore, the nitrate concentrations of the treated samples T1–T5 decreased to between 4 and 9 mg/L (Figure 5b). Therefore, the nitrate contents in the industrial wastewater samples before and after treatment were within the permissible limit.

Phosphates

The main sources of phosphates in water are sanitation agents, fertilizers, insecticides, and industrial effluents. Phosphate-containing rocks and solid or liquid discharges are examples of natural sources. Both plants and animals depend on phosphorus for their growth. Polyphosphates are harmful because they obstruct the flocculation, coagulation, and lime soda treatment processes. All of the industrial wastewater samples (S1–S5) in this study contained phosphate concentrations above the WHO and CPCB standard of 5 mg/L. The industrial wastewater samples exhibited phosphate concentrations between 5.7 and 6.9 mg/L (Figure 5a). The treated samples (T1–T5) showed 2.7–3.6 mg/L of phosphates (Figure 5b). Phosphates promote the development of aquatic plants and planktons, which provide food for fish and other aquatic life forms, thereby increasing the fish population and enhancing the quality of marine life. Eutrophication may occur if there is an overabundance of phosphates as numerous fish and aquatic creatures are affected. Because phosphorous attaches to particles easily, it is frequently biologically inaccessible in water. Phosphates are a form of soluble phosphorus that can be absorbed; phosphates are not toxic to humans or animals unless when present in extremely high amounts. All samples that were treated with the TiO 2 nanoparticles had phosphate levels below the 5 mg/L maximum allowable limit set by the CPCB and WHO.

Toxic metal ions

AAS was employed to assess the concentrations of toxic metals in industrial wastewater samples, including Cr, Ni, Cd, and Pb. The concentrations of these toxic metal ions were evaluated in both untreated and treated wastewater. In the untreated samples S1–S5, the Cd level was found to be 0.001–0.04 mg/L, Cr level was 0.2–0.64 mg/L, Pb level was 0.01–0.51 mg/L, and Ni level was 0.012–0.48 mg/L. As per the CPCB norms, the permissible limits for Cd, Cr, Pb, and Ni ions are 0.005 mg/L, 0.5 mg/L, 0.3 mg/L, and 0.1 mg/L, respectively. The treated samples T1–T5 showed a Cd level of 0.001–0.0035 mg/L, Cr level of 0.07–0.64 mg/L, Pb level of 0.01–0.03 mg/L, and Ni level of 0.012–0.1 mg/L (Figure 6).

The samples treated with the synthesized TiO 2 nanoparticles (50 μg/mL) for 5 h showed very promising results. Overall, more than 72% of the toxic metal ions present in the industrial wastewater samples (Cd: 91%–100%, Cr: 90%–100%, Pb: 94%–100%, Ni: 72%–96%) were adsorbed (Figure 7a).

Desorption and reuse efficiency

The mechanism of metal-ion adsorption on a nanoparticle surface is reversible. Following metal ion desorption, the nanoparticles may be subsequently reused. To remove the metal ions from the exterior surfaces of the nanoparticles, we used 2.0 mL of a 1:1 mixture of methanol and NaOH (0.1 mol/L) to elute the adsorbent ten times. Numerous elution cycles were performed to assess the adsorbent’s capacity for reuse. It was discovered that after 5–10 adsorption–desorption cycles, the regenerated adsorbent could remove over 90% of the metal ions from the sample solution (Figure 7b). Equation 8 was used to calculate the adsorption efficiency after the adsorption–desorption study was performed 5–10 times.

Toxicity assays

Phytotoxicity assay

Plant growth and seed germination assays were used to assess the responses of P. sativum to both untreated and treated samples of industrial effluents. The control sample (distilled water) had a germination rate of 80%. When P. sativum was exposed to the TiO 2 nanoparticles at different concentrations (25, 50, 75, and 100 μg/mL), the germination rate reached 100%; here, we observed 100% germination rates for 25 and 50 μg/mL of nanoparticles, 60% germination rate for 75 μg/mL of nanoparticles, and 40% germination rate for 100 μg/mL of nanoparticles. In contrast to the controls, seeds treated with TiO 2 nanoparticles showed 90%–100% germination rates (Figure 8a). The flaws in the seeds also had major impacts on the lengths of the shoots and roots. Table 3 shows the plumule and radicle lengths of the P. sativum samples.

Algal growth inhibition assay

Algal species like Chlorella sp. and Volvox sp. cultured under standard conditions exhibited the characteristic growth phases, namely lag, log, stationary, and decline. Figure 7c shows the growth curves from the conventional algal growth inhibition tests over 72 h for TiO 2 nanoparticles applied at concentrations of 25–100 μg/mL, where the controls were maintained using distilled water. We observed that the algal log stage was normal even upon administration of the TiO 2 nanoparticles, which indicated that the synthesized nanoparticles did not affect the growth of the algal species (i.e. cyanobacteria). Figure 8c also shows that the TiO 2 nanoparticles are effective bioremediating agents because they show minimal algal inhibition.

MTT assay

Cell viability analysis following exposure to varying concentrations of TiO 2 nanoparticles (20–100 μg/mL) (Figure 8b) revealed minimal cytotoxicity toward mammalian cells over 24 h. The viability ranged from 87.78% to 99.07%, indicating excellent cellular tolerance. At an optimal dose of 50 μg/mL, the 3T3-L1 fibroblast cells maintained approximately 96% viability, highlighting the biocompatibility of the prepared nanoparticles. Even at the highest concentration tested (100 μg/mL), no significant cytotoxic effects were observed as the cell viability remained above 87%, confirming the biocompatibility. Comparatively, NIH 3T3 cells exposed to the standard drug ampicillin reportedly had a viability of 90% (Meiyazhagan et al., 2015), further supporting the safety of the prepared TiO 2 nanoparticles. According to the fibroblast cytotoxicity experiment, the TiO 2 nanoparticles are extremely non-toxic within the studied concentration range, showing an EC 50 value of 100.57 μg/mL for the MTT assay (Figure 8b). An EC 50 value of 100.57 µg/mL (Figure 8d) was obtained from this MTT assay, suggesting that TiO 2 nanoparticles are extremely non-toxic within the studied concentration range. These findings suggest that TiO 2 nanoparticles synthesized via a biological route are non-toxic to mammalian cells even at elevated concentrations and can be considered environmentally safe. As a result, they hold promise as effective and sustainable agents for the removal of heavy metals from industrial effluents.

A.-penicillioides -mediated TiO 2 nanoparticle synthesis and characterization were reported in our earlier research (Yamini and Devi Rajeswari, 2024; Vinayagam and Vijayarangan, 2025). Using a phylogenetic tree, the fungus in the earlier study was identified as A. penicillioides and was reported in the NCBI gene sequence repository as A. penicillioides VDRVYF with the accession number PQ570604.1 (https://www.ncbi.nlm.nih.gov/nuccore/2847402264). The earlier works briefly explored the optimization and kinetic studies of TiO 2 nanoparticles to increase the rate of Ni and Pb adsorption. Moreover, the biocompatibility of the synthesized nanoparticles was assessed through a hemolytic assay (Yamini and Devi Rajeswari, 2024). Figure 1 shows the role of A. penicillioides VDRVYF biomolecules in the nanoparticle formation. The proteins, polysaccharides, and other biological compounds are responsible for the nucleation and growth of TiO 2, where peptide-mediated formation occurs first followed by surface stabilization via biopolymer-coated TiO 2 and adsorption selectivity for metal-ion adsorption.

Optimization was conducted to improve the adsorption rate of the synthesized TiO 2 nanoparticles. Numerous factors were examined for the TiO 2 nanoparticle optimization, including pH, TiO 2 nanoparticle concentration, concentrations of toxic metal ions (Cr and Cd), and irradiation source type. The results of our investigation show that the optimal circumstances for Cd adsorption are pH 6, a nanoparticle concentration of 2 μg/mL, Cd concentration of 60 μg/mL, and exposure to sunshine as the irradiation source (Figure 2a). In the case of optimal Cr adsorption, the Cr content is 60 μg/mL, pH is 6, nanoparticle concentration is 10 μg/mL, and sunlight is used as the irradiation source (Figure 2b). The reactions were carried out at RT for 5 h with stirring. At best, we found that 70% of the Cd and 75% of the Cr contents could be eliminated.

As the concentration of the TiO 2 nanoparticles increased, more active sites were accessible for binding the target particles, which then increased the adsorption efficiency. Additionally, numerous variables like temperature, irradiation source type, reaction time, and pH may affect the adsorption process. Hence, the reactions should be carried out with the ideal parameter values to achieve greater adsorption rates (Kaliyamoorthy et al., 2023).

Adsorption isotherms that can also identify the surface heterogeneity, adsorption capacity, and type of adsorption (monolayer or multilayer) were used to study the interactions between the adsorbates and adsorbent surfaces under constant conditions. The kinetics studies indicate that it is essential to regulate the procedures and rate of the adsorption process through physisorption or chemisorption (Rajabi et al., 2024). The results of the present investigation were thus correlated to the Freundlich adsorption isotherm and pseudo-second-order kinetics. The Freundlich isotherm indicates that the hazardous metal ions are adsorbed on heterogeneous surfaces by the multilayer TiO 2 nanoparticles. Additionally, the fact that the adsorption occurs via an adsorptive photocatalytic reduction process (a synergistic mechanism employed to adsorb or convert environmental contaminants) is exploited by the pseudo-second-order kinetics (Figures 3a,b) (Rajabi et al., 2024).

A laboratory-scale investigation was used to further evaluate the efficiency rate of the optimized TiO 2 nanoparticles. Using AAS, the Cd and Cr concentrations were ascertained; the findings showed that the concentrations of specific metals were lower in the treated samples than the untreated samples. The physicochemical properties of the industrial wastewater samples that were collected and treated with TiO 2 nanoparticles were analyzed, and the results were compared to the standard values set by the CPCB. Both treated and untreated industrial wastewater samples were analyzed for the physicochemical parameters (pH, temperature, color, TDS, TSS, TOC, turbidity, conductivity, total alkalinity, BOD, COD, sulfates, nitrates, and phosphates) along with the concentrations of toxic metals (Pb, Cd, Ni, and Cr), and the results were compared with the CPCB limits.

The toxic metal-ion concentrations and most of the physicochemical parameters of the industrial wastewater samples were higher than the permissible limits established by the CPCB. Therefore, it is not possible to discharge these effluents directly into the environment. Remarkably, apparent results were observed for the samples treated with the TiO 2 nanoparticles (50 μg/mL) for 5 h. The physicochemical parameters also adhered to the CPCB permitted limits, with over 72% of the toxic metal ions being eliminated (Pb: 94%–100%, Ni: 72%–96%, Cd: 91%–100%, and Cr: 90%–100%) after treatment with the nanoparticles. Ma et al. (2020) collected food wastewater samples from different places and measured various physicochemical parameters, including the concentrations of heavy metal ions; their results showed that the samples exceeded the CPCB allowable levels. Thus, the researchers proposed a wastewater treatment method and stated that it was not feasible to release the effluents directly into the environment (Ma et al., 2020).

Research on the desorption and reuse of the TiO 2 nanoparticles shows that the nanoparticles can be used for bioremediation treatments for up to six cycles. We found that the adsorption rate of the first cycle was 94.2%, which decreased to approximately 65.2% by the sixth cycle. The production of TiO 2 metal complexes after adsorption is a significant secondary resource as these are used for bioremediation treatment. Prior research suggests that TiO 2 nanoparticles can be recovered via controlled desorption using mild acids or chelating agents; this will enable partial regeneration of the nanoparticles and restore its active surface sites for adsorption (Gupta and Nayak, 2012).

The phytotoxicity analysis of the TiO 2 nanoparticles prepared in this study shows a germination rate of 90%–100%. Kalpana et al. (2018) used dyes to conduct a phytotoxicity investigation, and their findings indicated a 40% plant germination rate. The log stages of Chlorella sp. and Volvox sp., which were normal when administered the synthetic TiO 2 nanoparticles, were confirmed by algal growth inhibition. Takeuchi et al. (2014) used eight distinct industrial wastewater samples to conduct a 72-h algal growth inhibition experiment; here, the samples did not exhibit toxicity for the first 6 h but later became toxic within 24 h. To identify the contaminants in the acquired samples (industrial effluents), the researchers recommended assessments through fast toxicity assays; additionally, they recommended remediation before releasing the effluents into surface water (Takeuchi et al., 2014).

By administering varying concentrations of the produced TiO 2 nanoparticles, we demonstrated its low cytotoxicity toward mammalian fibroblast cells over 24 h through the MTT assay. The cell viability was maintained above 87%, indicating biocompatibility, and no discernible cytotoxic effects were observed even at the highest dosage tested (100 μg/mL). To further confirm the safety of the TiO 2 nanoparticles, NIH 3T3 cells were exposed to the common medication ampicillin and showed a 90% vitality rate (Meiyazhagan et al., 2015). These results demonstrate that the generated TiO 2 nanoparticles function as effective, biocompatible, and short-term environmentally friendly bioremediating agents for the treatment of industrial wastewater.

The current batch adsorption study results demonstrate the viability of the synthesized nanoparticles. For pilot-scale applications, a continuous-flow configuration must be maintained by employing either an immobilized TiO 2 bed reactor (Viggi et al., 2010) or a solid–liquid separation method (Chai et al., 2018); this would allow the reactor to maintain optimal time, pH, and temperature conditions while providing a continuous wastewater treatment facility.

The present study demonstrates the possibility of employing TiO 2 nanoparticles as a nanobioremediation agent to efficiently extract heavy metal ions from industrial wastewater. The high surface areas of the TiO 2 nanoparticles along with their potent photocatalytic activity and propensity for different functional groups make them effective adsorbents in water purification. More than 72% of the specified heavy metal ions were found to be adsorbed in this study through extensive tests using various industrial wastewater samples, demonstrating a notable rate of adsorption. Furthermore, the physicochemical properties of the treated wastewater were found to be within the CPCB permitted limits, demonstrating the practicality of the proposed approach for environmental rehabilitation. We additionally explored the reusability of the TiO 2 nanoparticles in this study and found that consistent performance could be achieved over six cycles; the adsorption rate in this experiment dropped from an initial value of 94.2% to 65.2% by the sixth cycle. Toxicity examinations involving the MTT assay, algal growth inhibition assay, and P. sativum seed germination assay demonstrated the short-term ecologically benign integrity of the produced nanoparticles. In conclusion, the incorporation of TiO 2 nanoparticles in wastewater treatment offers a viable, sustainable, and effective method of reducing heavy metal contamination while strengthening the potential for application to large-scale industrial wastewater treatment.