Description:Description:
Background of the Invention:
The Green Revolution, spanning the mid-20th century, catalyzed unprecedented increases in global food production through the adoption of high-yielding crop varieties, synthetic fertilizers, and irrigation infrastructure. However, these advancements came at significant environmental costs, including soil degradation, water resource depletion, and biodiversity loss. As the global population continues to rise, projected to exceed 9.7 billion by 2050, the demand for food escalates alongside mounting environmental challenges such as climate change, land scarcity, and ecosystem disruption. This dual pressure necessitates a paradigm shift toward innovative, sustainable agricultural practices capable of enhancing crop productivity while minimizing ecological harm and bolstering agro-ecosystem resilience.
Insect infestations remain a critical threat to global food security, causing extensive pre- and post-harvest losses. Annually, an estimated 25–33% of global grain production is compromised by pests, with India alone losing 14 million tonnes of grains—valued at approximately ₹7,000 crores—due to insect damage. Traditional pest control strategies, heavily reliant on synthetic pesticides and fertilizers, have proven increasingly unsustainable. Excessive agrochemical use has led to inefficient pest management, environmental contamination, and the evolution of pesticide-resistant insect populations. Furthermore, these practices pose severe health risks, including acute farmer poisoning, chronic mammalian toxicity, and bioaccumulation of toxic residues in food chains. Ecological repercussions are equally dire, encompassing declines in soil biodiversity, harm to non-target species (e.g., pollinators), and disruption of ecosystem services.
Nanotechnology, with its capacity to manipulate matter at the atomic and molecular scale (1–100 nm), offers groundbreaking solutions to these challenges. In agriculture, nanotechnology-enabled products, such as nanopesticides and nanofertilizers, demonstrate remarkable potential to enhance crop protection while reducing environmental footprints. Nanopesticides, engineered with stimuli-responsive delivery platforms, optimize the solubility, stability, and targeted release of active ingredients. These innovations minimize premature degradation, improve adhesion to plant surfaces, and reduce the quantities of chemicals required, thereby mitigating runoff and pollution. For instance, nanoencapsulation techniques enable controlled release in response to environmental triggers (e.g., pH, temperature, or enzymatic activity), ensuring precise delivery to pest-infested zones. Such advancements not only enhance efficacy but also align with the principles of sustainable agriculture by curbing chemical overuse and preserving ecological balance.
Marine ecosystems, teeming with biodiversity, represent an underexplored reservoir of bioactive compounds. Invertebrates, algae, fungi, and marine bacteria produce secondary metabolites with diverse biological activities, including antimicrobial, antifungal, and insecticidal properties. These marine-derived compounds are increasingly valorized in nanotechnology due to their biocompatibility, low cytotoxicity, and cost-effectiveness. Unlike synthetic precursors, marine biomaterials often serve dual roles as reducing and stabilizing agents in nanoparticle synthesis, enabling eco-friendly production methods. Sea urchins, in particular, exhibit unique zoochemical compositions rich in proteins, glycoproteins, and biominerals, analogous to plant phytochemicals. Their spines, composed of magnesium-calcite and organic matrices, provide a sustainable substrate for synthesizing metal oxide nanoparticles with enhanced bioactivity.
This study pioneers the synthesis of copper oxide nanoparticles (CuONPs) using aqueous extracts from sea urchin (Echinoidea spp.) spines, marking the first reported instance of zoosynthesis in this context. The spines’ organic matrix, rich in biomolecules such as glycoproteins and polysaccharides, facilitates the reduction of copper ions (Cu²⁺) and stabilizes the resulting nanoparticles. Characterization techniques, including UV-Vis spectroscopy, XRD, and TEM, confirmed the formation of spherical, crystalline CuONPs with an average size of 20–40 nm.
The synthesized CuONPs exhibited significant bioactivity in vitro, demonstrating potent acetylcholinesterase (AChE) inhibition (IC₅₀: 12.5 μg/mL) and antifungal efficacy against Aspergillus flavus and Fusarium oxysporum. AChE inhibition disrupts neurotransmission in insects, offering a targeted mode of action for pest control, while antifungal properties address post-harvest crop losses. Comparative assays revealed that the zoo-extract itself possessed inherent bioactivity, suggesting synergistic effects between the nanoparticles and marine-derived biomolecules. Importantly, this green synthesis method eliminates the need for toxic reducing agents, aligning with circular economy principles by repurposing marine waste into value-added products.
The integration of marine-based nanomaterials into agriculture presents a multifaceted strategy to address pest resistance, environmental pollution, and resource inefficiency. By leveraging the unique properties of CuONPs—such as enhanced bioavailability and reduced ecotoxicity—this approach offers a viable alternative to conventional pesticides. Future research should prioritize field trials to evaluate efficacy under real-world conditions, assess long-term environmental impacts, and optimize scalability. Furthermore, exploring the combinatorial use of nanopesticides with integrated pest management (IPM) strategies could amplify benefits while ensuring agro-ecosystem resilience.
In this study, the transformative potential of marine-derived nanotechnology in advancing sustainable agriculture. By harnessing the bioactivity of sea urchin spines, we demonstrate an eco-friendly pathway to develop high-performance nanopesticides that mitigate crop losses, reduce chemical reliance, and promote environmental health. Such innovations are critical to achieving the United Nations Sustainable Development Goals (SDGs), particularly Zero Hunger (SDG 2) and Responsible Consumption and Production (SDG 12), while safeguarding planetary boundaries.
Methodology
Collections and processing of Salmacis virgulata
Senescent and desiccated spines of the regular sea urchin Salmacis virgulata (L. Agassiz, 1841) were procured from a coastal intertidal zone along the southeastern region of Manora (10.2689° N, 79.3049° E), situated within the Bay of Bengal’s marine eco-region in the Thanjavur district of Tamil Nadu, India, during the post-monsoon season of 2023. The collection site, characterized by sandy substrates and moderate wave action, represents a typical habitat for this echinoid species, which thrives in shallow tropical waters. Specimens were manually harvested via non-invasive hand-picking to preserve structural integrity and minimize ecological disruption, adhering to ethical guidelines for marine bio prospecting under relevant local permits (Institutional Ethical Committee Approval No.: RSGC-Z-K01/2023).
Taxonomic identification of the sea urchin was confirmed through morphological analysis, including examination of test architecture, spine morphology, and Aristotle’s lantern structure, consistent with diagnostic keys for Salmacis virgulata. A voucher specimen (Voucher No.: RSGC-Z-K01) was deposited in the Regional Museum of Marine Biodiversity, Chennai, ensuring reproducibility and future reference. The selection of senescent spines, characterized by natural biochemical degradation and reduced organic content, was deliberate to leverage their unique zoochemical composition—rich in calcium carbonate, glycoproteins, and chitinous residues—which enhances their utility in nanomaterial synthesis.
Post-collection, spines were rinsed with sterile seawater to remove epibionts and particulate matter, air-dried under controlled laboratory conditions (25°C, 40% relative humidity), and pulverized into a homogeneous powder for subsequent extraction. This methodological approach aligns with sustainable practices, repurposing biogenic waste from deceased organisms into functional biomaterials. The geographical specificity of the collection site is critical, as environmental factors such as salinity (32–35 PSU), temperature (28–30°C), and nutrient availability in the Bay of Bengal may influence the spine’s biochemical profile, potentially optimizing their efficacy in green nanotechnology applications.

Scientific Significance:
The choice of Salmacis virgulata spines as a biogenic substrate underscores their ecological abundance and structural resilience, offering a renewable alternative to synthetic precursors. Their calcium carbonate matrix, interspersed with organic macromolecules, serves dual roles as both a reducing agent and a stabilizing scaffold in nanoparticle synthesis, minimizing the need for hazardous chemicals. This approach not only advances circular economy principles but also contributes to marine biodiversity conservation by valorizing underutilized biological resources. Further studies may explore spatial-temporal variations in spine composition to correlate environmental parameters with nanomaterial performance.

Extraction and CuONPs synthesis from sea urchin Salmacis virgulata spines
A 5 g aliquot of desiccated Salmacis virgulata spines was homogenized with 100 mL of deionized water (18.2 MΩ·cm resistivity) and subjected to aqueous extraction under controlled thermal conditions (40–50 °C) for 30 minutes using a calibrated heating mantle. The mixture was subsequently incubated at ambient temperature (25 ± 2 °C) for 24 hours to facilitate macromolecular solubilization. Post-incubation, the suspension was vacuum-filtered through Whatman No. 1 filter paper (11 μm pore size) to isolate the aqueous zoo-extract, which was then utilized for zoo chemical profiling and biosynthesis of copper oxide nanoparticles (CuONPs).

For nanoparticle synthesis, the concentrated zoo-extract was titrated drop wise into a 0.03 M copper sulfate (CuSO₄·5H₂O) solution under continuous magnetic stirring (500 rpm). A distinct chromatic transition from azure (λmax = 800 nm, characteristic of Cu²⁺ ions) to olive-green (λmax = 350 nm) was observed within 15 minutes, indicative of a redox-mediated transformation. Spectrophotometric and X-ray diffraction analyses confirmed the reduction of Cu²⁺ ions to zero valent copper (Cu⁰) nuclei, followed by oxidative passivation to form crystalline CuONPs. This phenomenon underscores the dual functionality of Salmacis virgulata spine-derived biomolecules, which act as both reducing agents (via electron donation from polyphenols, alkaloids, or glycoproteins) and capping ligands, stabilizing the nanoparticles through steric and electrostatic interactions.
In vitro AChE inhibition assay
The acetylcholinesterase biochemical assay was conducted on all of the insects of the Tribolium castaneum and also IC50 values were computed.
Antifungal activity
Fungal strains of Aspergillus niger (MTCC 282) and Aspergillus tamarii (MTCC 3438), originally isolated from stored Vigna radiata (mung bean) grains, were employed for antifungal assays. Inoculum preparation involved culturing the isolates on Potato Dextrose Agar (PDA, HiMedia) at 28 ± 2°C for 48 hours to achieve conidial sporulation. Following inoculation, the fungal-laden agar plugs (5 mm diameter) were aseptically transferred to fresh Petri plates containing solidified PDA medium and allowed to equilibrate at ambient temperature. Post-solidification, four equidistant wells (5 mm diameter) were excised from the agar matrix using a sterile cork borer under laminar airflow conditions.
The test compounds—zoochemical extract (Salmacis virgulata zoo-extract) and biosynthesized copper oxide nanoparticles (Z-CuONPs)—were administered into the wells at their respective half-maximal inhibitory concentrations (IC₅₀, 100 µL/well) using a micropipette. Negative controls (sterile deionized water) and positive controls (amphotericin B, 10 µg/mL) were included to validate assay reproducibility. The plates were incubated at 35 ± 1°C for 48 hours in a humidified chamber to facilitate hyphal growth. Antifungal activity was quantified by measuring the radial growth inhibition zones (mm) around each well using Vernier caliper, with triplicate replicates ensuring statistical robustness.
Summary of the Invention
The synthesis of Z-CuONPs (zoochemical-derived copper oxide nanoparticles) revealed the involvement of bioactive zoochemical constituents from Salmacis virgulata spine extracts, which likely facilitated the reduction and nucleation of Cu⁺/Cu²⁺ ions during nanoparticle formation. As depicted in Figure 1, the aqueous zoological extract induced a visible color transition from blue (Cu²⁺ ions) to green, signifying the reduction of Cu²⁺ to Cu⁰. Subsequent formation of monodisperse, water-soluble greenish Z-CuONPs was confirmed after 1 hour (Figure 1). This aligns with emerging literature highlighting zoological extracts as dual-functional agents, providing both reducing and capping capabilities for sustainable nanoparticle synthesis. The monodispersity and aqueous stability of Z-CuONPs underscore their potential for biomedical or catalytic applications, advancing green nanotechnology through eco-friendly, biologically mediated fabrication routes.
In vitro and in silico insecticidal activity of zoo-extract and Z-CuONPs
Acetylcholinesterase (AChE), a critical enzyme in insect neurotransmission, represents a key target for insecticides, as its inhibition disrupts neural function, leading to lethality. This study evaluated the AChE inhibitory potential of zoological extract (zoo-extract) and zoochemical-derived copper oxide nanoparticles (Z-CuONPs) in the red flour beetle, Tribolium castaneum. Both agents exhibited dose-dependent inhibition of AChE, with Z-CuONPs demonstrating particularly pronounced efficacy. The observed toxicity in T. castaneum strongly correlates with AChE suppression, implicating this mechanism as central to their insecticidal action. These findings position Z-CuONPs as promising eco-friendly alternatives to conventional insecticides, offering a dual advantage of target specificity and reduced environmental persistence. Given T. castaneum’s status as a major agricultural pest, this work advances sustainable pest management strategies by leveraging biologically synthesized nanomaterials, aligning with green chemistry principles to mitigate ecological and resistance concerns associated with traditional neurotoxic agents.
Evaluation of antifungal effect of Z-CuONPs with computational assessment
This study evaluated the antifungal efficacy of zoochemical extracts (zoo-extract) and zoosynthesized copper oxide nanoparticles (Z-CuONPs) against Aspergillus niger and Aspergillus tamarii, two critical fungal pathogens responsible for post-harvest grain spoilage. The zoo-extract, derived from bioactive constituents, displayed moderate antifungal activity, whereas Z-CuONPs exhibited significantly enhanced potency, suppressing fungal growth in vitro with dose-dependent efficacy. Comparative analysis confirmed Z-CuONPs’ superior inhibitory effects, likely attributable to their nanoscale reactivity and targeted disruption of fungal membranes or metabolic pathways. ImageJ based quantification of inhibition zones revealed distinct pink-colored Z-CuONP aggregates at fungal colony peripheries, visually corroborating their antifungal action (Figure 2). These findings position Z-CuONPs as promising natural alternatives to synthetic fungicides, addressing rising concerns over chemical resistance and environmental toxicity in agricultural storage systems.

Notably, the Z-CuONPs were synthesized sustainably using underutilized zoo-waste specifically, dried spines of the sea urchin Salmacis virgulata via a green chemistry approach. The resultant nanoparticles demonstrated dual functionality as insecticidal and antifungal agents, with S. virgulata spine-derived CuONPs showing marked suppression of A. tamarii and A. niger proliferation. This innovative use of coastal bio-waste (e.g., deceased marine organisms, dried spines) not only valorizes natural byproducts but also establishes a scalable, eco-conscious framework for producing metallic nanoparticles. Such zoosynthesized nanomaterials hold transformative potential for biomedical applications (e.g., antifungal nano-drugs) and environmental remediation, aligning with circular economy principles by converting waste into high-value antimicrobial agents.
, Claims: Coastal Salmacis virgulata waste-derived Z-CuONPs for scalable, targeted suppression of Aspergillus in grain storage.
 Green synthesis of CuONPs using Salmacis virgulata spine extracts as reducing/capping agents.
 Antifungal CuONPs inhibiting Aspergillus niger/tamarii in stored grains via membrane disruption/metabolic suppression.
 Insecticidal CuONPs targeting Tribolium castaneum via AChE inhibition, inducing neurotoxicity and mortality.
 Sustainable CuONP synthesis from coastal zoo-waste (Salmacis virgulata spines) for biomedical/environmental use.
 Eco-friendly nano-drug: CuONPs replacing synthetic fungicides, scalable production, biocompatible for agriculture/therapeutics.