مقایسه سمیت اکولوژیک نانوذرات اکسید مس و اکسید نیکل بر فعالیت‌های آنزیمی در دو خاک‌ با بافت متفاوت

Background and Objectives: In recent decades, the rapid advancement of nanotechnology has revolutionized various sectors, including agriculture, medicine, and electronics. Consequently, the release of engineered nanoparticles (ENPs) into the environment has become increasingly unavoidable. Soil serves as a primary sink for these pollutants and accumulates nanoparticles through the application of nano-fertilizers, pesticides, wastewater sludge, and atmospheric deposition. Among various nanomaterials, metal oxide nanoparticles (MO-NPs), particularly copper oxide (CuO) and nickel oxide (NiO), are widely used due to their unique catalytic and antimicrobial properties. However, their accumulation in soil ecosystems raises serious concerns regarding their potential ecotoxicity to non-target organisms. Soil health and fertility are closely linked to the activity of soil microorganisms and their extracellular enzymes. Enzymes such as urease and phosphatases play pivotal roles in the biogeochemical cycling of nitrogen (N) and phosphorus (P), respectively. Any disruption in their functionality can lead to the deterioration of soil quality and nutrient availability. The toxicity of nanoparticles in soil is a complex phenomenon governed not only by the type and concentration of the contaminant but also by the physicochemical properties of the soil, such as texture and organic matter content. While studies have assessed the toxicity of individual nanoparticles, comparative research on the time-dependent effects of CuO and NiO nanoparticles in calcareous soils with distinct textures remains limited. Therefore, this study aimed to comparatively evaluate the ecological toxicity of CuO and NiO nanoparticles on key soil biochemical indicators (urease, acid phosphatase, and alkaline phosphatase) in two soils with contrasting textural properties (Sandy Loam vs. Clay) to understand the interaction between soil matrix and nanoparticle toxicity kinetics.




Materials and Methods: To investigate the role of soil texture in modulating nanoparticle toxicity, two surface soil samples (0–30 cm) were collected from agricultural lands with distinct physicochemical characteristics. The first soil was characterized as Sandy Loam (Sand: 58.2%, Clay: 19.8%, Organic Carbon: 0.70%), while the second soil was classified as Clay (Sand: 18.2%, Clay: 49.8%, Organic Carbon: 1.37%). The study was conducted as a factorial experiment based on a Completely Randomized Design (CRD) with three replications under controlled laboratory conditions at 25°C. The experimental factors included Nanoparticle Type (Copper oxide ~40 nm and Nickel oxide 10–20 nm, both with 99% purity), Concentration (four levels: 0, 100, 500, and 1000 mg kg⁻¹ dry soil), and Incubation Time (three intervals: 7, 24, and 72 days) to assess toxicity kinetics. To simulate realistic exposure conditions and avoid any solvent-related interference, nanoparticles were added to the soil as dry powder (uncoated) and thoroughly mixed. Soil moisture was maintained at 60% of field capacity (FC) throughout the incubation. At each sampling time, enzyme activities were assayed using standard spectrophotometric methods: Urease activity was quantified by measuring the ammonium released during urea hydrolysis, while Acid and Alkaline Phosphatase activities were quantified by the release of p-nitrophenol (pNP) using p-nitrophenyl phosphate as the substrate. Statistical analyses, including Independent Samples t-test for baseline comparison and ANOVA followed by Duncan’s multiple range test for treatment effects, were performed to interpret the data.




Results: Independent t-test results revealed a significant inherent difference in the biological potential of the two soils. The baseline activity of acid phosphatase in the Clay soil (658 µg pNP g⁻¹ h⁻¹) was approximately 66% higher than that of the Sandy Loam soil (396 µg pNP g⁻¹ h⁻¹) (P < 0.01), emphasizing the role of clay and organic matter in supporting enzymatic pools. Analysis of variance revealed significant main effects and interactions (Time × Type × Concentration). CuO nanoparticles exerted consistently higher and more persistent toxicity than NiO across both soil types. In the Sandy Loam soil, the impact was severe due to low buffering capacity. At the highest concentration (1000 mg kg⁻¹) of CuO, urease activity plummeted by over 90% relative to the control by the end of the incubation period (day 72). This inhibition is primarily attributed to the relatively high solubility of CuO and the interaction of Cu²⁺ ions with thiol (-SH) groups in enzyme active sites. Conversely, NiO nanoparticles exhibited a time-lag toxicity pattern, where significant inhibition in Sandy Loam soil was delayed until day 72. In contrast, the inhibitory effects were notably ameliorated in the Clay soil. A key finding was the non-significant main effect of incubation time on urease activity (P > 0.05) in the clay soil. This suggests that the high clay content (49.8%) facilitated the formation of stable enzyme-clay-humus complexes, which protected urease enzyme from denaturation and proteolytic degradation over time. Interestingly, NiO nanoparticles at lower concentrations (100 mg kg⁻¹) occasionally demonstrated a hormetic effect (stimulation of activity), particularly for alkaline phosphatase in the early stages, contrasting with CuO which generally followed a strict dose-dependent inhibition pattern.




Conclusion: This study provides robust evidence that soil texture is a key determinant in the ecological risk assessment of engineered nanoparticles. Light-textured (Sandy Loam) soils exhibit significantly higher ecological sensitivity to metal oxide contamination compared to heavy-textured (Clay) soils, which possess a "protective effect" due to higher colloidal content. Among the tested pollutants, CuO nanoparticles pose a more severe and immediate threat to soil nutrient cycling than NiO due to their higher chemical reactivity. The findings suggest that environmental risk assessments for nanomaterials must account for site-specific soil properties and the dynamic nature of toxicity (aging effect) over time. Future research should focus on molecular‑level mechanisms and long-term field monitoring to further validate these interactions.