نوع مقاله : مقاله پژوهشی
عنوان مقاله English
نویسندگان English
The increasing entry of silver nanoparticles (Ag-NPs) into soil ecosystems threatens biochemical health and biogeochemical cycles. This study evaluated the ecotoxicity and temporal dynamics (aging) of Ag-NPs on soil enzyme activities across two contrasting soil textures. A laboratory incubation experiment was conducted as a factorial based on a completely randomized design (CRD). Treatments included soil texture (sandy loam and clay), Ag-NP concentration (0, 100, 500, and 1000 mg/kg), and incubation time (7, 24, and 72 days). Urease, acid phosphatase, and alkaline phosphatase activities were measured as biomarkers. Results showed that Ag-NP inhibition strongly depended on soil texture and time. In sandy loam soil, urease was highly vulnerable; 1000 mg/kg reduced its activity by 54% on days 7 and 24 compared with the control. In contrast, clay soil showed greater buffering capacity. A pronounced hormesis effect occurred for alkaline phosphatase in clay soil: 100 mg/kg on day 7 increased activity by 109% relative to the control, and stimulation stabilized at a persistent 21% increase on days 24 and 72. For acid phosphatase in clay soil, initial inhibition at 1000 mg/kg almost fully recovered by day 24 (8% decline), but a 16% decline reappeared by day 72. Clay colloids and organic carbon likely mitigated Ag-NP toxicity through chelation and reduced bioavailability, whereas light soils remained highly vulnerable without this protective barrier. These findings highlight the importance of soil properties in determining nanoparticle ecological impacts. Nevertheless, temporal monitoring showed that pollutant aging remains a silent long-term ecological risk, even in colloid-rich soils.
کلیدواژهها English
The rapid development and widespread application of nanotechnology in recent decades have inevitably led to the extensive release of engineered nanomaterials into the environment and biological cycles. Among these emerging materials, silver nanoparticles (Ag-NPs) have secured a prominent position in various sectors, including medicine, textiles, food packaging, wastewater treatment, and modern agricultural inputs, owing to their unique physicochemical properties, high surface-area-to-volume ratio, and exceptional antimicrobial capabilities. Recent global estimates indicate that the annual production of these nanoparticles has exceeded hundreds of tons. Consequently, a significant portion of these engineered nanoparticles eventually finds its way into the soil ecosystem through various pathways, such as the discharge of industrial and municipal wastewater, the application of sewage sludge as a soil amendment or fertilizer in agricultural lands, and the direct use of nano-based pesticides and fertilizers. Soil acts as the ultimate environmental sink for these modern contaminants. The accumulation of Ag-NPs in the surface horizons, where the highest concentration of biological activity occurs, poses a severe challenge to the functional stability, biodiversity, and biochemical health of terrestrial ecosystems. Soil quality, health, and fertility are directly linked to the dynamic activity of microbial communities and the secretion of extracellular enzymes. Soil enzymes, acting as biological catalysts, play a pivotal role in the decomposition of organic residues, the mineralization of essential nutrients, and the stabilization of soil organic matter. Among these, the urease enzyme, which plays a key role in urea hydrolysis and the nitrogen cycle, and phosphatases, which are responsible for releasing inorganic phosphorus from organic compounds in the phosphorus cycle, are considered some of the most sensitive and reliable biochemical indicators for assessing environmental toxicity. The primary mechanism of Ag-NP toxicity on these biocatalysts mainly occurs through the process of oxidative dissolution and the continuous, gradual release of silver ions (Ag+) into the soil solution. These free cationic ions have a strong thermodynamic affinity to interact with the functional groups of proteins, particularly the sulfhydryl or thiol (-SH) groups in the structure of the amino acids that make up the enzymes, leading to conformational changes (denaturation) and the blocking of the active site. Furthermore, Ag-NPs can induce oxidative stress through the excessive production of reactive oxygen species, leading to lipid peroxidation and the destruction of microbial cell membranes. Despite numerous toxicological studies, a major gap in the ecotoxicology of nanomaterials is the neglect of temporal dynamics, commonly referred to as the "aging" phenomenon. Toxicity in the heterogeneous soil environment is not a static phenomenon; it undergoes fundamental physical and chemical changes over time due to processes like aggregation, sulfidation, and surface coating by dissolved organic matter, which directly affect bioavailability. Furthermore, the severity of this toxicity is heavily influenced by the intrinsic properties of the soil, particularly its texture and colloidal content. Therefore, the present study was designed with the comprehensive aim of (1) monitoring the dose-dependent effects of varying levels of Ag-NPs on the key functional enzymes urease, acid phosphatase, and alkaline phosphatase, and (2) rigorously evaluating the role of pollutant aging (over sequential intervals of 7, 24, and 72 days) across two contrasting soil textures (sandy vs. clay). The overarching goal is to realistically assess the biochemical resilience and self-purification capacity of calcareous soils against this emerging nanotoxicity.
To monitor the ecotoxicological behavior of nanoparticles, composite sampling from the surface soil horizon (0-30 cm depth) was conducted in two agricultural regions with distinctly different pedological characteristics. The first soil was collected from agricultural lands in the suburbs of Karaj, while the second was obtained from the Chitgar area. Samples were air-dried in the shade at room temperature and passed through a standard 2-mm sieve. Physicochemical analysis revealed two contrasting ecological matrices: the first was a light-textured sandy loam soil (58.2% sand, 19.8% clay) characterized by a poor organic carbon content (0.70%); the second was a heavy-textured clay soil containing 49.8% clay and a relatively rich organic carbon content (1.37%), providing a significantly higher potential for surface interactions with pollutants. The stress-inducing agent used in this study was analytical-grade (99.99% purity) silver nanoparticle powder with a spherical morphology, a hydrodynamic diameter of approximately 20 nm, and a specific surface area ranging from 18 to 22 m²/g. To prevent laboratory errors and synergistic/antagonistic interferences from chemical solvents and stabilizers, and to accurately simulate the dry atmospheric deposition of pollutants, the uncoated nanoparticles were applied as a dry powder. The research was implemented as a microcosm incubation experiment organized in a factorial framework based on a completely randomized design (CRD) with three replications. The independent variables included soil texture (sandy and clay), Ag-NP concentration (0 as the control, 100, 500, and 1000 mg/kg of dry soil), and temporal dynamics or aging (sampled at 7, 24, and 72 days). The microcosms were prepared by adding the precise weight of Ag-NPs to 100 g of dry soil, followed by 30 minutes of mechanical mixing to ensure homogeneous distribution. Soil moisture was adjusted to 60% of the field capacity using distilled water. The experimental units were incubated in absolute darkness at a constant temperature of 25°C. At the specified intervals, destructive sampling was performed. Urease activity was assessed using the Zantua and Bremner protocol, where ammonium released from urea hydrolysis was measured via Nesslerization at 410 nm. Acid and alkaline phosphatase activities were determined based on the Eivazi and Tabatabai method, measuring the release of p-nitrophenol at 400 nm after incubation with p-nitrophenyl phosphate in specific buffers (pH 6.5 and 11, respectively). Prior to statistical modeling, data normality was confirmed. Due to the fundamental structural differences between the two soils and the confirmation of heterogeneous error variances by Bartlett's test, independent Analyses of Variance (ANOVA) were conducted for each soil matrix using SAS software. Mean comparisons were performed using Duncan's Multiple Range Test at the 5% and 1% probability levels.
The baseline biological potential evaluated via independent T-tests prior to nanoparticle treatment revealed that the sandy loam soil possessed significantly higher basal urease and alkaline phosphatase activities, whereas the clay soil exhibited greater basal acid phosphatase activity, indicating distinct inherent biochemical capacities. The ANOVA results demonstrated that the main effects of incubation time, Ag-NP concentration, and their interaction were highly significant for most enzymatic traits, proving that the inhibitory effect of silver nanoparticles is a highly dynamic process dictated by the soil matrix. In the light-textured sandy soil, urease showed the most severe dose-dependent vulnerability. On day 7, the application of 1000 mg/kg Ag-NPs plummeted urease activity by 57% compared to the control. This suppressive effect persisted robustly through day 24, resulting in a 52% decline. By day 72, the basal urease potential in the sandy soil had dropped significantly due to substrate depletion, and high concentrations of Ag-NPs maintained a chronic inhibitory state. Acid phosphatase in the sandy soil also displayed high susceptibility, particularly on day 24, where the 500 mg/kg dose caused a severe 55% reduction in catalytic activity. Conversely, the heavy clay soil demonstrated a remarkable and robust buffering capacity. Although urease activity in the clay soil experienced an initial dose-dependent decline on day 7 (dropping by up to 67% at the highest dose), it showed a fascinating stabilization over time. By day 72, the activities at 100 and 500 mg/kg Ag-NPs converged entirely with the control, showing less than a 7% fluctuation, thus confirming the protective role of clay colloids. More strikingly, the presence of Ag-NPs in the clay soil induced a clear hormesis effect on alkaline phosphatase. The application of 500 mg/kg Ag-NPs led to an astonishing 82% surge in alkaline phosphatase activity relative to the control on day 7. As the pollutant aged (days 24 and 72), this stimulation stabilized into a persistent 24% to 31% increase, indicating a strong compensatory response from the microbial community to overcome the initial oxidative stress. For acid phosphatase in the clay soil, the initial toxicity was fully neutralized by day 24, achieving a 100% recovery even at the maximum dose of 1000 mg/kg; however, the cumulative effects of aging and continuous ion dissolution led to a delayed, chronic toxicity, reducing its activity again by 24% on day 72.
The findings of this comprehensive study verify that the introduction of engineered silver nanoparticles into soil ecosystems poses a critical, complex, and time-dependent threat to biochemical health. The ecological toxicity of Ag-NPs is not merely a function of contaminant concentration; rather, it is profoundly modulated by the pedological characteristics of the receiving matrix and the aging duration. Light-textured sandy soils, suffering from colloidal poverty and low organic carbon, exhibit acute ecological sensitivity. In the absence of sufficient binding sites, silver ions remain highly bioavailable in the soil solution, leading to severe and irreversible biochemical degradation, specifically crippling the nitrogen cycle as evidenced by the dramatic collapse of urease activity. On the other hand, heavy clay soils act as potent ecological buffers. The high specific surface area, negative charge of clay minerals, and abundance of humic substances facilitate physical stabilization, surface adsorption, and intense chelation of free silver ions. This mechanism drastically reduces bioavailability and allows microbial communities to execute adaptive responses, such as hormesis and medium-term functional recovery. However, the temporal monitoring clearly illustrated that pollutant aging does not uniformly equate to detoxification. In heavy soils, while medium-term recovery is possible, the continuous oxidative dissolution of high-dose nanoparticles eventually overwhelms the soil's protective barriers, manifesting as delayed, chronic toxicity in the long term. Conclusively, this study emphasizes that environmental risk assessments and regulatory frameworks for the disposal of nanomaterial waste must transition from relying on short-term, acute toxicity paradigms. Instead, standards must be explicitly tailored to incorporate site-specific buffering capacities and long-term aging dynamics to prevent the prolonged degradation of agricultural soil fertility and safeguard terrestrial biogeochemical cycles.
This work was supported by the Department of Soil Science, Ka. C., Islamic Azad University, Karaj, Iran as a M.Sc. project of the first author of the manuscript. The founder had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.
Conceptualization Sh. R. and A. Kh., methodology, Sh. R., software, A. Kh., validation, Sh. R. and A. Kh., formal analysis, A. Kh., investigation, Sh. R., resources, Sh. R., data curation, Sh. R., writing—original draft preparation, Sh. R. and A. Kh., writing—review and editing, Sh. R. and A. Kh., visualization, Sh. R., supervision, Sh. R., project administration, Sh. R., funding acquisition, Sh. R. and A. Kh., All authors have read and agreed to the published version of the manuscript.
Generative AI and AI-assisted technologies were not used in writing this article.
The data that support the findings of this study are available from the corresponding author, upon reasonable requests.
The authors would like to thanks the Karaj branch of Islamic Azad University for kindly providing the equipment used in this study.
Ethical principles were considered by all authors during the conducting and publishing of this study.
The authors declare no conflict of interest.
