In vitro cytotoxicity and microbial safety assessment of a novel nano-nitrogen fertilizer formulation

Introduction

The rising global demand for food has led to increased use of nitrogen fertilizers in modern agriculture. Although conventional nitrogen fertilizers are essential for achieving high crop productivity, their nitrogen use efficiency (NUE) is often relatively low, resulting in substantial nitrogen losses through ammonia volatilization, nitrate leaching, runoff, and denitrification (Galloway et al., 2008; Guo et al., 2010; Saurabh et al., 2024) [18,10] . These losses not only reduce nitrogen availability to crops but also contribute to groundwater contamination, eutrophication, and greenhouse-gas emissions, particularly nitrous oxide (N₂O) emissions (Galloway et al., 2008; Lassaletta et al., 2014; Channab et al., 2024) [18,20,1] . In recent years, nanotechnology has emerged as a promising approach for improving nutrient-use efficiency and developing nano-enabled fertilizers with enhanced nutrient delivery characteristics (DeRosa et al., 2010; Dimkpa and Bindraban, 2018; Zulfiqar et al., 2019) [4,17,27] . Nano-nitrogen (Nano-N) fertilizers are specially designed formulations intended to improve nitrogen delivery, nutrient uptake, and nitrogen-use efficiency through controlled nutrient release and enhanced interaction with plant surfaces and tissues. Their physicochemical characteristics, including particle size, surface properties, composition, and nutrient-release behaviour, can influence nutrient availability and plant uptake (DeRosa et al., 2010; Liu and Lal, 2015; Dimkpa and Bindraban, 2018) [16,21,17] . Several studies have reported that nano-enabled fertilizers can improve crop growth and nutrient-use efficiency while potentially reducing the quantity of fertilizer required compared with conventional formulations (Kah et al., 2018; Zulfiqar et al., 2019) [19,27] . However, the increasing application of nanoscale materials in agriculture has also raised concerns regarding their potential effects on soil microorganisms, particularly beneficial microorganisms that are essential for nutrient cycling, soil fertility, and plant health (Simonin and Richaume, 2015; Kah et al., 2018) [24,19] .

Soil microorganisms, including beneficial bacteria and fungi such as Azotobacter, Rhizobium, Bacillus, and Trichoderma species, perform important functions in agricultural soils, including biological nitrogen fixation, nutrient solubilization, phytohormone production, pathogen suppression, organic-matter decomposition, and maintenance of soil health (Vessey, 2003; Bhattacharyya and Jha, 2012; Woo and Pepe, 2018) [25,14,26] . Consequently, adverse effects of nano-fertilizers on beneficial microorganisms could potentially alter microbial community structure and soil biochemical processes, thereby affecting soil ecological balance and long-term agricultural sustainability (Simonin and Richaume, 2015; Rajput et al., 2018) [24,23] . Model microorganisms such as Escherichia coli are also widely used in nanoparticle toxicity studies to investigate cellular responses, oxidative stress, membrane damage, and changes in bacterial viability (Auffan et al., 2009; Bondarenko et al., 2013) [13,15] . Nanoparticles can exert toxic effects on microorganisms through several mechanisms, including the generation of reactive oxygen species (ROS), oxidative stress, interaction with and disruption of cell membranes, release of toxic ions, intracellular accumulation, and interference with essential metabolic processes (Nel et [22,24,15] . Their small size and high specific surface area can increase their interaction with microbial cell surfaces and biological components, potentially resulting in membrane damage, altered membrane permeability, enzyme inhibition, and impaired cellular metabolism (Nel et al., 2006; Bondarenko et al., 2013) [22,15] . Although numerous studies have reported beneficial effects of nano-fertilizers on crop growth and nutrient-use efficiency, comprehensive evaluations of their potential effects on beneficial soil microorganisms remain comparatively limited (Kah et al., 2018; Rajput et al., 2018) [19,23] . Therefore, controlled laboratory investigations are necessary to evaluate microbial responses to nano-fertilizer exposure, including concentration-dependent effects on microbial growth, viability, oxidative stress, membrane integrity, and cellular morphology. Such assessments are important for establishing the biosafety profile of nano-enabled fertilizers and supporting the development of appropriate application strategies for sustainable agriculture. The present study therefore aimed to evaluate the microbial safety of a novel nano-nitrogen fertilizer formulation using standardized microbiological and cellular assays. The study investigated concentration-dependent effects on microbial viability and growth, colony-forming ability, oxidative stress responses, membrane integrity, and cellular morphology in selected beneficial microorganisms. These assessments were undertaken to determine the compatibility of the Nano-N formulation with beneficial soil microbiota and to provide evidence for its biosafety and potential suitability for sustainable agricultural application.

Materials and Methods

Microbial Toxicity

The study aimed to visualise the toxicity in different microorganisms. PPFM subjected to different ppm of nanofertilizer from 200ppm to 1600 ppm.

Lactose DeHydrogenase (LDH) assay

The lactose dehydrogenase assay determines the number of nonviable cells by detecting the lactate dehydrogenase enzyme produced by the cells under adverse conditions. Lactate dehydrogenase is an enzyme that converts the stored form of energy (lactate) into active energy (pyruvate). During this conversion, NAD+ is reduced to NADH. As a result of the proton generated by the oxidation of NADH to NAD+, the exogenously supplied diaphorase enzyme can transform the INT tetrazolium salt into a red-colored formazan product. The red-colored formazan product was measured at 490 nm. The absorbance increases with an increase in the number of non-viable cells and vice versa. The substance-mediated cytotoxicity assay on Bacillus subtilis and Azotobacter chroococcum bacteria was determined using the LDH assay (EZcount TM Lactose dehydrogenase cell assay kit). Add 10 μl of 10% neutral red reagent to all treatments, which are then incubated in an orbital shaker at 37°C, 80 rpm for 2-4 hours. All the samples were centrifuged and the pellet was used for the assay. To the collected pellet, add 100 μl of neutral red solubilization solution and incubate the 96-well plate at room temperature for 10 minutes, then observe OD at 540 nm. The cell viability percentage was calculated using the formula after background correction.

𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦% =

𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷 -𝑇𝑒𝑠𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 𝑂𝐷 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷 𝑋 100

Results and Discussion

The neutral red uptake (NRU) assay was used to assess the viability and functional integrity of PPFM cells following treatment with the nano-fertilizer formulation (Table 1). The NRU assay is based on the uptake and accumulation of neutral red within the lysosomes of viable cells and is widely used as an indicator of cellular viability (Repetto et [9,6,7] . In PPFM, the highest cell viability was observed in the untreated control (100%), followed by 200 ppm (99.5%), 400 ppm (98.7%), and 1200 ppm (94.9%). At the highest concentration of 1600 ppm, cell viability decreased to 92.9%. The progressive reduction in NRU response at higher concentrations may indicate alterations in lysosomal function and cellular integrity (Repetto et al., 2008; Godoy et al., 2021) [9,5] . However, the maintenance of more than 90% cell viability even at 1600 ppm indicates that the formulation exerted only a limited effect on PPFM viability. The lactate dehydrogenase (LDH) assay was employed to evaluate cellular damage based on the release of intracellular LDH following disruption of the plasma membrane (Table 1 [12,7] .

Conclusion

Overall, the NRU and LDH assays demonstrated that the nanonitrogen fertilizer formulation had minimal adverse effects on PPFM viability and cellular integrity under the tested conditions. PPFM maintained 92.9% viability even at the highest concentration of 1600 ppm, while LDH analysis showed only 8.9% cell death at the same concentration. The concentrationdependent decline in NRU response and corresponding increase in LDH release indicate a mild cellular stress response at higher concentrations; however, the overall effect remained limited. The consistent results obtained from both assays indicate low microbial toxicity and good compatibility of the nano-nitrogen formulation with PPFM, supporting its potential microbial safety for agricultural application.

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