Synthesis, optimization, and biosafety assessment of nanozeolite-based slow-release nitrogen fertilizers for spinach beet (Beta vulgaris var. bengalensis)

This study investigates the efficacy of synthesized nanozeolite-based nitrogen fertilizers (NZUF and SNZUF) in enhancing nutrient use efficiency, providing controlled-release properties, and ensuring crop safety in Spinach Beet (Beta vulgaris var. bengalensis). Nanozeolites (NZ), due to their high surface area and unique ion-exchange capabilities, offer promising alternatives to conventional nitrogen fertilizers. The research involved synthesizing NZ, characterizing their physicochemical properties through FTIR, XRD, SEM, TEM, TGA, and Zeta potential, toxicity, and slow-release studies, followed by pot experiments using the spinach beet crop to evaluate their effect on plant growth, yield, and nutrient uptake. Results indicated that nanozeolite-based fertilizers enhanced nitrogen use efficiency, maintained a sustained nutrient supply, and extended 75% ammonium and nitrate release up to ~48-50 and ~45-48 days, respectively, compared to 10 -15 days for urea. Over two years, T4 (100% SNZUF) yielded 8.67% more than T2 (urea), while T5 (100% NZUF) and T6 (75% SNZUF) had slightly higher yields of 0.87% and 1.09%, respectively, compared to T2. Importantly, no adverse toxic effects were observed in Spinach Beet, providing a strong reassurance of the safe application of these nanofertilizers. This study underscores the potential of nanozeolite-based fertilizers (especially SNZUF) to enhance nitrogen use efficiency, improve crop yield and soil health, and offer a sustainable, eco-friendly alternative to conventional urea for climate-smart agriculture.

The scarcity of nitrogen (N) in Indian soils is widespread, with approximately 99% of soils showing a positive response to N application. As an essential component of proteins, N plays a critical role in sustaining life on Earth, adhering to Liebig’s law of minimum, where its deficiencies must be addressed before introducing other nutrients to avoid futile efforts. The usage of N-based fertilizers has experienced significant growth over the years, surging from a mere 0.45 kg ha-1 in 1950-51 to nearly 123 kg ha-1 in 2020-21 (1, 2). Conventional N fertilizers are significant contributors to greenhouse gas (GHG) emissions through the production of nitrous oxide (N2O), which leads to low N use efficiency (NUE) (3). Low NUE has agronomic implications, such as increased fertilizer costs, lower yields, soil health degradation, and nutrient imbalances, as well as environmental implications, including water pollution, greenhouse gas emissions, and biodiversity loss (4–6).

Zeolites, naturally occurring minerals with a unique structure characterized by a three-dimensional framework of interconnected channels and cages, have long been recognized for their potential in agriculture. These pores can trap and store molecules such as N, which are slowly released to the plant roots over time (7). The dominant process in N adsorption on these zeolites is influenced by the electric field created by the charge-balancing cations in the pores and by hydrogen bonding with the surface (8). In the growing need for sustainable agricultural practices, using nanozeolite-based fertilizers emerges as a practical solution (9). Nanozeolites, with particles smaller than 100 nm, due to their high surface area and unique ion-exchange properties, have shown the potential to improve the efficiency of nutrient delivery to plants (10). Nanozeolite-based fertilizers have gained attention as potential solutions to improve agricultural nutrient use efficiency. Nanozeolites, porous aluminosilicates with high ion-exchange and adsorption capabilities, can be carriers for fertilizers, pesticides, and insecticides (11). Their unique structure, featuring micro-, meso-, and macropores, allows for the controlled release of nutrients, potentially reducing leaching and environmental contamination (12). Nanozeolites can be synthesized from waste materials, aligning with sustainable development principles. Nanotechnology in fertilizers, including zeolite-based composites, offers promising advancements over conventional polymer-coated fertilizers, with the potential for enhanced nutrient delivery through plant surfaces (13).

By incorporating N into nanozeolite structures, it is possible to create a slow-release mechanism that ensures that N is available to the plant over an extended period, reducing the risk of nutrient leaching into the soil and groundwater (14). The slow release of N from nanozeolite-based fertilizers can lead to several agronomic benefits, including improved nutrient use efficiency and prolonged N availability to plants. This slow-release mechanism aligns with the nutrient uptake patterns of crops, potentially leading to better growth and higher yields (15). Additionally, such fertilizers can reduce fertilizer application frequency, saving farmers costs and labor. This study goes beyond conventional approaches by integrating the development, characterization, and application of nanozeolite-based slow-release fertilizers within a single framework. While previous studies have largely focused on the structural properties and nutrient release behavior of nanozeolites, the present work extends this by evaluating both agronomic performance and potential environmental risks simultaneously (15). Specifically, surface-modified nanozeolite fertilizers (SNZUF) were developed and compared with unmodified systems (NZUF) and conventional nitrogen sources under different application levels. In addition to assessing plant growth and yield, this study uniquely incorporates toxicity and phytotoxicity evaluations, including their effects on earthworms as indicators of soil health.

Materials

Sodium aluminate, sodium silicate solution (extra pure), and hexadecyltrimethylammonium bromide (HDTMA-Br), also known as cetyltrimethylammonium bromide (CTAB), a cationic surfactant, were sourced from Merck Specialities Pvt. Ltd., Mumbai. Urea ammonium nitrate (liquid), used as a reagent for loading nanozeolite and surfactant-modified zeolite, was sourced from MAPCO Fertilizer Industries Pvt. Ltd., Noida, Uttar Pradesh. It contains 32% total N by weight, comprising up to 16.5% urea N (NH 2 -N), at least 7.5% ammonium N (NH 4 + -N), and at least 7.5% nitrate N (NO 3 -- N). The specific gravity of the liquid is between 1.30 and 1.35 at 15 °C. All chemicals utilized in this research were of analytical grade and were used without additional purification. All aqueous solutions were prepared using deionized water. The filtration was done using Qualigens’ 640 de, slow filter speed filter paper.

Synthesis of nanozeolite

The nanozeolite was prepared using the methodology that Hosokawa and Oki (16) described with few modifications. A total of 2.49 g of sodium aluminate was weighed and taken in a 50 mL glass vial, in which 10.7 mL of sodium hydroxide solution (≥ 97% NaOH, 8g per 100 mL) was added and stirred for 30 min at a maintained temperature of 40˚C. Another solution with 1.87 g of sodium silicate solution and 10.4 mL of distilled water was prepared and shaken vigorously. Then, the resulting solution was further stirred for 5 min and heated to 100˚C over two hrs. The resultant zeolite particle aqueous slurry was filtered and washed with distilled water. The filter cake was dried overnight at 60 ˚C and crushed with a mortar and pestle, yielding a white powder.

Synthesis of HDTMA-Br modified nano zeolite

HDTMA-Br, a cationic surfactant, was used to modify the surface of nanozeolite following the procedure by Schick et al. (17) in which 5 g of nanozeolite was treated with 25 mL of 162.6 mmol L -1 HDTMA-Br solution at 25 °C for 16 hrs. The mixture was continuously stirred at 150 rpm to achieve equilibrium. After filtration, the sample was washed with 50 mL of distilled water to remove excess HDTMA-Br, and it was then dried at 70 ˚C overnight.

Experimental design for loading N to nanozeolite and HDTMA-Br modified nanozeolite

The Box Behnken design, which is significant for its fine factor levels, is a tool in Response Surface Methodology obtained from Stat-Ease ® 360 software, Version: 22.0.6, Stat-Ease Incorporation, Minneapolis, USA. The experiment matrix was generated from three independent variables comprising temperature, contact time, and stirring speed to give 13 experimental runs in a standard order. RSM is a statistical approach used to maximize the loading process by optimizing operational variables. The interactive effects of the loading process were also determined using statistical techniques. NZUF and SNZUF were synthesized by treating 5 g of nanozeolite and HDTMA-Br-modified nanozeolite, respectively, with UAN solution containing 32% N at a solid-to-liquid ratio of 1:10.The solution was kept in a BOD incubator shaker at the temperature of 25 °C to 60 °C for a period of 6 to 10 hrs and stirred continuously at 150 to 200 rpm to optimize using Box-Behnken design (Table 1). Then, the solution was filtered and washed with 50 mL of distilled water to remove excess soluble N loaded on the extra lattice framework and dried at 40 °C for 24 hrs. Then, batch experiment samples were analyzed for total N content using the Kjeldahl method (18).

Characterization of nanoparticles

Nanoparticle synthesized in this study were characterized for FTIR, Zeta potential, particle size and morphological assessment, crystallinity and thermal stability using standard techniques for nanomaterial characterization. These techniques included FTIR (Bruker ALPHA II) for functional group analysis; TEM (JEOL JEM-1011) for particle size and morphological assessment; zeta potential analysis (Horiba SZ-100) for determining surface charge and dispersion stability; SEM (ZEISS EVO 18 S) for surface morphology evaluation (19); PXRD (Rigaku Ultima IV) for crystallinity and phase identification; and TGA (Mettler Toledo TGA2) for thermal stability assessment (20). These techniques collectively provided insight into the structural, morphological, and physicochemical properties of both the synthesized nanozeolite and its HDTMA-Br-modified form.

Analysis of N profile

After optimization using response surface methodology, the Kjeldahl method determined the total N and available N content in the synthesized fertilizers (18). The NH 4 + -N and NO 3 - -N were analyzed through the KCl extraction method (21) using a T80+ UV/VIS spectrophotometer (PG Instruments Ltd.).

Swelling ratio and equilibrium water content analysis

The swelling ratio (SR) is calculated by measuring the increase in the weight of the sample following water immersion (22, 23). During this experiment, 1 g of the prepared fertilizer was precisely weighed and then submerged in 200 mL of distilled water. Subsequently, the sample was allowed to undergo swelling over a 24-hr period under standard ambient temperature and pressure conditions. Upon completion of this swelling period, the sample was recovered through filtration, and the weight of the moist sample was carefully recorded for analysis.

The SR and Equilibrium Water Content (EWC) for NZUF and SNZUF were calculated using Equations 1, 2.

where,

Sw is the soaked weight of NZUF/SNZUF

Dw is the dry weight of NZUF/SNZUF

Water retention capacity

Water retention capacity was determined using three pre-weighed cups labeled A, B, and C. Cup A contained 50 g of sieved soil (sandy clay loam texture with 36% maximum water holding capacity, analyzed using the Hilgard apparatus), mixed with 30 mL of distilled water. Sieved soil (50 g) was used for the experiment. In contrast, cups B and C had 2 g of NZUF and SNZUF, combined with 50 g of soil, followed by 30 mL of distilled water. They were reweighed (W1) after allowing water to seep through all three cups for 24 hrs. Subsequently, daily weightings (W2) were conducted for 30 days by placing the cups in a glass box, following the procedure outlined by Khan et al. (22) and Mikhak et al. (23). WRC was calculated using Equation 3.

Evaluation of biosafety of nanozeolite N fertilizers

Germination test for spinach beet seeds

In this study, 20 seeds of Spinach beet underwent sterilization using a 10% (v/v) sodium perchlorate solution for 10 minutes, followed by two rinses with deionized water to remove residual chemicals. The sterilized seeds of Spinach beet were soaked in varying concentrations of NZUF (150, 300, 450, and 600 ppm) and SNZUF (125, 250, 375, and 500 ppm) solutions for 15 minutes. Control samples were treated with deionized water, following the method outlined by Srivastav et al. (24). After soaking, the treated seeds were transferred to Petri plates with filter papers containing 5 mL of the solution. The plates were sealed and placed in incubators set at 21 ± 2 °C. Seed growth and response were monitored and recorded on the 5 th and 10 th days of incubation. The seed vigor index was determined by assessing the germination percentage and the length of seedlings from the same batch of seeds (25).

Paper contact toxicity test for earthworm

The acute toxicity test was conducted according to the Organization for Economic Cooperation and Development-207 guidelines for testing chemicals (26) with few modifications. A flat-bottomed glass vial (2 cm in diameter and 10 cm in length) was lined with filter paper (7.0 cm × 9.5 cm) and covered with a perforated parafilm ‘M.’ NZUF and SNZUF were dissolved in 100 mL distilled water at concentrations of 0, 150, 300, 450, 600 mgL -1 and 0, 125, 250, 375, 500 mg L -1 /L, respectively. The concentration ranges for NZUF and SNZUF were selected based on their differing nitrogen contents (15% and 18%, respectively), and doses were adjusted accordingly to ensure comparability in terms of nitrogen exposure. These solutions were sonicated for 15 minutes using Labman’s Probe Sonicator. One milliliter of the sonicated solution was pipetted into each glass vial. The control vials were treated with 1 mL of deionized water. Earthworms collected from the compost pit were kept on moist filter paper for three hours for acclimatization. Individual worms were introduced into each vial. Mortality was recorded as the primary endpoint, with earthworms considered dead when they showed no response to gentle stimulus. Each treatment has ten replicates in the incubator for two days at a constant temperature of 20 ± 1˚C in dark conditions. The LC 50 values were calculated using probit analysis and regression models using a data analysis tool in Microsoft Excel.

Artificial soil contact test on earthworm

An artificial soil mixture was formulated for the study, comprising 10% finely ground dry sphagnum peat, 20% kaolin clay, and 70% river sand. The pH of the mixture was carefully regulated to 6.0 ± 0.5 by adding calcium carbonate. The experimental setup adhered to the guidelines specified in OECD 207 (26), maintaining a consistent water content of 35 percent throughout the investigations. NZUF was incorporated into the soil by blending various doses: 112.5, 225, 337.5, 450, and 562.5 mg per 750 g of dry artificial soil. Similarly, SNZUF was introduced into the soil at 93.8, 187.5, 281.3, 375, and 468.8 mg per 750 g of dry artificial soil. After keeping the earthworms in uncontaminated artificial soil for 24 hours, they were rinsed gently with distilled water, and excess surface water was removed and weighed. Ten worms were placed in 1 L wide-mouth bottles with soil under constant light at 20 ± 1 °C for 14 days. Regular water addition maintained 80% air moisture. Bottles, each with four replicates, were covered with perforated plastic film.

Slow-release studies

A 60-day column study was conducted using four glass columns (620 mm × 50 mm), each filled with 400 g of sieved soil with a bulk density of 1.37 g cm -3 and a saturated hydraulic conductivity of 0.435 cm hr -1 (27). Three columns received 10 g of nitrogen (N) applied in the form of urea, NZUF, and SNZUF, respectively, and were thoroughly mixed. To saturate the soil, 180 ml of distilled water was added. At regular intervals, 50 mL of water was collected at 24-hour intervals, and 100 mL of water was added to maintain constant moisture. The collected water samples were analyzed for the release patterns of Ammonical N and Nitrate N from the nanocomposite in the soil using a UV spectrophotometer (27).

Soil pot culture experiment

Details of treatments

The pot trial was conducted at the Division of Environmental Sciences, IARI, New Delhi, using a variety of beet leaves from Pusa Bharati in 14-inch diameter plastic pots from 2023 to 2024. The experiment followed a completely randomized design (CRD) with nine treatments and three replications. The treatments were as follows: T-1 (no N, only P 2 O 5 at 60 kg ha -1 through SSP and K 2 O at 80 kg ha -1 through MOP), T-2 (90 kg ha -1 Recommended Dosage of N (RDN) through urea), T-3 (90 kg ha -1 RDN through urea ammonium nitrate), T-4 (90 kg ha -1 RDN through SNZUF), T-5 (90 kg ha -1 RDN through NZUF), T-6 (67.5 kg ha -1 RDN through SNZUF), T-7 (67.5 kg ha -1 RDN through NZUF), T-8 (45 kg ha -1 RDN through SNZUF), and T-9 (45 kg ha -1 RDN through NZUF). Each treatment included P 2 O 5 at 60 kg ha -1 through SSP and K 2 O at 80 kg ha -1 through MOP.

Characteristics of experimental soil

For this experiment, surface soil (0-15 cm depth) was collected from the IARI farm in New Delhi, classified under the soil order Inceptisol. The collected soil was dried, sieved through a 2 mm sieve, and analyzed for physicochemical properties using standard analytical methods. Particle size analysis was performed using the Bouyoucos hydrometer method (28), identifying the soil as sandy clay loam according to the textural triangle. The pH was measured at a 1:2.5 soil-to-water ratio, and electrical conductivity (EC) was determined using a conductivity bridge (29). The cation exchange capacity (CEC) was assessed using the neutral standard ammonium acetate method (28). The Walkley and Black method (30) measured organic carbon content, and total N was determined using the Kjeldahl method (31). Available N was analyzed using the alkaline permanganate method (32) with NH 4 + -N and NO 3 -- N extracted using the KCl extraction method (21). Available phosphorus and potassium were measured using Olsen’s method (33) and the ammonium acetate method (34).

Soil health analysis

Soil urease activity was measured 45 days after sowing (DAS) of Spinach beet. Soil urease activity was measured following Bremner and Douglas (35), based on urea hydrolysis by estimating residual urea after incubating soil with urea solution at 37 °C. Activity was calculated from the difference between urea added (B) and urea recovered (A) after incubation. Urea was determined colorimetrically using diacetylmonoxime (DAM) with thiosemicarbazide (TSC), phosphoric acid (H 3 PO 4), and sulfuric acid (H 2 SO 4), producing a red-colored complex measured at 527 nm. Urease activity was calculated as (B − A) × t, where B is urea added (mg), A is urea measured (mg), t is incubation time (h), and x is oven-dry soil weight (g), and expressed as mg urea g - ¹ soil h - ¹. Dehydrogenase enzyme activity was determined by the triphenyltetrazolium chloride method (36). Soil microbial counts, including fungi and bacteria, were assessed using the serial dilution technique (37). The fumigation method (38) measured soil microbial biomass carbon and Soil microbial biomass N.

Plant sample analysis

The fresh biomass of plant parts (leaves, roots) was measured using an electronic balance after harvest. The plants were then placed in an oven at 60 °C for 48 hours. After drying, the plants were weighed to record the dry mass. Once properly dried, the plant samples were ground into a powder using a blender, sieved through a 2 mm sieve, and used for further analysis. Nitrogen content in the leaves and roots was determined using the Kjeldahl method (18).

Statistical analysis

The pot trial was conducted using a completely randomized design (CRD) with three replicates per treatment. Data were statistically analyzed using the general linear model procedure in SPSS (Version 16.0) (39) to assess the significant effects of nanozeolite-based N fertilizers on Spinach beet. The results were analyzed using a one-way analysis of variance (ANOVA), and means were separated using Duncan’s multiple range test (DMRT) at α = 0.05. Graphs were created using Origin Pro 2023b (40) software.

Synthesis and optimization of nanozeolite-based N fertilizers

The maximum total N loading capacity of NZUF and SNZUF was 15.5% and 18%, respectively. The maximum loading in NZUF was obtained with an initial temperature of 42.5˚C, contact time of 10 hours, and stir speed of 200 rpm. The maximum loading of SNZUF was obtained with an initial temperature of 60˚C, contact time of 6 hours, and stir speed of 175 rpm, as predicted by the Design-Stat-Ease 360 software used for work. The statistical model was verified with the analysis of variance (ANOVA) for the response surface quadratic model. The surfactant modification of the surface of nanozeolite reversed the negative charges on the framework to positive and allowed the loading of oxyanion, i.e., nitrate. The results indicated higher nitrate and ammonium ion loading in SNZUF compared to NZUF by 231% and 14.5%, respectively.

Fourier transform infrared spectroscopy of synthesized nanomaterials

The functional groups in the Fourier Transform Infrared (FTIR) spectra of nanozeolite, surface-modified nanozeolite, NZUF, and SNZUF were examined (Figure 1A-D). In all four samples, prominent peaks emerged at nearly 1000 cm -1 and 660 cm -1, attributed to -XO 4 and O-X-O groups, respectively, where X signifies Si or Al. Distinct frequencies (ranging from 1003 to 984 cm -1) indicated T-O-T stretching bands on the zeolite’s surface. However, a reduction in the intensity of this band after modification and nitrogen loading was observed, which can be attributed to surface coverage and interaction of nitrogen species with the zeolite framework, leading to attenuation of T-O-T vibrations. Similar behavior has been reported due to reversible deformation of the zeolitic lattice upon nitrogen interaction (41). Within the 640 to 714 cm -1 wavelength range, a cluster of vibrations displayed several peaks corresponding to the robust asymmetric stretching of T-O (T = Si and Al) owing to the internal vibrations within (Si, Al) O 4 tetrahedral units. Distinct frequencies (ranging from 1003 to 984 cm -1) indicated T-O-T stretching bands on the zeolite’s surface (41). The spectral band at approximately 1426 cm -1 was attributed to the vibration of the trimethyl ammonium quaternary group, denoted as C-N(CH 3) 3 in SNZ (Figure 1B) and SNZUF (Figure 1D). NZUF exhibited spectral peaks at 829.74 cm -1 and 1443 cm -1, indicative of the nitrate ion, and distinct stretching bands for N-H were observed at 3699 cm -1, 3502 cm -1, 2920 cm -1, 1646 cm -1, 1515 cm -1, and 1396 cm -1 (Figure 1C). SNZUF also displayed similar spectral peaks at 828.42 cm -1 and 1432 cm -1 attributed to the nitrate ion, along with N-H stretching bands observed at 3678 cm -1, 3499 cm -1,2360cm -1, 1646 cm -1,1515 cm -1, and 1395 cm -1, signifying the existence of amino and ammonium groups on the surface of zeolite structures. A similar study by Mohanraj et al. (42) confirmed the identification of NH 4 + -N and NO 3 -- N through distinct peaks in NH 4 + –N-loaded nanozeolite and NO 3 - –N-loaded surfactant-modified zeolite, indicating effective N sorption. These findings align with earlier research by Lateef et al. (27), Khan et al. (22), and Manikandan and Subramanian (8), further supporting the structural and functional integrity of modified NZ.

Transmission electron microscopy of synthesized nanomaterials

For nominal size measurement, Transmission Electron Microscopy (TEM) is the sole method through which individual particles are directly observed and measured. Figure 2A-D illustrates the high-resolution TEM images of NZ, SNZ, NZUF, and SNZUF. It depicts the various degrees of dispersion and agglomeration of nanoparticles. On appearance, they appear to be spherical with a wide range of diameters. The particle size (length) data was obtained by randomly selecting particles. The initial mean particle size of synthesized nanozeolite was 50.26 ± 23.78 nm. Due to the hydrothermal process of synthesis of SNZ, NZUF, and SNZUF, the mean particle size has been reduced to 30.55 ± 11.93 nm, 18.02 ± 5.75 nm, and 21.87 ± 13.29 nm, respectively.

The reduction in particle size after the hydrothermal process observed in SNZ, NZUF, and SNZUF indicates the successful refinement of NZ. The initial mean particle size of 50.26 ± 23.78 nm, decreasing to 18.02 ± 5.75 nm for NZUF, is consistent with previous studies that report NZ around 40-50 nm (11, 43). The smaller particle sizes observed in modified NZ likely result from enhanced dispersion, increasing surface area, and thus improving their potential application as nanofertilizers. Studies on other nanomaterials, such as nanozeolite, nanohydroxyapatite (nHA), and polystyrene nanospheres, have shown similar reductions in size with enhanced functionality (23). Furthermore, slight particle size increases in some modifications (SNZUF) align with findings by Goyal et al. (44), where surfactant induced stabilization, maintained nano-scale dimensions. These findings confirm the feasibility of controlled particle size reduction for improved efficiency in agricultural applications.

Scanning electron microscopy of synthesized nanomaterials

The Scanning Electron Microscopy (SEM) analysis provided a detailed examination of the morphology of the synthesized nanozeolite samples. The initial SEM characterization of the synthesized nanozeolite revealed a predominantly cubical structure, with particles ranging from cubic to slightly rounded shapes (Supplementary Figure 1A). This uniform morphology is characteristic of well-crystallized nanozeolite, indicative of successful synthesis. Upon modification with Hexadecyltrimethylammonium-Bromide (HDTMA-Br), a noticeable transformation in the morphology was observed. The SEM images of the SNZUF (Supplementary Figure 1B) showed a departure from the initial cubical shapes to more irregular forms. This alteration can be attributed to the adsorption of the cationic surfactant molecules onto the surface of the nano-zeolite, which disrupts the original structural regularity. Further SEM analysis of the NZUF (Supplementary Figure 1C) displayed a similar trend of morphological irregularity. The originally cubic structures appeared more distorted and porous, suggesting the introduction of N influenced the structural integrity, possibly by creating additional voids or channels within the zeolite framework. The interaction between N and zeolite frameworks has been extensively studied, revealing significant effects on adsorption properties and selectivity. N incorporation into zeolite structures preserves the framework while adding to surface and interior sites, crucial for designing biomass-refinement catalysts (45, 46). The most significant morphological change was observed in the SNZUF (Supplementary Figure 1D). The SEM images revealed a highly irregular, spongy structure. This pronounced sponginess is likely due to the combined effects of surfactant adsorption and N loading (47). The surfactant molecules create a less compact structure, and the subsequent N loading introduces further porosity and irregularity (48). SEM analysis shows that irregular morphology may be associated with surface roughness, agglomeration, or uneven particle distribution on the surface observed for the SNZ, NZUF, and SNZUF. TEM, on the other hand, provides nanoscale high-resolution internal structure imaging. The spherical nature with an extensive range of diameters observed in TEM indicates that, although the surface morphology is irregular, the internal structure of these particles is relatively uniform and often forms spherical nanostructures. Together, these techniques provide a comprehensive understanding of both the surface morphology and internal consistency of the material (49).

X-ray diffraction analysis of synthesized nanomaterials

X-ray diffraction (XRD) analysis was instrumental in examining the crystallinity and phase purity of the synthesized nanozeolite, surface-modified nanozeolite, and their derived N fertilizers (Figures 3A–D). In the NZ sample (Figure 3A), sharp peaks at 2θ values of 7.3, 10.1, 12.53, 18.42, 20.42, and others indicate a crystalline structure typical of NZ. The SNZ sample (Figure 3B) shows an increase in peak intensity, particularly at 7.18 and 16.1, suggesting enhanced crystallinity or additional phases due to HDTMABr modification and for NZUF (Figure 3C), peaks at 7.24, 11.2, 18.34, and 20.34 show peak positions and intensity changes, indicating possible interactions between the nanozeolite and urea ammonium nitrate. Similarly, SNZUF (Figure 3D) presents peaks at 7.24, 10.42, 18.34, and 20.34, showing a similar pattern to NZUF but with differences in peak intensity and position, reflecting the influence of HDTMA-Br modification. The close resemblance between the adsorbents, fabricated fertilizers, and N-loaded samples indicates that the structural integrity of the nanozeolite is retained post-loading, corroborating findings from previous studies (42, 50).

The diffraction data of all zeolite materials; without (NZ, SNZ) and with N impregnation (NZUF, SNZUF) showed peaks at 2θ values of 7.3, 18.34, and 29.94 (Figures 3A–D). The retention of prominent peaks at 2θ values of 7.3, 18.34, and 29.94 in both NZUF and SNZUF samples corroborates findings from previous research, such as that by Mohanraj et al. (42). In addition, other peaks are observed at 2θ values of 20.34 and 25.62, representing quartz and albite associated with the zeolite. The surfactant-modified nanozeolite showed a similar diffraction pattern to the synthesized nanozeolite except for increased peak intensity observed at 2θ values of 7.18 and 16.1, suggesting enhanced crystallinity or additional phases due to HDTMA-Br modification. This demonstrated the material’s structural stability before and after the surfactant treatment, consistent with earlier findings. For instance, while comparing the diffraction patterns of surfactant-modified forms of clinoptilolite zeolite with their corresponding unmodified counterparts, previous studies found a close similarity (22, 47). Moreover, loading the nanozeolite with a N source didn’t affect the nanozeolite structure (8, 42). Furthermore, a very minute change in peaks representing the N forms is observed in the N-loaded nanozeolites. However, the peak intensities observed around 2θ values of 7.3, 18.34, and 29.94 declined in the case of NZUF and SNZUF, possibly due to the incorporation of N.

Photon correlation spectroscopy of synthesized nanomaterials

The Dynamic Light Scattering analysis unveiled notable distinctions in the zeta potential and electrokinetic properties across the four examined samples, as depicted in Supplementary Figure 2. Nano Zeolite exhibited a mean zeta potential of -59.1 mV, reflecting its strong negative surface charge. Upon surfactant modification, the zeta potential decreased to -35.9 mV, indicating a reduction in surface charge density. Correspondingly, the electrophoretic mobility decreased from -0.000458 cm²/V for NZ to -0.000279 cm²/V for the surfactant-modified NZ, suggesting altered particle behavior under an electric field. With the loading of N forming NZUF, the mean zeta potential shifted to -47.1 mV, indicating further changes in surface charge distribution, while the electrophoretic mobility decreased to -0.000366 cm²/V. Similarly, the SNZUF displayed a mean zeta potential of -46.4 mV and electrophoretic mobility of -0.00036 cm²/V. The zeta potential reflects the sample’s stability and shows minimal aggregation in the fabricated fertilizers. The Z-Average hydrodynamic diameter (nm) and Polydispersity Index (PI) are: NZ (1099.7 ± 54, 1.547), SNZ (1049.9 ± 63, 1.035), NZUF (994 ± 58, 1.237), and SNZUF (922.6 ± 46, 1.196). The nanozeolite particles appeared more significant in size when measured by dynamic light scattering (DLS) due to their tendency to form clusters. This explains the higher Z-Average hydrodynamic diameter and Polydispersity Index (PI). DLS measures particle groups, unlike TEM, which typically shows individual particles, resulting in a wider size range (15).

Thermogravimetric analysis and differential thermogravimetric analysis of synthesized nanozeolite-based fertilizers

The TGA and DTG curves for the nanozeolite-based N fertilizer, i.e., NZUF and SNZUF, reveal distinct thermal degradation behaviors. For the NZUF sample (Figure 4A), the TGA curve shows three weight loss stages: the initial loss at 47.50 °C due to evaporation of physically adsorbed water and volatile components, a significant degradation at 165.17 °C likely representing the decomposition of organic or labile Nitrogenous compounds within the nanozeolite matrix, and a third phase at 213.50 °C indicating further breakdown of more stable nitrogenous materials or the zeolitic framework. The final residue at 800 °C is 48.18%, or 1.9244 mg, showing substantial remaining stable, non-volatile compounds. The DTG curve aligns with these stages, confirming the observed thermal events. In comparison, SNZUF (Figure 4B) also exhibits three weight loss stages: the initial loss at approximately 49 °C from adsorbed water and volatiles, a significant step at 170.83 °C indicating the decomposition of the HDTMA-Br surfactant and associated organic modifications, and a third stage at 219.17 °C representing the breakdown of remaining nitrogenous components and possibly the zeolitic framework. The final residue at 800 °C is 33.46%, or 1.1240 mg, less than that of NZUF, indicating a higher degree of decomposition. The DTG curve for SNZUF corroborates these findings, highlighting the influence of HDTMA-Br modification on the thermal stability and decomposition pathway of the nanozeolite-based N fertilizer (36, 51).

N profile of synthesized nanofertilizers

The primary forms of N in straight N fertilizers are urea-N, nitrate-N, and ammonical-N. The total N content of NZUF and SNZUF was 15.5% and 18%, respectively, with available N at 5.04% and 6.72%. NZUF contained 1.17% ammonium and 0.16% nitrate, while SNZUF had 1.34% ammonium and 0.53% nitrate. A standard zeolite has a surface area of 648 m² g -1; in its nano form, it increases to 1126 m² g -1. This larger surface area helps the nanozeolite retain more N, enhancing the N content in the fertilizer (52). Adding the surfactant, HDTMA-Br, increased the total N content to 18%. These findings align with Manikandan et al. (53), who reported that zeo-urea synthesized using naturally available zeolite and nanozeolite urea had total N contents of 18.5% and 28%, respectively. Manikandan et al. (8) and Kaushik et al. (54) both reported that 1:1 ratios of natural zeolite:urea achieved 18.5% total N content, while nanozeolite:urea reached 28% total N content. Guan et al. (55) demonstrated that surfactant-modified zeolites (SMZs) using HDTMA-Br removed 8-18 times more nitrate than raw zeolites, supporting the surfactant enhancement observed in your SNZUF formulation.

Physical parameters test of synthesized nanozeolite-based fertilizers

To evaluate the soil’s water retention (WR) capacity incorporating 2 g of NZUF and SNZUF, we calculated WR values for both the control and fertilized soil on the 5 th, 10 th, and 15 th days. The NZUF-mixed soil exhibited WR values of 78.1%, 71.5%, and 64.8%, while the SNZUF-mixed soil showed 84.6%, 79.8%, and 66.8% on the respective days. In contrast, the control soil recorded WR values of 65.8%, 57.2%, and 39.2%. The NZUF and SNZUF-mixed soils significantly demonstrated 18.7% and 28.6% higher WR rates, indicating superior water-holding capacities. This attribute is crucial for water conservation and promoting plant health. Moreover, we explored the equilibrium water content (EWC) and swelling ratio (SR) of NZUF and SNZUF. The measured EWC values for NZUF and SNZUF were 68.4% and 74.5%, respectively, while the SR values for NZUF and SNZUF were 0.551 and 1.287, respectively. These results are consistent with Khan et al. (22), who reported a 27% higher WR rate in nanozeolite-based composite fertilizer (NZCF) mixed soil, indicating superior water-holding capacity due to the porous structure of NZ. The higher WR helps conserve water and ensures its availability for plant uptake, particularly in arid and water-scarce environments. Similarly, Lateef et al. (27) observed an 18% higher WR rate for soil mixed with zeolite nanocomposite (ZNC), attributed to zeolite’s high absorbency and porosity. The ability of ZNC to retain water enhances moisture availability for crops and supports slow nutrient release, improving plant health and yield in dry regions. In our study, SNZUF, with its higher WR and EWC, similarly reflects the capacity to retain moisture, which can contribute to improved plant growth and soil health in water-limited conditions. Additionally, the swelling ratio of 1.287 for SNZUF and the high water absorbance observed in line with Lateef et al. (27) demonstrate the capability of these materials to absorb and hold water, making them highly effective in agricultural applications aimed at water conservation and nutrient management.

Ni

N release pattern of synthesized nanozeolite-based N fertilizers

The cumulative release of ammoniacal N (NH 4 + -N) over 60 days, as depicted in Figure 5A, shows that urea experienced the fastest release, with its cumulative release quickly reaching approximately 4.84 mg kg - ¹ and plateauing early in the experiment. In contrast, the NZUF and SNZUF exhibited significantly slower release rates. The NZUF and SNZUF gradually increased their cumulative losses, reaching around 3.5 mg kg - ¹ and 3.8 mg kg - ¹, respectively. This indicates a more controlled and extended release compared to urea. Figure 5B presents the cumulative loss of nitrate N (NO 3 - -N) over the same period. Urea again showed the fastest release, with its cumulative loss stabilizing around 63.4 mg kg - ¹. In contrast, the SNZUF treatment exhibited a slower release initially but ultimately reached a higher cumulative release of around 81.2 mg kg - ¹. The NZUF showed an intermediate release pattern, peaking at approximately 45.8 mg kg - ¹. Based on the release profiles, urea reached 75% N release within ~10–15 days for both NH 4 + -N and NO 3 - -N. In contrast, NZUF reached 75% release at ~33–35 days (NH 4 + -N) and ~50–55 days (NO 3 - -N), while SNZUF required ~48–50 days (NH 4 + -N) and ~45–48 days (NO 3 - -N), confirming a significantly prolonged nutrient release compared to urea. The enhanced surface area and high stability indicated by the zeta potential contribute to its capability to hold and gradually release N, making it an efficient and reliable material for controlled-release fertilizer applications (56, 57). The amount of nitrate released from SZNUF increased as the number of days increased compared to NZUF, which became constant after 26 days. It has been demonstrated that SNZ exhibits a strong ability to adsorb and retain nutrients within the nanopores. This capability arises from forming a surfactant bilayer on the NZ surface. The positively charged surface of SNZ serves as a binding site for anions such as phosphate and nitrate (58).

Evaluation for biosafety of the nanozeolite N fertilizers

Effect of nanozeolite-based fertilizers on phytotoxicity via germination test

The effects of NZUF and SNZUF on the germination percentages of Spinach beet were evaluated to assess potential phytotoxic effects. Germination data for Spinach beet were collected 5 and 10 days after incubation, as shown in the figure, and results are presented in Figure 6; Supplementary Figure 3. The control group exhibited a germination percentage of 33.3% and 60%, 5 DAI and 10 DAI, respectively, which served as the baseline for comparison. After 10 DAI, the application of NZUF significantly increased the germination percentage in Spinach beet. Specifically, germination percentages were 66.67% for N-150, 75% for N-300, 81.67% for N-450, and 96.67% for N-600. This demonstrates a clear dose-response relationship, where higher concentrations of the N fertilizer correlated with increased germination rates (59, 60). In a study, the phytotoxic effects of nanozeourea were tested on maize seeds over five days, revealing no detrimental impact on germination. Seeds treated with 500 mg of nanozeourea showed the highest germination rate (100%). In comparison, even at higher concentrations (1250 mg and 1500 mg), germination remained high (96.7%), indicating the safety of nanozeourea for maize with no adverse effects observed (53). Similarly, germination percentages for SNZUF treatments were 76% for S-125, 80% for S-250, 96.67% for S-375, and 46.67% for S-500. At initial concentrations, the doses significantly increased the germination percentage compared to NZUF. After modification with the cationic surfactant HDTMA, zeolite (SNZ) significantly increased the germination percentage at initial concentration doses compared to NZUF due to its enhanced ability to adsorb, hold, control, and release anionic nutrients. The charge reversal from negative to positive on SNZ surfaces facilitates the adsorption of anionic nutrients essential for germination and plant growth (61). However, germination was adversely affected when the concentration increased to 500 mg kg -1 (62).

Effect of nanozeolite-based fertilizers on phytotoxicity via seedling vigor test

The Seedling Vigor Index (SVI) of spinach beet was significantly influenced by nanozeolite-based N fertilizer and surfactant-modified nanozeolite-based N fertilizer applied at various concentrations. The control group, which received no fertilizer treatment, exhibited an SVI of 506. Applying NZUF resulted in a progressive increase in SVI with increasing concentrations up to a point. Specifically, NZUF-2 increased the SVI to 691, NZUF-3 to 860, and NZUF-4 reached the highest value of 1064. However, at the highest concentration (NZUF-5), the SVI decreased to 593, indicating potential phytotoxic effects at this level. In contrast, SNZUF treatments enhanced the SVI but with a different trend. SNZUF-2 showed a significant improvement with an SVI of 1007, while SNZUF-3 and SNZUF-4 had SVIs of 931 and 986, respectively. However, SNZUF-5 led to a substantial decline in SVI to 289, suggesting severe phytotoxic effects at this concentration. This decrease can be attributed to several mechanisms. Excessive N application can lead to soil acidification, reducing soil pH. Lower pH levels can negatively impact the availability of essential nutrients, particularly phosphorus and potassium, which are crucial for plant development (63). Consequently, this nutrient imbalance can inhibit seedling growth. Moreover, high N levels can induce physiological stress in seedlings, potentially damaging their root and shoot systems (63). Research has shown that moderate N levels can alleviate stress and improve SVI in other crops, such as wheat (64). This highlights the importance of applying fertilizers at appropriate rates.

Effect of nanozeolite-based fertilizers on earthworms via paper contact toxicity test

The paper contact toxicity test was conducted to assess the lethal concentration 50% (LC 50) values of two treatments, NZUF and SNZUF, on earthworms over two durations: 48 hours and 72 hours. Table 2 depicts the LC 50 of NZUF and SNZUF on earthworms exposed to different concentrations. After 48 hours of exposure, the LC 50 for NZUF was determined to be 1066.6 ppm with an R 2 value of 0.73, indicating a moderate correlation between the observed data and the model. In comparison, the LC 50 for SNZUF was lower at 910.9 ppm, with an R 2 value of 0.82, suggesting a stronger and more firm fit and higher toxicity of SNZUF. After the72-hours, the toxicity of both treatments increased, as evidenced by the reduced LC 50 values. The LC 50 for NZUF decreased to 835.1 ppm with an R 2 value of 0.76, while the LC 50 for SNZUF further dropped to 636.1 ppm, with an R 2 value of 0.87. These results consistently demonstrate that SNZUF is more toxic than NZUF, requiring a lower concentration to achieve the same lethal effect on earthworms. The higher R 2 values at 72 hours indicate improved reliability of the model predictions over the extended exposure period. As the recommended dosage of N generally followed as per the Indian Council of Agricultural Research (ICAR) for most crops falls between 100-150 kg/ha, the application of NZUF (150-300 ppm) and SNZUF (125-250 ppm) remains well within this range and significantly below the LC 50 threshold, indicating minimal toxicity risk to earthworms (65).

Effect of nanozeolite-based fertilizers on earthworms via artificial soil test

The artificial soil test was designed to evaluate the toxicity of NZUF and SNZUF on earthworms over extended exposure periods of 7 days and 14 days; results are shown in Table 2. After seven days, the LC 50 for NZUF was calculated to be 1279.8 ppm with an R 2 value of 0.71, indicating moderate model accuracy. In contrast, SNZUF exhibited a significantly lower LC 50 of 775.5 ppm with an R 2 value of 0.77, indicating higher toxicity and a more reliable model fit than NZUF. At the 14-day exposure period, the toxicity of both treatments further intensified. The LC 50 for NZUF decreased to 910.3 ppm with an R 2 value of 0.94, showing a strong correlation and high model accuracy. Similarly, the LC 50 for SNZUF dropped to 551.5 ppm, with an R 2 value of 0.94, demonstrating the highest observed toxicity in this study and an excellent fit of the data. These results clearly illustrate that SNZUF is significantly more toxic to earthworms than NZUF over prolonged exposure, with consistently lower LC 50 values and robust model reliability (Figure 7). The use of HDTMA-Br as a cationic surfactant for modifying nanozeolite was selected based on its effectiveness in improving the nitrate loading and surface modification of nanozeolite, making them more suitable for slow-release fertilizer applications. As per ICAR guidelines (100-150 kg ha -1), the applied doses of NZUF (150-300 ppm) and SNZUF (125-250 ppm) are within safe limits and not harmful to earthworms. However, it was observed that higher concentrations (more than optimal application (SNZUF-5, 468.8 mg per 750 g of dry artificial soil)) of bromide (Br) ions could potentially pose environmental risks, such as toxicity to aquatic ecosystems and soil organisms (62, 65).

Initial physicochemical properties of experimental soil

The soil analysis revealed a sandy clay loam texture consisting of 42% sand, 26% silt, 24% clay, and a slightly alkaline pH of 7.8. This texture indicates good drainage characteristics; as sandy clay loam typically allows for effective water movement while retaining adequate moisture for plant growth (66). Additionally, the electrical conductivity of 0.361 dS m -1 suggests low salinity, which is conducive to healthy crop development. The cation exchange capacity (CEC) of 16.28 C mol (p+) kg -1 reflects a moderate nutrient-holding capacity, enabling the soil to retain essential cations such as calcium, magnesium, and potassium. However, the low organic carbon content (0.401 g kg -1) and total N levels (0.021%) indicate that the soil’s fertility is limited and may require supplementation to improve nutrient availability and overall soil health. While the soil exhibits good drainage and a balanced nutrient-holding capacity as evidenced by its texture and CEC, the low organic carbon and N levels underscore the need for appropriate fertilization strategies to enhance fertility and optimize crop yields (67, 68).

Effect of nano zeolite-based N fertilizer on soil health

Soil organic carbon of experimental soil

Figure 8A illustrates the impact of various treatments on soil organic carbon (SOC) percentages over two years, 2023 and 2024. Soil organic carbon ranged from about 0.44% to 0.55% in 2023 and 0.46% to 0.55% in 2024; the trend remained consistent with negligible increases in SOC across all treatments. The average SOC over two years increased significantly by 7.72% for T4, 10.74% for T5, 6.38% for T6, and 7.05% for T7 compared to T2. The control (T-1) had the lowest SOC, highlighting the necessity of N for soil carbon augmentation. The data suggest that applying N, mainly through specific formulations like SNZUF and NZUF at higher rates, substantially enhances soil organic carbon content (69). Nanozeolite-based N fertilizers increase soil organic carbon due to their unique properties. These enhance nutrient use efficiency and slow N release, reduce leaching and volatilization losses, improve plant growth and increase biomass production, and contribute to higher soil organic carbon levels (70, 71). Furthermore, the structure of NZ aids in the retention and slow release of organic molecules and nutrients, supporting the development of stable organic matter fractions in the soil and raising the organic carbon content of the soil (72).

Soil enzyme activity of experimental soil

In 2023, urease activity ranged from 9.48 mg urea g - ¹ soil h - ¹ in T1 to 17.88 mg urea g - ¹ soil h - ¹ in T2, with the highest activity in T2, followed by T6, T7, and T8. In 2024, it ranged from 11.63 mg urea g - ¹ soil h - ¹ in T1 to 23.82 mg urea g - ¹ soil h - ¹ in T2, maintaining the same trend as the previous year, as depicted in Figure 8C. Slow-release nanozeolite-based N fertilizers can reduce the rapid hydrolysis of urea by urease enzymes. This controlled release slows the conversion of urea to ammonia (NH 3), reducing the risk of N loss through volatilization. By moderating urease activity, these fertilizers ensure a steadier supply of N in the form of ammonium (NH 4 +), which is less prone to leaching than nitrate (NO 3 -). Dehydrogenase enzymes are indicators of overall microbial activity and soil health. Dehydrogenase activity ranged from 0.88 µg TPF g - ¹ soil h - ¹ in T1 to 2.26-µg TPF g - ¹ soil h - ¹ in T-5, peaking in T5, T4, T6, and T7. In 2024, the values ranged from 1.04 µg TPF g - ¹ soil h - ¹ in T1 to 2.74-µg TPF g - ¹ soil h - ¹ in T5. Similar trends persisted, with T4, T5, and T6 showing the highest enzyme activities, while T-1 and T8 consistently showed the lowest, as illustrated in Figure 8C. The average soil urease activity for treatments T4, T5, T6, and T7 decreased by 22.17%, 28.28%, 2.34%, and 6.99%, respectively, compared to T2, while the average dehydrogenase activity increased significantly by 30.56%, 38.33%, 29.44%, and 23.89%, respectively. These enzymes are involved in the oxidative phosphorylation processes of soil microorganisms and are directly linked to the microbial respiration rate (73). Traditional N fertilizers like urea can lead to high concentrations of ammonium and nitrate, potentially causing toxicity to soil microbes and inhibiting enzyme activities (74, 75). In contrast, slow-release fertilizers mitigate this issue by avoiding the sharp fluctuations in nutrient levels that could harm microbial populations and enzyme activities, including dehydrogenase (76).

Soil microbial count of treated and untreated soil

As shown in Figure 8B, in 2023, bacterial counts were highest across treatments, ranging from approximately 22 × 10 6 CFU g - ¹ soil in the control (T1) to 36 × 10 6 CFU g - ¹ soil in treatments T4. The bacterial count was consistently lower in the control compared to the other treatments, indicating the positive impact of N application on bacterial proliferation. Fungal counts ranged from about 28 × 10 4 CFU g - ¹ soil in the control (T1) to 40 × 10 4 CFU g - ¹ soil in T4, peaking in T4, T5, T6, and T7 treatments in 2023. The highest fungal counts were observed in T4, reflecting the significant influence of SNZUF at 90 kgha -1. Actinomycetes counts in 2023 varied from approximately 11 × 10³ CFU g - ¹ soil in the control (T1) to over 17 × 10³ CFU g - ¹ soil in T4. The highest counts were recorded in T4, while the control and lower N rate treatments (T8 and T9) had the weakest actinomycetes counts. In 2024, the microbial count trends remained consistent with those observed in 2023. Bacterial counts were highest in T4, T5, and T6, while fungal and actinomycetes counts were maximized in T4 and T5. The data indicate that nanozeolite-based N fertilizer application significantly influences soil microbial counts, enhancing bacterial, fungal, and actinomycetes populations without causing any detrimental effect on soil microorganisms (77). This steady nutrient availability promotes microbial proliferation, leading to higher counts of bacteria, fungi, and actinomycetes than in soils treated with conventional fertilizers that release nutrients rapidly and unevenly (78, 79). Ibrahim et al. (80) reported that zeolite, when mixed with soil, increased the arbuscular mycorrhizal fungi colonization in their study on a combination of biochar, zeolite, and mycorrhiza inoculation on soil properties, heavy metal availability, and cowpea growth in multi-contaminated soil. In the present study, treatments with surfactant-modified nanozeolite (SNZUF) at lower concentrations did not negatively impact soil microflora, as indicated by the proliferation of beneficial microorganisms. However, at higher concentrations, HDTMA-Br may pose risks to soil microbial communities, as reported in previous studies. HDTMA-Br desorbed from surfactant-modified zeolites has been shown to exhibit toxicity towards certain microorganisms (65). Specifically, concentrations of HDTMA-Br ≥10 -5 M have been toxic to MS2 bacteriophage, ≥10 -4 M to Escherichia coli, and ≥10 -6 M to Bacillus subtilis (65). Nevertheless, no apparent toxic effect on the overall soil microflora was observed in the current study. Further research is necessary to explore the long-term impacts of higher concentrations of HDTMA-Br on soil microbial diversity and function.

Soil microbial biomass carbon and N of experimental soil

The study investigates the impact of various N treatments on soil microbial biomass carbon (MBC) and microbial biomass N (MBN) over two years, 2023 and 2024 (Figure 8D). The results reveal that treatments T2, T4, and T5 consistently achieved the highest MBC, while T4 and T5 outperformed in MBN values across both years, demonstrating their superior effectiveness in enhancing soil microbial biomass. Moderate MBC and MBN levels were observed in treatments T7, and T9, suggesting adequate but less optimal N availability for microbial growth compared to T4 and T5. The lowest MBC and MBN values were recorded in T1 (no N) and T8, implying that insufficient N application limits microbial biomass. Khati et al. (81) found that nano-compounds like nano-clay, nano-chitosan, and nanozeolite can improve soil health by supporting microbial populations. However, urea-treated soils showed reduced microbial biomass carbon and N, suggesting increased organic matter utilization, which depleted available organic carbon resources for microbial growth.

Crop growth and yield parameters of spinach beet

The results revealed that all growth, quality, and yield parameters were significantly influenced by different nitrogen treatments at p ≤ 0.05, as indicated by Duncan’s Multiple Range Test (DMRT), with acceptable levels of variability as reflected by standard error of difference (SE(d)) and coefficient of variation (CV%) (Supplementary Table 1).

At 20 DAS, T7 (12.72 ± 0.47 cm in 2023; 13.29 ± 0.65 cm in 2024) and T6 (12.29 ± 0.39 cm; 13.31 ± 0.49 cm) were significantly superior (p ≤ 0.05) over all other treatments (group ‘d’), whereas T1 remained significantly lowest (group ‘a’). At 40 and 60 DAS, T5 recorded the highest plant height (26.45 ± 1.13 to 31.54 ± 1.21 cm), which was statistically at par with T7 and T6 (groups ‘c–e’) but significantly higher than urea (T2) and UAN (T3). The low SE(d) values (0.543–1.412) and CV (3.49–6.04%) indicate high precision and reliability of the treatment effects. This improvement is likely due to the slow-release nature of NZUF, providing consistent N availability and improving N use efficiency. However, SNZUF-treated plants showed slightly lower growth, possibly due to interactions with soil microbial dynamics or nutrient availability. Overall, NZUF proved effective in promoting growth, offering a sustainable solution for improving N efficiency and reducing environmental impact in agriculture.

Significant differences (p ≤ 0.05) were observed in leaf area across treatments. In 2023, T7 and T2 at 40 DAS (59.21 ± 5.70 and 59.34 ± 5.05 cm²; group ‘d’) were significantly superior to T3 (36.26 ± 3.91 cm²; group ‘b’). At 60 DAS, T5 (69.79 ± 1.37 cm²; group ‘d’) was significantly higher than all treatments except T7. In 2024, T7 and T4 consistently recorded the highest leaf area (69.02–70.55 cm²; group ‘d’), significantly outperforming T3 and T8. The SE(d) (2.467–3.807) and CV (4.78–9.55%) suggest moderate but acceptable variability. Putri et al. (82) in their study on nanotechnology-based liquid organic fertilizer, increased plant height to 37.20 cm and leaf area to 19.95 cm 2 in red spinach hydroponic cultivation. The smaller leaf size observed in T3 (urea ammonium nitrate (UAN)), despite an increase in the number of leaves and moderate yield, can be attributed to the specific N release dynamics of UAN. It provides a readily available N source, which can lead to rapid initial growth, promoting the production of more leaves (83, 84). However, this fast N release may result in a quicker depletion of available N or cause an imbalance in nutrient uptake. As a result, the energy required to expand individual leaves could be diverted to produce more but smaller leaves, leading to the observed outcome. This rapid N availability may also cause water stress or nutrient competition among the growing leaves, further limiting their size while maintaining overall leaf number and yield (85).

The plant height of maize plants fertilized with a novel fertilizer formulation that was obtained by physically mixing urea with zeolite or nanozeolite or by fusing through intercalation processes rose dramatically, particularly during the crop’s active development period (60 & 90 DAS) (53, 86).

Leaf and root nitrogen content differed significantly (p ≤ 0.05) among treatments. T7 recorded the highest leaf N content (3.44 ± 0.20% in 2023; 3.45 ± 0.22% in 2024; group ‘g’), which was significantly higher than all other treatments, followed by T6 (group ‘f’). Root N content was highest in T6 (1.04 ± 0.03% and 1.03 ± 0.02%; group ‘d’), significantly superior to T1, T8, and T9 (groups ‘a–b’). The low SE(d) (0.03–0.14) and CV (<6.5%) confirm the consistency of nitrogen uptake results. (Supplementary Table 1).

The low root N content observed in T9 (NZUF at 45 kg ha -1) during 2023, despite applying 50% of the recommended N, may stem from the slow-release nature of nanozeolite-based fertilizers, which can limit immediate N availability. Environmental factors such as soil moisture levels and microbial activity can impede optimal N uptake (87). In comparison, T1 (control) recorded a N content of 0.83 ± 0.02%, likely due to residual soil N or organic matter. T1’s consistently lower N levels over two years underscore the adverse effects of long-term N deficiency (88) compared to more effective NZUF treatments. The observed patterns in N uptake in beet leaves, and roots highlight varying treatments’ influence on spinach beet’s N accumulation. Kráľová and Jampílek (89) found that nanozeolite-based N fertilizers enhanced N assimilation efficiency and supported overall plant growth in crops like spinach. They reported that plants treated with these fertilizers exhibited higher N content in both leaves and roots than those receiving traditional fertilizers. Additionally, findings from Dimkpa et al. (6) indicated that NZ facilitate a gradual and controlled N release, ensuring sustained availability for plants while minimizing N loss to the environment.

Protein content showed significant variation (p ≤ 0.05), with T7 recording the highest values (21.47 ± 1.24% and 21.58 ± 1.38%; group ‘g’), followed by T6 (group ‘f’) and T4/T5 (group ‘e’). These were significantly superior to urea (T2; group ‘d’) and control (T1; group ‘a’). The SE(d) (0.823–0.885) and CV (~6%) indicate reliable treatment effects. (Supplementary Table 1). These findings indicate consistent trends in protein accumulation across both years, highlighting the effectiveness of specific treatments in enhancing protein content in spinach beet. The crude protein content of maize grains was highest in nanozeolite urea fertilized plants, regardless of soil texture. The highest crude proteins were found in black and red soils, with 26.1% and 36.1% higher than urea fertilized treatments (50). Chaudhary et al. (90) reported in their study that combined treatment of nanozeolite and bio-inoculants significantly increased the protein and phenol contents in maize compared to the control. Nano-chelated N fertilizer increased protein content in wheat by 52% compared to urea. Nanozeolite and plant growth-promoting rhizobacteria (PGPR) treatment positively increased total protein in maize up to twofold (91). The N content of maize plants was significantly influenced by fertilization with various zeolite-based fertilizers. The highest N content was found in the roots of maize plants fertilized with nanozeourea (0.32%), while urea-fertilized plants had only 0.26%. The highest N content was found in grains harvested from nanozeourea-fertilized maize plants. The total N content was higher in nanozeourea than in other treatments, with a 28% higher content in grains. The slow-release pattern of nanozeourea fertilizer may be responsible for enhanced N uptake. Zeolites mixed with chemical fertilizers help retain nutrients in the root zone, improving soil quality and nutrient absorption.

Yield was significantly affected by N treatments (p ≤ 0.05), with T4 producing the highest yield (243 ± 11 and 257 ± 3 g pot - ¹; group ‘d’), which was significantly superior to T2 (219–242 g pot - ¹; group ‘cd’) and T3 (198–200 g pot - ¹; group ‘b’). Treatments T5 and T6 were statistically at par with T4 in some cases (groups ‘cd–de’), indicating comparable performance. The control (T1) recorded the lowest yield (165–182 g pot - ¹; group ‘a’). The SE(d) (9.661–11.9) and CV (~7%) confirm acceptable experimental variability. (Supplementary Table 1), demonstrating the efficacy of slow-release N formulations. These results suggest enhanced-efficiency fertilizers, particularly slow-release N at higher rates, can significantly improve N use efficiency and crop yield. Khan et al. (22) found that applying Nanozeolite Composite Fertilizer improved the soil’s physical, chemical, and biological properties. This included decreased soil pH, increased total dissolved solids (TDS), and enhanced water retention capacity. These improvements facilitated nutrient availability in the soil, ensuring plant roots readily absorbed essential elements. Additionally, Salako et al. (41) compared zeolite A-based fertilizer with conventional NPK 15:15:15 fertilizer and noted that the former resulted in significantly higher lettuce yield. Specifically, plants treated with zeolite A-based fertilizer achieved a mean yield of 84.69 units, whereas those treated with NPK 15:15:15 fertilizer yielded an average of 69.34 units. These findings underscore the efficacy of nanozeolite-based N fertilizer in enhancing crop yield through improved soil conditions and sustained nutrient availability compared to conventional fertilizers.

Correlation analysis of soil health parameters along with N content of soil and crop yield

The correlation analysis highlights significant positive relationships among key variables, as explained in Supplementary Figure 4. Soil organic carbon correlates moderately to strongly with dehydrogenase activity (0.85023), microbial biomass carbon (0.69634), microbial biomass N (0.79786), and total yield (0.66423). Dehydrogenase activity shows strong positive correlations with SOC (0.85023), MBC (0.78763), MBN (0.89648), and total yield (0.71964), indicating its role in microbial activity and crop yield. MBC and MBN are positively correlated (0.8081) and with dehydrogenase, emphasizing their interdependency. Soil available N correlates positively with SOC (0.55669), dehydrogenase (0.52275), MBC (0.51678), and total yield (0.72023), influencing microbial activity and crop productivity. Total yield correlates positively with SOC (0.66423), dehydrogenase (0.71964), MBC (0.78001), MBN (0.59928), and soil available N (0.72023), reflecting the combined effects of soil properties and microbial activities on crop yield. These findings affirm that the use of nanozeolite-based N fertilizers enhances microbial biomass and activity, leading to improved N availability and increased crop productivity.

This study demonstrates that surfactant-modified nanozeolite fertilizer (SNZUF) is a highly effective and sustainable option for Spinach Beet cultivation. SNZUF achieved a total nitrogen loading of 18% with 6.7% available N, outperforming NZUF (15.5% total N, 5% available N), and supplied ammonium (1.34%) and nitrate (0.53%) in a balanced manner. Seed germination improved from 60% in control soils to 96.7% with moderate SNZUF doses, while the seedling vigor index reached ~1064, highlighting enhanced early growth. Post-harvest soil nitrogen was highest under SNZUF (221 mg/kg total N), 7.6% higher than urea, ensuring sustained nutrient availability. Water retention increased to 67% and swelling capacity to 1.28, supporting better soil–plant water relations. Controlled-release patterns demonstrated prolonged nutrient supply, with 75% ammonium release occurring at ~48–50 days and nitrate release at ~45–48 days, compared to 10–15 days for urea, minimizing nutrient losses. At practical agronomic doses, SNZUF maintained biosafety while improving microbial biomass, soil enzymatic activity, and nitrogen use efficiency by 20–30% over urea. These findings highlight SNZUF as a precision, eco-friendly fertilizer that enhances Spinach Beet yield and quality while safeguarding soil health. Future applications integrating nanozeolite fertilizers into climate-smart and resource-efficient agricultural systems could redefine sustainable vegetable production.