Effect of NPS Fertilizer, Spraying Nano-NPK+TE and Cerium Oxide NPs on the Qualitative Properties and Bioactive Components of Rice Grains (Oryza Sativa L.)

The aim of this research was to identify the effects of NPS fertilizer with the spraying of Nano-NPK+TE and Cerium Oxide NPs on the active substances in rice grains, amber 33 cultivar. This field study was carried out in the Tali’a region, 41-Husainiya, Hilla city, Babylon province, Iraq, from 14/6/2022 to 25/11/2022, The study treatments were randomly distributed on the experimental units according to a simple and one-way experiment by a randomized complete block design (RCBD). The study treatments consisted of adding NPS fertilizer at four levels (0, 100, 200, and 300 kg ha-1) and spraying with nanomaterials of Nano-NPK+TE and Cerium Oxide NPs at concentrations of (2.5 ml L-1) and (50 mg L-1), respectively, with four sprays for each of them. A total of 16 experimental units were used. The Statistical analysis using the least significant difference test revealed that the process of fertilizing with NPS fertilizer and nanomaterials led to significant differences and an increase in the contents of volatile oils, saturated and unsaturated fatty acids among the dietary components of rice grains, so the findings from gas chromatography-mass spectrometry (GC-MS) analysis indicated that soil fertilization with NPS mineral fertilizer at level (300 kg ha-1) achieved the highest level of stearic acid content with a value of (9.460%), as well as the highest improvement in Palmitic acid content (24.240%) recorded by the treatment (NPS 100 kg ha-1). Moreover, the bi-combination of NPS (200 kg ha-1) + Spray CeO2 NPs gave the highest content of Oleic acid content in percentage (10.310%), and Linoleic, another type of unsaturated fatty acid, indicated a content of (10.020%) by the combination of (NPS (300 kg ha-1) + Spray nano-NPK+TE). Additionally, the highest percentage of Volatile oils showed at the foliar spraying combination (Spray nano-NPK+TE + CeO2 NPs), which gave a value of (46.50%).


Introduction
Rice is a grain crop that generally needs large amounts of nutrients, mineral fertilizers, and large amounts of water during the various stages of growth.Because of immersion irrigation methods large amounts of conventional fertilizers are exposed to washing, volatilization, and fixation in the soil [1], must trying to find a balance of nutrients and compensate for the lost ones, especially when cultivating grain crops such as Yellow Corn and Rice, in addition, agricultural soils are exposed to consumption Fixed oils can be defined as esters of fatty acid united with glycerol, which are insoluble in water, do not volatilize, and dissolve in alcohol and other organic solvents.They are stored in large quantities in the seeds and in smaller quantities in the rest of the plant parts.They also get volatilization when exposed to heating or sometimes at room temperature, so they are called Volatile Oils are chemical compounds that consist of a liquid part consisting of hydrocarbon compounds, and a part that represents solids in the liquid part called (Stearoptenes) consisting of compounds derived from Hydrocarbon Compounds that make up the liquid part [22].In general, the use of Nano-fertilizers as a modern technology and the most effective way to provide the plant with its needs and reduce losses.In addition to enhancing the economic benefits, their joint use with traditional mineral fertilizers contributes to creating a balance of continuous nutrition in different stages of plant growth, and reducing dependence on chemical fertilizers [23,24], which is what the current study aims to determine by evaluating the fertilizer groups used from NPS fertilizer at three levels.As an addition on soil, integrated Nano-NPK +TE fertilizer, and Cerium Oxide NPs by foliar spraying on the vegetation cover of Rice with the aim of knowing the effect of these fertilizers on the qualitative characteristics and biologically active components of rice grains ,Amber 33 variety.

Experiment Location
This field experiment was carried out in Tali'a region, 41-Husainiya, Hilla city, Babylon province, Iraq, in the interval between 14/6/2022 to 25/11/2022, Within the geographical coordinates of latitude (N-3560050) and longitude (E-481907), which is the required time period for the completion of all growth stages in the rice plant.

Experience Design
The experiment of current study was carried out with randomly distribution of the study treatments on the experimental units according to a simple and one-way experiment by a randomized complete block design (RCBD), and the number of treatments reached (16) within each replicate with three replicates for the field experiment, Consequently, the total number of experimental units increased to 48, according to [25].The treatments consist of adding NPS fertilizer at four levels (0, 100, 200, and 300 Kg ha -1 ) and spraying with nanomaterials of Nano-NPK+TE and Cerium Oxide NPs at concentrations of (2.5 ml L -1 ) and (50 mg L -1 ), respectively, with four sprays for each of them.
Table 1.Provides a list of the fertilizers and nanomaterials utilized in the experiment with their respective components.

Experiment Field Preparations
The experimental field unit was selected from agricultural land and subjected to perpendicular tillage, smoothing, and levelling processes.A representative sample for field soil was taken at a depth of 20 cm from five locations surrounding and the center of experimental field, to characterize the classification of agricultural soil, that was classified as Entisols according to the latest American classification [26] with clay loam texture.Also we found that soil have the following properties: Electrical Conductivity (Ec) = 3.02 ds m -1 , pH = 7.56, N= 21.11 mg N kg -1 soil, Available P= 9.32 mg P kg -1 soil, Available K= 261.24 mg K kg -1 soil, available S = 297.53mg S kg -1 soil, Organic matter = 1.44%.The total study area of the field was (300 m 2 ), the area divided into three equal replicates, each

Plantation and Crop Management
The dry rice seeds of (Ambar-33) variety, were approved by Rice Research Station located in Al-Mishkhab District of Al-Najaf governorate, for planting directly into previously prepared soil on (14/6/2022), at a rate of (120 kg ha -1 ).The soil was covered after planting to prevent erosion by irrigation water and consumption by birds.During the patching stage (1-4/8/2022), irrigation water depth was maintained at approximately (10 cm) to ensure adequate water availability for the plants to attain full maturity, in addition to weeding while doing the patching process.Irrigation of the field was stopped for 14 days before harvesting and weeding while doing the patching process.

Fertilization
The soil of experimental units were initially fertilized with Tron fertilizer NPK (20:20:20) at a rate of (200 kg ha -1 ), then the fertilization process with NPS fertilizer at four levels (0, 100, 200, and 300 kg ha -1 ) applied to the soil (Table 3).The calculated quantities of addition were depending on the area of the experimental unit.In addition, the foliar spraying with nanomaterials combine of Nano-NPK +TE fertilizer at a concentration of (2.5 ml L -1 ) and Cerium Oxide NPs at a concentration of (50 mg L -1 ).The process of spraying Nano-materials was carried out according to concentrations with four sprays, and the period between one spray and another was 7 days, starting from pre-flowering stage to 50% flowering.These foliar sprays were carried out at the crack of dawn applying at a proportion of (400 liters ha -1 ) (Table 3).The size of particles of Nano-NPK +TE and Cerium Oxide NPs was determined using scanning electron microscope (FE-SEM), this analysis was performed at the physics department's central laboratory of the University of Tehran.to confirm that the particle size was within the Nano-dimension range (1-100 nm).

Harvest
On 11/25/2022, the rice crop was harvested at full plant maturity for all treatments.Moisture percentage in grain at harvest was measured to be within the range of 18-25%, as mentioned in [27].

Quantify the bioactive components in rice grains through Gas Chromatography-Mass Spectrometry (GC-MS)
Rice grain samples were extracted using a modified version of the method proposed by reference [28,29], Firstly, (5 g) of dried powder of rice grains from every treatment were added to (5 ml) of 50% ethanol and constantly stirred.The mixture was filtered with filter sheets, and the filtrate was collected while the sediment was re-extracted using ethanol.The two filtrates were combined and intensified at (40 °C) for 24 hours before adding (10 ml) of hexane and (10 ml) of chloroform.After drying, the desiccated substance was solubilized in (5 ml) of 20% methanol, and (1 ml) of the extracted solution was taken for analysis.The GCMS-QP2010 Ultra device equipped with the AOC-20i auto-identifier system was used to analyze 2 microliters of the extract in accordance with these parameters: a 100% Dimethyl Polysiloxane chromatographic column with measurements of 0.25 nm x 30 x 1μm df, a helium carrier gas at a flow volume of (1 ml min -1 ), an injector temperature of (250ºC), and an ionic source temperature of 200ºC.An automatic temperature programming of the oven was performed to achieve a thermal gradient, the temperature program started at an initial temperature of (40 ºC), which was maintained for a duration of 3 minutes.The temperature was subsequently increased in increments of (15 ºC) every minute until it reached (180 ºC), followed by a further increase of (10 ºC) every 3 minutes until the temperature reached (300 ºC).The temperature was then held steady at (300 ºC) for the remainder of the analysis.The total analysis time for each sample was (28 minutes).The mass spectra were acquired using an ionization energy of (70 eV), with a time period of (0.5 seconds) and a scan range of (40-450 Daltons).The resulting spectra were compared with those stored in the National Institute of Standards and Technology (NIST) database to identify the components.This testing was performed in The Gas Chromatography Laboratory located at Kashan University in the Islamic Republic of Iran.

Statistical Analysis
The data of the obtained results were assessed using statistical methods by variance analysis (ANOVA), using the tabulation program, and the statistical analyses of the data included the Analysis ToolPak, then the differences between the averages of treatments were assessed by using the least significant difference test, at the level of probability (P ≤ 0.01) and (P ≤ 0.05), to determine the significant differences, as mentioned in [25].

Effect of NPS Fertilizer, Spraying Nano-NPK +TE and Cerium Oxide NPs regarding the percentage of Saturated fatty acids (Palmitic Acid and Stearic Acid)
By noting the results shown in Table No. (1) and Figure (2), the single treatment by adding fertilizer NPS at level (300 kg ha -1 ) achieved a substantial level on their counterparts in the content of palmitic saturated fatty acid, which recorded quantity of (24.240 %), then the level (200 kg ha -1 ) of NPS as soil addition which noted a value of (22.750 %), in addition to the other treatments in combination (NPS (100) +CeO 2 ), (NPS (300) +NPK +TE +CeO 2 ), (NPS (300) +NPK +TE ), (NPS (300) +CeO 2 ) and (NPS (100) +NPK +TE +CeO 2 ) was achieved with consecutive values of (17.430, 15.580, 13.600, 8.570 and 8.320) % respectively, in comparing with The treatment with the least effect (NPS (200) +NPK +TE +CeO 2 ) registered (3.800 %), Moreover, bi and single treatments Nano-NPK +TE and Cerium Oxide NPs showed that the single treatment (NPK +TE ) achieved progress in Palmitic fatty acid with the quantity of (19.650 %), while the treatment of spraying CeO 2 nanoparticles amounted (11.420 %), and the combination treatment of these nanomaterials gave significant notable response by recording (9.750 %).Regarding to the content of Stearic fatty acid, through Table No. (1) and Figure (3), we note that also the single treatment by adding fertilizer NPS at level (300 kg ha -1 ) had a distinct with its observed a reading of (9.460 %) when comparing with their counterparts, and the treatments of foliar spraying achieved highest notable progress by recording (6.360, 5.400, 9.130) % for the following treatments (CeO 2 ), (NPK +TE ) and (NPK +TE +CeO 2 ) respectively.also other integrated treatments (NPS (100) +CeO 2 ), (NPS (200) +CeO 2 ), (NPS (100) +NPK +TE ), (NPS (100) +NPK +TE +CeO 2 ) and (NPS (200) +NPK +TE ) and (NPS (300) +CeO 2 ) distinguished by the readings for studied trait, reaching (5.420, 5.260, 3.760, 3.440, 3.330 and 2.920) % respectively, when comparing with the weakest value of the treatment (NPS (200) +NPK +TE +CeO 2 ) which registered (1.390 %), Although, it is worth noting that there is a noticeable increase in the creation of Stearic acid, which is formed through changes in the configuration of Palmitic acid by means of the beta-oxidation pathway.This suggests that the use of nanoparticles as catalysts has an overall positive impact on enhancing the nutritional quality of rice grains [30,31].) also observed marked progress at Oleic unsaturated fatty acid with values (6.470, 6.150, 6.140, 5.420 and 4.850)% in comparing with the weakest treatment observed a reading of (2.610 %), Also, the single treatment of ground fertilization with (NPS) mineral fertilizer at level (300 Kg ha -1 ) gave notable progress in Oleic acid content with quantity of (6.670 %), while the treatments of foliar spraying of Nano-materials with (CeO 2 , NPK +TE ) reached a substantial value level of (6.340, 5.400)% respectively, and combined treatment (NPK +TE + CeO 2 ) was recorded value of (4.410 %).
From the results in Figure (4) we noted that the highest significant response of combined fertilizers at Linoleic acid content in the grains exhibited in the bi-treatment (NPS (300) +NPK +TE ) which observed a reading of (10.020 %) when comparing with the weakest treatment of (NPS (200) + CeO 2 NPs) observed a reading of (2.140 %), and the other combinations (NPS (100) +CeO 2 , NPS (100) +NPK +TE + CeO 2 , NPS (300) +NPK +TE +CeO 2 and NPS (100) + NPK +TE ) also noted an exceptional performance in Linoleic fatty acid by recording quantities of (9.770, 9.650, 8.690 and 5.040) %, respectively.While the single foliar spraying treatments (NPK +TE and CeO 2 ) showed values of percentage in Linoleic acid (6.390 and 5.380) % respectively, and also showed a significant increase in comparing with control treatment, significant values were also observed in the treatment with combined nanomaterials (NPK +TE +CeO 2 ) by recording (4.030 %) in comparing with the weakest treatment value, these results indicate that the increase in Oleic and Linoleic acids came as a result of the catalytic action of the producing reactions of unsaturated fatty acids, which may explain the abiotic tension that occurred on the rice plant as a result of spraying these nanocomposites.The reaction was the production of antioxidant compounds, including unsaturated fatty acids, it is worth noting that the difference in the production of Linoleic and Oleic acids have reached a doubling the production of the amount of these bioactive substances by two and three times, and this is an improvement in the nutritional content of rice grains [32,33].

Effect of NPS Fertilizer, Spraying Nano-NPK +TE and Cerium Oxide NPs regarding the percentage of Volatile Oils
Through the results in Table (3) and Figure (6), we can notice a notable progress in the content of Volatile oils in the treatments with the effectiveness of the combined treatment of foliar spraying (NPK +TE +CeO 2 ), which was distinct from the rest of the treatments with a value of (46.500 %).Then the treatment (NPS (100) +NPK +TE +CeO 2 ) observed a reading of (39.340 %), and about bicombinations, the treatment (NPS (200) +NPK +TE ) achieved a notable progress over its counterparts by a quantity of (35.520 %) in comparing with the weakest treatment (NPS (300) +CeO 2 NPs) that noted readings of (8.470 %), while the treatments of foliar spraying for nanomaterials by the single treatment (CeO 2 NPs) reached a substantial value level of (27.260 %), then the treatment (NPK +TE ) was registered (10.350 %).These results are consistent with the sensory evaluations to examine the rice grains obtained during this research experiment.This confirms and enhances the characteristic attributes that amplify the appeal of rice grains for consumers, especially the grains that distinguished by their aroma [34,35].

Conclusions
It was found that the results of the current study are recording the largest quantities and attaining progress in the proportions of bioactive ingredients, Volatile oils and Fatty acids of high nutritional value in rice grains, and this is reflected in the quality of the rice crop represented by grains.This was achieved well improve when treat the plant with different fertilization formulations, such as ground fertilization at different levels, foliar spraying with nutrients and Nano-catalysts represented by Nanofertilizer NPK +TE and Cerium Oxide NPs.And this due to what these materials possess of a very small size and the high surface area of these particles and Nano-nutrients.This shows the contribution role of fertilizers in supplying integrated nourishment for the plant, which in turn attained the highest levels of the percentage of active ingredients in the rice grains of Amber 33.Therefore, these combinations can help reduce the amount of fertilizers added to the crop and reduce Ecological degradation due to wrong fertilizer management and inappropriate cultivation practices.Nevertheless, there is a demand for a detailed physiological and molecular understanding of the influence of these fertilizers, Mainly, the effect of nano-sized particles on rice, and we note that the largest quantities were noted by the effect of soil addition and nanomaterials in the bioactive components, and improvement in the nutritional content of rice grains.

Figure 1 .
Figure 1.Particles of Cerium Oxide NPs by FE-SEM scanning electron microscope.

Figure 2 .
Figure 2. Content percentage% of Palmitic saturated fatty acid in the rice grains.

Figure 4 .
Figure 4. Content percentage% of Oleic unsaturated fatty acid in the rice grains.

Figure 5 .
Figure 5. Content percentage% of Linoleic unsaturated fatty acid in the rice grains.

Table 2 .
A description of Soil Fertilization Treatments with ground NPS fertilizer and levels of dosages, the nanomaterials used, the concentration employed and Number of Sprays.

Table 3 .
Effect of NPS and spray of Nano-NPK+TE fertilizer and Cerium Oxide NPs sprays on the content percentage % of bioactive components of Saturated and Unsaturated fatty acids and Volatile oils in rice grains.