Nanoparticulate Fertilizers Increase Nutrient Absorption Efﬁciency and Agro-Physiological Properties of Lettuce Plant

: The extensive use of chemical fertilizers is responsible for numerous environmental problems including low food quality, soil degradation, and toxicity to beneﬁcial living organisms in the soil. Nano-fertilizers (NFs) application may be a promising solution for combat these challenges. The current study focused on the efﬁciency of applying small amounts of NFs incorporated with conventional nitrogen, phosphorus, and potassium (NPK) fertilizers to reduce the quantities of conventional fertilizers (CFs) in lettuce cultivated in sandy soil. This study evaluated the effect of these incorporations on plant growth, yield, phytochemical accumulation, leaf nutrient, and leaf nitrate. A pot experiment was conducted during the winter seasons of 2020/2021 and 2021/2022 using the following treatments: CF100: 100% CFs, CF75NF25: 75% CFs + 25% NFs, CF50NF50: 50% CFs + 50% NFs, CF25NF75: 25% CFs + 75% NFs, and NF100: 100% NFs (=10% of CFs). Our ﬁndings displayed that the CF75NF25 and CF50NF50 treatments recorded the highest plant growth parameter values (plant length, root length, number of leaves, and fresh and dry biomass). The maximum of chlorophyll ﬂuorescence measurements (photosystem II efﬁciency) were obtained in plants fertilized with CF75NF25, followed by CF50NF50 and CF100. The improvement ratios of photosynthetic pigments (Chlorophyll (Chl) a, b, and total) for CF75NF25 were 23.77, 50, and 23.72% in the ﬁrst season and 10.10, 51.0, and 24.90% in the second season for Chl a, b, and total, respectively, as compared with the CF100 treatment. A similar tendency was observed for the CF50NF50 treatment. Generally, CF75NF25 signiﬁcantly raised the content of total phenolic compounds (TPC), total ﬂavonoid content (TFC), and antioxidant activity (AOA) in lettuce plants by 36.09, 47.82, and 40.16% in the ﬁrst season and by 30.39, 37.53, and 32.43% in the second season, respectively, compared with CF100. In addition, the levels of leaf nutrient content and uptake of N, P, and K were signiﬁcantly higher in plants fertilized with CF75NF25 compared to the other treatments, whereas CF25NF75 had the lowest values among the different treatments across both seasons for most of the tested traits. The nitrate content in lettuce leaves (NO 3 − ) for both seasons was lower than the acceptable level for human consumption. These results indicate that incorporating a low concentration of NFs into CFs could be a promising strategy to reduce the amount of CFs to 75% or 50% of lettuce NPK requirements without signiﬁcant adverse effects on the growth and productivity of lettuce plants cultivated in sandy soil.


Introduction
Lettuce (Lactuca sativa L.) is one of the most common and popular leafy vegetables worldwide.It belongs to the family Compositae, and is a good source of fiber, folate, vitamin C, antioxidant, and several dietary minerals required for human nutrition [1].Lettuce is low in calories, fat, and sodium content and rich in other beneficial bioactive compounds [2].In Egypt, lettuce is cultivated for local consumption and exportation.The acreage of lettuce in Egypt was 3895 ha in 2020, while production was 79,045 tons in the same year, with an average yield of 20,3 tons per ha.The exportation quantity increased from 12,877 in 2016 to 43,715 tons in 2020 [3].
The agricultural sector has encountered many challenges that limit the sustainability of cropping systems, including declining crop productivity, low fertilizer use efficiency, nutrient depletion, climate change, and low water availability.In addition, increasing food demand due to the rapid growth of the global population, which is expected to increase to 9 billion by 2050, indicates that food production will need to rise by 25-70% compared to current levels [4,5].In addition to all aforementioned challenges, there is a great increase in the prices of conventional fertilizers (CFs) as well as extensive use of fertilizer quantities by farmers.However, many environmental problems have been reported as a result of the heavy use of fertilizers, including low food quality, soil degradation, and toxicity toward beneficial living organisms in the soil [6][7][8].In addition, there can be a large amount of loss of most CFs to the surrounding environment via irrigation under traditional methodologies, depending on soil properties.Around 40-70% of N, 80-90% of P, and 50-70% of K in CFs are lost and cannot be absorbed by plants [9,10].Meanwhile, macronutrient consumption is predicted to rise to 263 million tons by 2050 [11].
The world is turning to new strategies to protect food security and fill gaps in sustainability [12].These efforts are being made to synchronize nutrient availability and improve nutrient use efficiency values in agricultural systems without a further deterioration of surrounding environments [13].Nanotechnology has the potential to develop new innovative types of fertilizers such as nano-fertilizers (NFs) [14].NFs can be defined as nanomaterials or nanoparticles of essential or beneficial nutrients that can be delivered to plants at the nanoscale (1-100 nm) to support plant growth and improve production [15,16].
Compared to CFs, NFs have unique characteristics that make them more efficient due to their positive impacts on the productivity and nutritional quality of crops as a result of rapid uptake by the plant roots, penetration into cells, and transport and representation within plant tissues when applied as foliar or soil application [17][18][19].In addition, the application of NFs reportedly improves nutrient use efficiency by 20%, and increases nutrients' dispersion in soil and availability to plants due to their small particle size, high surface area, and high solubility in water [16,20,21].In this respect, NFs play a critical role in improving the growth, yield, nutrient content, and bioactive compounds of various crops, including French bean [22], wheat [23], green pepper [9], cucumber [24], and green bean [25].
Slow-release NFs can be used for a continuous supply of nutrients to reduce nutrient losses [26].Chitosan-based NFs are considered slow-release NFs, which can be used efficiently to supply nutrients to plants over a long time [27].Moreover, many researchers reported that the NFs of NPK based on chitosan improved the growth, productivity, and chemical constituents of various crops [23,28,29].
Several studies have affirmed that NFs obtained promising results on productivity, physiological, and biochemical parameters of lettuce, i.e., Zn [30], B [31], Fe, and Si [32].For instance, Nofal et al. [33] found that all lettuce vegetative growth parameters, yield, and marketable yield of lettuce were positively enhanced by nano N application, even though nano K treatment significantly affected the head quality and biochemical and nutrient content.In the context of sustainable agriculture, there is no doubt as to the recent progress in the use of NFs to enhance crop quality and productivity, which has had a positive impact on the agricultural sector [16].However, one of the major concerns with the use of NFs is the toxicity caused to plants, microbes, and animals [34][35][36].Moreover, direct disease transmission from the use of NFs to human beings has not been reported at present, but the use of supra-optimum application rates may lead to the deposition of NFs and cause nanotoxicity [37].Furthermore, there are great apprehensions that the extensive release of NFs into the environment and the food chain may pose a risk to human health in the long-term of use [16].These concerns can limit the use of this technology in the agricultural sector [38].Due to those safety concerns, the risks of NFs application should be carefully examined before use, and the optimum dose determination of NFs is required for a correct and safe application [37].Many investigators reported that a small amount of NPK nano-fertilizers (10%) significantly improved agro-physiological traits, bioactive compounds, yield quantity, and quality of various crops including wheat, potato, French beans, and pepper, as compared to the high doses of nano fertilizers and chemical fertilizers [22,23,29,39,40].
Therefore, the incorporation between CFs and NFs could be a good strategy to manage the extensive use of either CFs or NFs.We thus concluded that there is a need to expand research in this area.This study aimed to reduce the amount of CFs through using NFs (10% of CFs) and, in addition, evaluate the effects of single and combined application of those fertilizers (NFs and CFs) on agro-physiological characteristics, i.e., Chl content, photosynthetic parameters, phytochemical compounds, and macro-and micronutrients of lettuce plants cultivated in sandy textured soil.

Experimental Condations and Plant Material
A pot experiment was conducted in a net greenhouse in the Department of Vegetable Crops, Faculty of Agriculture, Cairo University, Giza, Egypt (Longitude 31.2067• with a Latitude 30.0171 • ) during the winter seasons of 2020/2021 and 2021/2022.The average temperature was 15.8 ± 2 and 14.1 ± 2 • C, and relative humidity was 61.91 and 66.57% in the first and second season, respectively.Lettuce cv. Green Wave (Condor seed production company, Somerton, AZ, USA) seeds were sown in 209-cell seedling trays filled with a commercial medium of peat moss and vermiculite (1:1 v/v) in the nursery on 4 October 2020 and 2021.The transplanting date was at the second week of November for both seasons.Plastic pots (35 L volume) were filled with sand (Typic torri psamment).The physicochemical properties of the used soil before planting are shown in Table 1.The seedlings were transplanted at the 2nd unfolded true leaf (12 BBCH Scale) [41].Two seedlings were planted in each pot, with 12 plants per m 2 .Five treatments were tested with six replicates (six pots per replicate and 36 pots per each treatment), as shown in Table 2.The treatments consisted of NFs (NPK) alone or in combination with CFs (NPK); recommended doses of NPK fertilizers were used as control.Irrigation was applied twice per week and other agricultural practices took place according to recommendations of the Ministry of Agriculture and Land Reclamation (MALR), Egypt.The experiment was laid out in a Completely Randomized Design (CRD), was repeated in both seasons.All NFs were purchased from Nanotech for Photo Electronics (Nanotech, Giza, Egypt) and used according to the manufacturer's instructions.The NFs were synthesized according to the bottom-up method using an alkylation technique based on crosslinking with dialkyl halide followed by alkylation with octyl halide and further methylation with methyl iodide.Rutin-loaded chitosan nanoparticles were prepared by dissolving rutin in ethanol (70%) and mixing with the chitosan solution, followed by loading with NFs of N, P, and K. Tripolyphosphate was subsequently added dropwise to the chitosan-rutin mixture with continuous stirring.The colloidal suspension was stirred for 2 h for particle hardening [42].A transmission electron microscope (TEM) (JEOL-JME 2100, Tokyo, Japan) was used to determine the size and shape of the used NFs (Figure 1A-F).

Fertilizers Treatments and Concentrations
In this experiment, the CFs and NFs were applied to the soil via irrigation individually and/or incorporated in five treatments, as presented in Table 2.The treatments were applied when a lettuce plant unfolded at the 3 rd true leaf (13 BBCH Scale).The NFs were prepared from the same sources of applied CFs.The CF100 represents the NPK dose for lettuce production in sandy soil according to recommendations of the Ministry of Agriculture and Land Reclamation (MALR), Egypt.The NF100 represents only 10% of CF100.The quantity of CFs (CF100) were 318.8 kg/ha of ammonium nitrate, 333.2 kg/ha of potassium sulfate, and 48.8 L/ha of phosphoric acid, while the quantity of NFs (NF100) (10% of CF100) were 32 kg/ha, 33.35 kg/ha, and 5 L/ha, of NPK NFs, respectively.

Plant Growth Parameters
Upon reaching 80% of the leaf mass typical of the variety (48 BBCH Scale), lettuce plants were gently removed from pots.The root system was then washed with tap water and dried at room temperature.Plant growth parameters, including plant length, root length, number of leaves, and plant fresh and dry biomass, were recorded [43].

Photosynthetic Pigments and Chl Fluorescence Parameters
Chl a, b, and Total Chl and Chl Fluorescence Chl a, b, and total chl were extracted by acetone (80%, v/v) from green leaves at 80% of the leaf mass typical of the variety reached (48 BBCH Scale) by adding 0.5 g of green

Fertilizers Treatments and Concentrations
In this experiment, the CFs and NFs were applied to the soil via irrigation individually and/or incorporated in five treatments, as presented in Table 2.The treatments were applied when a lettuce plant unfolded at the 3rd true leaf (13 BBCH Scale).The NFs were prepared from the same sources of applied CFs.The CF100 represents the NPK dose for lettuce production in sandy soil according to recommendations of the Ministry of Agriculture and Land Reclamation (MALR), Egypt.The NF100 represents only 10% of CF100.The quantity of CFs (CF100) were 318.8 kg/ha of ammonium nitrate, 333.2 kg/ha of potassium sulfate, and 48.8 L/ha of phosphoric acid, while the quantity of NFs (NF100) (10% of CF100) were 32 kg/ha, 33.35 kg/ha, and 5 L/ha, of NPK NFs, respectively.

Measurements 2.3.1. Plant Growth Parameters
Upon reaching 80% of the leaf mass typical of the variety (48 BBCH Scale), lettuce plants were gently removed from pots.The root system was then washed with tap water and dried at room temperature.Plant growth parameters, including plant length, root length, number of leaves, and plant fresh and dry biomass, were recorded [43].

Photosynthetic Pigments and Chl Fluorescence Parameters
Chl a, b, and Total Chl and Chl Fluorescence Chl a, b, and total chl were extracted by acetone (80%, v/v) from green leaves at 80% of the leaf mass typical of the variety reached (48 BBCH Scale) by adding 0.5 g of green leaves to 20 mL of acetone (80%, v/v) and incubating in dark bottles for 3 days.The absorbance was then measured at 470, 646, and 663 nm with a spectrophotometer (model UV-2401 PC, Shimadzu, Milano, Italia) according to Costache et al. [44].The values were expressed as mg/g F.W.
Chl fluorescence parameters were measured using a portable Optic-Science OS-30p + Fluorometer (Opti-Sciences, Inc., Hudson NH, USA) at 80% of the leaf mass typical of the variety reached (48 BBCH Scale).Before taking the measurements, the leaves were adapted to darkness using clips for 20 min.After adaptation in the dark, the maximum efficiency of the photosystem (PSII, Fv/Fm) and maximum primary yield of PSII photochemistry (Fv/Fo), were estimated [45].

Phytochemical Components Ascorbic Acid, Total Flavonoid Content, and Total Phenolic Compounds
The plants were harvested for analysis upon reaching 80% of the leaf mass typical of the variety (48 BBCH Scale).
Ascorbic acid (AA) was determined using the titrimetric method with 2, 6-dichlorophenol indophenol as stated in AOAC 2000 [46] and presented as mg /100 g lettuce fresh weight.
Total Flavonoid Content (TFC) was determined using the method described by Meda et al. [47] with minor modifications.A 0.25 mL of sample (1 mg/mL) was added to a tube containing 1 mL of double-distilled water.Next, 0.075 mL of 5% of sodium nitrate, 0.075 mL of 10% aluminum chloride, and 0.5 mL of 1 M sodium hydroxide were added at 0, 5, and 6 min, sequentially.Finally, the volume of the reacting solution was adjusted to 2.5 mL with double-distilled water.The absorbance of the solution at a wavelength of 410 nm was detected using an Ultrospec 2100 Pro spectrophotometer.The data are shown as mg/g of fresh weight.Total phenolic compounds (TPC) were measured by the spectrophotometric method using Folin-Ciocalteu reagent, according to Singleton and Rossi [48].The results of lettuce were defined as gallic acid mg/g of lettuce dry weight (mg GAE/g dry weight).

Antioxidant Activity
The effect of different applications on lettuce antioxidant activity (AOA) was assessed according to the technique described by Fang et al. [49].Lettuce samples (10 g) were homogenized in 200 mL of distilled water, and then filtered using Whatman No.1 filter paper, after which 5 mL of filtrate was diluted into 25 mL of distilled water.Lettuce extract (1 mL) was added to 3 mL of methanol and 1 mL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (0.012 g DPPH in 100 mL -1 of methanol).The mixture was shaken in the dark at room temperature for 10 min.DPPH inhibition percentage (DPPH I N%) was determined spectrophotometrically at 517 nm (model UV-2401 PC, Shimadzu, Milano, Italia) and calculated using the following equation [50]: Antioxidant activity (%) = ((A 517nm of DPPH solution − A 517nm of sample)/A 517nm of DPPH solution) × 100 2.4.Nutrient Composition and Uptake 2.4.1.Plant Analysis NO 3 − (mg/100 g) was determined in distilled water extracts of lettuce leaves tissue (48 BBCH Scale) according to Cataldo et al. [51].The plant material was dried in an electric oven at 70 • C for 24 h according to Helrich [52] and then finely ground for chemical determination of the elements.The wet digestion of 0.2 g of plant material with sulfuric and perchloric acids was carried out on samples by adding concentrated sulfuric acid (5 mL).The mixture was heated for 10 min, followed by the addition of 0.5 mL perchloric acid, with heating continued until a clear solution was obtained [52,53].Total N content was determined using the modified micro-Kjeldahl method, as described by Helrich [52].Total P was determined colorimetrically by using the chloro-stannous molybdophosphoric blue color method in sulfuric acid according to Jackson [53].Total K, Ca, and Mg concentrations were determined using a flame photometer apparatus.The concentrations of micro-nutrients (Zn, Fe, and Mn) were determined in the same previous digested solutions using atomic absorption spectrophotometry.

Soil Analysis
The mechanical analysis of the pre-planting soil sample was performed using the dry sieve method [55] to obtain the percentage of different grain sizes contained within the soil.Before chemical analysis, samples of pre-planting soil were collected and air-dried, ground by a Wiley mill, and passed through a 2-mm mesh screen.The pH was measured in a 1:2.5 (w/v) soil-water suspension using a glass electrode pH meter.EC was measured in a 1:5 (w/v) soil-water suspension using an electrical conductivity meter to obtain dS/ m [52].Soluble cations (K + , Na + , Ca +2 , and Mg +2 ) as well as soluble anions (HCO 3−, SO 4 −2 , and Cl − ) have been measured using standard methods [53].Soluble cations (K + , Na + , Ca +2 , and Mg +2 ) were extracted from the soil by water in a 1:5 (w/v) and measured using flame photometer.Carbonates (CO 3 ) and bicarbonates (HCO 3 − ) were determined in a 1:5 (w/v) soil-water extract by titration 10 mL of soil-water extract using 0.01 N standard H 2 SO 4 in the presence of 1 drop of phenolphthalein and 2 drops of methyl orange indicators.Soluble SO 4 −2 was extracted using 0.15% CaCl 2 .2H 2 O (1:5 w/v) and measured in 10 mL of the extract by a turbidimetric method using 1 g of barium chloride in the presence of 1 mL of HCl + 5 mL of sorbitol followed by read the absorbance on a spectrophotometer at 470 nm wavelength.Cl − was determined in 1:5 (w/v) soil-water extract using silver nitrate titration method in the presence of potassium chromate solution (K 2 CrO 4 ).Nutrient concentrations were determined of soil samples using the standard methods.Total N content in soil was determined using the Kjeldahl procedure according to Chapman and Pratt [56] by the wet digestion of 1 g soil sample in 15 mL of concentrated H 2 SO 4 with a catalyst mixture followed by steam distillation, using excess NaOH.Then, the distillate is collected in saturated H 3 BO 3 , and titrated with dilute H 2 SO 4 .Available N (NH 4 and NO 3 ) was extracted using 2 M KCl solution (1:5 ratio) and determined by steam distillation of ammonia (NH 3 ) using 0.2 g heavy MgO for NH 4 and 0.2 g Devarda's Alloy for NO 3 , followed by collecting the distillate in a saturated H 3 BO 3 and titrated to pH 5.0 with dilute H 2 SO 4 [57].After digestion of the 2 g soil sample with 30 mL perchloric acid (HClO 4 ), total P was determined in the digested solution in a 50 mL flask by adding 10 mL of ammoniumvanadomolybdate solution to 5 mL of the filtrated digested solution and then measured on a spectrophotometer at 410 nm wavelength as described by Olsen and Sommers [58].Available P was determined using a spectrophotometer at 882 nm wavelength after being extracted by 100 mL NaHCO 3 (0.5N, pH 8.5) as described by Olsen et al. [59].Available K (soluble + exchangeable) was extracted by 1: 5 (w/v) using a neutral ammonium acetate solution (1 N) and measured by the flame photometer as described by Jackson [60].

Statistical Analysis
The results are expressed as means ± standard error.All data were analyzed in six replications for each parameter.Data were analyzed statistically utilizing analysis of variance by using the Statistica 7 program (version 2004).Differences between means were considered significant (p < 0.05) at a 95% confidence level according to the Tukey test.Pearson's correlation between tested variables were performed using Statistica 7 software (2004, TIBCO Software Inc., Palo Alto, CA, USA).

Plant Growth Parameters
The number of leaves and plant length values were not differed significantly among CF75NF25, CF50NF50, and CF100 during both seasons, as shown in Table 3.In contrast, lower values were observed for CF25NF75 and NF100 treatments compared with CF100.However, CF75NF25 and CF50NF50 recorded the significantly highest values of root length and fresh and dry weights as compared to CF100.CF75NF25 treatment significantly raised the values of root length and fresh and dry weights in lettuce plants by 14.40, 11.61, and 9.90% in the first season and by 35.09, 19.04, and 20.18% in the second season, respectively, compared with CF100.Meanwhile, CF50NF50 improved the values of root length and fresh and dry weights in lettuce plants by 10.90, 15.49, and 9.69% in the first season and by 28.71, 6.93, and 11.88% in the second season, respectively, compared with CF100.In contrast, lower values were noted with treatments CF25NF75 and NF100 compared with the control (CF100) in plant fresh weight, however, insignificant differences were obtained between CF25NF75 and NF100 compared with the control (CF100) in root length and plant dry weight in both seasons.

Photosynthetic Pigments and Chl Fluorescence Parameters
As shown in Figure 2A-C, incorporated treatments except CF25NF75 significantly influenced the photosynthesis pigments (Chl a, b, and total).In both seasons, CF75NF25 had the highest values of Chl a, b, and total compared with the other treatments, although these values were not differed significantly compared with CF50NF50 in Chl a at both seasons or in Chl b and total in the second season.The improvement ratios, for CF75NF25, were 23.77, 50, and 23.72% in the first season and 10.10, 51, and 24.90% in the second season for Chl a, b, and total, respectively, as compared with the CF100 treatment.A similar tendency was observed for CF50NF50 treatment, without significant differences compared with CF100 in Chl a, b, and total in both seasons.In contrast, CF25NF75 and NF100 revealed the lowest values of Chl (a, b, and total) compared with the control treatment (CF100) but without significances in Chl a or Chl b in the first and second season, respectively.Such similar results were observed in the Chl fluorescence measurements (Figure 2D,E).The highest values of the maximum primary yield of PSII photochemistry (Fv/Fo) and the maximum efficiency of the photosystem II (PSII, Fv/Fm) were recorded in plants treated with CF75NF25 compared with the other treatments.Compared with CF100, CF75NF25 increased the maximum primary yield of PSII photochemistry (Fv/Fo) and maximum efficiency of photosystem II (PSII, Fv/Fm) by 14.81 and 12.01% in the first year (2020/2021) and by 13.59 and 10.1% in the second year (2021/2022), respectively.
ments (Figure 2D,E).The highest values of the maximum primary yield of PSII photochemistry (Fv/Fo) and the maximum efficiency of the photosystem II (PSII, Fv/Fm) were recorded in plants treated with CF75NF25 compared with the other treatments.Compared with CF100, CF75NF25 increased the maximum primary yield of PSII photochemistry (Fv/Fo) and maximum efficiency of photosystem II (PSII, Fv/Fm) by 14.81 and 12.01% in the first year (2020/2021) and by 13.59 and 10.1% in the second year (2021/2022), respectively.

Phytochemical Components
The content of AA, TPC, TFC, and AOA in the leaves of treated lettuce plants is shown in Figure 3.The lettuce plants fertilized with CF75NF25 showed the highest content of all studied bioactive compounds and AOA of all treatments.This treatment (CF75NF25) significantly raised the content of TPC, TFC, and AOA in lettuce plants by 36.09,47.82, and 40.16% in the first season and by 30.39, 37.53, and 32.43% in the second season, respectively, compared with CF100.The content of AA was higher in CF75NF25 by 11.95% and 4.27% in the first and second season, respectively, compared with C100 but without significances differences.These phytochemical compounds (TPC, TFC, and AOA)

Phytochemical Components
The content of AA, TPC, TFC, and AOA in the leaves of treated lettuce plants is shown in Figure 3.The lettuce plants fertilized with CF75NF25 showed the highest content of all studied bioactive compounds and AOA of all treatments.This treatment (CF75NF25) significantly raised the content of TPC, TFC, and AOA in lettuce plants by 36.09,47.82, and 40.16% in the first season and by 30.39, 37.53, and 32.43% in the second season, respectively, compared with CF100.The content of AA was higher in CF75NF25 by 11.95% and 4.27% in the first and second season, respectively, compared with C100 but without significances differences.These phytochemical compounds (TPC, TFC, and AOA) were also significantly increased in plants fertilized with CF50NF50 and NF100, but to a lesser degree than in plants fertilized with CF75NF25 treatment, as compared with CF100.

N, P, and K Uptake
The combination of nano and conventional NPK fertilizers affected NPK uptake (Figure 4).The highest N, P, and K uptake was found in lettuce plants fertilized with the CF75NF25 treatment compared with the CF100 treatment, CF75NF25 increased N, P, and K uptake by 30.70%, 23.56%, and 27.11% in the first season and by 36.04%,17.85%, and 10.11% in the second season, respectively.Meanwhile, the lowest NPK uptake values were noted in plants treated with CF25NF75.
were also significantly increased in plants fertilized with CF50NF50 and NF100, but to a lesser degree than in plants fertilized with CF75NF25 treatment, as compared with CF100.

N, P, and K Uptake
The combination of nano and conventional NPK fertilizers affected NPK uptake (Figure 4).The highest N, P, and K uptake was found in lettuce plants fertilized with the CF75NF25 treatment compared with the CF100 treatment, CF75NF25 increased N, P, and K uptake by 30.70%, 23.56%, and 27.11% in the first season and by 36.04%,17.85%, and 10.11% in the second season, respectively.Meanwhile, the lowest NPK uptake values were noted in plants treated with CF25NF75.

Nitrate Content of Leaves
The concentration of NO3 − in the lettuce ranged from 490 to 1730 mg/kg fresh weight in the first season and from 300 to 1780 mg/ kg fresh weight in the second season (Figure 5).These concentrations were lower than the acceptable NO3 -content for human con-

Nitrate Content of Leaves
The concentration of NO 3 − in the lettuce ranged from 490 to 1730 mg/kg fresh weight in the first season and from 300 to 1780 mg/ kg fresh weight in the second season (Figure 5).These concentrations were lower than the acceptable NO 3 − content for human consumption, which ranges from 3500 to 4500 mg/kg fresh weight.Data from both seasons showed that the NO 3 − concentration was significantly increased with CF75NF25, followed by NF100 and CF50NF50; this is when compared with CF100 and CF25NF75, which represented the lowest NO 3 − values.Different letters indicate a significant difference between treatments (Tukey test at 95%).FW= Fresh Weight.

Nutrient Content in Plant and Soil
The data presented in Table 4 show the effect of various CFs, NFs, and combination treatments on the mineral composition of lettuce plants.Compared with the control (CF100), the maximum accumulation of several essential nutrients (N, P, K, Ca, Mg, Fe, Mn, and Zn) was found in lettuce plants treated with CF75NF25 followed by NF100 and CF50NF50.In both seasons, the lowest element accumulations were observed in lettuce plants fertilized with CF25NF75 treatment.The behavior of the treatments in both seasons was in the following order: CF75NF25 > NF100 > CF50NF50 > CF100 > CF25NF75.
From Supplementary Table (1), the results showed that the incorporation of NFs and CFs affected soil NPK content.The application of NFs, alone or combined with CFs, increased soil NPK content significantly more than CF100.The maximum content of soil N, P, and K were recorded in soil fertilized with CF50NF 50 followed by CF75NF25 compared with soil fertilized with CF100, in both seasons.Compared with CF100 treatment, CF75NF25 and CF50NF50 treatments increased soil N content by 31.58 and 36.96%,P content by 31.62 and 36.48%, and K by 48.65 and 64.71%, respectively, in the first season.Both treatments also improved the soil N content by 31.45 and 39.51%, P content by 24.28 and 33.42%, and K by 29.1 and 47.77%, respectively, in the second season.Table 4. Effect of nano and conventional NPK fertilizers either alone or incorporated, CF100: 100% conventional fertilizers (CFs) (recommended dose), CF75NF25: 75% CF + 25% nano-fertilizers (NFs), CF50NF50: 50% CFs+ 50% NFs, CF25NF75: 25% CFs + 75% NFs, and NF100: 100% NFs on N, P, K, Ca, Mg (%), Fe, Mn, and Zn (mg/kg) of lettuce during the 2020/2021 and 2021/2022 seasons.

Season Treatment
Macronutrients

Nutrient Content in Plant and Soil
The data presented in Table 4 show the effect of various CFs, NFs, and combination treatments on the mineral composition of lettuce plants.Compared with the control (CF100), the maximum accumulation of several essential nutrients (N, P, K, Ca, Mg, Fe, Mn, and Zn) was found in lettuce plants treated with CF75NF25 followed by NF100 and CF50NF50.In both seasons, the lowest element accumulations were observed in lettuce plants fertilized with CF25NF75 treatment.The behavior of the treatments in both seasons was in the following order: CF75NF25 > NF100 > CF50NF50 > CF100 > CF25NF75.
From Supplementary Table S1, the results showed that the incorporation of NFs and CFs affected soil NPK content.The application of NFs, alone or combined with CFs, increased soil NPK content significantly more than CF100.The maximum content of soil N, P, and K were recorded in soil fertilized with CF50NF 50 followed by CF75NF25 compared with soil fertilized with CF100, in both seasons.Compared with CF100 treatment, CF75NF25 and CF50NF50 treatments increased soil N content by 31.58 and 36.96%,P content by 31.62 and 36.48%, and K by 48.65 and 64.71%, respectively, in the first season.Both treatments also improved the soil N content by 31.45 and 39.51%, P content by 24.28 and 33.42%, and K by 29.1 and 47.77%, respectively, in the second season.

Correlation Study
The Pearson's correlation analysis (Table 5 and Figure 6) showed that leaf N content was positively correlated with the plant growth parameters (fresh and dry biomass of plants, Chl a, total Chl).Furthermore, a similar correlation was found between leaf N content and Chl fluorescence parameters (Fv/Fm and Fv/Fo).For bioactive compounds, leaf N content, leaf K content, and leaf Fv/Fm (PSII) were positively linked to ascorbic acid, total phenols, total flavonoids, and total antioxidant activity.A positive association was also observed between leaf N content and P, K, Ca, Mg, Fe, and Mn in the lettuce leaves.Table 4. Effect of nano and conventional NPK fertilizers either alone or incorporated, CF100: 100% conventional fertilizers (CFs) (recommended dose), CF75NF25: 75% CF + 25% nano-fertilizers (NFs), CF50NF50: 50% CFs+ 50% NFs, CF25NF75: 25% CFs + 75% NFs, and NF100: 100% NFs on N, P, K, Ca, Mg (%), Fe, Mn, and Zn (mg/kg) of lettuce during the 2020/2021 and 2021/2022 seasons.Mean values in the same column with different small letters indicate significant differences according to Tukey test (p < 0.05).Values reported are the means ± standard error (n = 6).

Table 5.
Pearson's correlation analysis between the agro-physiological traits of lettuce plants cultivated in sandy soil and treated with nano and conventional NPK fertilizers either alone or incorporated.P.L: plant length, R.L: root length, FW: fresh weight, DW: dry weight, and Chl: chlorophyll.Fv/F0: maximum primary yield of PSII photochemistry, FV/Fm: maximum efficiency of the photosystem, NO 3, AA: Ascorbic acid, AOA: antioxidant activity, TPC: Total phenols, TFC: Total flavonoids and plant nutrients (P, K, Ca, Mg, Fe, Mn, and N).Values in bold are differ from 0 with a significance level alpha = 0.05.

Discussion
The overuse of CFs, especially NPK fertilizers, is a result of the increased world demand for food.These types of fertilizers may improve plant growth and productivity, but they have harmful impacts on the ecosystem and human health [61].Therefore, the conversion of NPK fertilizers from CFs to NFs is considered one of the most promising alternatives for agriculture systems [40].In NFs, the macronutrients (such as N, P, and K) are linked alone or in combination with nano-adsorbents, which release nutrients quite slowly as compared to CFs.This approach not only improves the uptake and use efficiency of NPK nutrients but also minimizes their losses via leaching [40,59].However, although the advantages of NFs are certainly opening new approaches toward sustainable agriculture, their limitations should also be carefully considered before implementation on a large

Discussion
The overuse of CFs, especially NPK fertilizers, is a result of the increased world demand for food.These types of fertilizers may improve plant growth and productivity, but they have harmful impacts on the ecosystem and human health [61].Therefore, the conversion of NPK fertilizers from CFs to NFs is considered one of the most promising alternatives for agriculture systems [40].In NFs, the macronutrients (such as N, P, and K) are linked alone or in combination with nano-adsorbents, which release nutrients quite slowly as compared to CFs.This approach not only improves the uptake and use efficiency of NPK nutrients but also minimizes their losses via leaching [40,59].However, although the advantages of NFs are certainly opening new approaches toward sustainable agriculture, their limitations should also be carefully considered before implementation on a large scale in agriculture production [62].These limitations could be due to a lack of information on the optimum doses, nutrient bioavailability, safety, and toxicity of those fertilizers [63].
In particular, several scientific publications have shown that the application of NFs at high doses can generate toxicological impacts on economic crops such as tomato, lettuce, wheat, and cucumber, to mention just a few [64][65][66][67].Other researchers stated that the vast release of NFs into the environment cause a risk to human health [62].In this context, on a local scale, we suggested applying NFs of NPK with low concentration (about 10%), alone or in combination with CFs, to convince gradually the Egyptian farmers to use those fertilizers.In addition, we recommend studying their effectiveness on nutrient uptake, growth performance, and productivity of lettuce plants.
The current study showed that soil application of CF75NF25 (75% CFs + 25% NFs) and CF50NF50 (50% CFs + 50% NFs) significantly improved plant growth measurements compared with CF100 treatment (100% CFs), as presented in Table 3.Furthermore, the lettuce plants treated with CF75NF25 and CF50NF50 increased the plant length by 5 and 4.23%, root length by 11.21 and 22.73%, fresh weight by 11.73 and 8.29%, and dry weight by 8.92 and 13.71%, respectively, compared with the CF100 treatment, even though the amount of CFs was decreased to 75 or 50% with CF75NF25 and CF50NF50, respectively.In addition, Pearson's correlation showed that leaf N content correlated to plant fresh and dry weights (Table 5).The favorable impact of NFs on these tested features could be related to the fact that they have smaller particles (<100 nm), higher surface area, and higher absorption and slowly release their ions in a timely manner to cope with crop demand [20,21].These improvements in the studied vegetative parameters could be associated with an enhancement in the nutrient uptake, leaf photosynthetic pigments, and photosynthesis rates that increase total carbohydrate accumulation, which is considered the main component of dry biomass [68].Similar findings were observed by several investigators who reported that upon application of NFs with low concentration (about 10%), concentration significantly increased plant growth, yield, and quality of different crops, as compared with the CFs-treated plants [15,22,40,[69][70][71][72].
The lettuce plants fertilized with CF25NF75 (25% CFs + 75% NFs) and NF100 (100% NFs) treatments had the lowest values for all plant growth parameters (Table 3).These findings may be due to the rate of NFs release being lower than the plant's needs, especially when using a small amount of NFs with or without small amount of CFs in CF25NF75 or NF100, respectively.Wu and Liu [73] and Duhan et al. [10] noted the slowness of dissolution and release of NFs.This rate declines with nano-coating.In addition, prolonged release from coated fertilizer leads to more absorption via plant roots.It has been reported that the use of slow-release NFs prolongs nutrients availability by slow and sustained release efficiency [74].Furthermore, Bhardwaj [75] studied the release duration of NO 3 − and NH 4 + from CFs or NFs and found an increase in the release duration of NO 3 − and NH 4 + , from 12 to 20 days, in favor of NFs.Ha et al. [76] tested the nutrients release kinetics from chitosan -NPK NFs applied to coffee plants.They found that N was released slowly after more than 48 h to about 13%, then increased to 60% after 72 h, and remained constant with a slight increase to 66.7% after more than 192 h.K kinetics also showed a release after the first 72 h of about 55.4%, becoming constant after the next 240 h at 58%, whereas P represented the slowest release kinetics at 3%.
The present study revealed that the photosynthesis pigments (Chl.a, b, and total) and Chl fluorescence parameters of lettuce plants were improved in plants fertilized with CF75NF25 and CF50NF50 compared with the CF100 (Figure 2).This enhancement in the photosynthesis pigments (Chl.a, b, total) and Chl fluorescence parameters of lettuce plants could be related to the high uptake of N, P, and K (Figure 4).Furthermore, Pearson's correlation showed that leaf N content was positively associated with leaf Chl a, total Chl, and photosystem II content (Table 5).In agreement, Peng et al. [69] reported that the application of N fertilizer enhances photosynthetic pigment content, light energy capture, photochemical efficiency, and promotes the quantum efficiency and self-protection ability of PSII.This mechanism may be linked to the catalytic impact of N fertilizer on the activity of light-activated enzymes in plant leaves, which improves the energy capture efficiency of the PSII reaction center [77].In contrast to fertilizers, which have a positive impact on pigment content in lettuce, photosynthetic dyes can be reduced by pesticides used in lettuce cultivation or phytopathogenic fungi [78].On the other hand, lettuce plants treated with CF25NF75 or NF100 treatments recorded the lowest values of leaf Chl content, which could be attributed to the low nutrient uptake, especially N content, which was inadequate to promote or enhance photosynthetic pigments as well as PSII (Figures 2 and 4).
Regarding the bioactive compounds, the highest accumulations of AA, TPC, TFC, and AOA were obtained in the leaves of lettuce plants fertilized with CF75NF25 (Figure 3).These improvements in the bioactive compounds in plants might be due to the enhancement of total non-structural carbohydrates [79,80].This could be linked to the role of N and K in stimulating photosynthesis activity and the role of K in increasing the translocation of carbohydrates to plant leaves [81,82].The increase in translocation indirectly enhanced the biosynthesis of AA, AOA, TPC, and TFC in plants with high K uptake.N and K had a high positive correlation with AA, AOA, TPC, and TFC (Table 5).The current findings are in agreement with those of Ibrahim et al. [83] and Elmogy et al. [84].They found that the increase in AA, TPC, and TFC in Labisia pumila leaves was due to an increase in the production of total non-structural carbohydrates under high K application.
Lettuce plants have a great ability to accumulate NO 3 − in their leaves, which have adverse effects to human health [85,86].NO 3 − is a potential carcinogenic nitrosamine precursor [87,88].Therefore, the estimation of NO 3 − concentration in leafy vegetables, particularly in lettuce plants, is quite important for human health.The European Union reported that the highest permissible concentration of NO 3 − is 3500-4500 mg of N-NO 3 − /kg fresh weight for winter crops and 2500 mg of N-NO 3 /kg for summer crops [88].In the present study, the concentration of NO 3 − in leaf tissues of all treated lettuce plants ranged from 490 to 1730 or 300 to 1780 mg/kg fresh weight in the first and second season, respectively (Figure 5).The highest concentration of NO 3 − was recorded in the leaves of plants treated with CF75NF25 (Figure 5).This finding could be attributed to the high uptake of N (Figure 4).Concentrations of NO 3 − in the lettuce leaves fertilized with different treatments were lower than the acceptable NO 3 − content for human consumption.Our results showed that the highest accumulation of nutrients (N, P, K, Ca, Mg, Fe, Mn, and Zn) was found in the leaf tissues of lettuce plants fertilized with CF75NF25 compared with other treatments.This positive effect could be related to high uptake of N, P, and K (Figure 4).Furthermore, the improvement in the accumulation of Mg, Ca, Fe, Zn, and Mn in plant leaves could be due to the increasing root growth of lettuce plants treated with CF75NF25.Our findings are consistent with those of Abdel-Aziz, [40], who reported that the content of essential elements (N, P, K, Ca, Fe, and Mn) in the fruit of Capsicum annuum was significantly higher after receiving applications of NFs compared with plants fertilized with traditional fertilizers.In addition, Sharaf-Eldin [72] found that the application of NFs (N) increased the fertilizer use efficiency at a lower dose than recommended dose.In their study, the highest N uptake, apparent recovery efficiency %, and N use efficiency were obtained when lettuce was fertilized with 75% of N demand as fertigation plus 25% as foliar application in NFs.These results suggested that the incorporation of NFs and CFs, at a ratio of 75% CFs + 25% NFs or 50% CFs + 50% NFs, significantly improved the nutrient uptake (NPK), vegetative growth, photosystem II, and metabolites accumulation, which consequently promoted the head weight and total yield of lettuce.
Results regarding the soil macro nutrient (NPK) content after harvesting of lettuce plants are presented in Supplementary Table S1.The improvement in the content of the available NPK in the soil fertilized by NFs, alone or in combination with CFs, can be related to the unique chemical and physical properties of NFs that are characterized by their slow release, fine targeting, absorption speed by roots, and penetration of living membranes, as well as protection from adsorption and sedimentation reactions than CFs [89].These results were in harmony with the findings reported by several researchers [90-93].

Conclusions
The combined application of NFs and CFs to lettuce plants proved a useful tool to enhance vegetative growth characteristics, photosynthesis, nutritional quality, and yield.Furthermore, this application also improved soil NPK content and nutrient uptake.The magnitude of the impact appeared to be the ratio-dependent concentration and combination between NFs and CFs.The combinations of NFs and CFs at a ratio of 75% CFs + 25% NFs (CF75NF25) or 50% CFs + 50%NFs (CF50NF50) led to better agro-physiological properties and nutritional quality, thus suggesting a benefit to incorporating a low amount of NFs into CFs to reduce or save the amount of CFs up to 25% or 50%.Further toxicological experiments are needed to confirm that the edible parts of plants treated in this manner are not poisonous to humans and animals.In addition, there is a need for further research with long-term field studies focused on soil and groundwater-NFs interactions based on innovative, safe, and cost-effective methods or techniques.

Figure 6 .
Figure 6.Pearson's correlation analysis between the agro-physiological traits of lettuce plants cultivated in sandy soil and treated with nano and conventional NPK fertilizers either alone or incorporated.P.L: plant length, R.L: root length, FW: fresh weight, DW: dry weight, and Chl: chlorophyll.Fv/F0: maximum primary yield of PSII photochemistry, FV/Fm: maxi-mum efficiency of the photosystem, NO3, AA: Ascorbic acid, AOA: antioxidant activity, TPC: Total phenols, TFC: Total flavonoids and plant nutrients (P, K, Ca, Mg, Fe, Mn, and N).

Figure 6 .
Figure 6.Pearson's correlation analysis between the agro-physiological traits of lettuce plants cultivated in sandy soil and treated with nano and conventional NPK fertilizers either alone or incorporated.P.L: plant length, R.L: root length, FW: fresh weight, DW: dry weight, and Chl: chlorophyll.Fv/F0: maximum primary yield of PSII photochemistry, FV/Fm: maxi-mum efficiency of the photosystem, NO3, AA: Ascorbic acid, AOA: antioxidant activity, TPC: Total phenols, TFC: Total flavonoids and plant nutrients (P, K, Ca, Mg, Fe, Mn, and N).

Table 1 .
Physicochemical properties of used soil before planting.
Mean values in the same column with different small letters indicate significant differences according to Tukey test (p < 0.05).Values reported are the means ± standard error (n = 6).