Integrated soil-foliar strategy using biochar and Zinc Oxide Nanoparticles reduces cadmium bioavailability and improves growth, antioxidant defense, and yield of Maize (Zea mays L.)

Integrated soil-foliar strategy using biochar and Zinc Oxide Nanoparticles reduces cadmium bioavailability and improves growth, antioxidant defense, and yield of Maize (Zea mays L.) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integrated soil-foliar strategy using biochar and Zinc Oxide Nanoparticles reduces cadmium bioavailability and improves growth, antioxidant defense, and yield of Maize (Zea mays L.) Muhammad Waseem, Saqib Ali, Mamoona Munir, Muhammad Saim Talal, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9008412/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims Agricultural soils contaminated with cadmium (Cd) pose a serious threat to food safety and crop yield. Various conventional methods are employed to mitigate toxic effects; however, biochar amendments with ZnO NPs are particularly suitable for Cd remediation. Methods In this study, we examined the ability of biochar (BC) (1% and 2% w/w as a soil amendment) and zinc oxide nanoparticles (ZnO NPs; 250 and 500 ppm as a foliar application) to mitigate Cd-induced stress in maize (Zea mays L.). Results Exposure to Cd increased oxidative stress markers while significantly decreasing plant biomass, chlorophyll content, and photosynthetic efficiency. By increasing chlorophyll a/b ratios, improving growth performance, and increasing antioxidant enzyme activity, the application of ZnO NPs and BC, both separately and in combination, reduced oxidative damage. Interestingly, compared with Cd-stressed controls, the combination treatment significantly (p < 0.05) decreased Cd accumulation in maize roots, shoots, and grains. When ZnO NPs and BC were combined, they exhibited a synergistic effect that enhanced plant physiological tolerance to heavy metal stress and promoted redox equilibrium. The highest SOD, POD, and CAT values (98.8%, 99%, and 72%) were observed for T4 compared to the control. Conclusion Our findings suggest that BC and ZnO NP applications improve maize crop growth and yield and effectively mitigate the toxic effects of Cd. Cadmium nutrients Zea mays biochar zinc oxide concentration of chlorophyll antioxidant enzymes Figures Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Highlights Biochar and ZnO NPs reduce cadmium levels in soils, will reduce crop contamination, and decrease human health risks. It improves the quality of the soil by including nutrients and increasing beneficial microorganisms. Using biochar and ZnO NPs is an inexpensive and sustainable way to gain greater food production safety. 1. Introduction Soil pollution, particularly with respect to toxic metals like cadmium (Cd), is a global environmental concern. Farming soils are already stuffed with nasties that have fallen there from the atmosphere, from geological substrates, and as a result of human activity (Khan et al, 2016 ; Gaspéri et al., 2018 ). Cd contamination of agricultural soil is mainly a consequence of human activities like the application of sewage waste sludge, fertilizer, pest control aerosols, and fossil fuel combustion (Alengebawy et al., 2021 ). Cd decreases the photosynthetic efficiency, biomass, and yield of plants. Morphological, biological, and molecular levels at which disease hinders plant function (Bayçu et al., 2017 ; Qayyum et al., 2017 ). Through the food chain, Cd toxicity creates many health issues in humans. Therefore, sustainable and eco-friendly remediation strategies are necessary to mitigate cadmium (Cd) accumulation and toxicity in food crops. In recent decades, various physicochemical techniques have been employed to remediate Cd-contaminated soils and mitigate Cd uptake by crops, yet these approaches exhibit notable limitations (Noman et al., 2020; Ahmed et al., 2023 ). Among these, the application of biochar (BC) in heavy metal immobilization has gained attraction as a cost-effective and environmentally sustainable solution (Irshad et al., 2024c; Algethami et al., 2024). It functions by reducing the mobility and bioavailability of Cd in the soil via several physicochemical mechanisms, including precipitation, passivation, adsorption, ion exchange, and complexation (Yu et al., 2024). Previous research has demonstrated that incorporating BC into contaminated soils effectively remediated Cd pollution while enhancing both crop yield and quality (Xu et al., 2014 ). However, recent investigations have highlighted the limitations of the Cd adsorption capacity of BC, suggesting a critical need for advancements to bolster its efficacy in this regard (Yang et al., 2023). Therefore, the efficiency of BC in reducing the toxicity of heavy metals to plants can be enhanced by increasing its surface area and surface functional groups through different techniques, such as impregnation with minerals, combined application with other materials (Zhang et al., 2020). These days, nanotechnology is one of the fastest-growing and most widely applied technologies for remediation of heavy metals from contaminated soil and water media. The agricultural applications of nanomaterials have increased over the past decade because of their smaller size and larger surface area, making them valuable tools for adsorbing toxic metals (Kubra et al., 2021 ; Khan et al., 2022 ). The advantages of the environment for synthesizing ZnO nanoparticles with green materials such as plants and bacterial extracts are immense (Jayabalan et al., 2019 ). In particular, ZnO nanoparticles have received attention as they cause low toxicity and exhibit high biocompatibility, and are easy to bind with others with high binding energy and low cost, and also have a higher ability to degrade pollutants (Ali et al., 2019 ; Hussain et al., 2021 ; Hussain et al., 2024 ). Various studies demonstrated that ZnO NPs are used for the reduction of water and soil pollution and significantly decrease the toxic effects of Cd in maize (Kamali et al., 2021 ; Sana et al., 2024 ). Recently, the modification of BC by mixing with nanoparticles has become a hot area due to better activity, lower toxicity, and more environmentally-friendly properties of this eco-friendly nanocomposite BC (Kamali et al., 2021 ; Ahuja et al., 2022 ; Hussain et al., 2022 ). Thus, with the large area of ionic conversion and porosity, it acts as a brilliant support for metal remediation. Many researchers have also focused on soils treated with modified BC and different NPs that would trap particular contaminants, such as Cd (Xing et al., 2015 ; Yuan et al., 2019 ). A modified BC medium may provide a stable environment for the in-situ production and stabilization of zinc oxide nanoparticles (ZnO NPs), which could work in concert to limit the bioavailability of Cd in the soil-plant system and immobilize it (Saleem et al., 2023 ). The circular economy is further supported by using agricultural leftovers as feedstock for BC production and nanoparticle synthesis, which offers a resource-efficient and nature-based method of sustainable remediation. Furthermore, by boosting antioxidant defense mechanisms, combining BC with ZnO NPs improves plant physiological resistance in addition to providing a workable nanotechnology-based method for lowering Cd toxicity in crops (Rizwan et al., 2017 ; Xu, Liu, Zhao, Li, & Deng, 2014 ). Maize (Zea mays) is a staple food for animals and the most significant source of food for humans. Maize was the first food to be domesticated, and thus, historical. It is also the third most important feed crop in the world, with an estimated global yield of 10.9 billion bushels per year, with much of it estimated to be used for food. The economics of this thickens, calling it also being used in biofuel production (Wan et al., 2020 ; Qian et al., 2022 ; Li et al., 2023 ; Tan et al., 2023 ). Moreover, Cd-contaminated maize also shows similar phenomena that reveal enhanced accumulation of Cd heavy metals (Zulfiqar et al., 2022 ). When grown in heavy metal-rich soils, maize can absorb the toxic metals into its stems, leaves, and kernels. As a result, they can be bioaccumulated by animals of the food chain and can cause significant health problems to humans (Ahmed et al., 2023 ). Specific agricultural soil toxicity includes ROS formation due to the accumulation of Cd in maize shoots, which results in a decrease of chlorophyll content, impeding the biosynthesis of chlorophyll due to reduced yield and growth. Soil and rhizosphere Cd elimination as a means to improve the yield of Zea mays (Ahmed et al., 2023 ; Saleem et al., 2023 ). Research on the combined effectiveness of BC and ZnO NPs in Cd-stressed maize systems is still lacking, despite these encouraging viewpoints (Zhu, Li, & Ge, 2020 ). In this study, we prepared BC and ZnO NPs applications to investigate their efficiency for Cd heavy metal adsorption and mitigation for plant uptake and phytotoxicity. We hypothesized that a synergistic application of BC and ZnO NPs in a composite form would efficiently mitigate Cd stress in maize by enhancing biochemical and physiological functions. The objectives of this study were to (i) appraise the efficacy of BC and ZnO NPs composite in the reduction of Cd uptake and accumulation in maize, (ii) identify the role of BC and ZnO NPs in the management of Cd-induced oxidative stress in maize, and (iii) determine the impact of BC and ZnO NPs applications on enzymatic and non-enzymatic antioxidant defence in maize. The findings of the current study provide valuable insights into alleviating Cd toxicity in maize and may suggest that BC and ZnO NPs combined treatments are a promising strategy for sustainable agriculture in heavy metal-polluted soils. 2. Materials and Methods 2.1. Biochar (BC) synthesis Dried wheat straw was collected from an agricultural field used for BC production. The straw was washed with distilled water to remove any dust and dried for 48 h at 60°C, blasted, agitated, and run through a 0.6 mm mesh filter. In short, wheat stalk biomass was pyrolyzed with an electric furnace with limited oxygen, at a temperature of 400°C for 4h; the produced BC was separated using a 2mm mesh and stored in a plastic flask. These composites were then sieved to 100 mesh and placed into Hoover freezing bags. 2.2. ZnO Nanoparticles Synthesis The leaf extract of Conocarpus (Conocarpus Eractus) plants is used for the synthesis of ZnO NPs and intermediate salts, such as zinc. They are cost-effective, biocompatible, safe, and acceptable in the environment. In this case, chloride was used as a precursor salt in the preparation of nanoparticles from zinc sulphate heptahydrate (ZnSO 4 ·7H 2 O). The synthesis of ZnO NPs was performed by following the method reported in (Jayabalan et al., 2019 ). Table 1 Physicochemical properties of soil used in this study. Characteristics Soil pH 7.7 ± 0.2 Soil texture Clay loam EC (dS m − 1 ) 3.6 ± 0.1 CEC (c mol kg − 1 ) 8 ± 1.6 Total N (g kg − 1 ) 1.10 ± 0.05 Total K (g kg − 1 ) 0.6 ± 0.05 Total P (g kg − 1 ) 0.007 ± 0.0001 2.3. Experimental design A completely randomized experimental design with seven treatments plus a control was undertaken. The soil used in the experimentation was collected from the fields of the Agriculture University, Faisalabad, through a random collection of soil samples for air drying and sieving through a 2mm wire mesh. Soil was spiked with Cd (a toxic heavy metal) at 5 mg kg − 1 . The soil pH in distilled water and the electrical conductivity (EC) of the soil were determined in a solid-liquid ratio (1:2.5 (w/v) using a pH meter and an EC meter, respectively. The concentration of Cd was determined by digesting the soil samples with HNO 3 and HCl and analyzing them via an atomic absorption spectrometer (TAS-900, China). Soil pH was 7.7, and 28 pots were used, and each pot contained 8 kg of soil. The treatment is described in Table 1 . Table 2 Treatments plan. No Treatments CK No treatment T1 Biochar 1% w/w T2 Biochar 2% w/w T3 Biochar 1% w/w + 250 ppm ZnO NPs T4 Biochar 2% w/w + 500 ppm ZnO NPs T5 200 ppm ZnO NPs T6 500 ppm ZnO NPs Diammonium phosphate, urea, and potassium sulphate were applied before crop sowing, as per the required 250-300- 300 kg ha-1. P&K applied basal before sowing, whereas N was applied in two splits, one before sowing and the other after crop establishment. 2.4. Harvesting of the plant Plants were taken, and roots, shoots, and grains were separated. We measured the height of one plant, then counted the number of leaves for each plant. Roots and shoots were washed three times with distilled water at the same time to rid them of any impurity and detritus. To obtain a direct constant weight using a weight balance, the samples were dried in a hot air oven at 65°C. 2.5 Measurement of chlorophyll values and gas exchange parameters All maize plants were completely saturated before harvest, with leaf samples taken for chlorophyll analyses. To ascertain the chlorophyll structure within the maize leaves, pigments were extracted with an acetone solution (covered from light), and chlorophyll a and b were quantified. Transpiration rate, photosynthesis rate, and stomatal conductance were determined in an open system portable infrared gas analyzer (Lichtenthaler, 1987 ). 2.6. Antioxidant enzyme activities and oxidative stress measurement The measurement of antioxidant enzyme activities was examined in maize plant leaves after 70 days of plant growth. Then, samples of plant leaf (0.25g) were normalized in phosphate buffer (0.1 M, pH 7.0) under a nitrogen atmosphere. After that collected mixture was centrifuged at 4500rpm for 30 minutes at 4◦C and the supernatant was collected for further analysis. The catalase enzyme (CAT) activities were checked by the following method (Aebi, 1984 ). And the peroxidase (POD) and superoxide dismutase (SOD) activities were measured by the following method (Zhang, 1992 ). The following methods evaluated the content of oxidative stress parameters malonaldehyde MDA, hydrogen peroxide (H 2 O 2 ), and electrolyte leakage (EL) in the leaf of a maize plant. The MDA content and the H 2 O 2 are measured according to (Heath & Packer, 1968 ). The EL values were calculated by applying (Dionisio-Sese & Tobita, 1998 ) method. 2.7. Cd concentration measurement in plants The maize plants (roots, shoots, and grains) were harvested, dried, and digested in a 2:1 HNO 3 :HClO 4 ratio. An Atomic Absorption Spectrophotometer (AAS) was used to detect Cd. 2.8. Statistical analysis Statistical significance was determined using one-way ANOVA. For the calculation of average and standard deviation, descriptive statistics were used. There were 7 treatments and 4 replicates, 28 for each time point. The least significant difference, or LSD, test was used to compare the treatment means at a significance level of 0.05. 3. Results Characterization of ZnO NPs : 3.1 UV/ vis spectroscopy As the particle size decreased, the optical properties of ZnO nanoparticles increased. The study was performed by UV-visible spectroscopy to investigate the radiation-enhanced optical absorption of ZnO. Change in supernatant color correlates with the absorbance peaks in the range of 200–500 nm, which are characteristic of the ZnO nanoparticles synthesis (ZnO nanoparticles formation) after 24 h of salt addition. (also validated by UV/Vis spectroscopy (Fig. 1 ) and subsequently analyzed by UV/V is spectroscopy (Fig. 1 ). Confirmed the formation of Zinc Oxide nanoparticles with a typical peak in the 350–500 nm range. 3.2. X-ray diffraction (XRD) The X-ray diffractogram is depicted in (Fig. 2 ), where the incident beam of X-ray scattering in multiple directions occurs when the X-ray beam is irradiating the crystal of a dense structure. Six distinct peaks and widths indicate the XRD data of ZnO nanoparticles. The low crystallinity of the material is confirmed by a 220° XRD diffraction pattern with low relative intensity. A XRD displayed peaks observed at 220°, 101°, 110°, and 103° conformation was determined for the ZnO nanoparticles. 3.3. Fourier transform infrared spectroscopy (FTIR) Fourier-transform infrared (FTIR) spectroscopy is a common analytical technique employed to identify specific molecules and materials by characterizing their characteristic vibrational and rotational modes. Its effectiveness as an identification and characterization tool for composites makes it a unique tool in its market. The FTIR spectra of ZnO nanoparticles are shown in Fig. 3 . The FT-IR spectra of synthesized nanoparticles were performed on a Barker Spectrum at the wave number of 500–4000 cm⁻¹ for 87 cycles. The band at 3330 cm − 1 was assigned to the stretching vibration of ZnO. The peaks appearing in ZnO nanoparticle spectra at frequencies are 3330, 2329, 1630, and 600 cm − 1 . The highest absorption is at 3330 cm⁻¹. 3.4 EDX and SEM of ZnO NPs The morphology and particle size of ZnO nanoparticles were analyzed by scanning electron microscopy (SEM). The SEM images in 2a and b confirm that the morphology of ZnO is highly porous and rough (Fig. 5 ). The Topography of the ZnO NPs, SEM images of the samples with EDX data depicting their elemental compositions of ZnO nanoparticles (Figs. 4 & 5 ). 3.5. Growth parameters of the maize plant The results demonstrated that the impacts of ZnO NPs and BC amendments on maize growth and biomass were recorded (Fig. 6 ). ZnO NPs and BC application significantly affected growth parameters. The minimum values of all growth parameters (root and shoot length, root dry weight, and shoot dry weight) were recorded in the control treatment, where no application was made. These growth parameters gradually increased as the concentration of ZnO NPs and BC amendments increased. However, the maximum values of root length were measured at the highest level of ZnO NPs and BC amendments T4 as compared to the control. BC amendments significantly increased the root and shoot dry weight and the root and shoot length. Whereas ZnO NPs further enhanced the growth parameters than their respective control. Maize root and shoot dry weight were enhanced by 75% and 84% at T4, the maximum level of ZnO NPs and BC amendments. 3.6. Measurement of chlorophyll contents and photosynthesis The chlorophyll a and b content and photosynthetic rate in maize plants are shown in Fig. 7 . Chlorophyll a and b maximum increased by 84.8% and 91.7% over the control, respectively, at a high concentration of the amendment T4. The highest values of photosynthetic rate of the maize plants were also observed in the T4 application (Fig. 7 ). All interventions had a positive effect on the photosynthetic rate. The maximum photosynthetic rate (92.5%) of the maize plant was shown at T4 treatments. 3.7. Gas exchange parameter measurements In Fig. 8 , the concentration of stomatal conductance, intracellular CO 2, and transpiration rate is examined. The highest values of stomatal conductance, intracellular CO 2, and transpiration rate were enhanced by 76.5%, 91.9%, and 97.7% at T4, and the lowest values are at the control. Amendments in soils also improved the stomatal conductance and transpiration rate of maize plants. In treatment T4 (500ppm + 2% BC w/w), the rate of gas exchange parameters was increased compared to the control. Transpiration rates significantly increased with both biochar alone and combined treatments of biochar + ZnO. 3.8. Oxidative parameters measurements The effects of the ZnO NPs and BC amendments on MDA, H 2 O 2, and EL contents in maize plants were illustrated in Fig. 9 . The highest values of MDA were recorded by 65.5% at the control, and the lowest was 25.7% at T4. Moreover, the values of H 2 O 2 and EL were also decreased by increasing the ZnO NPs and BC applications. The maximum values of H 2 O 2 and EL were measured at 51.9% and 73.7% were observed, in the control. And minimum values of H 2 O 2 and EL by 13.9% and 28.7% by T4. A negative correlation was recorded between MDA, H 2 O 2, EL, and growth attributes, and a positive correlation was recorded between Cd concentration in various tissues of maize plants. 3.9. Antioxidative enzyme activities In Fig. 10 , the effects of the ZnO NPs and BC amendments on CAT, SOD, and POD contents in maize plants are shown. The CAT, SOD, and POD activities were enhanced in different applications of ZnO NP and BC. The highest values were recorded by 98.8, 99, and 72% shown by T4 over the control. ZnO NPs and BC amendments showed a significant increase in CAT, SOD, and POD in maize plants relative to the control. Overall, the findings showed that combined treatments of ZnO NPs and BC yielded the highest results compared to applications of BC and ZnO NPs alone. 3.10. Values of Cd in the maize plant These findings imply that biochar, zinc oxide nanoparticles, and their mixtures can effectively mitigate Cd toxicity in maize roots, shoots, and grains (Fig. 11 ). Cd had the maximum concentration in the respective control group. Maize plants treated with the highest dose of ZnO nanoparticles and biochar exhibited the lowest Cd concentration. At T4, the concentration of Cd in maize roots decreased by 53.52% compared with the control. Increase in yield response was the same for Cd content (in the Zea mays plants). The uptake of Cd in the maize plant shoot and in grains decreased (by 36.2% and 55.9% respectively) during the use of T4, compared to the control. 4. Discussion Our results depicted that the growth of maize was increased by applying ZnO NPs, and these effects were further increased by the treatments of BC application combined with ZnO NPs. The minimum growth was recorded in the control (Fig. 6 ). Photosynthetic values were reduced due to higher Cd concentrations in the plant, which reduced plant development (Rizwan et al., 2019 ). The treatments of ZnP NPs increased the growth parameters (Fig. 6 ). The foliar application of ZnO-NPs increased the physical properties and nutritional values of grains in plants according to (Kolenčík et al., 2019 ). Previous research demonstrated that co-composting BC increased crop health (Agegnehu & Bird, 2017; Kammann et al., 2015 ). Our findings exposed that the combined applications of BC and ZnO NPs further increased the root and shoot dry weight of maize plants (Fig. 6 ). Simultaneously applying BC with foliar ZnO NPs enhanced the growth of maize plants (Rizwan et al., 2019 ). The maximum level of growth and biomass was recorded at higher treatments of BC and ZnO NPs, and the lowest values were recorded at the control. The maize plant biomass reduced at no treatment might be due to the enhanced values of MDA, H 2 O 2 , and EL in leaves (Fig. 9 ). Previous results showed that BC enhanced the soil structure, nutrient retention, plant height, surface of the leaf, and overall biomass of plants (Liu et al., 2021 ; Silva & Bettiol, 2020). Our research showed that alone and combined applications improved the chlorophyll values in leaves of maize plants under Cd stress by applying both amendments (Fig. 7 ). ZnO-NPs increased the chlorophyll values when used as a foliar treatment (Rizwan et al., 2019 ). ZnO NPs treatments (250 and 500 ppm) and BC (1 and 2% w/w) enhanced the chlorophyll content values. Similarly, photosynthetic rate and gas exchange parameters were also increased in Cd stress by applying soil amendments. Changing the structure of the chloroplast due to the toxicity of Cd may be the reason for decreased photosynthetic pigments in plants (Sharma & Dietz, 2009 ). According to the foliar and priming treatments, ZnO NPs significantly increased the chlorophyll values. Moreover, the rate of photosynthesis was also enhanced by amendments of BC and NPs (Ahmad et al., 2024 ). Biochar and ZnO NP treatments enhanced the rate of photosynthesis by decreasing oxidative stress parameters in maize plants (Fig. 7 ). ZnO NP treatments under heavy metal stress decreased oxidative stress indices in Leucaena leucocephala plants (Venkatachalam et al., 2017 ). The BC application also reduced the oxidative stress parameter in many species of plants (Abbas et al., 2018 ; Ali et al., 2018 ). Previous research demonstrated that the combination of BC contributed to the balance of chlorophyll values and improved the photosynthetic rate under salinity (Chen et al., 2023 ; Helaoui et al., 2023 ). The treatments of ZnO-NPs alone or combined with BC reduced the oxidative stress in plants. The (250 and 500ppm) treatments of ZnO-NPs reduced the MDA (6.4, 14.9%), H 2 O 2 (3.5, 4.3%), and EL (5.2, 17.6%) values as compared to the control. And the 1 and 2% w/w BC application were reduced the (25.7 and 51.9%); and (13.9 and 22.4%); and (28.7 and 47.2%), as compared to control shown in (Fig. 9 ). The concentrations of antioxidant enzymes like CAT enhanced (98.8%); SOD, (99.2%), and POD (72%) at BC 2% w/w + 500ppm ZnO-NPs as compared to control. Previous research predicted that ZnO NP treatments would reduce oxidative stress in plants (Venkatachalam et al., 2017 ). A previous study has explained that ZnO NPs improved the antioxidant system and enhanced the activities of CAT, SOD, and POD (García-López et al., 2019 ; Ulhassan et al., 2022 ). Antioxidant enzymes are a plant defense mechanism against reactive oxygen stress caused by metals. Additionally, a previous study found that adding NPs to soil improved the enzymatic activity of removing reactive oxygen species (ROS) (Rastogi et al., 2017 ; Sytar, Kumari, Yadav, Brestic, & Rastogi, 2019 ). BC is a carbonated organic material that can decrease the presence of heavy metals in soil (Palansooriya et al., 2022 ). BC application, combined with other soil amendments, reduced the accessibility of metals in soil (Yin et al., 2017 ). Therefore, using a combined application could be an effective method for the remediation of Cd from polluted soil. The treatments of 1 and 2% w/w BC reduced the root (19.1 and 25.3%) and shoot (15.1and 20.5%) Cd from Cd-contaminated soil. The combined effects of both BC and ZnO NPs reduced the Cd contents from the soil as compared to the control (recorded in Fig. 11 ). Recent studies have shown that exogenously applied ZnO NPs decreased the values of heavy metals and oxidative enzymes in numerous crops (Salam et al., 2022 ; Sharifan, Ma, Moore, Habib, & Evans, 2019 ). BC can immobilize the toxic materials in the soil due to changes in the pH of the soil and metal speciation (O'Connor et al., 2018 ). The amount of Cd in the roots, shoots, and grain of maize plants was decreased by the application containing BC and ZnP NPs (Fig. 11 ). According to Abbas et al. ( 2018 ), BC lowers the bioavailability of metals in the soil. Furthermore, applying NPs may result in a decrease in bioavailable Cd, as maize plants acquire the highest amounts of Cd due to the increase in plant biomass. On the other hand, maize plants treated with the greatest ZnO NPs had reduced Cd values. Cd levels in maize were lowest at the control, and highest at BC 2% w/w + 500 ppm ZnO NPs (Fig. 11 ). The primary mechanism for the production and accumulation of Cd in grains is the transfer of Cd metals through soil to grains (Liang et al., 2017 ). 5. Conclusion In the current study, BC with ZnO NP application has emerged as a naturally friendly approach for reducing the bioavailability of Cd from polluted soil. The application of BC with ZnO NPs showed a positive response to the development and yields of maize plants and reduced the Cd contents in the root, shoots, and grains of maize. All these treatments increased the antioxidant enzyme activities and reduced the oxidative stress parameters. The increase in plant growth and yields with reduced Cd contents showed the efficacy of BC combined with ZnO NPs in Cd-contaminated soil. The findings of this research indicated a sustainable approach. This is a profitable technique for the remediation of contaminants from soil and a green method for the future. We recommend this as a circular economy and nature-based solution for the future, utilizing industrial byproducts or plant extracts for BC synthesis to reduce dependence on synthetic inputs. Further research is needed on bacterial strain-synthesized NPs for the remediation of heavy metals. Declarations Conflict of interest statement: All the authors have no conflict of interest. Data availability Data that support the findings of this study are available within the article. Fundings The authors received no financial support (funding) for the research, authorship, and publication of this article. References Abbas, T., Rizwan, M., Ali, S., Adrees, M., Zia-ur-Rehman, M., Qayyum, M. F., . . . Murtaza, G. (2018). Effect of biochar on alleviation of cadmium toxicity in wheat (Triticum aestivum L.) grown on Cd-contaminated saline soil. 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Science of the Total Environment, 659 , 473-490. Zhang, X. (1992). The measurement and mechanism of lipid peroxidation and SOD, POD and CAT activities in biological system. Research methodology of crop physiology , 208-211. Zhu, Q., Li, X., & Ge, R.-S. (2020). Toxicological effects of cadmium on mammalian testis. Frontiers in genetics, 11 , 527. Zulfiqar, U., Ayub, A., Hussain, S., Waraich, E. A., El-Esawi, M. A., Ishfaq, M., . . . Maqsood, M. F. (2022). Cadmium toxicity in plants: Recent progress on morpho-physiological effects and remediation strategies. Journal of Soil Science and Plant Nutrition, 22 (1), 212-269. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9008412","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634825436,"identity":"813d8886-ab83-486f-91ed-02218ece4241","order_by":0,"name":"Muhammad 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of the ZnO NPs.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/7ed709a8c4006c4dcb72012a.png"},{"id":109296454,"identity":"9e7e99ef-3a4e-43ab-9f82-2823645c273c","added_by":"auto","created_at":"2026-05-15 08:47:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73614,"visible":true,"origin":"","legend":"\u003cp\u003eZnO NPs FTIR spectrum.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/7dfc9cce70190814f56dc399.png"},{"id":109289236,"identity":"b165ebfa-1610-4afb-8070-b67d60304e4d","added_by":"auto","created_at":"2026-05-15 06:15:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":173409,"visible":true,"origin":"","legend":"\u003cp\u003eEDX of Zn NPs.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/2d642c0af941d503fdf24b6e.png"},{"id":109289230,"identity":"4e43def0-ee52-4222-86b5-b8302a99a1bb","added_by":"auto","created_at":"2026-05-15 06:15:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":278542,"visible":true,"origin":"","legend":"\u003cp\u003eSEM of Zn NPs\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/89dcd44bf964d48e1d367d0d.png"},{"id":109289233,"identity":"ba1c7e0f-e29c-4b62-b50a-dfac3d8f1456","added_by":"auto","created_at":"2026-05-15 06:15:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53760,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent treatments of BC, ZnO nanoparticles, and the combination effects of both on morphological attributes in Cd-contaminated soil. (A) root and (B) shoot length, (C) root length, and (D) shoot length. The bars characterize the standard error for 4 replicates, and the bar letters demonstrate significant differences between different treatments (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/8fe13d0c1e84a39636e8ab73.png"},{"id":109296320,"identity":"b904590e-dbd7-484c-88b8-c126eadbae40","added_by":"auto","created_at":"2026-05-15 08:46:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":60112,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different concentrations of biochar (BC), zinc oxide (ZnO) nanoparticles, and their combination on (A) chlorophyll a, (B) chlorophyll B\u003csub\u003e,\u003c/sub\u003e and (C) Photosynthetic rate, in cadmium-contaminated soil. All of the bars are mean standard error of the four replicates; bars with different letters are significantly different for treatment (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/d8ee90993475876a68359efa.png"},{"id":109289234,"identity":"4cb7932b-e9f1-47d1-9f4f-b38703c78406","added_by":"auto","created_at":"2026-05-15 06:15:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":44729,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different concentrations of biochar (BC), zinc oxide (ZnO) nanoparticles, and their combination on (A) stomatal conductance, (B) Intracellular CO\u003csub\u003e2,\u003c/sub\u003e and (C) transpiration rate in cadmium-contaminated soil. All of the bars are mean standard error of the four replicates; bars with different letters are significantly different for treatment (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/b74a8e04f12d7908fb463dbd.png"},{"id":109296088,"identity":"a4a2db3a-4b0c-46eb-a7a0-89546615374a","added_by":"auto","created_at":"2026-05-15 08:45:25","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":40151,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different applications of BC, ZnO nanoparticles, and BC + ZnO nanoparticles on (A) MDA, (B) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and (C) EL, maize plants in Cd-contaminated soil. The bars characterize the standard error for 4 replicates, and the bar letters demonstrate significant differences between different treatments (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/eba2a6e9ee73bca2474d4b8c.png"},{"id":109296517,"identity":"d805ed84-0110-4381-b60e-f3f24e6cc22f","added_by":"auto","created_at":"2026-05-15 08:47:45","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":44387,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different applications of BC, ZnO nanoparticles, and BC + ZnO nanoparticles on (A) CAT, (B) POD, and (C) SOD, maize plants in Cd-contaminated soil. The bars characterize the standard error for 4 replicates, and the bar letters demonstrate significant differences between different treatments (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/42924fc5c4bd11e06986b914.png"},{"id":109289237,"identity":"ba1f2d95-fe37-4245-a5f5-7d7c56429937","added_by":"auto","created_at":"2026-05-15 06:15:28","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":123092,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different levels of BC, ZnO nanoparticles, and BC + ZnO nanoparticles on (A) Cd in root and (B) Cd in shoot and (C) Cd in grains in maize plant in Cd-contaminated soil. The bars characterize the standard error for 4 replicates, and the letters on the bars demonstrate significant differences between different treatments (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/c1e0faffcfda2ce199b0d342.png"},{"id":109481780,"identity":"349ba9e8-3e4f-4508-8be8-a56ab6af15cf","added_by":"auto","created_at":"2026-05-18 15:19:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1119665,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9008412/v1/d7f81442-ab23-42bb-a041-f40c756dbbe2.pdf"}],"financialInterests":"","formattedTitle":"Integrated soil-foliar strategy using biochar and Zinc Oxide Nanoparticles reduces cadmium bioavailability and improves growth, antioxidant defense, and yield of Maize (Zea mays L.)","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eBiochar and ZnO NPs reduce cadmium levels in soils, will reduce crop contamination, and decrease human health risks.\u003c/li\u003e\n \u003cli\u003eIt improves the quality of the soil by including nutrients and increasing beneficial microorganisms.\u003c/li\u003e\n \u003cli\u003eUsing biochar and ZnO NPs is an inexpensive and sustainable way to gain greater food production safety.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eSoil pollution, particularly with respect to toxic metals like cadmium (Cd), is a global environmental concern. Farming soils are already stuffed with nasties that have fallen there from the atmosphere, from geological substrates, and as a result of human activity (Khan et al, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gasp\u0026eacute;ri et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Cd contamination of agricultural soil is mainly a consequence of human activities like the application of sewage waste sludge, fertilizer, pest control aerosols, and fossil fuel combustion (Alengebawy et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cd decreases the photosynthetic efficiency, biomass, \u0026ensp;and yield of plants. Morphological, biological, and molecular levels at which disease hinders plant function (Bay\u0026ccedil;u et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Qayyum et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Through the food chain, Cd toxicity creates many health issues in humans. Therefore, sustainable and eco-friendly remediation strategies are necessary to mitigate cadmium (Cd) accumulation and toxicity in food crops.\u003c/p\u003e \u003cp\u003eIn recent decades, various physicochemical techniques have been employed to remediate Cd-contaminated soils and mitigate Cd uptake by crops, yet these approaches exhibit notable limitations (Noman et al., 2020; Ahmed et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among these, the application of biochar (BC) in heavy metal immobilization has gained attraction as a cost-effective and environmentally sustainable solution (Irshad et al., 2024c; Algethami et al., 2024). It functions by reducing the mobility and bioavailability of Cd in the soil via several physicochemical mechanisms, including precipitation, passivation, adsorption, ion exchange, and complexation (Yu et al., 2024). Previous research has demonstrated that incorporating BC into contaminated soils effectively remediated Cd pollution while enhancing both crop yield and quality (Xu et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, recent investigations have highlighted the limitations of the Cd adsorption capacity of BC, suggesting a critical need for advancements to bolster its efficacy in this regard (Yang et al., 2023). Therefore, the efficiency of BC in reducing the toxicity of heavy metals to plants can be enhanced by increasing its surface area and surface functional groups through different techniques, such as impregnation with minerals, combined application with other materials (Zhang et al., 2020).\u003c/p\u003e \u003cp\u003eThese days, nanotechnology is one of the fastest-growing and most widely applied technologies for remediation of heavy metals from contaminated soil and water media. The agricultural applications of nanomaterials have increased over the past decade because of their smaller size and larger surface area, making them valuable tools for adsorbing toxic metals (Kubra et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Khan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The advantages of the environment for synthesizing ZnO nanoparticles with green materials such as plants and bacterial extracts are immense (Jayabalan et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In particular, ZnO nanoparticles have received attention as they cause low toxicity and exhibit high biocompatibility, and are easy to bind with others with high binding energy and low cost, and also have a higher ability to degrade pollutants (Ali et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hussain et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hussain et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Various studies demonstrated that ZnO NPs are used for the reduction of water and soil pollution and significantly decrease the toxic effects of Cd in maize (Kamali et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sana et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, the modification of BC by mixing with nanoparticles has become a hot area due to better activity, lower toxicity, and more environmentally-friendly properties of this eco-friendly nanocomposite BC (Kamali et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ahuja et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hussain et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, with the large area of ionic conversion and porosity, it acts as a brilliant support for metal remediation. Many researchers have also focused on soils treated with modified BC and different NPs that would trap particular contaminants, such as Cd (Xing et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A modified BC medium may provide a stable environment for the in-situ production and stabilization of zinc oxide nanoparticles (ZnO NPs), which could work in concert to limit the bioavailability of Cd in the soil-plant system and immobilize it (Saleem et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The circular economy is further supported by using agricultural leftovers as feedstock for BC production and nanoparticle synthesis, which offers a resource-efficient and nature-based method of sustainable remediation. Furthermore, by boosting antioxidant defense mechanisms, combining BC with ZnO NPs improves plant physiological resistance in addition to providing a workable nanotechnology-based method for lowering Cd toxicity in crops (Rizwan et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xu, Liu, Zhao, Li, \u0026amp; Deng, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMaize (Zea mays) is a staple food for animals and the most significant source of food for humans. Maize was the first food to be domesticated, and thus, historical. It is also the third most important feed crop in the world, with an estimated global yield of 10.9\u0026nbsp;billion bushels per year, with much of it estimated to be used for food. The economics of this thickens, calling it also being used in biofuel production (Wan et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Qian et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Tan et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, Cd-contaminated maize also shows similar phenomena that reveal enhanced accumulation of Cd heavy metals (Zulfiqar et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When grown in heavy metal-rich soils, maize can absorb the toxic metals into its stems, leaves, and kernels. As a result, \u0026ensp;they can be bioaccumulated by animals of the food chain and can cause significant health problems to humans (Ahmed et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Specific agricultural soil toxicity includes ROS formation due to the accumulation of Cd in maize shoots, which results in a decrease of chlorophyll content, impeding the biosynthesis of chlorophyll due to reduced yield and growth. Soil and rhizosphere Cd elimination as a means to improve the yield of Zea mays (Ahmed et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Saleem et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Research on the combined effectiveness of BC and ZnO NPs in Cd-stressed maize systems is still lacking, despite these encouraging viewpoints (Zhu, Li, \u0026amp; Ge, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we prepared BC and ZnO NPs applications to investigate their efficiency for Cd heavy metal adsorption and mitigation for plant uptake and phytotoxicity. We hypothesized that a synergistic application of BC and ZnO NPs in a composite form would efficiently mitigate Cd stress in maize by enhancing biochemical and physiological functions. The objectives of this study were to (i) appraise the efficacy of BC and ZnO NPs composite in the reduction of Cd uptake and accumulation in maize, (ii) identify the role of BC and ZnO NPs in the management of Cd-induced oxidative stress in maize, and (iii) determine the impact of BC and ZnO NPs applications on enzymatic and non-enzymatic antioxidant defence in maize. The findings of the current study provide valuable insights into alleviating Cd toxicity in maize and may suggest that BC and ZnO NPs combined treatments are a promising strategy for sustainable agriculture in heavy metal-polluted soils.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.1. Biochar (BC) synthesis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eDried wheat straw was collected from an agricultural field used for BC production. The straw was washed with distilled water to remove any dust and dried for 48 h at 60\u0026deg;C, blasted, agitated, and run through a 0.6 mm mesh filter. In short, wheat stalk biomass was pyrolyzed with an electric furnace with limited oxygen, at a temperature of 400\u0026deg;C for 4h; the produced BC was separated using a 2mm\u0026ensp;mesh and stored in a plastic flask. These composites were then sieved to 100 mesh and placed into Hoover freezing bags.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. ZnO Nanoparticles Synthesis\u003c/h2\u003e \u003cp\u003eThe leaf extract of Conocarpus (Conocarpus Eractus) plants is used for the synthesis of ZnO NPs and intermediate salts, such as zinc. They are cost-effective, biocompatible, safe, and acceptable in the environment. In this case, chloride was used as a precursor salt in the preparation of nanoparticles from zinc sulphate heptahydrate (ZnSO\u003csup\u003e4\u003c/sup\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO). The synthesis of ZnO NPs was performed by following the method reported in (Jayabalan et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of soil used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSoil\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSoil texture\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClay loam\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEC (dS m\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCEC (c\u003c/b\u003e\u003csub\u003e\u003cb\u003emol\u003c/b\u003e\u003c/sub\u003e \u003cb\u003ekg\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal N (g kg\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal K (g kg\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal P (g kg\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental design\u003c/h2\u003e \u003cp\u003eA completely randomized experimental design with seven treatments plus a control was undertaken. The soil used in the experimentation was collected from the fields of the Agriculture University, Faisalabad, through a random collection of soil samples for air drying and sieving through a 2mm wire mesh. Soil was spiked with Cd (a toxic heavy metal) at 5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The soil pH in distilled water and the electrical conductivity (EC)\u0026ensp;of the soil were determined in a solid-liquid ratio (1:2.5 (w/v) using a pH meter and an EC meter, respectively. The concentration of Cd was determined by digesting the soil samples with HNO\u003csub\u003e3\u003c/sub\u003e and HCl and analyzing them via an atomic absorption spectrometer (TAS-900, China). Soil pH was 7.7, and 28 pots were used, and each pot contained 8 kg of soil. The treatment is described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTreatments plan.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo treatment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiochar 1% w/w\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiochar 2% w/w\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiochar 1% w/w\u0026thinsp;+\u0026thinsp;250 ppm ZnO NPs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiochar 2% w/w\u0026thinsp;+\u0026thinsp;500 ppm ZnO NPs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200 ppm ZnO NPs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500 ppm ZnO NPs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDiammonium phosphate, urea, and potassium sulphate were applied before crop sowing, as per the required 250-300- 300 kg ha-1. P\u0026amp;K applied basal before sowing, whereas N was applied in two splits, one before sowing and the other after crop establishment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Harvesting of the plant\u003c/h2\u003e \u003cp\u003ePlants were taken, and roots, \u0026ensp;shoots, and grains were separated. We measured the height of one plant, then counted the number of leaves for each plant. Roots and shoots were washed three times with distilled water at the same time to rid them of any impurity and detritus. To obtain a direct constant weight using a weight balance, the samples were dried in a hot air oven at 65\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.5 Measurement of chlorophyll values and gas exchange parameters\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAll maize plants were completely saturated before harvest, with leaf samples taken for chlorophyll analyses. To ascertain the chlorophyll structure within the maize leaves, pigments were extracted with an acetone solution (covered from light), and chlorophyll a and b were quantified. Transpiration rate, photosynthesis rate, and stomatal conductance were determined in an open system portable infrared gas analyzer (Lichtenthaler, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1987\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Antioxidant enzyme activities and oxidative stress measurement\u003c/h2\u003e \u003cp\u003eThe measurement of antioxidant enzyme activities was examined in maize plant leaves after 70 days of plant growth. Then, samples of plant leaf (0.25g) were normalized in phosphate buffer (0.1 M, pH 7.0) under a nitrogen atmosphere. After that collected mixture was centrifuged at 4500rpm for 30 minutes at 4◦C and the supernatant was collected for further analysis. The catalase enzyme (CAT) activities were checked by the following method (Aebi, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). And the peroxidase (POD) and superoxide dismutase (SOD) activities were measured by the following method (Zhang, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe following methods evaluated the content of oxidative stress parameters malonaldehyde MDA, hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and electrolyte leakage (EL) in the leaf of a maize plant. The MDA content and the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are measured according to (Heath \u0026amp; Packer, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1968\u003c/span\u003e). The EL values were calculated by applying (Dionisio-Sese \u0026amp; Tobita, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Cd concentration measurement in plants\u003c/h2\u003e \u003cp\u003eThe maize plants (roots, shoots, and grains) were harvested, \u0026ensp;dried, and digested in a 2:1 HNO\u003csub\u003e3\u003c/sub\u003e:HClO\u003csub\u003e4\u003c/sub\u003e ratio. An Atomic Absorption Spectrophotometer (AAS) was used to detect Cd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical significance was determined using one-way ANOVA. For the calculation of average and standard deviation, descriptive statistics were used. There were 7 treatments and 4 replicates, 28 for each time point. The least significant difference, or LSD, test was used to compare the treatment means at a significance level of 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of ZnO NPs\u003c/b\u003e:\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 UV/ vis spectroscopy\u003c/h2\u003e \u003cp\u003eAs the particle size decreased, the optical properties of ZnO nanoparticles increased. The study was performed by UV-visible spectroscopy to investigate the radiation-enhanced optical absorption of ZnO. Change in supernatant color correlates with the absorbance peaks in the range of 200\u0026ndash;500 nm, which are characteristic of the ZnO nanoparticles synthesis\u0026ensp;(ZnO nanoparticles formation) after 24 h of salt addition. (also validated by UV/Vis\u0026ensp;spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and subsequently analyzed by UV/V is spectroscopy\u0026ensp;(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Confirmed the formation of Zinc Oxide nanoparticles with a typical peak in the 350\u0026ndash;500 nm range.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. X-ray diffraction (XRD)\u003c/h2\u003e \u003cp\u003eThe X-ray diffractogram is depicted in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), where the incident beam of X-ray scattering in multiple directions occurs when the X-ray beam is irradiating the crystal of a dense structure. Six distinct peaks and widths indicate the XRD data of ZnO nanoparticles. The low crystallinity of the material is confirmed by a 220\u0026deg; XRD diffraction pattern with low relative intensity. A XRD displayed peaks observed at 220\u0026deg;, 101\u0026deg;, 110\u0026deg;, and 103\u0026deg; conformation was determined for the ZnO\u0026ensp;nanoparticles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Fourier transform infrared spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFourier-transform infrared (FTIR) spectroscopy is a common analytical technique employed to identify specific molecules and materials by characterizing their characteristic vibrational and rotational modes. Its effectiveness as an identification and characterization tool for composites makes it a unique tool in its market. The FTIR spectra of ZnO nanoparticles are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe FT-IR spectra of synthesized nanoparticles were performed on a Barker Spectrum at the wave number of\u0026ensp;500\u0026ndash;4000 cm⁻\u0026sup1; for 87 cycles. The band at 3330 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned to the stretching vibration of ZnO. The peaks appearing in ZnO nanoparticle spectra at frequencies are 3330, 2329, 1630, and 600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The highest absorption is at 3330 cm⁻\u0026sup1;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 EDX and SEM of ZnO NPs\u003c/h2\u003e \u003cp\u003eThe morphology and particle size of ZnO nanoparticles were analyzed by scanning electron microscopy (SEM). The SEM images in 2a and b confirm that the morphology of ZnO is highly porous and rough (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The Topography of the ZnO NPs, SEM images of the samples with EDX data depicting their elemental compositions of ZnO nanoparticles (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Growth parameters of the maize plant\u003c/h2\u003e \u003cp\u003eThe results demonstrated that the impacts of ZnO NPs and BC amendments on maize growth and biomass were recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). ZnO NPs and BC application significantly affected growth parameters. The minimum values of all growth parameters (root and shoot length, root dry weight, and shoot dry weight) were recorded in the control treatment, where no application was made. These growth parameters gradually increased as the concentration of ZnO NPs and BC amendments increased. However, the maximum values of root length were measured at the highest level of ZnO NPs and BC amendments T4 as compared to the control. BC amendments significantly increased the root and shoot dry weight and the root and shoot length. Whereas ZnO NPs further enhanced the growth parameters than their respective control. Maize root and shoot dry weight were enhanced by 75% and 84% at T4, the maximum level of ZnO NPs and BC amendments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Measurement of chlorophyll contents and photosynthesis\u003c/h2\u003e \u003cp\u003eThe chlorophyll a and b content and photosynthetic rate in maize plants are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Chlorophyll a and b maximum increased by 84.8% and 91.7% over the control, respectively, at a high concentration of the amendment T4. The highest values of photosynthetic rate of the maize plants were also observed in the T4 application (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). All interventions had a positive effect on the photosynthetic rate. The maximum photosynthetic rate (92.5%) of the maize plant was shown at T4 treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Gas exchange parameter measurements\u003c/h2\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the concentration of stomatal conductance, intracellular CO\u003csub\u003e2,\u003c/sub\u003e and transpiration rate is examined. The highest values of stomatal conductance, intracellular CO\u003csub\u003e2,\u003c/sub\u003e and transpiration rate were enhanced by 76.5%, 91.9%, and 97.7% at T4, and the lowest values are at the control. Amendments in soils also improved the stomatal conductance and transpiration rate of maize plants. In treatment T4 (500ppm\u0026thinsp;+\u0026thinsp;2% BC w/w), the rate of gas exchange parameters was increased compared to the control. Transpiration rates significantly increased with both biochar alone and combined treatments of biochar\u0026thinsp;+\u0026thinsp;ZnO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Oxidative parameters measurements\u003c/h2\u003e \u003cp\u003eThe effects of the ZnO NPs and BC amendments on MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e and EL contents in maize plants were illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. The highest values of MDA were recorded by 65.5% at the control, and the lowest was 25.7% at T4. Moreover, the values of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and EL were also decreased by increasing the ZnO NPs and BC applications. The maximum values of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and EL were measured at 51.9% and 73.7% were observed, in the control. And minimum values of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and EL by 13.9% and 28.7% by T4. A negative correlation was recorded between MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e EL, and growth attributes, and a positive correlation was recorded between Cd concentration in various tissues of maize plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Antioxidative enzyme activities\u003c/h2\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, the effects of the ZnO NPs and BC amendments on CAT, SOD, and POD contents in maize plants are shown. The CAT, SOD, and POD activities were enhanced in different applications of ZnO NP and BC. The highest values were recorded by 98.8, 99, and 72% shown by T4 over the control. ZnO NPs and BC amendments showed a significant increase in CAT, SOD, and POD in maize plants relative to the control. Overall, the findings showed that combined treatments of ZnO NPs and BC yielded the highest results compared to applications of BC and ZnO NPs alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.10. Values of Cd in the maize plant\u003c/h2\u003e \u003cp\u003eThese findings imply that biochar, zinc oxide nanoparticles, and their mixtures can effectively mitigate Cd toxicity in maize roots, shoots, and grains (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Cd had the maximum concentration in the respective control group. Maize plants treated with the highest dose of ZnO nanoparticles and biochar exhibited the lowest Cd concentration. At T4, the concentration of Cd in maize roots decreased by 53.52% compared with the control. Increase in yield response was the same for Cd content (in the Zea mays plants). The uptake of Cd in the maize plant shoot and in grains decreased (by 36.2% and 55.9% respectively) during the use of T4, compared to the control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur results depicted that the growth of maize was increased by applying ZnO NPs, and these effects were further increased by the treatments of BC application combined with ZnO NPs. The minimum growth was recorded in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Photosynthetic values were reduced due to higher Cd concentrations in the plant, which reduced plant development (Rizwan et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The treatments of ZnP NPs increased the growth parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The foliar application of ZnO-NPs increased the physical properties and nutritional values of grains in plants according to (Kolenč\u0026iacute;k et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Previous research demonstrated that co-composting BC increased crop health (Agegnehu \u0026amp; Bird, 2017; Kammann et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Our findings exposed that the combined applications of BC and ZnO NPs further increased the root and shoot dry weight of maize plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Simultaneously applying BC with foliar ZnO NPs enhanced the growth of maize plants (Rizwan et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe maximum level of growth and biomass was recorded at higher treatments of BC and ZnO NPs, and the lowest values were recorded at the control. The maize plant biomass reduced at no treatment might be due to the enhanced values of MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and EL in leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Previous results showed that BC enhanced the soil structure, nutrient retention, plant height, surface of the leaf, and overall biomass of plants (Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Silva \u0026amp; Bettiol, 2020).\u003c/p\u003e \u003cp\u003eOur research showed that alone and combined applications improved the chlorophyll values in leaves of maize plants under Cd stress by applying both amendments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). ZnO-NPs increased the chlorophyll values when used as a foliar treatment (Rizwan et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). ZnO NPs treatments (250 and 500 ppm) and BC (1 and 2% w/w) enhanced the chlorophyll content values. Similarly, photosynthetic rate and gas exchange parameters were also increased in Cd stress by applying soil amendments. Changing the structure of the chloroplast due to the toxicity of Cd may be the reason for decreased photosynthetic pigments in plants (Sharma \u0026amp; Dietz, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). According to the foliar and priming treatments, ZnO NPs significantly increased the chlorophyll values. Moreover, the rate of photosynthesis was also enhanced by amendments of BC and NPs (Ahmad et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Biochar and ZnO NP treatments enhanced the rate of photosynthesis by decreasing oxidative stress parameters in maize plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eZnO NP treatments under heavy metal stress decreased oxidative stress indices in Leucaena leucocephala plants (Venkatachalam et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The BC application also reduced the oxidative stress parameter in many species of plants (Abbas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ali et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Previous research demonstrated that the combination of BC contributed to the balance of chlorophyll values and improved the photosynthetic rate under salinity (Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Helaoui et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe treatments of ZnO-NPs alone or combined with BC reduced the oxidative stress in plants. The (250 and 500ppm) treatments of ZnO-NPs reduced the MDA (6.4, 14.9%), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (3.5, 4.3%), and EL (5.2, 17.6%) values as compared to the control. And the 1 and 2% w/w BC application were reduced the (25.7 and 51.9%); and (13.9 and 22.4%); and (28.7 and 47.2%), as compared to control shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The concentrations of antioxidant enzymes like CAT enhanced (98.8%); SOD, (99.2%), and POD (72%) at BC 2% w/w\u0026thinsp;+\u0026thinsp;500ppm ZnO-NPs as compared to control. Previous research predicted that ZnO NP treatments would reduce oxidative stress in plants (Venkatachalam et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A previous study has explained that ZnO NPs improved the antioxidant system and enhanced the activities of CAT, SOD, and POD (Garc\u0026iacute;a-L\u0026oacute;pez et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ulhassan et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Antioxidant enzymes are a plant defense mechanism against reactive oxygen stress caused by metals. Additionally, a previous study found that adding NPs to soil improved the enzymatic activity of removing reactive oxygen species (ROS) (Rastogi et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sytar, Kumari, Yadav, Brestic, \u0026amp; Rastogi, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBC is a carbonated organic material that can decrease the presence of heavy metals in soil (Palansooriya et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). BC application, combined with other soil amendments, reduced the accessibility of metals in soil (Yin et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, using a combined application could be an effective method for the remediation of Cd from polluted soil. The treatments of 1 and 2% w/w BC reduced the root (19.1 and 25.3%) and shoot (15.1and 20.5%) Cd from Cd-contaminated soil. The combined effects of both BC and ZnO NPs reduced the Cd contents from the soil as compared to the control (recorded in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Recent studies have shown that exogenously applied ZnO NPs decreased the values of heavy metals and oxidative enzymes in numerous crops (Salam et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sharifan, Ma, Moore, Habib, \u0026amp; Evans, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). BC can immobilize the toxic materials in the soil due to changes in the pH of the soil and metal speciation (O'Connor et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe amount of Cd in the roots, shoots, and grain of maize plants was decreased by the application containing BC and ZnP NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). According to Abbas et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), BC lowers the bioavailability of metals in the soil. Furthermore, applying NPs may result in a decrease in bioavailable Cd, as maize plants acquire the highest amounts of Cd due to the increase in plant biomass. On the other hand, maize plants treated with the greatest ZnO NPs had reduced Cd values. Cd levels in maize were lowest at the control, and highest at BC 2% w/w\u0026thinsp;+\u0026thinsp;500 ppm ZnO NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The primary mechanism for the production and accumulation of Cd in grains is the transfer of Cd metals through soil to grains (Liang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn the current study, BC with ZnO NP application has emerged as a naturally friendly approach for reducing the bioavailability of Cd from polluted soil. The application of BC with ZnO NPs showed a positive response to the development and yields of maize plants and reduced the Cd contents in the root, shoots, and grains of maize. All these treatments increased the antioxidant enzyme activities and reduced the oxidative stress parameters. The increase in plant growth and yields with reduced Cd contents showed the efficacy of BC combined with ZnO NPs in Cd-contaminated soil. The findings of this research indicated a sustainable approach. This is a profitable technique for the remediation of contaminants from soil and a green method for the future. We recommend this as a circular economy and nature-based solution for the future, utilizing industrial byproducts or plant extracts for BC synthesis to reduce dependence on synthetic inputs. Further research is needed on bacterial strain-synthesized NPs for the remediation of heavy metals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData that support the findings of this study are available within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no financial support (funding) for the research, authorship, and publication of this article. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbas, T., Rizwan, M., Ali, S., Adrees, M., Zia-ur-Rehman, M., Qayyum, M. F., . . . Murtaza, G. (2018). 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Cadmium toxicity in plants: Recent progress on morpho-physiological effects and remediation strategies. \u003cem\u003eJournal of Soil Science and Plant Nutrition, 22\u003c/em\u003e(1), 212-269.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cadmium, nutrients, Zea mays, biochar, zinc oxide, concentration of chlorophyll, antioxidant enzymes","lastPublishedDoi":"10.21203/rs.3.rs-9008412/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9008412/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eAgricultural soils contaminated with cadmium (Cd) pose a serious threat to food safety and crop yield. Various conventional methods are employed to mitigate toxic effects; however, biochar amendments with ZnO NPs are particularly suitable for Cd remediation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, we examined the ability of biochar (BC) (1% and 2% w/w as a soil amendment) and zinc oxide nanoparticles (ZnO NPs; 250 and 500 ppm as a foliar application) to mitigate Cd-induced stress in maize (Zea mays L.).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eExposure to Cd increased oxidative stress markers while significantly decreasing plant biomass, chlorophyll content, and photosynthetic efficiency. By increasing chlorophyll a/b ratios, improving growth performance, and increasing antioxidant enzyme activity, the application of ZnO NPs and BC, both separately and in combination, reduced oxidative damage. Interestingly, compared with Cd-stressed controls, the combination treatment significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decreased Cd accumulation in maize roots, shoots, and grains. When ZnO NPs and BC were combined, they exhibited a synergistic effect that enhanced plant physiological tolerance to heavy metal stress and promoted redox equilibrium. The highest SOD, POD, and CAT values (98.8%, 99%, and 72%) were observed for T4 compared to the control.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings suggest that BC and ZnO NP applications improve maize crop growth and yield and effectively mitigate the toxic effects of Cd.\u003c/p\u003e","manuscriptTitle":"Integrated soil-foliar strategy using biochar and Zinc Oxide Nanoparticles reduces cadmium bioavailability and improves growth, antioxidant defense, and yield of Maize (Zea mays L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 06:15:22","doi":"10.21203/rs.3.rs-9008412/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c03b9823-c3e9-4373-837b-637b36f55d0f","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject without review","date":"2026-05-18T11:17:47+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-05T13:00:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-01T01:42:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-04-30T16:31:32+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T15:19:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 06:15:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9008412","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9008412","identity":"rs-9008412","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}