Biologically and chemically synthesized ZnO nanoparticles for textile wastewater treatment and phytotoxicity alleviation in Vigna radiata

Abstract

This study reports the development of biologically and chemically synthesized zinc oxide nanoparticles (ZnO-NPs) using a leaf extract of Conocarpus erectus and sodium hydroxide, respectively. The synthesized nanomaterials were characterized to determine their morphology, functional groups, and crystalline nature using SEM, FTIR, and XRD. The performance of ZnO-NPs was evaluated for the photocatalytic treatment of synthetic azo dye solutions and real textile wastewater. Furthermore, they were assessed for the mitigation of phytotoxicity in Vigna radiata. The results demonstrated that a lower catalyst dose of ZnO(B)-NPs showed higher efficiency for decolorizing Congo red as compared to ZnO(C)-NPs. However, dye concentration, light sources (sunlight and UV) and reducing agents had a significant effect on decolorization rates. In actual textile wastewater treatment, ZnO(B)-NPs reduced the pH, EC, TDS, sulfate, phosphate, color intensity, and COD more efficiently than ZnO(C)-NPs, showing enhanced remediation potential. Subsequently, phototoxicity studies revealed significant improvements in seed germination, growth parameters, photosynthetic content, and antioxidative enzymatic activity in Vigna radiata under wastewater stress. In contrast, ZnO(B)-NPs reduced the levels of oxidative stress indicators, such as hydrogen peroxide and malondialdehyde, and increased the activities of superoxide dismutase, catalase, and peroxidase. Multivariate analyses further confirmed the consistent and better performance of ZnO(B)-NPs in wastewater remediation and plant stress alleviation response metrics. Overall, this study suggests that Conocarpus derived ZnO-NPs represent green and sustainable high-performance materials to mitigate textile effluent toxicity and improve crop performance under stress conditions.

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Environmental significance

Dyes are commonly used in the textile industry and pose a critical environmental concern because of their unregulated discharge, which releases persistent, carcinogenic, and toxic compounds into ecosystems. So, the removal of dyes from textile wastewater has become a challenge owing to rising water and soil pollution. Herein, the current study was designed to compare the efficacy of Conocarpus erectus-derived ZnO-NPs with chemically synthesized counterparts for synthetic and real wastewater treatment and to evaluate their potential to mitigate phytotoxicity in Vigna radiata under stress. This research demonstrates the potential use of ZnO-NPs for efficient wastewater treatment and sustainable agricultural production by bridging the gap between wastewater treatment and crop health restoration.

1 Introduction

Water is one of the most important pillars of the environment, required not only for sustaining life on Earth but also for maintaining the balance of ecosystems. Although there are plentiful natural water resources, only a fraction of them are suitable for human consumption. Industrial activities discharge toxic organic and inorganic pollutants, such as heavy metals, dyes, salts, and other compounds, into water bodies. Among chemical industries, the textile sector is considered a major contributor, releasing huge concentrations of dyes and heavy metals into freshwater bodies without any proper treatment.¹ It has been reported that the manufacture of 1 g of fabric requires approximately 200 L of water and 90 g of dyes, discharging 280 000 tons of dye-contaminated water into freshwater annually.^1,2 Textile wastewater contains toxic dyes, heavy metals, surfactants, anions, and other organic compounds such as azo compounds.^1,3 These non-biodegradable pollutants resist conventional treatments and persist in the environment, reducing water quality, impairing photosynthesis, and increasing chemical and biological oxygen demands.⁴ This wastewater degrades agricultural land when used for irrigation, thus necessitating efficient treatment before disposal.

Conventional physicochemical methods for wastewater treatment are costly and energy-intensive, utilize toxic chemicals, and produce secondary byproducts such as sludge.^5,6 Although biological methods use microorganisms,⁷ they are less effective due to the complex nature of microorganisms and the survival challenge. Thus, advanced oxidation processes have emerged as great alternatives for wastewater treatment by producing reactive oxygen species such as superoxide and hydroxyl radicals for organic compound oxidation.^7–10 Different photocatalysts, such as copper,¹¹ iron,⁴ nickel¹² and titanium,¹³ generate active oxygen species under light, which react with dyes and convert them into harmless byproducts.

On the other hand, zinc oxide nanoparticles (ZnO-NPs) are particularly useful in agricultural and environmental applications due to their semiconducting nature,¹⁴ photocatalytic and antibacterial properties and use as a fertilizer.^15–17 Various physicochemical methods, such as mechanochemical,¹⁸ polyol,¹⁹ solvothermal,²⁰ hydrothermal,²¹ and thermal decomposition,²² have been used for the synthesis of ZnO-NPs. Although these are effective methods for preparing nanoparticles with different morphologies, shapes, and ecotoxicities, they require toxic reducing agents and high-energy fabrication. In contrast, biological methods use microorganisms such as Serratia nematodiphila²³, Bacillus foraminis²⁴, Lactococcus lactis²⁵, Cordyceps militaris²⁶, and plants like Ziziphus spina-christi²⁷, Thymbra Spicata²⁸, Capparis spinosa²⁹, Moringa oleifera³⁰, and Allium saralicum³¹ for the fabrication of ZnO-NPs. Microbial-based fabrication of NPs is environmentally friendly, but the synthesis rate is low, making large-scale production difficult. However, plant-based synthesis can overcome the issues of the physicochemical and microbial fabrication of nanoparticles due to a fast production rate and limited use of toxic chemicals and stabilizing agents such as phenolics, flavonoids, and alkaloids⁴ for the synthesis of ZnO-NPs.

Agriculture systems that are being irrigated with textile effluent pose a significant threat to food security. Mung bean, termed Vigna radiata, is a leguminous, nutrient-rich crop, whose yield and production are affected by textile wastewater in countries such as Pakistan. Kothari et al.³² reported a study on the germination and growth of Vigna radiata under heavy metals and dyes stress present in textile wastewater. Genotoxic and cytotoxic contaminants of textile effluent decreased the mitotic index, enhanced chromosomal abnormalities and affected plant health.³³ Previously, different microbial species, including Citrobacter³⁴ and Bacillus,³⁵ have been used to overcome the phytotoxicity of textile effluents. In addition, iron oxide nanoparticles⁴ and titanium oxide nanoparticles³⁶ have also been reported recently. Numerous nanomaterials have been tested for wastewater treatment and promotion of plant growth, however limited data is available regarding the effects of textile wastewater and ZnO-NPs on the germination and growth of mung beans. The present study was designed to synthesize efficient and sustainable nanomaterials for the treatment of textile effluents with better agricultural yield and productivity under wastewater stress. First, the ZnO-NPs were synthesized chemically using NaOH and biologically using Conocarpus erectus leaves for synthetic and actual textile wastewater treatment. Furthermore, this study optimized the threshold dosage of synthesized materials by evaluating their influence on the germination of mung beans. Finally, the optimum dosage of ZnO-NPs was determined, demonstrating their effective application in alleviating wastewater-induced phytotoxicity in Vigna radiata.

2 Materials and methods

2.1 Materials and chemicals

Fresh leaves of Conocarpus erectus were obtained from the botanical garden of Government College University, Faisalabad, Pakistan. All chemicals and reagents, including zinc sulfate heptahydrate, sodium hydroxide, sodium borohydride, acetone, and sulfuric acid, were of analytical grade and purchased from Sigma-Aldrich.

2.1.1 Biosynthesis of ZnO-NPs. For the green synthesis of ZnO-NPs, the fresh leaves of Conocarpus were initially washed with tap water, subsequently with distilled water to remove dust particles, and then dried in an oven at 50 °C. The dried leaves were ground into a fine powder and used for extraction of their extract, following the method described in Batool et al.⁴ After the extract was obtained, 75 mL of 0.1 M solution of ZnSO₄·7H₂O was prepared and mixed with 25 mL of extract with continuous stirring for around 3 hours on a hot plate. This reaction mixture was sonicated for 30 minutes and centrifuged (10 minutes at 7000 rpm) to remove the supernatant. The NP pellet was rinsed with deionized water to remove any surface impurities, dried in an air oven and ground into a fine powder with the aid of a ceramic mortar and pestle. The proposed mechanism behind the Conocarpus based synthesis of NPs is shown in Fig. 1.

Fig. 1 Schematic mechanisms of biosynthesis of ZnO-NPs using the leaf extract of Conocarpus erectus.

2.1.2 Chemical synthesis of ZnO-NPs. For chemical production of ZnO-NPs, a 0.25 M solution of ZnSO₄·7H₂O was prepared (eqn (1)) and slowly hydrolyzed with a 0.25 M solution of NaOH (eqn (2) and (3)) with continuous stirring for around 24 hours. This reaction mixture was sonicated for 30 minutes and centrifuged (10 minutes at 7000 rpm) to remove the supernatant. The NP pellet was rinsed with deionized water to remove any unreacted residuals, dried in an air oven, and ground into fine powder with the aid of a ceramic mortar and pestle.

ZnSO₄·7H₂O → Zn²⁺ + SO₄²⁻ + 7H₂O … (1)

Zn²⁺ + 2NaOH → Zn(OH)₂↓ + 2Na⁺ … (2)

Zn(OH)₂ ^Δ→ ZnO + H₂O … (3)

2.1.3 Characterization of ZnO-NPs. The synthesized ZnO nanomaterials were analyzed using different characterization techniques. An ultraviolet-visible spectrophotometer was used to confirm the synthesis of ZnO-NPs. After confirmation, the synthesized nanomaterials were subjected to scanning electron microscopy (SEM) for estimation of their shape. The determination of functional groups on the surface of ZnO-NPs was confirmed through a Fourier Transform Infrared (FTIR) spectrophotometer. Finally, the size and crystalline nature of the NPs were confirmed by X-ray diffraction (XRD) analysis.

2.2 Application of ZnO-NPs for the treatment of dye-laden synthetic wastewater

The biologically and chemically synthesized ZnO-NPs were used for the decolorization of five dyes, namely methylene blue (MB), Congo red (CR), reactive black-5 (RB-5), malachite green (MG), and reactive red-2 (RR-2), in synthetic water under different conditions. The proposed mechanism for the decolorization of the dye is shown in Fig. 2.

Fig. 2 Schematic mechanisms behind the decolorization of dyes using ZnO-NPs.

2.2.1 Optimization of ZnO-NPs for the decolorization of Congo red dye. The synthesized ZnO-NPs were initially optimized for the removal of Congo red dye. Different concentrations of NPs like 0.5, 1, 1.5, and 2 mg mL⁻¹ were added to a 50 ppm dye solution and placed under sun incubation with an uninoculated control. This experiment was performed in triplicate (n = 3). About 2 mL samples were taken from each reaction mixture and subjected to spectrophotometry for the estimation of the optimum concentration of NPs, after a specific time interval. The % dye decolorization was calculated using the formulae described in eqn (4),

Dye decolorization (%) = (C − S/C) × 100 … (4)

The optimized concentrations of nanomaterials were further used for the treatment of synthetic wastewater under different conditions. However, their experimental details and results have been added to supplementary information (Section S.1–S.2).

2.3 Application of ZnO-NPs for the treatment of actual wastewater

The efficacy of the optimized dosage of ZnO-NPs was additionally estimated for real textile wastewater treatment. The wastewater samples were obtained from the Paharang drain at two locations along Samundri (N-31.398127 and E-73.079553) and Sargodha (N-31.526904 and E-73.118483) roads, Pakistan. The wastewater samples were mixed and filtered initially to separate the suspended particles. After filtration, the filtrate was enriched with CR dye to have considerable dye concentration and color intensity. The dye was also added to ensure the consistent spectrophotometric evaluation of the dye removing potential of the prepared ZnO-NPs while maintaining the other physicochemical properties of textile wastewater. The optimized concentrations of biologically and chemically synthesized ZnO-NPs were added to the dye laden wastewater sample (n = 3) keeping the untreated control sample independent, for wastewater treatment. The reaction solution was shaken for an hour and different parameters, like pH, color intensity, electrical conductivity (EC), Cr-reduction, chemical oxygen demand (COD), total dissolved solids (TDS), phosphate, and sulphate, were estimated before and after the treatment.

2.4 Optimization of ZnO-NPs for the growth of mung beans (Vigna radiata)

After the treatment of textile wastewater, different concentrations (0, 25, 50, 75, and 100 mg L⁻¹) of ZnO-NPs were applied to Vigna radiata for evaluating their threshold level. This experiment was conducted in Petri plates (n = 3). About 13 seeds were added to each Petri plate and treatments were applied on alternative days for 7 days. Different parameters, such as total germination (G%), chlorophyll content, superoxide dismutase (SOD), and hydrogen peroxide (H₂O₂), were considered for the selection of the optimum dosage of biologically and chemically synthesized ZnO-NPs following the protocol described in Batool et al.³⁷ and Shafqat et al.³⁸

2.5 Effect of optimized dosage of ZnO-NPs on germination of Vigna radiata (mung bean) under textile wastewater stress

After evaluating the threshold of ZnO-NPs, the effect of the optimum dose of ZnO-NPs (biologically and chemically synthesized) along with treated and untreated effluent on the seed germination of mung beans was studied. The complete randomized experiment (n = 3) was again conducted in Petri plates in a growth chamber having temperatures between 26 and 28 °C with a 16 h light/8 h dark photoperiod. Around 13 seeds were put into each Petri plate after dipping in D. H₂O and wastewater. All the selected treatments (T) were foliarly applied on intermittent days, and the treatment layout is presented in Table 1. The germinated seeds were counted daily, and the collected data were used to estimate different seed germination parameters, such as time to 50% germination (50% G), germination % (G%), mean emergence time (MET), coefficient of uniformity of emergence (CUE), emergence index (EI), and length using the protocol described in Batool et al.⁴

Table 1 Treatment layout designed for the growth and germination of mung beans (Vigna radiata) under wastewater stress

Treatments	Description
T1	Positive control (D. H2O)
T2	D. H2O + ZnO(C)-NPs
T3	D. H2O + ZnO(B)-NPs
T4	Negative control (wastewater (WW))
T5	WW + ZnO(C)-NPs
T6	WW + ZnO(B)-NPs
T7	Treated wastewater (TWW) using ZnO(C)-NPs
T8	TWW using ZnO(B)-NPs
T9	TWW using ZnO(C)-NPs + ZnO(C)-NPs
T10	TWW using ZnO(B)-NPs + ZnO(B)-NPs

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2.6 Estimation of the effect of the optimized dosage of the synthesized nanomaterials and treated effluent on the growth of Vigna radiata under stress

2.6.1 Experimental details. The pot experiment was conducted in a growth chamber having a temperature of about 26–28 °C and photoperiod of 16 h light and 8 h dark in Government College University, Faisalabad. The seeds (10 seeds per pot) of Vigna radiata were procured from the Ayub Agriculture Research Institute, Pakistan. The sandy loam textured soil was also collected from the research institute having an organic content of 0.87, an EC of 2.59, a potassium content of 159, a phosphorus content of 11.9, and a nitrogen content of 0.71. The experiment was arranged in a complete random design (CRD) with three replicates (The total number of pots = 30) of each treatment. All the designed treatments (Table 1) were applied intermittently for 21 days. After harvesting, the Vigna radiata seedlings were tested for growth (root length (RL), shoot length (SL), root weight (RW) and shoot weight (SW)), photosynthetic (chlorophyll a, b, total chlorophyll, and carotenoids), enzymatic antioxidants (catalase CAT, peroxidase POD, and superoxide dismutase SOD), and oxidative stress (malondialdehyde (MDA) and hydrogen peroxide (H₂O₂)) attributes.

2.7 Statistical analysis

All results were analyzed statistically using Statistix 8.1 software, and graphs were created using GraphPad Prism 8. One-way ANOVA was applied to examine the difference between the treatments and the post hoc test (LSD) was utilized for the comparison of means. The difference between the applied treatments was considered statistically significant at p ≤ 0.05. For multivariant analysis, R-studio (4.2.2) was used to generate heat maps and perform principal component analysis (PCA).

3 Results and discussion

3.1 Synthesis and characterization of biologically and chemically synthesized ZnO-NPs

The Conocarpus and NaOH derived ZnO-NPs were synthesized and characterized for the determination of their structure, crystalline nature, and presence of different functional groups. During synthesis, after the addition of precursor salt solution to the plant extract and NaOH, the reaction mixture color was changed from yellowish to pale brown and transparent to milky, as shown in Fig. S4. This was the first confirmation of the production of ZnO(B)-NPs and ZnO(C)-NPs. The UV-vis spectra of ZnO(B)-NPs and ZnO(C)-NPs showed an absorption peak at 398 nm (Fig. 3A) and 352 nm (Fig. 3B), respectively. In contrast, Ogunyemi et al.³⁹ synthesized ZnO-NPs using leaf extracts of Matricaria chamomilla L., Olea europaea, and Lycopersicon esculentum and observed their peaks at 384, 380, and 386 nm, respectively. They also reported that phytochemicals such as flavonoids, glycosides, and tannins are responsible for the reduction of zinc salt to nanoparticles. The spectrum of FT-IR confirmed the presence of distinct functional groups on the surface of ZnO-NPs. The transmittance peak (Fig. 3C) at 3527.9 cm⁻¹ was due to the presence of the OH group, while the peak at 2925.3 cm⁻¹ was due to the stretching of the C–H bond. Phosphines of C=C and C≡C primary amine groups were observed at 2400.7 and 2150.0 cm⁻¹. The peaks at 1698 and 1151.7 cm⁻¹ were due to the presence of C=O and C–F groups. The alkyl amine and C–O–H bonds were observed at 1104.7 and 1050.2 cm⁻¹. The peak at 1000.8 cm⁻¹ was due to the C–F bond and 935 cm⁻¹ was due to ZnO stretches. On the other hand, the FT-IR spectrum of ZnO(C)-NPs (Fig. 3D) showed peaks at 3361 and 2890 cm⁻¹, which were due to N–H and C–H bonds. The peaks at 2370.2 and 2195 cm⁻¹ confirmed the presence of the C≡N and C≡C bonds. The CH₂ bending was noted at 1430 cm⁻¹. The peaks at 1104 and 805 cm⁻¹ belong to alkyl amide and C–Cl groups. Datta et al.⁴⁰ observed the peaks of ZnO-NPs at 3458, 2451, 2270, 2245, 1643 and 424 cm⁻¹ that were assigned to OH, C≡N, and C≡C groups. The FTIR spectrum confirmed that enzymes present in the extract are responsible for the stability, reduction and capping of nanomaterials. Meanwhile, the XRD pattern of the ZnO-NPs displayed characteristic diffraction peaks and confirmed the hexagonal wurtzite phase of NPs, including prominent reflections at 2θ values of approximately 31.8°, 34.4°, and 36.2° (Fig. 3E and F), indexed to the (100), (002), and (101) planes, respectively. The results of XRD analysis are consistent with the findings of Stan et al.;⁴¹ they also confirmed the hexagonal wurtzite structure of ZnO-NPs. However, the peaks of ZnO(B)-NPs were relatively broader and less intense, indicating a smaller crystallite size and lower crystallinity, which can be attributed to the presence of bio-organic compounds during synthesis. In contrast, the chemically synthesized ZnO-NPs exhibited sharp and well-defined peaks at the same 2θ positions, confirming the formation of a highly crystalline, phase-pure ZnO structure. Finally, the SEM images of ZnO-NPs (Fig. 3G and H) revealed irregular, aggregated particles with a rough and blocky morphology.

Fig. 3 Characterization of biologically (UV-vis (A), FTIR (C), XRD (E), and SEM (G)) and chemically (UV-vis (B), FTIR (D), XRD (F), and SEM (H)) synthesized ZnO-NPs.

3.2 Application of ZnO-NPs for the treatment of synthetic wastewater

3.2.1 Optimization of ZnO-NPs for the decolorization of Congo red dye. The decolorization potential of ZnO(C)-NPs and ZnO(B)-NPs was studied using four different concentrations under solar irradiation of about 4 hours (Fig. 4). The decolorization efficiency of ZnO-NPs increased with time and concentration. It was found that at 0.5 mg mL⁻¹, the efficiency of ZnO(C)-NPs (Fig. 4A) increased from 13.88 ± 0.64% after 1 hour to 36.34 ± 1.24% after 4 hours. The higher concentrations resulted in more decolorization with 87.99 ± 3.06% at 2.0 mg mL⁻¹ after 4 hours of solar incubation. A parallel trend was observed at 1.0 and 1.5 mg mL⁻¹, suggesting that ZnO(C)-NPs require a higher dosage to reach optimal performance. The results for ZnO(C)-NPs are aligned with those of Ahmed et al.⁴² They described that the decolorization potential of ZnO-NPs increased with increasing catalyst concentrations. It might be due to the increasing number of active sites present on the surface of nanoparticles, which generate more photogenerated holes and hydroxyl radicals for the degradation of dyes.⁴³ Conversely, ZnO(B)-NPs exhibited the highest decolorization efficiency at the lowest concentration of 0.5 mg mL⁻¹, achieving 80.41 ± 1.95% after 4 hours (Fig. 4B). Although 1.0, 1.5, and 2.0 mg mL⁻¹ also showed considerable dye removal, their potential remained slightly lower than that of 0.5 mg mL⁻¹. The observed trend might be attributed to the aggregation of nanomaterials as reported by ref. 44, which may reduce the surface area available for the attachment of dye molecules.⁴⁵ It reduces the absorption of photons and the production of reactive oxygen species like superoxide anions and hydroxyl radicals required for the degradation of dyes.^{40,42,43,46,47} These results support the better performance of ZnO(B)-NPs at 0.5 mg mL⁻¹ and for ZnO(C)-NPs, it was 2.0 mg mL⁻¹, respectively.

Fig. 4 Removal of Congo red (CR) dye using multiple concentrations (0.5, 1, 1.5, and 2 mg mL⁻¹) of (A) chemically and (B) biologically prepared ZnO-NPs. Bar graphs represent the decolorization of dyes, while the (C) heat map and (D) PCA demonstrate the multivariant analysis. Error bars represent the standard deviation (n = 3).

Meanwhile, cluster heatmap analysis was carried out to identify the most effective and optimum concentration of ZnO-NPs for decoloring dyes from synthetic wastewater. It (Fig. 4C) shows the clustering of nanoparticles (Y-axis) over distinct time intervals and concentrations (X-axis). In this analysis, dark green color indicates higher activity, and light colors suggest lower decolorization potential. The results confirmed that ZnO(C)-NPs showed maximum decolorization at a concentration of 2.0 mg mL⁻¹ and ZnO(B)-NPs displayed optimal decolorization at 0.5 mg mL⁻¹ especially after 4 hours. Additionally, principal component analysis (PCA) was carried out to evaluate the effect of each concentration of ZnO(B)-NPs and ZnO(C)-NPs on the decolorization of CR (Fig. 4D). In the database, DIM 1 and DIM 2 showed 99.8% contribution (DIM 1 contributed 97.9% and DIM 2 contributed about 1.9%). The analysis revealed that different nanomaterial concentrations significantly affected CR dye decolorization.

3.3 Application of ZnO-NPs for the treatment of actual wastewater

After synthetic wastewater treatment, the biologically and chemically synthesized ZnO-NPs were used to treat the actual wastewater of textile industries (Table 2). The visual representation of wastewater treatment is shown in Fig. 5A. It was observed that untreated wastewater was characterized by high pH (9.01 ± 0.05), EC (4036 ± 7.25), TDS (2650 ± 10), color intensity (0.432 ± 0.01), sulphate (757.1 ± 9.38), phosphate (27.69 ± 0.30), and COD (124.71 ± 1.39) levels. The application of ZnO(B)-NPs improved the quality of wastewater significantly by reducing tested parameters. It lowers the pH of effluent to 7.85 ± 0.06, while ZnO(C)-NPs reduced it to 8.17 ± 0.04, compared to untreated wastewater. The EC of effluent was reduced to 3416 ± 5.29 by ZnO(B)-NPs and 3980 ± 9.01 by ZnO(C)-NPs. Additionally, the remediation of textile wastewater with ZnO(B)-NPs lowered the color intensity, sulfate, phosphate, and COD by 72.58%, 43.54%, 79.37%, and 71.30%, respectively. Conversely, ZnO(C)-NPs reduced the color intensity by 67.28%, sulphate by 15.75%, phosphate by 60.88%, and COD by 46.58%, compared to wastewater. The reduction in color intensity is due to the production of ROS (reactive oxygen species) as discussed earlier, while the decrease in pH, TDS, COD, and EC might be due to the oxidation and photo-transformation of pollutants initiated by the production of oxidizing species after absorbing sunlight at the surface of ZnO-NPs.^4,48 The decrease in phosphate and sulphate contents may be because of the precipitation process, started by solar light.⁴⁹

Table 2 Treatment of real textile wastewater with ZnO(B)-NPs and ZnO(C)-NPs

Treatments	Variables
	pH	Electrical conductivity (µS cm^−1)	Total dissolved solids (mg L^−1)	Color intensity (abs)	Sulphate (mg L^−1)	Phosphate (mg L^−1)	Chemical oxygen demand (mg L^−1)
Control	9.01 ± 0.05	4036 ± 7.25	2650 ± 10	0.434 ± 0.01	757.16 ± 9.38	27.69 ± 0.30	124.71 ± 1.29
ZnO(B)-NPs	7.85 ± 0.06	3416 ± 5.29	1894 ± 3.60	0.119 ± 0.003	427.44 ± 3.93	5.71 ± 0.10	35.785 ± 1.85
% Removal	—	—	28.52	72.58	43.54	79.37	71.30
ZnO(C)-NPs	8.17 ± 0.04	3980 ± 9.01	2090 ± 4	0.142 ± 0.01	637.86 ± 9.44	10.83 ± 0.37	66.619 ± 2.77
% Removal	—	—	21.13	67.28	15.75	60.88	46.58

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Fig. 5 (A) Visual demonstration of untreated and treated textile wastewater using ZnO-NPs. Multivariate analysis, including (B) heat map, (C) PCA of parameters, and (D) PCA of treatments shows the impact of synthesized nanomaterials on wastewater treatment.

During multivariant analysis, the heat map (Fig. 5B) clearly demonstrated the presence of high levels of pollutants in untreated wastewater (control), indicated by the blue color. The biosynthesized ZnO-NPs treated the wastewater more efficiently as compared to ZnO(C)-NPs as shown by the red color (positive relationship). The PCA of parameters (Fig. 5C) showed that most of the parameters were strongly correlated and contributed to DIM-1, which accounted for 92.3% of the total variance. In contrast, DIM-2 accounted for 7.7% of the variance. The PCA of treatments (Fig. 5D) additionally confirmed that ZnO(B)-NPs have superior potential for treating effluent as they were located on the positive side of DIM-1 and ZnO(C)-NPs have moderate potential, as confirmed by intermediate positioning.

3.4 Optimization of ZnO-NPs for the growth of Vigna radiata

After textile wastewater treatment, different concentrations of biologically and chemically prepared ZnO-NPs were used to evaluate their threshold level for the growth of Vigna radiata. It was noticed that the highest germination was seen at 75 mg L⁻¹ and 100 mg L⁻¹ of ZnO(B)-NPs followed by 50 mg L⁻¹ and 25 mg L⁻¹ (Table 3). Conversely, the application of chemically synthesized counterparts showed the maximum germination at 50 and 75 mg L⁻¹. Similarly, the maximum total chlorophyll content was observed at 100 mg L⁻¹ by ZnO(B)-NPs and 50 mg L⁻¹ by ZnO(C)-NPs. Briefly, it was noticed that the chlorophyll content was increased in Vigna radiata with the increase in the concentration of ZnO(B)-NPs, but the application of ZnO(C)-NPs increased the chlorophyll content until 50 mg L⁻¹, and a further increase in NPs induced phytotoxicity. Additionally, the H₂O₂ content was also estimated, and it was observed that minimum oxidative stress was observed at 75 and 50 mg L⁻¹ of ZnO(B)-NPs and 50 mg L⁻¹ of chemically synthesized nanomaterials. The maximum antioxidative stress (SOD) was observed at 100 mg L⁻¹ of ZnO(B)-NPs and 50 mg L⁻¹ of ZnO(C)-NPs. The findings of the study are consistent with the results of Shafqat et al.³⁸ They applied different concentrations of ZnO-NPs on cotton and reported that 100 ppm of biosynthesized nanomaterials was the optimal dosage for the growth of crops. The difference in the optimal dosages of biologically and chemically synthesized nanomaterials might be due to the selection of reducing agents. The toxicity of chemically synthesized NPs at high concentrations may lead to the displacement of magnesium ions in the chlorophyll molecules and inhibition of enzymes, such as aminolaevulinic acid dehydratase (ALAD), leading to chlorosis. Conversely, the higher threshold of ZnO(B)-NPs may be due to the natural capping agents, which may slowly release the zinc ions and prevent the chloroplast membrane from being disrupted by the sudden influx of ions.⁵⁰ The findings of this experiment were further confirmed using multivariate analysis. The heat map in Fig. 6A shows a clear difference in the applied treatments, where ZnO(B)-NPs cluster together at 75 and 100 mg L⁻¹ and show a positive relationship (red color) for all tested parameters. Meanwhile, the highest concentration of ZnO(C)-NPs showed a negative relationship (blue color), affecting physiological parameters and elevating stress. The PCA of the parameters (Fig. 6B) also confirmed this trend, where germination, chlorophyll content, and SOD load followed a positive direction along DIM-1 (81.8% of the total variance), whereas the hydrogen peroxide was negatively correlated, indicating it as a stress indicator. Meanwhile, the PCA of treatments (Fig. 6C) confirmed that the application of ZnO(B)-NPs showed a strong and consistent positive physiological response. Therefore, 100 mg L⁻¹ ZnO(B)-NPs and 50 mg L⁻¹ ZnO(C)-NPs were determined as the optimal dosages for the efficient growth of Vigna radiata.

Table 3 Impact of multiple concentrations of biologically and chemically synthesized ZnO-NPs on growth of Vigna radiata

Parameters	0 mg L^−1	25 mg L^−1		50 mg L^−1		75 mg L^−1		100 mg L^−1
	Distilled water	ZnO(B)-NPs	ZnO(C)-NPs	ZnO(B)-NPs	ZnO(C)-NPs	ZnO(B)-NPs	ZnO(C)-NPs	ZnO(B)-NPs	ZnO(C)-NPs
Total germination	9 ± 3	10 ± 2	9 ± 1	11 ± 1	12 ± 3	13 ± 2	12 ± 3	13 ± 1	9 ± 2
Total chlorophyll (mg g^−1)	2.1 ± 0.006	3.19 ± 0.035	2.96 ± 0.07	3.47 ± 0.05	3.59 ± 0.04	3.81 ± 0.008	2.11 ± 0.01	4.01 ± 0.07	1.37 ± 0.03
Hydrogen peroxide (nmol g^−1 FW)	10.7 ± 0.1	18.77 ± 0.43	15.01 ± 0.09	10.46 ± 0.28	9.11 ± 0.81	6.84 ± 0.73	18.29 ± 0.69	6.80 ± 0.61	27.38 ± 0.29
Superoxide dismutase (mg^−1 protein)	30.0 ± 0.02	29.51 ± 0.06	24.62 ± 0.82	35.32 ± 0.49	31.52 ± 0.21	43.11 ± 0.36	26.39 ± 0.04	47.18 ± 0.18	19.98 ± 0.02

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Fig. 6 Multivariant analysis, including (A) heat map, (B) PCA of parameters, and (C) PCA of treatments showing the impact of multiple concentrations of biologically and chemically prepared ZnO-NPs on the growth and biochemical attributes of Vigna radiata.

3.5 Effect of the optimized dosage of ZnO-NPs on seed germination (SG) of Vigna radiata under wastewater stress

After evaluating the threshold of biologically and chemically synthesized ZnO-NPs, the optimum dosage of each nanomaterial was used to study their effect on the germination of mung beans under wastewater stress (Fig. 7). It was monitored that T3 application decreased the days to 50% G by 65% in contrast to the positive control (distilled water application T1), while the T2 application improved the days to 50% G by 26% (Fig. 7A). This improvement in germination by ZnO-NPs might be due to the fact that zinc is a vital component of seed metabolism, especially in the activation of hydrolytic enzymes like alpha-amylase, which may increase the mobilization of seed reserves and fast prominence of the radicle.⁵¹ The wastewater (T4) application significantly enhanced the time of 50% G as compared to the positive control (T1), which might be due to the presence of dyes, heavy metals, and other organic pollutants that induce phytotoxicity. However, the synthesized ZnO-NPs with different treatments reduced the time of 50% G under effluent stress. The applications of T6, T8, and T10 decreased in the days to 50% G by 54%, 58%, and 77% as compared to the wastewater (T4) application. Meanwhile, the applications of T5, T7, and T9 decreased the effect of effluent by decreasing the days to 50% G by 48%, 56%, and 61%. The G% was also calculated and it was observed that maximum germination appeared in the application of T3, and the minimum germination was noticed in the wastewater (negative control T4) application (Fig. 7B). However, T5–T10 applications significantly decreased their impact by increasing the germination %. In the case of mean emergence time, the application of T2 increased it by 14% but the ZnO(B)-NPs (T3) decreased the MET by 27.4% in comparison to the positive control (T1) (Fig. 7C). The application of ZnO-NPs (T5-T10) decreased the impact of effluent and significantly reduced the MET as compared to T4. Meanwhile, the emergence index was also calculated (Fig. 7D), and it was noticed that the highest EI was observed in the application of T3, and the least EI was observed in the application of wastewater. The T5–T10 applications lowered the wastewater (T4) impact by increasing EI, especially the application of green synthesized NPs outperformed its chemically synthesized counterpart by showing a high EI. This improvement in the observed parameters under stress may be due to natural phytochemical capping, which improves the stability of nanoparticles, controls the release of Zn²⁺ as mentioned by Singh et al.,⁵² and strengthens the antioxidant defense mechanisms, thereby reducing oxidative damage and promoting uniform emergence.⁵³ Similar results were observed in the case of CUE (Fig. 7E) and length (Fig. 7F).⁵⁴ The observed results were also confirmed by multivariant analysis. According to the PCA of the variables (Fig. 7G), all the observed parameters were clustered together and pointed towards the right side, showing a positive correlation between them. Meanwhile, the PCA of treatments (Fig. 7H) showed that the control and treatments labelled as T3, T8, T9, and T10 are clustered together and show prominent results. While T4 was positioned far to the left, it confirmed the negative impact of wastewater on the germination of Vigna radiata. From the heat map (Fig. 7I), it was clearly visualized that the application of T3 and T10 showed the most prominent results, as indicated by the red color (positive relationship). Conversely, wastewater induced phytotoxicity and showed a negative relationship (blue). Meanwhile, the application of T5 to T9 attempted to reduce the phytotoxicity of wastewater, as shown by the pink to dark red colors.

Fig. 7 Effect of different treatments on the (A–F) seed germination attributes of Vigna radiata under wastewater stress. Bar graphs represent the different germination parameters, while (G) PCA of parameters, (H) PCA of treatments, and the (I) heatmap demonstrate the multivariate relationships among the treatments. Error bars represent the standard deviation (n = 3) and distinct letters show significant differences among treatments.

3.6 Estimation of the effect of the optimized dosage of the synthesized nanomaterials and treated effluent on the growth of Vigna radiata under stress

After evaluating the impact of the synthesized nanomaterials and treated wastewater on germination, they were again foliarly applied to Vigna radiata grown in soil medium under the stress of textile wastewater (Fig. 8Q). During the analysis of growth attributes, it was noticed that the application of ZnO(B)-NPs (T3) improved the RL (Fig. 8A), SL (Fig. 8B), RW (Fig. 8C), SW (Fig. 8D) by 16.8%, 14.5%, 23.09%, and 28.98%, as compared to the application of distilled water (T1), whereas chemically synthesized ZnO-NPs increased them by 8.94%, 4.6%, 22.87%, and 7.39%, respectively. The wastewater (T4) application notably decreased the RL, SL, RW, and SW by 49%, 26.0%, 65.33%, and 73.1%, compared to the positive control (T1). However, T5 and T6 increased the RL by 31.7% and 37.3%, SL by 10.0% and 18.0%, RW by 52.2% and 54.9%, and SW by 44.4% and 65.2% as compared to the application of the negative control (wastewater T4). The T7 and T8 applications further reduced the impact of wastewater by improving RL by 41.9% and 46.7%, SL by 21.7% and 25.2%, RW by 60% and 63.3%, and SW by 60.6% and 69.3%. The applications of T9 and T10 improved the RL by 45% and 52%, SL by 26% and 29%, RW by 67% and 73%, and SW by 68% and 75%, respectively, as compared to the negative control (T4). Different studies have reported the impact of ZnO-NPs on enhancing crop growth under different stress conditions.^55–58 Mainly, zinc is an essential micronutrient, which is required for the synthesis and regulation of auxin.^57,58 The sufficient availability of Zn may increase the division and elongation of cells, which improves shoot and root development, even under stress conditions.⁵⁹

Fig. 8 Effect of different treatments on the (A–D) physiological and (E–M) biochemical parameters of Vigna radiata under wastewater stress. Bar graphs represent physiological and biochemical parameters, while (N) PCA of parameters, (O) PCA of treatments, and the (P) heatmap demonstrate the multivariate analysis. Error bars represent the standard deviation (n = 3) and distinct letters show significant differences among treatments. (Q) Visual growth response of Vigna radiata under different treatments.

For photosynthetic content (Chl a, b, total Chl, and carotenoid), it was noted that ZnO(B)-NPs increased the Chl a (Fig. 8E), b (Fig. 8F), total Chl (Fig. 8G), and carotenoid (Fig. 8H) by 1.89%, 36.79%, 22.42%, and 9.75% relative to the application of distilled water (positive control). The application of wastewater reduced the photosynthetic content by reducing total Chl and carotenoid by 56% in comparison to the application of distilled water (T1). However, T5 and T6 decreased the impact of wastewater by enhancing Chl a by 24.5% and 35.17%, Chl b by 66.07% and 64.03%, total Chl by 48% and 50.03%, and carotenoid content by 31.91% and 36%, respectively. Similarly, T7 and T8 also lowered the phytotoxicity of textile effluent by increasing chlorophyll and carotenoid content in comparison to the wastewater application (negative control). Meanwhile, nanomaterial application along with treated wastewater (T9 and T10) further decreased the impact by increasing Chl content by 60% and 63% and carotenoids by 33.3% and 60.49%, respectively. According to García-López et al.,⁶⁰ ZnO-NPs increase the photosynthetic content in ionic form; it is the cofactor of several enzymes, mainly isomerase, ligase, transferase and hydrolase, which enhance the cellular performance when available in sufficient amounts. Meanwhile, zinc is an important ion for osmoregulation, water relations, and mineral uptake.^61,62 It can reduce ionic phytotoxicity, which ultimately increases the gaseous exchange in the affected plants.⁶³

During the estimation of antioxidative attributes, it was noted that T3 application enhanced the CAT (Fig. 8I), SOD (Fig. 8J), and POD (Fig. 8K) by 0.52%, 16.6%, and 28.7% as compared to the application of distilled water (T1). Conversely, the application of wastewater significantly decreased CAT, SOD, and POD by 51.9%, 66.3%, and 71.14%, as compared to the positive control (T1). It might be due to the production and accumulation of reactive oxygen species, which is responsible for the impairment of the antioxidative defense system.^64,65 The applications of T5 and T6 decreased the effect of wastewater by enhancing CAT, SOD, and POD by 21.3%, 57.8%, and 46.05%, respectively, compared to the application of wastewater. Additionally, T7 and T8 applications further decreased the impact of wastewater by increasing antioxidative stress parameters. Meanwhile, the application of T9 and T10 enhanced the CAT by 44% and 48.3%, SOD by 63.6% and 67%, and POD by 68.1% and 77%, respectively, under effluent stress. This improvement in the antioxidative stress system might be because ZnO-NPs may scavenge the ROS and help maintain cellular redox balance. Briefly, the improvement in SOD activity may facilitate the conversion of superoxide radicals into H₂O₂. In contrast, CAT and POD activities may further neutralize H₂O₂ and prevent oxidative damage in plants.^60,63

The impact of different treatments on oxidative stress parameters in Vigna radiata under wastewater stress was also investigated. The application of T3 decreased H₂O₂ (Fig. 8L) by 44.5% and MDA (Fig. 8M) by 83% compared to distilled water application (T1), whereas chemically synthesized counterparts decreased H₂O₂ by 23.7% and MDA by 74%. Conversely, the application of wastewater increased H₂O₂ and MDA levels by 53% and 67%, respectively, compared to the positive control (T1). This may be due to the production of ROS, which damages the cell membrane and increases lipid peroxidation.^37,66 The application of T5 and T6 reduced the impact of effluent by reducing H₂O₂ by 13% and MDA by 51.9% and 62.3% relative to the negative control (T4). The treatments T7 and T8 further reduced the impact of effluent; however, the application of T9 and T10 decreased the H₂O₂ content by 62% and 70.2% and MDA content by 74.4% and 79%, respectively. The findings of the whole study are compared with those of other studies and summarized in Table S2. Meanwhile, these findings are consistent with those of Haidri et al.,⁶⁶ who confirmed that ZnO-NPs have the potential to reduce the impact of wastewater by decreasing oxidative stress attributes. The decrease in H₂O₂ and MDA levels with the application of nanoparticles might be due to the detoxification of ROS, which inhibits the production of hydrogen peroxide and consequently inhibits lipid peroxidation. Meanwhile, the decline in oxidative stress attributes might be due to the application of ZnO-NPs, which stabilize the membrane integrity and reduce oxidative damage. This study confirmed the potential of nanomaterials in strengthening the intrinsic plant defense mechanisms against wastewater stress.

During multivariate analysis, the PCA of parameters (Fig. 8N) showed that the growth, photosynthetic, antioxidant, and oxidative attributes had a positive relationship along the first principal component, where DIM-1 contributed approximately 91.2% and DIM-2 approximately 2.9% of the total variance. The PCA of the treatment (Fig. 8O) further confirmed the results, where the application of ZnO(B)-NPs and ZnO(C)-NPs clustered on the positive side towards Dim-1 and were clearly separated from the wastewater. The heat map (Fig. 8P) clearly shows that the application of ZnO-NPs improved the growth of Vigna radiata, as indicated by the red color (positive relationship), by reducing the oxidative stress attributes. The wastewater application inhibits the growth of plants by inducing phytotoxicity (blue color-negative relationship). Overall, it was observed that biosynthesized ZnO-NPs had greater potential to reduce the phytotoxicity of textile effluent than chemically synthesized ZnO-NPs.

4 Conclusion

This work focuses on the comparative analysis of biologically (Conocarpus erectus) and chemically prepared (NaOH) ZnO-NPs for the nano-remediation of textile wastewater and reducing its phytotoxicity against Vigna radiata. The optimum dosage of biosynthesized ZnO-NPs (0.5 mg mL⁻¹) showed more potential and served as an environmentally friendly alternative for remediating synthetic and actual wastewater. They reduced the color intensity, sulfate, phosphate, and COD by 72.58%, 43.54%, 79.37%, and 71.30% as compared to their chemically synthesized counterparts (2 mg mL⁻¹), which reduced the color intensity by 67.28%, sulphate by 15.75%, phosphate by 60.88%, and COD by 46.58%. Furthermore, the optimal dosages of ZnO(C) (100 mg L⁻¹) and ZnO(B) (50 mg L⁻¹) NPs showed a promising bio-stimulant effect under controlled conditions, as they not only increased the germination of Vigna radiata but also enhanced its growth by bolstering photosynthetic efficiency (Chl content by 60% and 63% and carotenoids by 33.3% and 60.49%) and antioxidative defense mechanisms (CAT by 44% and 48.3%, SOD by 63.6% and 67%, and POD by 68.1% and 77%) against oxidative damage imposed by wastewater irrigation (increased H₂O₂ by 53% and MDA by 67%). Therefore, this study highlights the application of ZnO-NPs for wastewater treatment and for improving plant health under wastewater stress.

Consent for publication

All authors consent to the publication of this original article.

Author contributions

Fatima Batool: writing – original draft, methodology, validation, and formal analysis; Muhammad Zubair: methodology, writing – review and editing, and visualization; Faisal Mahmood: methodology, writing – review and editing, and visualization; Muhammad Shahid: methodology, formal analysis, and writing – review and editing; Tanvir Shahzad: methodology, data curation, and visualization; Weitao Liu: data curation, formal analysis, and validation; Aman Ullah: investigation, methodology, validation, and supervision; Sabir Hussain: conceptualization, resources, investigation, methodology, funding acquisition, and supervision.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: additional materials and methods, experimental results, figures (Fig. S1–S4), and tables (Tables S1 and S2) related to nanoparticle characterization, dye decolorization experiments, wastewater treatment, and comparative analyses. See DOI: https://doi.org/10.1039/d6va00120c.

Acknowledgements

The authors extend their appreciation to the HEC Pakistan for funds under project number 8206/Punjab/NRPU/R&D/HEC/2017 by the Higher Education Commission of Pakistan, Government College University Faisalabad, and the University of Alberta for providing laboratory facilities.

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