Humic Acid Alleviates the Toxicity of Nanoplastics towards Solanum lycopersicum

Abstract

Nanoplastics (NPs) are emerging pollutants that contaminate agricultural produce. The present study investigates the impact of polystyrene (PS) and humic acid (HA) individually and in combination on the germination and growth of seeds of Solanum lycopersicum (tomato). Here we report the formation of eco-corona upon the interaction of PS with humic acid at 24 h with a significant increase in hydrodynamic size. Seed germination, plant growth, and chlorophyll content increased in the coronated PS. In addition, we report that the treatment of seeds with PS + HA resulted in the germination of 90% of seeds, while treatment with only PS resulted in the germination of only 65.8% of seeds. A quantitative analysis of chlorophyll (a, b, and a + b) revealed that HA-treated groups and PS + HA-treated groups showed significantly high chlorophyll (a, b, and a + b) contents of (PS: 3.48 mg g⁻¹, 2.12 mg g⁻¹, and 4.19 mg g⁻¹, HA: 5.76 mg g⁻¹, 3.88 mg g⁻¹, and 6.41 mg g⁻¹, PS + HA: 4.17 mg g⁻¹, 3.23 mg g⁻¹, and 6.58 mg g⁻¹)respectively compared to PS treated groups. Similarly, ROS levels were comparatively low in HA and PS + HA-treated groups than in only-PS-treated groups. Furthermore, we observed a decline in the level of antioxidant enzyme (superoxide dismutase and catalase) activity in HA and PS + HA treated groups than that in only-PS treated groups. The results indicate that HA significantly reduces PS-induced toxicity and improves germination and growth of seeds of Solanum lycopersicum; the corresponding reduction in toxic effects may be due to eco-corona formation on the PS. We understand that eco-corona is a way to protect plants from xenobiotics concerning nanoplastics.

1. Introduction

Nanoplastics (NPs) have been identified as one of the major environmental threats [1] and plastic waste has become one of the most serious environmental issues [2]. Plastic is extensively used in agriculture, manufacturing, construction, and other areas, and human activities are responsible for the terrestrial contamination of nanoplastics [3]. As a result, global plastic usage was reported to have exceeded 311 Tg (million metric tonnes) in 2014, and it is expanding at a rate of 20 Tg per year [4]. Plastics are xenobiotic substances that are rapidly accumulating in the environment [5]. Initially, general littering, dumping, and improper management of landfills were mainly focused on as the major source of terrestrial contamination of nanoplastics [6]. However, over the last few years, agricultural tools and techniques have become a growing environmental concern, and agricultural practices like the use of greenhouse covers, water pipes, and disposal of plastic mulch are the extended sources of nanoplastics contamination in agricultural fields [7]. Furthermore, the use of sludge resulting from wastewater treatment plants as fertilizers on agricultural soil has now become a common practice [8]. Wastewater treatment plants efficiently remove nanoplastics and concentrate them in the sludge, which could be a potent source of nanoplastics contamination in agricultural soils [9]. Polyethylene (PE), polypropylene (PP), polyester (PE), polystyrene (PS), polyurethane (PU), polyvinylchloride (PVC), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), acrylics, and polycarbonates (PC) are some of the most frequently used synthetic plastics [10]. Microplastics and nanoplastics (MNPs) have been reported to have a greater impact on both marine [11] and terrestrial ecosystems [12]. Nanoplastics, post-deposition on the soil surface, are incorporated into the soil via several routes, which also include biological activity [13]. Moreover, since the accurate decomposition rate of plastics in the soil is still unclear, it is only scientifically reasonable to assume that these plastics remain persistent and tend to accumulate in the soil [14]. This paved the way for researchers to study the impact of NPs contamination on terrestrial ecosystems [15]. Initial results of the studies have reported the adverse effect of NPs on the soil biota, including earthworms and alterations in the biophysical properties of the soil like water holding capacity, soil aggregation, and bulk density [16]. In this work, we intended to study the impact of NPs on Solanum lycopersicum, commonly known as a tomato plant, an edible berry, a high-value vegetable crop extensively grown during summer and spring seasons in India with an average productivity of around 17.7 t/ha [17]. Nanoplastics can be consumed by soil organisms or act as transporters for a variety of other toxins in the environment, and their long-term presence in the soil can change soil characteristics [18].

Humic acid lightens the soil, enabling easier water absorption and root development. When sprayed on a soil surface, it provides an organic supply and improves water stagnation capacity [19] and seed germination, water retainment, drought tolerance, and nutrient uptake [20]. Beneficial microorganisms act by dissolving nutrients in the soil and are nourished by humic acid [21]. Humic acid’s main benefit is that it reduces the influence of chemical fertilizers [22]. It carries a net negative charge and also has a massive molecular size. Because of these properties, humic acids can bind to soil particles, allowing microbes and healthy root development to thrive [23].

When microplastics come into contact with organic biomolecules, they compete for attachment to the microplastic’s hydrophobic surfaces, resulting in the formation of eco-corona (EC) [24]. An EC is formed when nanoplastic enters an environmental medium and biomolecules such as proteins are adsorbed on the particle surface. These biomolecules are mostly by-products of agricultural organism’s metabolic activities (e.g., Extracellular Polymeric Substances (EPS)) [25]. There is a lot of research on eco-corona formation over plastic particles [26]; in a few studies, scientists found that humic acid could improve plant growth of Solanum lycopersicum [27,28]. However, it is unclear how eco-corona attenuates PS-induced toxic effects in plants upon interaction with humic acid. Moreover, the underlying mechanism of humic acid-mediated attenuation of PS-induced toxic effects remains an enigma.

Based on a critical examination of previous studies with nanoplastics, it is clear that the effects of nanoplastics on tomato plants are not well studied, and the role of reactive oxygen species generation in toxicity is not studied adequately. In the literature, only a few studies have been conducted on eco-corona formation over plastic particles, but it has not been tested whether it can modify the toxic effects on tomato seeds and plants. A major goal of this study was to investigate the toxic effects of PS, HA, and PS + HA on tomato seeds to plants Solanum lycopersicum, and to assess how the formation of an eco-corona over the particles may mitigate the impacts. In the experiment, we measured seed germination, root and shoot length, microscopical images (seeds and plants), chlorophyll estimation, reactive oxygen species generation, and antioxidant enzyme activity after interactions with PS, HA, and eco-coronated PS + HA with Solanum lycopersicum.

2. Materials and Methods

2.1. Seed Collection

The Solanum lycopersicum seed used in this study was collected from the VIT School of Agriculture and Advanced Learning (VAIAL), VIT University, Vellore, Tamil Nadu, India. Seeds were soaked in distilled water for 10–15 min. The seed selection process was done based on a float/sink assay. Seeds that floated on the surface were discarded and those that sunk at the bottom were selected for further studies. Selected seeds were cleaned with distilled water followed by immersing in 80% ethanol for 2 min at room temperature. After rinsing with ethanol, they were again rinsed with deionized water 5 times and immersed with 60% ethanol, and then again rinsed with deionized water. Seeds were thus surface sterilized to avoid fungal infection. All the experiments were carried out in triplicate.

2.2. Experimental Design

Polystyrene nanoplastics (PSNPs—100 nm size) were obtained from Corpuscular Inc., New York, NY, USA, and Humic acid (HA) from an organic source was obtained from Sigma Aldrich, India. The experimental concentration of PSNPs used was 5 mg/L prepared from a stock solution of 25,000 mg/L and humic acid (HA) used was 5 mg/L. To study the interaction of nanoplastics and humic acid, both were taken in a ratio of 1:1. Around 6 sterile Solanum lycopersicum seeds were placed on Whatman filter paper (125 mm) in a sterile glass Petri dish (diameter, 100 × 15 mm). Treatment solution containing polystyrene with and without humic acid was added at the top and the bottom of the filter paper (each in a separate plate in triplicates) using the hydroponic method. The lids were closed, and the plates were kept at room temperature with a relative humidity of 50%. The experimental set-up was left undisturbed until the seeds germinated. The experiment was carried out for a period of one to two weeks with regular replenishment of treatment solution (by adding superficially) twice a day. Deionized water served as a control.

2.3. Characterization Study

2.3.1. Particle Size Determination

The hydrodynamic size of the particle was analyzed using the dynamic light scattering (DLS) method, a diode-pumped frequency double laser at 532 nm, (10 mW) light scattering at an angle of 173° were used to estimate the particle size of the PS, and PS + HA. The (SZ-100 series software) provided by the manufacturer was used to collect and analyze the data. Similarly, the zeta potential (Z—potential) of the PS and PS + HA, the electrophoretic mobility (cm²/V-s) of the particles was converted to zeta potential (millivolts-mV) and assessed using the (SZ-100 series software) provided, both DLS and Z—the potential was analyzed using nanoparticle analyzer (Horiba, Tokyo, Japan).

2.3.2. Transmission Electron Microscopy

The size and shape of the PS, HA, and PS + HA were determined using transmission electron microscopy. Samples (PS, HA, and PS + HA) dispersed in water were placed on a copper grid with a carbon coating, and one section of the suspension was imaged using Transmission electron microscopy (TEM; JEOL 1010, JEOL Ltd., Tokyo, Japan—HRTEM).

2.3.3. FTIR—Fourier Transform Infrared Spectroscopy Analysis

Samples (PS, HA, and PS + HA) dispersed in water were subjected to FTIR analysis for the validation of n-characteristic functional groups and were examined using an FTIR-6800, (Jasco, Tokyo, Japan) at a specific resolution (scan) of 4 cm⁻¹. The analysis was performed in the spectral range between 4000 and 400 cm⁻¹.

2.3.4. XRD—X-ray Diffraction Analysis

X-ray diffraction analysis was performed to evaluate the structure of crystal structure of the samples. Bruker, Karlsruhe, Germany) source 2.2-kilowatt Cu-anode ceramics tube was used to evaluate the samples (PS, HA, and PS + HA) dispersed in water. A Lynx Optic Detection System (Silicon Strip Detection Technique) with a Reflectance Detector (for low-angle detection) was used for the instrument.

2.4. Seeds and Plants Growth

Solanum lycopersicum seeds grown using the hydroponic method in Petri dishes were assessed for different growth parameters, such as seed germination, root length, and shoot length. The surface of the seeds, root, and shoot was observed under the optical microscope (Leica DM-2500, Wetzlar, Germany) at 40× magnification.

2.5. Plant Physiology

Photosynthetic Pigment Measurement

The role of chlorophyll in photosynthesis is to absorb light; there are two forms of chlorophyll: a and b. The chlorophyll-a is an electron donor, while chlorophyll-b permits organisms to absorb more blue light for photosynthesis. Around 0.2 g of leaves that germinated from tomato seeds were finely cut and taken in sterile glass tubes separately. About 2.5 mL of 80% acetone, along with 0.03125 g of softly crushed magnesium carbonate powder was added to each tube. The leaves were gently ground using a mortar and pestle. The samples were then incubated for 3 h at 4 °C. After incubation, all the samples were centrifuged at 2500× g rpm for 5 min. The aqueous phase (supernatant) was transferred to a sterile glass tube, which was then filled to 2 mL with 80% acetone and used for chlorophyll quantification. The absorbance of the solutions was measured by UV-visible spectrophotometer (Hitachi, U-2910, Tokyo, Japan) at 645 nm and 663 nm (λmax for chlorophyll a and b respectively) using acetone solution of 80% as blank. The chlorophyll concentration was estimated by averaging the results of three measurements. The following equations were used to compute the quantities of chlorophyll a, b, and a + b [29].

$$\mathrm{chlorophyll} a (\mathrm{mg}/\mathrm{g} \mathrm{tissue})=\frac{12.7\left(\mathrm{A}663\right)-2.695\left(\mathrm{A}645\right)\times \mathrm{V}}{1000\times \mathrm{W}}$$

$$\mathrm{chlorophyll} b (\mathrm{mg}/\mathrm{g} \mathrm{tissue})=\frac{22.9\left(\mathrm{A}645\right)-4.68\left(\mathrm{A}663\right)\times \mathrm{V}}{1000\times \mathrm{W}}$$

$$\mathrm{total} \mathrm{chlorophyll} a+b (\mathrm{mg}/\mathrm{g} \mathrm{tissue})=\frac{20.2\left(\mathrm{A}645\right)+8.02\left(\mathrm{A}663\right)\times \mathrm{V}}{1000\times \mathrm{W}}$$

here, A—is the absorbance at a specific wavelength; V—final volume of chlorophyll extract in 80% acetone; W—the fresh weight of tissue extracted.

2.6. Oxidative Stress Analysis

2.6.1. Sample Preparation for Oxidative Stress Analysis

About 0.2 g each of the leaf, root, and shoot of all the (treated) and control (untreated) samples were finely cut and taken in mortar and pestle. About 2 mL of 0.5 M phosphate buffer was added to each sample to homogenize. Samples were collected from plants at regular intervals of 0, 24, 48, and 72 h and were used for analyses of oxidative stress (Overall Reactive Oxygen Species (ROS), Superoxide dismutase (SOD), and Catalase).

2.6.2. Overall Reactive Oxygen Species (ROS)

2′-7′dichlorofluorescin diacetate (DCFH-DA), a cell-permeable fluorescent dye that detects reactive oxygen species (ROS), was used to assess the total ROS generated in different treatment groups [30]. Samples from tomato plants (leaf, shoot, and root) treated with PS, HA, and PS + HA, prepared using the above-mentioned procedure, were mixed with 100 µL of (100 µM) DCFH-DA and incubated in a dark atmosphere for 30 min. The fluorescence intensity of the samples was measured using a spectrofluorometer (Jasco FP-8300, Tokyo, Japan)) at an excitation and emission wavelength of 485 and 530 nm, respectively. The fluorescence spectrum results of all treated samples were compared with control samples.

2.6.3. Superoxide Dismutase (SOD)

In this study, the function of superoxide dismutase in the leaf, shoot, and root of tomato plants was measured (leaf, shoot, and root) [31]. The test is based on the ability of superoxide dismutase to inhibit the interaction of superoxide and Nitro Blue Tetrazolium (NBT). Samples prepared as previously mentioned were centrifuged at 13,000× g rpm for 20 min and the supernatants were collected separately. A chemical concoction containing 50 mM of Na₂CO₃, 96 mM of NBT, 20 mM of hydroxylamine hydrochloride, and 0.6% of Triton X-100 was added to 100 µL of each supernatant and incubated in the UV zone at 37 °C for 20 min. The absorbance intensity of the samples was determined using UV-Visible spectroscopy at a wavelength of 560 nm (Hitachi, U-2910, Tokyo, Japan). The results were represented concerning control.

2.6.4. Catalase

Catalase activity was evaluated in the tomato plant (leaf, shoot, and root) using the protocol developed by Yilancioglu [32]. Samples prepared as previously mentioned were centrifuged at 13,000× g rpm for 20 min and supernatants were collected separately. About 2 mL of 10.8 mM H₂O₂ solution was added to 100 µL of each supernatant. The hardness of the phosphate buffer solution with H₂O₂ was used as a baseline template for this experiment. The absorbance intensity of the samples was determined using UV-visible spectroscopy at a wavelength of 560 nm (Hitachi, U-2910, Tokyo, Japan).

2.6.5. Statistics

All data were statistically analyzed using Graph Pad Prism (version. Number: 5.0) software (San Diego, CA, USA). GraphPad Prism was used to measure the difference between the control and the other NPs-interacted samples, as well as the discrepancy between polystyrene (nanoplastic), humic acid, and polystyrene-humic acid combos. Two-way ANOVA was performed for statistical comparisons of data. p values less than 0.05 were considered to be statistically significant. All data represented are the standard error of the mean (S.E.M.) of at least three independent experiments in triplicate.

3. Results

3.1. Characterization Study

3.1.1. Particle Size

PS particle had a diameter of 149.5 nm, and the PS + HA complex was 168.3 nm respectively, and the difference between PS and PS + HA is 18.8 while the size of the PS particle is increasing as it combines with HA. The z-potential value of PS and PS + HA was observed to be 11.7 mV, and −41.9 mV, respectively (

Table 1. The particle size & Zeta potential of PS, & PS + HA.

Contents	PS	PS + HA
DLS size	149.5 nm	168.3 nm
ZETA potential (mV)	11.7 mV	−41.9 mV

).

3.1.2. Transmission Electron Microscope (TEM)

Structural characterization of samples using TEM revealed the shape of PS particles to be spherical, as indicated by the manufacturer. However, as expected, humic acid was observed as irregular shapes. Further, PS + HA showed a clear formation of eco-corona wherein humic acid was observed to be adsorbed onto the surface of PS (Figure 1A–C).

3.1.3. Fourier Transform Infrared (FTIR)

FTIR spectra showed signature bands for PS at (2365.26 cm⁻¹, 133.41 cm⁻¹, 1506.13 cm⁻¹, 1058.73 cm⁻¹ and 650.858 cm⁻¹). The (1506.13 cm⁻¹) band corresponds to C=C stretching vibrations in PS. On the other hand, HA showed its bands at (3270.68 cm⁻¹, 2347.91 cm⁻¹, 2118.42 cm⁻¹, 1633.41 cm⁻¹, 1250.61 cm⁻¹, and 964.233 cm⁻¹). The band at (2118.42 cm⁻¹ and 3270.68 cm⁻¹) reflected C=H and O-H stretching vibrations in humic acid. The IR signature bands of the PS + HA complex were (3304.43 cm⁻¹, 2117.46 cm⁻¹, and 3304.43 cm⁻¹). The band at (2117.46 cm⁻¹ and 3304.43 cm⁻¹) represented C=O and O-H stretching vibrations, respectively (Figure 2A–C).

3.1.4. X-ray Diffraction (XRD)

An XRD pattern was produced for PS, HA, and PS + HA (Figure 3). The peaks on the XRD angled (2θ) at 20.30° and 23.56° corresponding to (232) and (252) which are respective planes of the PS. The peaks located at angles (2θ) were 24.89°, 26.64°, 31.70°, and 45.52° which correspond to (307), (343), (448), and (730) respective planes of the HA. The peaks located at angles (2θ) 21.63°, 23.86°, and 31.65° correspond to (237), (284), and (445) which are respective planes of the PS + HA complex.

3.2. Effect of PS, HA, and PS + HA on Solanum lycopersicum (Seeds)

3.2.1. Seed Imaging by Optical Microscopy

Seeds that interacted with three different groups (PS, HA, and PS + HA) along with control were observed under an optical microscope. Control, PS, HA, and PS + HA treated seeds were shown in (Figure 4A–D). Seeds after interaction with PS + HA resulted in the eco-corona formation after 24 h. Due to the formation of an eco-corona layer on the seed surface, nanosized PS increased in size. Further, eco-coronated PS was impermeable to seed therefore the presence of humic acid-induced seed germination.

3.2.2. Seed Germination

The use of PS, HA, or both had a substantial impact on seed germination. On day 8, the control seeds started to germinate. However, seeds that had been exposed to PS germinated on day 9. PS exposure caused a one-day delay in seed germination. The seeds that had been given HA germinated on day 8 like the seeds in the control group. It’s interesting to note that seeds exposed to the PS + HA combination germinated on day 5 at a substantially earlier time point. The percentage of seed germination for the PS, HA, and PS + HA treated groups are 65.8%, 88%, and 90%, respectively, with the control showing 100% germination. This statement insists that HA reduces the toxic effect of PS on seed germination. However, eco-coronated PS increased seed germination when compared to PS-treated seed (Figure 5).

3.3. Effect of PS, HA, and PS + HA on Solanum lycopersicum (Plants)

3.3.1. Shoot and Root Length

On the ninth day, the average shoot and length of the plants of the control group were 5 cm and 14.7 cm, respectively. On the contrary, the PS-treated group showed shoot and root lengths of 3 cm and 9.4 cm, respectively. The HA-treated set showed shoot and root lengths of 7.3 cm and 16.2 cm, respectively. On the other hand, the PS + HA complex has a shoot and root length (Figure 6A,B) of 6.3 cm and 15.3 cm for the shoot and root length, respectively. The maximum shoot and root length were observed in the HA and PS + HA-treated groups (Figure 7).

3.3.2. Plant Imaging by Optical Microscopy

Microscopic image (Figure 8) showed hair-like structures called trichomes present on the surface of the control shoot, HA, and PS + HA treated groups. On the contrary, only a few trichomes were observed on the shoot of PS-treated plants. Further, observing the features of the root, well-grown root hairs were seen on the surface of the root in the control and HA groups. However, a very smaller number of root hairs were observed in the PS-treated groups indicating that PS had negatively impaired the root system impairing the development and growth of root hairs which are inevitable parts of the plant for the uptake of water and nutrients from the soil. This, in turn, could affect the growth and elongation of the shoot. Interestingly, this negative effect of PS on the development and growth of root hairs was reduced by treatment with humic acid. We observed that the development of root hairs in PS + HA-treated groups were as normal as that of the control group with a comparatively high number of root hairs indicating the protective role of humic acid after being coronated around PS. Further, the PS + HA group also displayed secondary roots. Humic acid, forming eco-corona around PS, had significantly abated the negative effect of PS on the root hair development and hence root health of the plant. Secondary roots were also observed in the PS + HA complex. On the contrary, the PS-treated plant’s presence of root hairs was very less.

3.3.3. Photosynthetic Pigment Estimation

Chlorophyll (Chl) a, b, and total chlorophyll (a + b) in the leaf were analyzed in control, PS, HA, and PS + HA complex-treated seedlings. Chlorophyll of PS treated plants showed lower chlorophyll content. The control showed Chl a and Chl b are 3.80 mg g⁻¹ and 2.82 mg g⁻¹ respectively and the total Chl (a + b) was 4.72 mg g⁻¹. In PS-treated samples, the Chl a and Chl b are 3.48 mg g⁻¹ and 2.12 mg g⁻¹, respectively and the total Chl (a + b) was 4.19 mg g⁻¹. In HA-treated samples, the Chl a and Chl b are 5.76 mg g⁻¹ and 3.88 mg g⁻¹ respectively and the total Chl (a + b) was 6.41 mg g⁻¹. In PS + HA treated samples, the Chl a, and Chl b are 4.17 mg g⁻¹ and 3.23 mg g⁻¹, respectively, and the total Chl (a + b) was found to be 6.58 mg g⁻¹. Visually, there was a conspicuous difference between the treated samples and the control leaf (Figure 9).

3.4. Oxidative Stress Analysis

3.4.1. ROS Production

The total ROS for PS, HA, and PS + HA treated plants (leaf, shoot, and root) were studied using DCFH-DA fluorescent dye (Figure 10). ROS production in all the samples (PS, HA, and PS + HA) significantly increased when compared to control (p < 0.001). When a comparison was made between the time points, ROS production in PS-treated samples at 0 h to 24 h, and 0 h to 72 h (leaf, shoots, and roots) was significantly higher (p < 0.001) when compared to samples at 48 h to 72 h (leaf) (p > 0.001), 48 h to 72 h (shoot and root) (p > 0.001). In HA-treated samples, ROS production decreased as follows 0 h to 24 h, and 0 h to 72 h (leaf, shoots, and roots) was significantly higher (p < 0.001) when compared to samples at 48 h and 72 h (leaf, shoots, and roots) (p > 0.001). The difference between the time points was observed highly significant decrease in overall ROS generation in 48 h and 72 h samples when compared to 24 h samples were to be significant (leaf, shoot, and root).

Furthermore, eco-coronation of PS decreased the ROS production at all the time points when compared to PS-treated samples. In eco-coronated PS, the samples showed a decrease in the ROS generation at 0 h to 24 h and 0 h to 72 h (leaf, shoot, roots) which are significantly (p < 0.001), at 48 h and 72 h decreasing significant changes were observed (p > 0.05), but in the case of shoots at 48 h and 72 h there no significant were changes observed (p > 0.05). However, in the graph, it is quite evident the eco-coronated treated plant results showed decreased total ROS (p < 0.001) with increasing time of treatment.

3.4.2. Effects on Superoxide Dismutase Activity

All the treated (PS, HA, and PS + HA) plants (leaf, shoots, and roots) showed a significant increase (p < 0.001) in the SOD activity when compared to the control samples (Figure 11). PS-treated samples (leaf, shoots, and roots) showed a highly significant increase (p < 0.001) when compared to 0 h to 24 h, 24 h to 48 h and 0 h to 72 h in the SOD activity with the control samples. In HA-treated samples, ROS production decreased as follows 0 h to 24 h, and 0 h to 72 h (leaf, shoots, and roots) was significantly higher (p < 0.001) when compared to samples at 48 h and 72 h (leaf and shoot) (p > 0.05) and 48 h and 72 h (root) (p < 0.001). The difference between the time points was observed highly significant decrease in overall ROS generation in 48 h and 72 h samples when compared to with 0 h to 24 h and 0 h to 72 h samples were to be significant (leaf, shoot, and root).

Furthermore, eco-coronation of PS decreased the SOD activity at all the time points when compared to the PS-treated samples. In eco-coronated PS, the samples showed a decrease in the SOD activity at 0 h and 24 h (leaf, shoots, and roots) which are significant (p ≤ 0.001). For leaf and root, at 48 h to 72 h, there were significantly no changes observed (p > 0.05), but in shoots, at 48 h to 72h, there was no significance observed (p < 0.05). However, in the case of eco-coronated PS-treated plants results showed positive effects in SOD activity with increasing time of treatment.

3.4.3. Effects on Catalase Activity

Catalase activity in the sample (PS, HA, and PS + HA) treated plants (leaf, shoots, and roots) is illustrated in (Figure 12). PS-treated plants (leaf, shoots, and roots) showed a significant increase (p ≤ 0.001) in catalase production with the control samples. Furthermore, it is observed that PS-treated plant (leaf, shoots, and roots) samples had higher significance when compared to their respective HA and eco-coronated PS-treated samples (p ≤ 0.001).

4. Discussion

Eco-coronated PS nanoplastics showed a progressive rise in size. These findings imply that HA-formed eco-coronated PS has better plant cell binding affinity. The stability and aggregation behavior of nanoplastics in the agricultural environment is regulated by their surface charge, which determines their colloid -behavior. Prior research suggests that blockage of pores can decrease water absorption in soybean (Glycine max) seeds [33] and garden cress (Lepidium sativum) [34]. The dynamic light scattering, and zeta potential (Table 1) indicated that the PS and PS + HA in deionized water formed a stable dispersion of the particles of nearly 149.5 nm, and 168.3 nm in size, respectively. The overall charge on the PS and PS + HA irrespective of their surface functionalization could be due to the involvement of PS- (carrying a positive charge), and eco-corona-formed PS- (carrying a negative charge) [35,36,37]. These findings indicate that negatively charged HA has a better binding affinity on the seed surface than positively charged PS. In another related study, the humic acid may easily attach to the hydrophobic surface of nanoparticles due to its heterogeneous functional group, as well as its capacity to form complexes [38]. The TEM image results PS (Figure 1A), HA (Figure 1B), and PS + HA (Figure 1C) reveal the eco-corona formation. Another study got a consistent TEM picture of eco-coronated on the surface of the PS result with nanoplastic in onion plant root Allium cepa [39] and nanoplastics in microalgae Chlorella sp. [40].

A similar study revealed that when HA complexes with silver nanoparticles (AgNPS), the HA changes the surface charge of the AgNPs, lowering their toxicity [41]. Infrared spectroscopy is presumed to be a qualitative tool for assessing the presence of functional groups in PS, HA, and PS + HA (Figure 2). The existence of C=C groups was discovered to be detected by the bands at 1668 cm⁻¹ area for (PS) [42]. HA existence of O ̶ H groups was discovered to be detected by the bands at 3270.68 cm⁻¹ [43]. In Figure 3 the PS XRD peak was observed at 23.56°, which was similar to another study where they got the peak at 18.8° [44]. The HA XRD peak was observed at 45.52°, which was similar to another study where the peak was at 47.45° [45]. Suspension of PS + HA interacted with tomato seed resulting in eco-corona formation on the seed surface after 24 h. Figure 4 and Figure 5 show plant seed germination of PS, HA, and PS + HA. Microscopical images showed that fewer healthy cells are present on the surface of the seed of PS in the case of HA and PS + HA higher number of healthy cells are present on the surface of the seed. It clearly shows that the toxic effects were reduced by humic acid interacting with PS due to the eco-corona layer on the surface. This is the first visible outcome of eco-corona development on the seed surface by microscopical image. Another intriguing study found that HA might minimize PS aggregation, as well as entanglement and body load to Daphnia Magna [46]. Importantly, humic acid enhances nutrient cycling that induces growth [47]. Another researcher found in Allium cepa root interaction with eco-coronated PS increased in size of the particle [39]. Figure 6 shows the seed germination rate, in Figure 7 shows the shoot length, and root length in Figure 8, the microscopical images showed a lesser number of trichomes in the shoot, root hair in the root, and leaf hair in the leaf in the PS-treated shoot, when compared to HA and PS + HA-treated plants. Therefore, the effect of humic acid on the plant was studied, and it helped to improve soybean yield in the field [48] and also another researcher found that humic acid improves plant growth in maize [49]. Compared to control plants, HA-treated and PS + HA-treated plants exhibited a higher number of trichomes in the shoot, root hair in the root, and leaf hair in the leaf was observed. Furthermore, secondary root formation was observed in PS + HA-treated plants. Root and shoot length were found to increase in PS + HA-treated plants compared to others due to eco-corona formation.

Regarding chlorophyll estimation, Chl a and Chl b and both Chl a + b were evaluated. Chl a, b, and a + b contents were higher in HA-treated and eco-coronated PS-treated plants, and they were significantly reduced in PS-treated plants (Figure 8). A similar result was observed in the study done by [50] where the PS reduced the chlorophyll in Lettuce (Lactuca sativa L.). A similar result was observed in which humic acid treatment increased the chlorophyll pigment production in the common bean plants (Phaseolus vulgaris L.) [51]. Because of the presence of carboxylic and phenolic groups in its structure, as well as an abundance of functional groups such as methoxyl, hydroxyls, ketones, and quinines, HA is likely to be highly adsorbed to plastic particles [52]. Similar results were seen in Lemna minor, where HA reduced the toxicity of AgNPs [41]. An additional investigation on the effects of PS on total ROS production in plant systems was required to fully understand the impacts of PS. Reactive oxygen species appear to be the primary mediator of oxidative stress [53]. ROS is produced in photosynthetic systems like plants as a result of the essential cellular process [54]. Antioxidant enzymes are indirect markers of cellular oxidative stress and Superoxide dismutase, and catalase are two examples of extensively utilized enzymes. Furthermore, PS-induced membrane damage is connected to generating [55]. Recent research with Scenedesmus obliquus found a comparable reduction in ROS generation by PSNPs in the presence of fulvic and humic acids (HA and FA) [26]. PS-induced extensive oxidative damage was observed in terms of ROS in Solanum lycopersicum, and HA reduced its toxicity. In parallel with this result, HA reduced the toxic effects of AgNPs in Lemna minor plants [41]. In this experiment, ROS activity was higher in PS-treated samples (leaf, shoot, and root) than in untreated (control) and a lesser quantity of ROS was observed in eco-coronated PS (leaf, shoot, and root) (Figure 9). A similar decrease in the production of ROS by PS in presence of HA was reported in a recent study with Scenedesmus obliquus [4]. Furthermore, following exposure to PS, there was an increase in CAT activity in tomato leaves, shoots, or roots, although SOD activity dramatically reduced. The eco-coronated form of PSNPs that causes a lower increase in both the ROS levels leads to a lower in the CAT activity and a lower in SOD activity as compared to the control. Therefore, larger extents of eco-coronation in PS cause a marked decline in SOD activity (Figure 10) and a reduction in CAT activity and SOD activity (Figure 11). The same trend of decreased CAT activity in Synechococcus sp. after Fulvic acid (a variant of NOM) coated iron nanoparticles treatment has been reported in the past [56]. The same trend of decreased SOD activity in Allium cepa root in eco-coronated PS has been reported [39].

Increased oxidative stress, as mentioned in the preceding sections, would increase the activities of the antioxidant enzyme catalase in the current investigation. When the coronated PS interacted with the seed cells, the enzyme activity significantly decreased. Because of the nano size of the PS, they could readily enter the seed cells, generating ROS and decreasing the plant physiology. The nanoplastic became impermeable to the seed as the size of the PS increased with corona development, increasing seed healthy cells, seed germination, ROS production as well as antioxidant enzyme activity.

5. Conclusions

The current study focuses on the phytotoxic effects of PS in higher plants tomato (Solanum lycopersicum) as a typical bioindicator. According to the study’s findings, eco-coronated PS and HA produced similar seed germination, chlorophyll estimate, root and shoot length, and a considerable reduction in oxidative stress when compared to PS. The results clarified that PS-treated plants were slower in overall growth rate compared to HA-treated plants and control plants. Further, adding HA to PS treatment of plants enhanced the growth rate and reduced the toxicity caused by nanoplastic.

The formation of an eco-corona on the PS probably reduced the uptake of PS by the surface of tomato seeds, potentially via enhancing particle aggregation. It was concluded that the toxicity of nanoplastic—one of the most significant emerging agricultural contaminants in recent years could be reduced by the natural organics humic acid found in agricultural soil. Overall, our findings are likely to contribute to a better understanding of the interactions between nanoplastic and organic sources (found in agricultural soils).