1. Introduction
Zinc (Zn) and copper (Cu) are crucial micronutrients in leafy vegetables and for human health. Zn plays a vital role in plant growth and development, and its presence in vegetables is influenced mainly by soil pH and fertilization [
1,
2]. Similarly, Cu is essential for enzymatic activities, cell metabolism, and signaling in plants, including leafy vegetables [
3]. However, Zn and Cu deficiencies are prevalent in the human population, with more than half being deficient in Zn [
4,
5]. Therefore, it is crucial to ensure the leafy vegetables’ biofortification of these micronutrients to address these deficiencies.
Nanoparticles (NPs, materials with a size less than 100 nm) have the potential to address micro- and macronutrient scarcity by enhancing nutrient mobilization and uptake in plants [
6,
7]. Considering the differences between NPs and bulk materials, such as the larger surface area determined by the smaller size of NPs, the zeta potential, and the homogeneity of the suspension, these factors may impact their penetration and movement in plants. Engineered nanomaterials (ENMs) can be used as nanoherbicides and nanopesticides to detect agro-pathogens onsite, post-harvest management, soil fertility, and irrigation management [
8]. Nanofertilizers, in particular, positively influence plant growth, development, and interactions with soil microflora [
9]. Furthermore, ENMs can suppress plant disease and enhance crop yield, potentially due to the greater availability of nutrients in the “nano” form [
10].
Singh [
11] and Goyal [
12] highlighted the potential of nano-enabled technology to enhance growth, nutritional quality, and Zn content in cereal crops and leafy vegetables. Sb [
13] further demonstrated that 50 nm of ZnO NPs at a concentration of 1000 ppm increased spinach’s leaf length, width, leaf area, protein, and dietary fiber content. The researchers [
14] determined the most appropriate concentrations of ZnO NPs application through the soil for lettuce—20 ppm, beet—225 ppm, wheat, bean, pea—450 ppm, maize, radish, tomato, and cucumber—900 ppm, plants based on the highest Zn content in the shoots of the studied plants. They highlighted that the effects varied depending on the plant species and especially on soil pH. However, Zhao [
15] noted that applying 800 mg kg
−1 of ZnO NPs led to the biggest Zn bioaccumulation in cucumber leaves, less in stems, and the least in fruits, suggesting a potential risk of excessive NP exposure. Dimkpa [
16] demonstrated that the foliar application of ZnO NPs (18 nm, 6 ppm) more effectively increased Zn amount in sorghum shoots than application through the soil. Sharifan [
17] reported that a 15–137 nm size in ZnO NPs at 100 ppm concentration significantly elevated the Zn contents in cilantro, parsley, and spinach. Ji [
18] found that ZnO NPs—200 mg kg
−1—and CuO NPs—25 mg kg
−1—concentrations enhanced absorption of certain nutrients by 3 and 2.5 times, respectively, in
Medicago polymorpha L compared with non-treated plants.
The effects of CuO NPs on leafy vegetables are complex and may vary depending on factors such as concentration and exposure duration. Pelegrino [
19] found that 0.2–20 µg ml
−1 concentrations of green synthesized CuO NPs (6.6 nm) can enhance lettuce growth, but 40–300 µg ml
−1 concentrations can lead to decreased plant weight, net photosynthesis level, and water content, as well as the inhibition of seed germination and radicle growth. However, Wang [
20] found that CuO NPs at 200 and 400 mg kg
−1 increased Cu amount in shoots and roots, lettuce photosynthesis rate, and productivity. Zafar [
21] noted that CuO NPs 53 nm in size and at 500 to 1500 ppm concentration inhibited root, stem, and leaf growth in
Brassica nigra seedlings. Researchers [
22] investigated that the highest amount of Cu was found after foliar application of CuO NPs (40–60 nm) at 250 ppm concentration exposure in lettuce and cabbage after 15 days. These findings collectively suggest that nanotechnology can be crucial in addressing Zn and Cu micronutrient scarcity in leafy vegetables.
It is worth noting that most of the research has been conducted with various plants popular in horticulture, and NPs are usually applied through the soil. However, in practice, the spraying technique is more convenient. Furthermore, the specific influence of lighting on plants’ uptake of ZnO and CuO NPs has not been explored yet. Considering the lack of knowledge, a study was conducted to investigate how different HPS and LED lighting combined with CuO and ZnO NPs influence the elemental composition of ice plants (
Mesembryanthemum crystallinum L., family Aizoaceae, Caryophyllales). The ice plant is a crucial research subject due to its unique ability to grow in salinity soil or water [
23,
24], switch from C3 photosynthesis to Crassulacean acid metabolism [
23], and it contains d-pinitol, which is important for people with diabetes because it contributes to the regulation of blood sugar, and is also a source of mineral elements and antioxidants [
25] to supplement the daily diet. The plant’s distribution and growth are influenced by various environmental factors, making it an essential species for ecological studies [
26]. Additionally, the ice plant’s potential as a highly salt-tolerant crop and its beneficial properties for human health make it a promising candidate for future agricultural use [
24].
The main research objectives were (i) to determine the possible accumulation of Zn and Cu in ice plants treated with ZnO and CuO NPs, and to find out how different lighting characteristics can lead to Zn and Cu accumulation; (ii) to investigate the possible risk assessment to human health of increased uptake of ZnO and CuO NPs in the edible parts of ice plants; and (iii) to determine the effect of ZnO and CuO NPs, by evaluating the correlation with other microelements, macroelements, and growth indicators. This study hypothesized that the accumulation of Zn and Cu in ice plant tissues can be intensified by exposure to ZnO and CuO NPs, and by adjusting lighting conditions while keeping the hazard quotients within safe limits.
2. Results
This research investigated the impact of lighting and NPs on the growth rates and elemental composition of ice plant leaves. The growth parameter results show that LED lighting significantly impacted the fresh shoot weight of the ice plant, leading to an increase of 12 to 80%, regardless of whether NPs were used. The highest fresh weight of the roots was observed under the influence of HPS lighting and CuO NPs, while the lowest was under the influence of ZnO NPs. From
Figure 1, it is evident that the effect of NPs under different lighting conditions is noticeable. Under HPS lighting, the CuO NP-suspension had a positive impact on ice plant growth by increasing the leaf area (by 70%), FW, and DW of both shoots (by 40 and 15%, respectively) and roots (by 42 and 53%, respectively) compared to the plants that were not treated. Meanwhile, ZnO NPs did not show statistically significant differences in growth rates from unaffected-plant NPs.
Under LED lighting, the treatment of CuO NPs showed statistically significant differences by reducing the FW of the shoot part by 12% and DW of roots by 25%, and increasing leaf area by 23% compared to plants that were not treated with NPs.
Moreover, a strong positive correlation (
Table 1) was found between the FW and DW of the shoot and root. Thus, the results correspond proportionally to each other in
Figure 1. A strong positive correlation was also found between leaf area, shoot, and root FW.
It is noticeable that the utilization of LED lighting has led to a statistically significant increase in magnesium (Mg) by 36–50% and potassium (K) by 3–16% content in the leaves of ice plants (
Table 2). On the other hand, HPS lighting combined with CuO NPs treatment increased Ca content by 39%. In addition, phosphorus (P) showed a strong positive correlation (
Table 1) with K and zinc (Zn) and a weaker positive correlation with Mg. Furthermore, Ca exhibited a positive correlation with copper (Cu) and sodium (Na) elements, while showing a strong negative correlation with K, Mg, and molybdenum (Mo).
LED lighting combined with ZnO NPs application had a particularly significant effect on Zn accumulation in plant leaves, increasing it by 66% compared to the influence of HPS and ZnO NPs treatment. However, HPS lighting combined with CuO NPs were more efficient, increasing Cu content in ice plant leaves by 40% compared to the effect of LED and CuO NPs. It should be noted that a strong positive correlation (
Table 1) was found between Zn, P, and B. A positive correlation was found between Cu and Ca, but a negative correlation was found with Mg.
The content of Fe and Mo increased in ice plant leaves under the influence of LED lighting and CuO NPs by 266 and 54%, respectively, compared to the HPS and CuO NPs treatment. A strong positive correlation of Fe with K, Mg, Mn, and Mo were found. Mo was strongly positively correlated with Fe, K, Mg, and Mn, while a negative correlation was found with B, Ca, and Na. Mn content increased by 49% in the leaves of ice plants under the combined effect of LED and ZnO NPs, compared to the effect of HPS and ZnO NPs. In addition, Mn positively correlated with Fe, K, Mg, and Mo (
Table 3).
The results revealed that LED lighting positively affected the leaf area, the DW and FW of shoots and roots, and the amounts of K, Mg, Na, Zn, Fe, Mn, and Mo. HPS lighting positively affected the Ca and Cu content in ice plant leaves, while P and B lighting had no effect. CuO NPs significantly positively affected the leaf area, the DW and FW of shoots, the FW of roots, and the amount of Cu and Mo content (
Table 4). The influence of NPs was not determined on root DW, Ca, K, Mg, Na, Fe, and Mn amounts. ZnO NPs significantly positively affected the amount of P, Zn, and B elements. Combined factors show that LED illumination without NPs (the number of values of a and ab in the interactions of the factors—10), with CuO NPs (6), with ZnO NPs (11), and HPS with CuO NPs (6) resulted in larger plants and the accumulation of more elements (
Table 4).
The ability of plants to absorb Cu was strongly determined by HPS lighting and the application of CuO NPs. HQ increased from 0.032 (HPSx0NPs) and 0.002 (LEDx0NPs) to 0.068 and 0.051, respectively, when using CuO NPs, indicating that as a metal, this amount would not harm human health and does not exceed the dangerous limit of 1.
Zn accumulation was positively affected by LED illumination combined with the application of ZnO NPs in ice plant leaves. BCF increased by 72% under combined exposure to LED and ZnO NPs than under combined exposure to HPS (
Table 5). HQ was found to be the highest at 0.075, when ice plants were grown under LED lighting and foliar applications of ZnO NPs, and 0.045 under combined HPS with ZnO NPs exposure, but the indices are still within safe limits. BCF
Cu and HQ
Cu found the strongest positive correlations (
Table 1) with the leaf area, Cu, and Ca; for BCF
Zn and HQ
Zn, the strongest positive correlations were found with P, B, and Zn.
4. Materials and Methods
4.1. Nanoparticles Preparation and Characteristics
The commercial zinc (ZnO) and copper (CuO) oxide nanoparticles (NPs) used for plant exposure in this study were purchased from US Research Nanomaterials (Inc., Houston, TX, USA). Suspensions of ZnO (size: 35–45 nm, 99% purity)—800 ppm and CuO (size: 40 nm, 99% purity)—30 ppm NPs were prepared in deionized water. The suspensions were prepared in 300 mL flasks by weighing ZnO and CuO NP powders, respectively, which were weighed using a highly sensitive balance (Radwag AS 220 R2 PLUS, RADWAG Balances and Scales, Torunska, Poland) and an antistatic ionizer (DJ-04 Antistatic Ionizer, RADWAG Balances and Scales, Torunska, Poland) to remove the static charge of the powder particles. The flasks with suspensions were placed in an ultrasonic bath (Sonerex super ultrasonic bath 80 W, Weidinger GmbH, Gernlinden, Germany) and suspended for 60 min. Immediately afterward, the NPs’ size and suspension stability were measured using a Delsa™Nano Submicron Particle Size (Beckman Coulter Instruments Corporation, Fullerton, CA, USA) and a Zeta Potential device (Dispersion Technology Inc., Bedford Hills, New York, NY, USA).
Table 6 shows the positive particle surface charge of the ZnO and CuO-NP suspensions. The suspensions were stable, according to the zeta potential. In addition, the polydispersity index (PDI) showed that NP suspensions were monodisperse.
Plants were sprayed immediately after the ultrasonic bath using automatic sprayers (Rechargeable electric sprayer,1 L, 3.6 V, Nozzle hole diameter: 13 mm, Yato, Haiyan, Jiaxing, China) to the full surface maturity during the first half of the day. Before spraying, the plant systems were covered with a plastic sheet to protect the hydroponic solution and roots from exposure to NPs. It should be noted that certain individuals who carried out the spraying followed all safety requirements, wearing a full protective suit, gloves, and a respirator.
4.2. Plant Growth Conditions, Lighting, and Nanoparticle Treatments
Experiments were conducted at the Institute of Horticulture in the Lithuanian Research Centre for Agriculture and Forestry. The study was performed in a controlled environment plant growth chamber measuring 4 m by 6 m with a height of 3.2 m. Seeds of the ice plant (
Mesembryanthemum crystallinum L.) were obtained from CN Seeds, Ely, UK. The 200 rockwool cubes measuring 2.5 cm × 2.5 cm × 3.0 cm were used as the growing medium. Before use, the rockwool cubes were soaked in deionized water with an adjusted pH of 5.0 by adding sulfuric acid and placed in a plastic tray. Germinated seedlings were grown in this setup for 29 days at a temperature of 25 ± 1 °C and humidity of 60 ± 5%. The germinated plants were watered with enough hydroponic solution to cover one centimeter of the bottom of the rockwool cubes daily. Then, they were transferred to Ebb-type hydroponic systems with 80 L containers containing a hydroponic solution made of deionized water and nutrients in the following quantities (mg L
−1): N (120), Ca (88), P (20), K (128), Mg (40), S (53), B (0.16), Mo (0.2), Mn (0.08), Cu (0.08), Fe (1.6), and Zn (0.8). The electrical conductivity (EC) of the nutrient solution was 1.4 mS cm
−1, and the pH was measured daily using a portable meter (GroLine HI9814, Hanna Instruments, Woonsocket, RI, USA) and adjusted to 6.0 using sulfuric acid or sodium bicarbonate. During the experiment, the plants were exposed to two different lighting conditions: a combination of white—380–760 nm (4000 K), blue—455 nm, and red—660 nm light-emitting diodes (LEDs, OSRAM Oslon SSL, Ecolight, Vilnius, Lithuania) at the ratio of 13:1:6, respectively, and high-pressure sodium lamps—2050 K (HPS, SON-T Agro, 400 W, Philips, Eindhoven, The Netherlands) at a photosynthetic photon flux density (PPFD) of 250 ± 5 μmol m
−2 s
−1 with a 16 h photoperiod. PPFD and spectra (
Figure 2) were measured at the plant growth level using a portable spectroradiometer (WaveGo, Wave Illumination, Oxford, Oxfordshire, UK). After eight days of ice plant cultivation in Ebb hydroponic systems, the plants were treated with ZnO and CuO-NP suspensions (preparation described in
Section 4.1) by spraying and were allowed to grow for five more days until the end of the experiment. Then, the plant growth parameters were measured, and raw materials for the analysis were collected.
4.3. Growth Characteristics
The growth characteristics of the ice plant were studied by measuring various parameters such as the number of leaves, fresh (FW) and dry weights (DW) of the shoots and roots, and leaf area. The study involved 10 adult plants per treatment. Adult plants were described as side shoots with secondary leaves but no flowers and with primary leaves [
44]. The FW was measured using an electronic scale (Mettler Toledo, ML104T/00; Mettler-Toledo, Columbus, OH, USA). The DW was determined by drying the divided samples of shoots and roots for 48 h in a drying oven (Venticell-222, Medcenter Einrichtungen, Gräfeling, Germany) at 70 °C. The leaf area was measured using a leaf area meter (CI-202 Laser Area Meter; CID BioScience, Camas, WA, USA).
4.4. Elemental Composition of Ice Plant
The macro-and microelement quantities in ice plant leaves were determined using microwave digestion combined with inductively coupled plasma optical emission spectrometry. The shoots of the ice plants were harvested, gently rinsed with ultrapure water, and dried at 70 °C for 48 h, then ground to powder using a centrifugal mill with a ZM 200 rotor at 15,500 rpm (Ultra Centrifugal Mill ZM 300, RETSCH GmbH, Haan, Germany). Complete digestion of dry ice plant material (0.3 g) was achieved with 8 mL of 65% HNO
3 using the microwave digestion system Multiwave GO (Anton Paar GmbH, Graz, Austria). The digestion program was as follows: (1) 170 °C reached within 3 min, digested for 10 min; and (2) 180 °C reached within 10 min, digested for 10 min. Full-digested samples were diluted to 50 mL with deionized water. The elemental profile was analyzed by an ICP–OES spectrometer (Spectro Genesis, SPECTRO Analytical Instruments, Kleve, Germany). The operating conditions employed for ICP-OES determination were 1300 W RF power, 12 L min
−1 plasma flow, 1 L min
−1 auxiliary flow, 0.8 L min
−1 nebulizer flow, and 1 mL min
−1 sample uptake rate. The analytical wavelengths chosen were 213.618 nm for P, 766.491 nm for K, 279.079 nm for Mg, 589,592 nm for Na, 445.478 nm for Ca, 324.754 nm for Cu, 257.611 nm for Mn, 259.941 nm for Fe, 213.856 nm for Zn, 249.773 nm for B, and 208,414 nm for Mo. The operating conditions employed for the ICP-OES were as follows: 1.3 kW RF power, 1.0 L min
−1 auxiliary argon (Ar) flow, 0.80 L min
−1 nebulizer Ar flow, 12 L min
−1 coolant Ar flow, and axial plasma configuration. Each sample was analyzed in triplicate. The calibration standards were prepared by diluting a stock multi-elemental standard solution (1000 mg L
−1) in 6.5% (
v/
v) nitric acid and by diluting stock phosphorus and standard sulfur solutions (1000 mg L
−1) in deionized water. The calibration curves for all the studied elements ranged from 0.01 to 400 mg L
−1. The contents of macro and microelements in the DW of ice plants are presented [
45,
46].
4.5. Bio-Concentration Factor and Intake Risk Assessment
Bioaccumulation is the ability of plants to absorb elements and retain them. The efficiency of this process depends on environmental conditions, and the plant type has the most influence on the elements’ retention. Due to the complexity of the methods, the bioconcentration factor (BCF) of specific elements was calculated to assess the environmental risk that may arise from the substances under investigation. Depending on the component, a high BCF value means a low element solubility in water and a high relative octanol–water partition coefficient besides a high soil adsorption coefficient.
Therefore, BCF was calculated as an index of the ice plants’ ability to accumulate zinc (Zn) and copper (Cu). It was calculated as the ratio of the Zn or Cu concentration (mg L
−1) in the hydroponic solution and the Zn or Cu concentrations (mg kg
−1) in the ice plant [
47]:
The average daily intake (mg kg
−1 day
−1) of potentially toxic metals by consuming leaves of ice plant after foliar application of ZnO NPs was calculated by the following equation [
48]:
Cm—the metal concentration in a plant (mg kg−1) on a dry weight basis.
Cf—the conversion factor (0.085) to convert the fresh to dry weight.
IR—the ingestion rate of vegetables.
Bw—the average body weight for an adult is 70 kg.
The average daily intake of leafy vegetables was estimated to be 100 g (0.1 kg person−1 day−1).
The risk of non-carcinogenic health effects is often evaluated from hazard quotients (HQ), which are the ratio of the daily intake (often the average daily intake, ADI) to a toxicological reference dose (RfD) according to the following equation [
48]:
RfD—the oral reference dose for Zn is 0.3 mg kg
−1 day
−1 [
49] and Cu 0.04 mg kg
−1 day
−1 [
50].
If the value of HQ is less than 1, it is assumed to be safe from the risk of non-carcinogenic effects. Conversely, if the HQ is equal to or higher than 1, it indicates a potential risk for some exposed individuals to experience adverse health effects.
4.6. Statistical Analysis
MS Excel Version 2010 and XLStat 2020 Data Analysis and Statistical Solution for Microsoft Excel (Addinsoft, Paris, France) statistical software were used for data processing. Analysis of variance (ANOVA) was carried out along with the Tukey multiple comparison test for statistical analyses (p ≤ 0.05).