Plasma-Activated Water as an Alternative Nitrogen Source: Effects on Lettuce Growth and Mineral Composition

Abstract

Plasma-activated water (PAW) is enriched with reactive oxygen and nitrogen species (RONS). Application of PAW in plant cultivation demonstrated that RONS promote seed germination and early plant growth, as well as stimulate plant defense mechanisms. The aim of this paper was to investigate the potential of reactive nitrogen species in PAW to partially replace urea fertilizer nitrogen in lettuce cultivation without resulting in a negative effect on growth and mineral composition. Lettuce was grown under two treatments: urea only and a combined treatment in which 10% of the urea-derived nitrogen was replaced by an equivalent amount of nitrogen supplied via plasma-activated water (PAW). Plant growth parameters of lettuce (number of leaves, head weight, rosette diameter and height, and dry matter weight) were measured. Concentrations of 21 elements in the plants were analyzed using inductively coupled plasma optical emission spectroscopy (ICP—OES). Results showed no significant difference in growth parameters between the two treatments, as well as no significant difference between treatments in the concentrations of most elements except magnesium, boron and sodium. The results demonstrate that PAW reactive nitrogen can partially substitute for nitrogen from synthetic fertilizer without negative effects on the growth and nutritional content of lettuce. The study contributes to the development of sustainable horticultural fertilization practices and the adoption of environmentally friendly technologies.

1. Introduction

Achieving a highly economic crop yield is a main goal of farmers in addressing the increasing demand associated with global food consumption, projected to grow by 35 to 56% in the period between 2010 and 2050, depending on multiple socioeconomic and climate factors [1]. Increasing crop yield on existing agricultural land presents a biodiversity-friendly alternative to clearing more land for farming [2]; however, achieving high yield depends on many factors, including plant genetics [3], soil and water management [4] and, not the least, crop fertilization [5]. Although the relationship between nutrient availability and plant growth is complex, increased supply of nutrients generally results in increased growth and yield. Thus, chemical fertilization of crops, with nitrogen fertilization being the limiting factor for increased biomass, constitutes basic practice in agriculture [6].

According to the 2025 FAOSTAT report, nitrogen fertilizer use in 2023 was estimated to be around 112 million tonnes [7], while IFA predicts the number to rise to 116 million tonnes in 2025 [8], with urea being the most common inorganic nitrogen fertilizer. However, most of the fertilizer used ends up as an excess in the soil, since plants can take up only a limited amount of nitrogen. Increasing awareness about ecotoxicity of excess fertilizers, such as diminished soil fertility and contamination of water from agricultural runoff, has driven the need for alternative ways of delivering nutrients to plants, from organic farming to biotechnologically advanced fertilization methods [9,10,11].

Promising green technology for sustainable agriculture comes from the field of plasma physics, in the form of cold or non-thermal plasma. Cold plasma is a type of plasma produced in atmospheric conditions with simple plasma discharge, resulting in active plasma species existing at low temperature, which makes it applicable in biotechnology: medicine, agriculture, and food processing. Depending on the technique of cold plasma production and the type of gas used, plasma consists of various reactive oxygen and nitrogen species (RONS). If cold plasma comes in close contact with an aqueous medium, RONS are incorporated into the medium. Such a product is plasma-activated water (PAW)—an aqueous solution of nitrite, nitrate, peroxide and ozone ions [12,13]. Studies of the use of PAW in biotechnology reported antifungal and antimicrobial properties, owing to the low pH of the solution and high concentration of reactive oxygen species (ROS). Possible uses of PAW’s antimicrobial properties include protection of the fruit from fungal infections [13,14], and some studies investigated the promotion of plant defense mechanisms after PAW treatment, such as resistance of tomato to bacterial leaf spot [15].

Most of the research on PAW application in agronomy has focused on the treatment of seeds and observing the germination rates and early sprouting [16], evidencing increased germination rate, enhanced growth of early seedlings, and higher yield [17,18].

In studies on lettuce, plant growth in greenhouses accompanied with PAW treatment resulted in a significant increase in leaf number and lettuce head mass, diameter, and height. Kučerová et al. [19] reported that PAW irrigation resulted in lettuce dry weight values comparable to those obtained under irrigation treatment with solutions containing similar nitrogen and H₂O₂ concentrations. However, PAW-irrigated plants displayed higher photosynthetic pigment content and higher photosynthetic activity [20]. In general, PAW treatment increased lettuce yield, but did not result in a significant increase in secondary metabolites (except proline) [21].

Other crops were also examined under various methods of PAW usage. Eggplant yield was increased with a PAW foliar spraying method [17], while experiments on maize showed no biomass increase and no effect on micro- and macronutrients in plants treated with PAW foliar spraying [22]. Research on tomatoes was conducted with PAW treatment applied throughout the whole life cycle of plants: from seed treatment to flowering, fruit development, and senescence. PAW treatment enhanced early seedling growth, resulted in a higher number of flowers, and increased the number and weight of tomato fruits. Senescence was also delayed in PAW-treated plants [23]. In hydroponic cultivation experiments on radish, PAW hydroponic solution treatment induced a non-significant increase in root length and shoot length and fresh weight comparable to radishes grown in conventional hydroponic solutions [24]. Microalgae cultivation revealed increase in specific growth and biomass of algae culture grown with PAW [25].

Studies of PAW’s effect on nutritional composition of plants investigated primarily amino acid composition, chlorophyll content, and secondary metabolites in lettuce [26,27,28], apples, spinach [29], and wheat, amongst others [30]. Results varied, and depend on many variables such as the length of the plasma exposure time of water, i.e., the final concentration of RONS in PAW. Some amino acids were shown to be significantly more synthesized in PAW-treated plants compared to chemical fertilizers, while others had significantly reduced concentrations [28]. Concentration of phenolic compounds also depends on the length of PAW generation and treatment, and influenced by oxidizing elements in the PAW [19].

Published studies on plant mineral composition under the influence of PAW indicate variability and lack a consistent trend. Wang et al. described increased total free amino acids, and higher values of Ca, P, K, S, Mg, Mn, Na, Fe and Zn in the plantlet juice of PAW-treated wheat shoots [30]. Experiments with plasma-treated water and wastewater reported that out of four macroelements (Mg, Ca, Na, K), only sodium concentrations significantly rise in plants treated with PAW [31]. PAW irrigation in tomato plants produced increased levels of P, S, Ca and Mg in the leaves of the plants, and resulted in increased resistance against attack from two-spotted spider mites (Tetranychus urticae Koch.) [32].

The aim of this work was to examine whether it is possible to substitute the nitrogen coming from urea fertilizer with nitrogen in the form of RONS in the PAW, without negatively influencing the growth and quality of lettuce. Most PAW studies to date have focused on seed germination and early growth responses, while comprehensive multi-element analyses and assessments of nutrient dynamics across plant developmental stages remain limited. We address this gap by performing a robust ICP—OES analysis of the total contents of 21 elements. The study evaluates the effects of plasma-activated water on plant nutrient composition across multiple stages of growth in soil-grown lettuce.

2. Materials and Methods

2.1. Plasma-Activated Water Preparation

Plasma-activated water used in this experiment was generated at the Institute of Physics in Zagreb (Croatia). PAW was generated using a single-electrode atmospheric-pressure plasma jet reactor. The plasma jet is constructed of a quartz tube (1.5 mm outer diameter, 1.0 mm inner diameter) into which a copper wire (100 μm diameter) is inserted and serves as the electrode.

Nitrogen gas (99.996% purity) was supplied to the plasma jet at a flow rate of 500 sccm. The electrode creates a discharge in the gas with a power of approximately 15 W, driven by a sinusoidal voltage waveform of 28 kHz with a maximum voltage of 12 kV (PVM500-2500 Plasma Power Generator, Information Unlimited, Amherst, NH, USA).

The 800 mL of commercially purified, pharmaceutical-grade water (Pharmacopoeia Europaea, Ph. Eur. 9, pH = 6.5, conductivity of 0.98 μScm⁻¹) was placed in a Berzelius beaker and brought into close contact with the plasma jet. The water surface was positioned at 5 mm from the quartz capillary opening during 70 min of plasma jet discharge.

Immediately after treatment and several times during storage, PAW was analyzed for pH and concentrations of ${\mathrm{NO}}_2^{-}$, ${\mathrm{NO}}_3^{-}$ and H₂O₂ using QUANTOFIX test strips. The strips were evaluated using the QUANTOFIX Relax unit optical reader (MachereyNagel GmbH, Düren, Germany). NaNO₂ and NH₄NO₃ solutions of known concentrations were used for the calibration, which was confirmed with UV–VIS absorption spectroscopy. The aging dynamics of PAW were evaluated as a function of pH, which was adjusted by introducing magnesium ions into the water. A 5 g piece of solid magnesium was inserted into water during plasma treatment and remained in the solution for one hour after treatment.

The values measured after treatment, which correspond to the values at the time of water use, were as follows: pH 6 ± 0.2, H₂O₂ = 4.4 ± 0.2 mgL⁻¹, ${\mathrm{NO}}_2^{-}$ = 5.5 ± 1 mgL⁻¹, and ${\mathrm{NO}}_3^{-}$ = 20 ± 2 mgL⁻¹.

2.2. Experimental Set-Up

The experiment was set up on 9 April 2025 at the experimental testing ground of the University of Slavonski Brod in Slobodnica, Croatia (N 45°9′58′′, E 17°57′8′′). Lettuce seeds of the Attraktion variety (Franchi sementi S.P.A., Grassobio, Italy) were sown in a modular tray with 60 sowing cells (50 mL volume each), in Potgrond P substrate (pH 6.0, N = 220 mgL⁻¹, P₂O₅ = 110 mgL⁻¹, K₂O = 220 mgL⁻¹, Mg = 80 mgL⁻¹) (Klasmann-Deilmann GmbH, Geeste, Germany).

Seedlings were grown in a nursery under controlled conditions (approximately 21 °C, 12 h photoperiod) until development of 3–4 true leaves. Healthy and uniform seedlings were transplanted into pots (3.75 L volume) filled with garden soil substrate (pH-KCl 6.42, pH-H₂O 6.8, Al-P₂O₅ = 23.57 mg/100 g soil, K₂O = 23.09 mg/100 g soil, CaCO₃ 9.6%, humic content 1.21%).

Plants were grown under greenhouse conditions, with a day temperature range of 8–14 °C and night temperature range of 6–8 °C. Carbon dioxide concentration ranged from 400 ppm to 550 ppm, and relative humidity from 50 to 65%.

Experimental set-up included plants grown under three different treatments. Control treatment plants were not supplemented with additional fertilizer during growth. Conventional fertilizer treatment plants were supplemented with urea three times (9 April, 30 April, 15 May), with 0.037 g of total nitrogen applied in each application. Plants in the experimental treatment were supplemented with combined treatment of urea fertilizer (0.031 g of nitrogen) and watered with 1 L of PAW on each occasion (9 April, 30 April, 15 May), where 10% of the nitrogen supplied in the urea treatment was replaced by an equivalent amount of nitrogen provided by PAW, and the total nitrogen input was kept constant. All plants were watered as required to reach a fully watered state.

Growth indicators (rosette height, number of leaves, head diameter and mass) were measured at three timepoints (30 April, 15 May, 26 May 2025) during the life cycle of the plants, with four replicates per treatment.

2.3. Dry Matter Weight

Plant samples for dry-matter weight measurements were taken at the same times as growth indicator measurements, in four replicates per treatment (control, urea, urea/PAW). The samples were oven-dried at 60 °C for 48 h (Memmert 100, Memmert GmbH + Co. KG, Schwabach, Germany), during which four measurements of total dry mass of samples were taken using an analytical balance. The total dry weight of all replicates per treatment was then divided by the number of plants to obtain the average dry mass of the individual plants.

2.4. Elemental Analysis by ICP—OES

Samples for elemental analysis were taken at the same times as growth indicator measurements. The sample collections consisted of four replicates per treatment (control, urea, urea/PAW). Samples were oven-dried (Memmert 100, Memmert GmbH + Co. KG, Schwabach, Germany) at 60 °C until constant weight and homogenized in a centrifugal mill (Retsch ZM 200, Retsch GmbH, Haan, Germany).

Homogenized samples (~200 mg each) were digested with 6 mL concentrated HNO₃ and 2 mL of 30% H₂O₂ in a microwave digestion system (Ethos UP, Milestone, Sorisole, Italy), rinsed and rounded up with ultrapure water to 25 mL volume per sample.

Analysis of macro- and microelements of the plant samples was performed by inductively coupled plasma optical emission spectroscopy (ICP—OES) (Shimadzu ICPE-9800, Shimadzu, Kyoto, Japan). In total, twenty-one elements were analyzed: Al, B, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, Si, and Zn.

Instrument calibration was performed using serial dilutions of single-element standard solutions (Inorganic Ventures, Christiansburg, VA, USA) and fitting the calibration curves with linear regression (R² > 0.999). To optimize for signal saturation, low-concentration elements were measured axially and macroelements were measured radially. Plasma was generated using argon gas at 99.999% purity (Linde Gases, Ananindeua, Brazil).

2.5. Data Analysis

All collected data were processed in TIBCO Statistica (v14.1.0.8). A one-way ANOVA was performed. Levene’s test was used for homogeneity of variance testing and Fisher’s post hoc test for significance and simultaneous confidence intervals. Least significant differences (LSD) at p < 0.05 were determined from comparisons of the mean values.

3. Results

3.1. Morphological Characteristics

Morphological characteristics of the plants were evaluated based on the number of leaves, height of the plant, diameter and mass of the lettuce head, and dry weight of the plant. All measurements were done on four biological replicates per treatment.

The mean values for each treatment in three measurements are presented in

Table 1. Mean values of morphological characteristics. C—control; U—urea; UP—urea + PAW; *—statistical significance; n.s.—no statistical significance; values for different treatments within one measurement followed by different lowercase letters are statistically significantly different (LSD) at p < 0.05.

Measurement	Treatment	Number of Leaves	Height (cm)	Head Diameter (cm)	Head Mass (g)	Dry Matter (g)
1st measurement	C	11.5 b	8.98 b	10.95 b	5.13 b	0.41 b
	U	16.25 a	12.93 a	15.1 a	16.32 a	1.31 a
	UP	16.5 a	11.33 a	15.83 a	13.8 a	1.14 a
F test		* (p < 0.05, F = 6.11)	* (p < 0.05, F = 11.68)	* (p < 0.05, F = 11.89)	* (p < 0.05, F = 13.34)	* (p < 0.05, F = 12.12)
2nd measurement	C	25.75	12.33	20.83 b	51.15	6.23
	U	27.5	14.83	21.25 b	61.82	7.8
	UP	29.75	13.23	24 a	66.79	8.58
F test		n.s. (p > 0.05, F = 0.97)	n.s. (p > 0.05, F = 2.33)	* (p < 0.05, F = 9.69)	n.s. (p > 0.05, F = 1.96)	n.s. (p > 0.05, F = 1.5)
3rd measurement	C	45.75	11.75	23.75	97.1	11.18 b
	U	51.75	12.5	27.13	150.46	15.91 a
	UP	52	15.5	22.5	135.21	18.01 a
F test		n.s. (p > 0.05, F = 0.49)	n.s. (p > 0.05, F = 0.67)	n.s. (p > 0.05, F = 0.96)	n.s. (p > 0.05, F = 2.63)	* (p < 0.05, F = 10.24)

. For every characteristic, mean values in the first measurement show a statistically significant difference between the control treatment plants grown without nitrogen supplementation and plants supplemented with urea and urea/PAW, where values of the former treatment are lower than that of the latter treatments. There is no statistically significant difference between plants grown with urea and those grown with urea/PAW.

In the second measurement, the characteristics show the same trend of lower values in the control treatment plants, but there is no statistically significant difference, except for the measurement of lettuce head diameter.

In the third measurement, there is again a slight trend of higher values in plants grown with urea and urea and PAW, but not significantly different except for the dry matter measurement. Dry matter value of the control treatment plants is significantly lower than of those grown with urea and urea/PAW.

3.2. Elemental Analysis

The ICP—OES analysis was performed on the samples collected at the three timepoints in the life cycle of the plants. In total, 21 elements were measured and the results were divided in three groups for the easier interpretation: essential macroelements (P, K, Ca, S and Mg), essential microelements (Fe, B, Mn, Zn, Cu, Mo and Ni), and non-essential elements, minerals, and heavy metals group (Al, Cd, Co, Cr, Li, Na, Pb, Se and Si). The results presented are mean concentration values of four replicates for each measurement for each of the treatments, expressed in mg kg⁻¹ on a dry weight basis.

Macroelement measurements are presented in

Table 2. Mean concentrations of essential macroelements measured by ICP—OES, expressed as mg kg⁻¹ on a dry weight basis. C—control; U—urea; UP—urea + PAW; *—statistical significance; n.s.—no statistical significance; values for different treatments within one measurement followed by different lowercase letters are statistically significantly different (LSD) at p < 0.05.

Measurement	Condition	P	K	Ca	S	Mg
1st measurement	C	3507	37,233	14,204	2643	4585
	U	3783	42,303	11,983	2560	4711
	UP	3850	43,603	11,786	2407	3865
F test		n.s. (p > 0.05, F = 0.44)	n.s. (p > 0.05, F = 2.01)	n.s. (p > 0.05, F = 4.1)	n.s. (p > 0.05, F = 0.5)	n.s. (p > 0.05, F = 0.86)
2nd measurement	C	2475	21,645	9643	1713	4410
	U	2548	24,632	8932	1768	4365
	UP	2756	28,014	8637	1351	2671
F test		n.s. (p > 0.05, F = 1.48)	n.s. (p > 0.05, F = 2.6)	n.s. (p > 0.05, F = 0.32)	n.s. (p > 0.05, F = 1.44)	n.s. (p > 0.05, F = 2.53)
3rd measurement	C	2849 a	24,639 a	9640 a	1347	2798 ab
	U	2268 b	17,740 b	7633 b	1429	3502 a
	UP	2573 ab	16,907 b	7641 b	1138	2328 b
F test		* (p < 0.05, F = 5.71)	* (p < 0.05, F = 19.74)	* (p < 0.05, F = 6.75)	n.s. (p > 0.05, F = 1.51)	* (p < 0.05, F = 5.23)

and show no statistically significant difference between the three treatments for the first two measurements. Concentrations of phosphorus and potassium are noticeably higher in plants supplemented with urea, and even higher in those supplemented with urea/PAW in the first two measurements, while concentrations of calcium and sulfur follow the opposite pattern: lowest values are in the urea/PAW plants. In the final measurement, K and Ca in the control plants are significantly higher than in both the urea and urea/PAW plants, which are not significantly different between each other. P concentrations are highest in the control plants and lowest in the urea-supplemented plants. Magnesium concentrations are highest in the urea-supplemented plants and lowest in the urea/PAW treatment, and that difference is statistically significant.

The results for microelements are presented in

Table 3. Mean concentrations of essential microelements measured by ICP—OES, expressed as mg kg⁻¹ on a dry weight basis. C—control; U—urea; UP—urea + PAW; *—statistical significance; n.s.—no statistical significance; values for different treatments within one measurement followed by different lowercase letters are statistically significantly different (LSD) at p < 0.05. Concentrations of Cu, Mo and Ni were below limits of detection (LOD) of the instrument.

Measurement	Treatment	Fe	B	Mn	Zn
1st measurement	C	380.58	9.98 b	60.45	24.67
	U	168.61	9.72 b	41.43	21.09
	UP	153.14	11.16 a	57.15	26.98
F test		n.s. (p > 0.05, F = 1.9)	* (p < 0.05, F = 5.91)	n.s. (p > 0.05, F = 4)	n.s. (p > 0.05, F = 0.46)
2nd measurement	C	53.64	10.61	43.74	18.15
	U	65.29	9.78	42.24	15.83
	UP	81.07	10.23	36.44	11.4
F test		n.s. (p > 0.05, F = 0.27)	n.s. (p > 0.05, F = 0.26)	n.s. (p > 0.05, F = 0.27)	n.s. (p > 0.05, F = 2.27)
3rd measurement	C	224.76	14.77 a	31.96	10.82
	U	181.02	9.3 b	28.53	13.03
	UP	230.08	9.97 b	22.17	9.08
F test		n.s. (p > 0.05, F = 0.21)	* (p < 0.05, F = 19.49)	n.s. (p > 0.05, F = 3.09)	n.s. (p > 0.05, F = 1.89)

, with levels of copper, molybdenum and nickel being below the limits of detection (LOD) of the ICP—OES instrument. Concentrations of boron in the first measurement are at the significantly highest level in plants supplemented with urea/PAW, but do not show a significant difference in the second measurement, and are highest in the control plants in the third measurement. Iron, manganese and zinc show no statistically significant differences between the treatments in all three measurements; however, Mn and Zn show a trend of lower mean concentration for plants supplemented with urea/PAW in the last two measurements.

Table 4. Mean concentrations of non-essential elements, minerals and heavy metals measured by ICP—OES, expressed as mg kg⁻¹ on a dry weight basis. C—control; U—urea; UP—urea + PAW; *—statistical significance; n.s.—no statistical significance; values for different treatments within one measurement followed by different lowercase letters are statistically significantly different (LSD) at p < 0.05. Concentrations of Cd, Co, Cr and Pb were below limits of detection (LOD) of the instrument.

Measurement	Treatment	Al	Li	Na	Se	Si
1st measurement	C	500.08	7.19	5492	1.1	437.59
	U	224.81	7.32	4711	1.64	358.11
	UP	199.42	6.94	4507	1.04	326.59
F test		n.s. (p > 0.05, F = 1.12)	n.s. (p > 0.05, F = 0.6)	n.s. (p > 0.05, F = 1.85)	n.s. (p > 0.05, F = 1.61)	n.s. (p > 0.05, F = 1.15)
2nd measurement	C	89.34	7.49	8505	1.61	237.32
	U	103.34	7.53	10,361	1.49	257.51
	UP	138.87	7.7	5859	1.72	312.34
F test		n.s. (p > 0.05, F = 0.42)	n.s. (p > 0.05, F = 0.25)	n.s. (p > 0.05. F = 4.16)	n.s. (p > 0.05, F = 0.34)	n.s. (p > 0.05, F = 1.52)
3rd measurement	C	351.27	7.71	5766 b	1.28	492.42
	U	270.74	7.99	9254 a	1.44	427.8
	UP	385.83	7.63	5296 b	1.23	449.23
F test		n.s. (p > 0.05, F = 0.47)	n.s. (p > 0.05, F = 1.95)	* (p < 0.05, F = 10)	n.s. (p > 0.05, F = 0.21)	n.s. (p > 0.05, F = 0.54)

presents non-essential elements, minerals and heavy metals. Cadmium, cobalt, chromium and lead concentration levels were below the limits of detection of the instrument in all of the samples. Sodium concentration levels are significantly different between the treatments in the third measurement, which, in addition to a non-significant difference in the first and second measurement, indicates a trend in sodium concentration levels being lowest in plants supplemented with urea/PAW.

Aluminum concentration levels show no significant differences between treatments, although they do show a slight trend of higher values in plants supplemented with urea/PAW during the progression of the life cycle of the plants. Lithium, selenium and silicon concentration levels show no significant differences or trends across measurements.

4. Discussion

Plasma-activated water is an aqueous solution of reactive oxygen and nitrogen species: hydrogen peroxide, nitrate and nitrite, nitric oxide, and ammonium, among others, depending on the process of production, duration of the treatment, type of gas used, etc. [33]. This makes it an interesting alternative for use in delivering nitrogen supplementation to crops.

In previous studies [20,21,34] of the PAW treatment on lettuce growth, biometric measurements of lettuce plants showed a significant positive effect of PAW supplementation on several parameters, such as the diameter and mass of the lettuce head, dry matter of the plants, heights of the rosette, and the number of leaves, in comparison to the control plants, which were not under any nitrogen treatment. This was in accordance with similar studies on radishes [24], tomatoes [23], microalgae [25], green leafy vegetables [35] and lettuce [27,36], among others, which found that PAW treatment promotes germination, early seedling growth, and biomass increase. Nitrates in PAW are a source of nitrogen that is easily accessible to plants. Nitrites in PAW are not an accessible source of nitrogen, but in combination with H₂O₂, nitrites could serve as a source of mild abiotic stress that promotes germination.

In the present experiment, the results of PAW treatment on lettuce growth and quality were compared to the results of the urea fertilizer treatment. Ten percent of the total supplemented nitrogen from urea was replaced with an equivalent amount of nitrogen from PAW. The morphological parameters of the plants treated with urea and urea/PAW were significantly higher in early growth than those of plants without any treatment (Table 1). There was no statistically significant difference between the two treatments. In later measurements and for most parameters, the plants under both treatments had non-significantly higher values than the control plants. Dry matter weight is the only parameter that was significantly higher in urea and urea/PAW plants compared to the control plants in the third measurement. These results are in accordance with past studies on lettuce and melon, which show positive effects of PAW on growth parameters and biomass of plants, and its ability to replace conventional fertilizer [19,31]. Experiments with replacing more than 10% of urea nitrogen with PAW could elucidate whether PAW treatment has more beneficial effects on lettuce growth than conventional nitrogen fertilizer treatment.

In previous studies on lettuce and spinach, concentrations of some macro- and microelements and secondary metabolites in plants under PAW treatment and different growing conditions (greenhouse and open field) were measured. No statistically significant effect of PAW was observed, though levels of some elements were observed to be higher in plants treated with PAW, such as phosphorus and potassium in spinach [37] and potassium, calcium and iron in lettuce, but these differences were also dependent on growing conditions [20].

In this research, analysis of elemental composition of plants under urea and PAW treatments was conducted. In total, 21 elements were analyzed, out of which five are essential plant macroelements: P, K, Ca, S and Mg. Nitrogen and carbon are not included in the analysis because of the constraints of the sample digestion method and ICP—OES detection limits. The values measured represent total elemental content in leaves and stems of lettuce plants, without information about the chemical species or bioavailability of the elements. Macroelement concentrations of potassium and calcium were significantly lower in treated plants compared to the control in the final measurement, and there was no significant difference between the two treatments for those two elements. Phosphorus concentration was also not significantly different for the two treatments in the same measurement, but it should be noted that the PAW-treatment levels were between the control and urea treatment levels. Low greenhouse temperatures could act synergistically with different regimes of fertilization (watering with PAW) to produce this observed difference [38]. In an experiment on melon plants treated with wastewater and plasma-treated wastewater, Čechová et al. [31] found that K concentrations in melon plants were significantly increased in only one treatment of plasma-treated tap water, but generally did not show a significant increase in wastewater and plasma-treated wastewater conditions. Most studies on PAW treatment reported increased concentrations of P and K in PAW-treated plants [26,30,32,39], although Škarpa et al. reported no effect on nutrient composition with foliar treatment of PAW [22]. Calcium levels were significantly higher in the control plants in the final measurement, but there was no significant difference in calcium levels between the two treatments.

Magnesium concentration levels were significantly lowest under PAW treatment in the final measurement and similar to the control plants. Previous research reported contradicting results of PAW influence on Mg levels, with Nicoletto et al. [26] reporting decreased levels of Mg²⁺ in root biomass of PAW-treated lettuce, while others observed either no effect [22] or positive effect of PAW on Mg concentrations in plants [31]. The decrease in Mg concentration under PAW treatment may be attributed to the chemical footprint of PAW, specifically its low pH and high concentration of H⁺ ions [35]. Increased proton activity can lead to a competitive inhibition of cation uptake at the root plasma membrane. Furthermore, the presence of RONS in PAW may trigger signaling pathways that regulate ion-specific channels [40]. Contradicting previous results on Mg levels are potentially due to variability in pH and RONS concentrations in plasma-activated water in different studies.

Out of the measured microelements, only boron showed a significant difference between the urea and urea/PAW treatments, although only during the early plant growth. In the third measurement, B levels in the treatment plants were significantly lower than the control, but there was no significant difference between the treatments. Other microelements were not significantly different in concentration levels between treatments (Table 3). Kuzin et al. reported increased B levels in apple leaves after PAW treatment and foliar supplementation with micronutrients, which corresponds with our early measurements [14]. Boron is shown to be more bioavailable in soil in conditions of lower pH, which is the case in PAW-treated plants [40], suggesting that PAW may initially enhance the permeability of root membranes or increase the expression of boric acid channels (NIPs). The subsequent decline to levels below the control in the third measurement might reflect a “dilution effect”, where PAW-stimulated biomass production outpaces the rate of boron acquisition, a common phenomenon in high-growth treatments.

Concentrations of elements that are toxic or cause stress in plants, and are considered pollutants (Al, Cd, Co, Cr, Pb, Li), were also measured, in addition to Na, Se and Si—non-essential elements but beneficial for plant growth. Sodium concentration in the urea/PAW plants was significantly lower than in the urea-treated plants (Table 4). Wang et al. [30] reported a significant increase in Na concentration in wheat plantlet juice in PAW-treated wheat, while Nicoletto et al. reported decreased Na content both in aerial and root biomass of lettuce, demonstrating contradicting results for this non-essential element [26]. Elevated Na concentrations are known to activate plant stress responses [41]. Given that hydrogen peroxide in PAW can function as a signaling molecule under low-stress conditions, the lower Na levels observed in PAW-treated plants may reflect redox-mediated priming of stress response mechanisms that reduce Na uptake. The observed decrease in Na is particularly interesting from an agronomic perspective, as it suggests PAW might alleviate salinity stress by restricting the accumulation of non-essential, potentially toxic ions, even if Mg uptake is slightly suppressed as a trade-off.

In the species-specific study on PAW treatment of several crop species, the positive effect of PAW on growth parameters and biochemical composition of plants varied highly between species. It also depended on the varied duration of plasma treatment of water [42]. This was reflected in the chemical composition of plants, i.e., the concentrations of anions and cations. So, for example, a positive effect of PAW on K⁺ ion concentrations was recorded for tomato, basil, Swiss chard and cabbage; on Na⁺ and Mg²⁺ ion concentrations in lettuce; and Ca²⁺ ion concentrations in basil, Swiss chard and lettuce.

The chemical composition of plants determines the nutritional value of crops, since all essential plant elements and some other trace elements in plants are also essential in humans and animals. It correlates with the commercial value of plants—extended shelf life and enhanced organoleptic characteristics of fruit [43,44]. Elemental composition also plays a role in protection from pests and microbial diseases [14,45].

Our study confirms that PAW can replace a portion of traditional fertilizer without a loss in the growth or mineral composition of lettuce. A relatively low level of nitrogen replacement (10%) with reactive nitrogen species from PAW could be the reason that no significant differences in treatment were observed for most of the studied characteristics. Significant differences in Mg, Na and B levels in PAW-treated plants, however, warrant further studies with higher levels of PAW-derived nitrogen replacement. Nevertheless, the observed effects on these three elements did not contribute to any negative effect on the lettuce yield.

5. Conclusions

The replacement of 10% of the total nitrogen supplemented by urea in the treatment with an equivalent amount of nitrogen from plasma-activated water did not negatively affect the growth parameters of lettuce plants. The plants supplemented with both urea and urea/PAW showed significantly higher values than the control plants in several growth parameters during the early life stage, and had higher values for the same parameters in the later growth phase, although these were not statistically significant. The results confirmed our hypothesis that replacing a portion of chemical fertilizers with PAW’s greener technology will not negatively affect lettuce growth.

Mineral composition measurements of the lettuce showed no significant difference between nitrogen treatments for almost all of the elements investigated. Literature on the effect of PAW on mineral composition of plants reports various and sometimes contradicting results, dependent on plant species, method of PAW generation, method of PAW application in plant growth, and other growth conditions. This study provides a comprehensive analysis of the plasma-activated water effects on 21 elements, across plant growth stages, representing one of the first broad elemental assessments in soil-grown plants. Observed limited effects of PAW for elements such as Mg, Na and B point to the influence of PAW on ionic regulation rather than overall nutrient accumulation. These findings have practical relevance for horticultural applications, as they indicate that PAW use may modify stress-related ion management. This study employed a conservative approach in PAW usage, with only 10% of PAW-derived nitrogen as a replacement. Therefore, examining the effect of higher PAW percentages in total nitrogen supplementation and evaluating species-specific mineral composition in response to the PAW treatment are necessary to better understand the influence of PAW on plant growth and element uptake.