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Article

Crystal Formation in Solanum lycopersicum L. Leaves Under Antibiotic Stress Reduced by Non-Thermal Plasma Treated Water

by
Marius Cicirma
1,2,
Aurora Daniela Neagoe
1,3,4,*,
Mirela Nedelescu
5,*,
Adrian Ionascu
1,
Marius Dumitru
2,
George Dinca
3,6 and
Sergiu Emil Georgescu
1
1
Faculty of Biology, University of Bucharest, Splaiul Independenței, No. 91–95, 050095 Bucharest, Romania
2
National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Str., 077125 Magurele, Romania
3
“Dan Manoleli” Research Centre for Ecological Services (CESEC) and “Dimitrie Brandza” Botanical Garden, University of Bucharest, Intrarea Portocalelor, No. 1–3, 060101 Bucharest, Romania
4
Research Institute of the University of Bucharest—ICUB, Panduri Road, No. 90–92, 050663 Bucharest, Romania
5
Department of Hygiene and Environmental Health, Faculty of Medicine, University of Medicine and Pharmacy Carol Davila, 020956 Bucharest, Romania
6
Geological Institute of Romania, Caransebes Street, No. 1, 012271 Bucharest, Romania
*
Authors to whom correspondence should be addressed.
Crops 2026, 6(2), 35; https://doi.org/10.3390/crops6020035
Submission received: 9 February 2026 / Revised: 12 March 2026 / Accepted: 16 March 2026 / Published: 20 March 2026
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Abstract

Calcium oxalate (CaOx) crystals in plants can form naturally within their idioblasts but may also be induced by other factors, such as environmental pollution. Here, we report qualitative and semiquantitative results obtained using scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) from two experiments in which tomato seedlings were moderately irrigated with Sulfamethoxazole (SMX) and Amoxicillin (AMX) solutions (0.25 mM). Abundant prismatic CaOx co-crystals appeared on the leaf surface induced by these two antibiotics compared to the distilled water (DW) control. Applying a non-thermal plasma (NTP) treatment for 20 min (T20) to the SMX initial solution led to a dramatic suppression of these crystals, with a shift toward spherical structures. Furthermore, the investigation into the composition of both crystal types, indicated different percentual levels of O, C, Ca, K, Mg, S, and Mn as main constituent minerals involved in crystal formation. However, crystal morphology was affected by each applied experimental condition, while detecting their constituent elements depended on their mineral homogeneity at the micro- or macro-field scales. Although both antibiotics induced crystal formation and T20 phenotypically reduced the abundance of the acicular–prismatic crystals by removing the effects of SMX, their mode of action has not yet been clarified.

1. Introduction

Water scarcity, especially in Mediterranean countries, has led to the use of reclaimed water from wastewater treatment plants in agriculture, although it may contain trace pharmaceuticals [1,2]. The accumulation of pharmaceuticals in crops, including antibiotics, is mainly caused by irrigation with improperly treated reclaimed wastewater and by the use of organic amendments of animal origin [3,4]. Consequently, these compounds have also increased in agricultural ecosystems through the use of this type of water for crop irrigation [5,6,7]. Although research solutions for wastewater treatment have been extensively documented, [8,9,10,11], the problems of microorganism resistance [12] in human disease treatments [13] are still unsolved.
As the removal capacity of such micropollutants in regular wastewater treatment plants is limited, the potential risk to humans from consuming vegetables contaminated with various pharmaceutical classes is a concern [3,14,15,16]. Although the bioaccumulation of xenobiotics in plants may not always be harmful if ingested [17], the occurrence of calcium oxalate crystals (CaOx) [18,19], as a consequence of soil pollution, might pose a real threat. Although this is one of many defense mechanisms in plants specialized to fight against organic pollutants (e.g., antibiotics) or inorganic pollutants (e.g., heavy metals), it can also be used against herbivorous animals [20,21,22].
Recent research has elucidated key genes involved in the formation of CaOx crystals in plants, which, in addition to their physiological role and broad ecological implications, also contribute to carbon sequestration [23]. Also, the formation of CaOx in plants is relevant to understanding Ca transport and regulation, oxalic acid biosynthesis, and Ca levels in plant tissues and organs. Given that most plants form CaOx crystals, it is interesting to observe to what extent their development can be affected by various soil treatments in which they grow.
It has been shown that SMX may induce abundant crystals on the surfaces of tomato leaves [24], and it has also been linked to the medical field [25]. However, for the study presented in this article, the aim was to understand the formation of these crystals (CaOx). Calcium crystal formation in plants is extremely poorly documented [26], and for this reason, it is also important to determine their mineral composition and how antibiotic irrigation treatment influences them. In contrast, the use of NTP treatment for polluted waters containing antibiotics (including SMX or AMX) on a laboratory scale, using ozonation and pulsed corona discharge systems, has been proven to be successful [27,28,29]. However, their potential applications are still under continuous improvement, considered a new environmentally friendly technique, and recommended for use in agriculture [30].
The objective of this research was to demonstrate whether the presence and abundance of crystals, as opposed to their shape and size, are due to their chemical composition and appearance as a consequence of the use of SMX or AMX irrigation solutions. To address this objective, the following working hypothesis was tested: In Solanum lycopersicum L. species irrigated with initial SMX or AMX solutions, a large amount of SMX and AMX will lead to an increased abundance of crystals, while plants irrigated with NTP-treated solutions are expected to have similar abundance and mineral compositions as controls.

2. Materials and Methods

Experimental design
Two toxicity experiments were performed on tomato seedlings exposed to 0.25 mM SMX (TCI, CAS 723-46-6, >98.0%) and AMX (TCI, CAS 61336-70-7, >98.0%) from Tokio Chemical Industry Co., Ltd. (Tokyo, Japan), for 30–40 days. Both experiments were performed using 500 mL polyethylene pots filled with 450 g of soil, in which 12 tomato seeds were sown (at a 2 cm depth), with 5 replicates for every applied irrigation treatment. The water-holding capacity (WHC) of the soil was measured, and the plants were irrigated daily to maintain the initial (T0) soil humidity. Treatments were applied on day 0, days 1–15, 18–20, and on day 25 after sowing. Between the applied solutions for irrigation, distilled water was used to alternate the watering treatments until day 40.
The first experiment was carried out under fully controlled conditions in a growth chamber (Daihan LabTech Co., Ltd., Namyangju, Republic of Korea) for 30 days (25/19 °C with a 16/8 h day/night cycle, light intensity of 5000 Lux, and 60% humidity). Six different treatments, with 5 replicates each, were applied using seeds of Solanum lycopersicum L., Zaraza variety (Four Agro, Bucharest, Romania).
The NTP technique promotes partial or total degradation of SMX, depending on the exposure time. Preparation of the NTP solutions was performed in a multi-wire-to-plate plasma reactor using pulsed filamentary corona discharge in a gas phase (oxygen) above the initial solution. The treatment solution was circulated continuously between the plasma reactor and the ozonation reactor by a peristaltic pump, with oxygen added at a flow rate of 300 mL min−1. The ozone generated in the gas phase of the discharge bubbled through the solution in the ozonation reactor, thereby enabling the in situ formation of reactive species. After treatment, the final irrigation solution acronyms were defined as follows: DW—distilled water control; SMX—initial solution (0.25 mM) dissolved in tap water; TW—tap water as solvent control; T5—SMX solution treated for 5 min using an NTP ozonation technique [31], containing approximately 15 mg L−1 SMX at the end of the plasma treatment; PAW—tap water treated by NTP for 20 min (plasma-activated water control); and T20—initial SMX solution treated with NTP for 20 min, with the parental compound completely degraded [31]. This NTP treatment of polluted water in a circulating ozonation system required 20 min for complete SMX degradation and additional time for handling the solutions. The final restrictive volume after each cycle was 330 mL of treated solution. The plants were allowed to grow to the seedling stage by alternating the prepared irrigation solutions with distilled water. This decision was made both to avoid excessive antibiotic exposure to the plants and to maintain soil moisture in accordance with its initially measured water-holding capacity.
In the second experiment, the same concentration of 0.25 mM was used for both the SMX and AMX initial solutions. No NTP water treatment was used. Seeds of Solanum lycopersicum L., Buzău 47 variety (S.C.D.L. Buzau, Buzau, Romania) were sown. The plants were irrigated for 40 days under greenhouse conditions (Dimitrie Brandza Botanical Garden, University of Bucharest), with antibiotic solutions alternating with distilled water. The experimental treatments were also selected based on the names of the irrigation solutions, as follows: DW—distilled water (control); SMX and AMX solutions, each with 5 replicates.
To ensure data quality, two tomato leaves were randomly collected from each experimental treatment of the two described experiments. The leaves were lyophilized to maintain the three-dimensional structure and minimize leaf shrinkage, thus preventing the appearance of deformation artifacts that occur during air drying. Before scanning, they were coated with gold [32], allowing the collection of high-resolution images using SEM and EDX techniques for qualitative and semi-quantitative results.
The leaf microarchitecture, crystal morphology, and element composition were investigated with a Hitachi TM3030 SEM (Hitachi, Tokyo, Japan) operated at an accelerating voltage of 15 kV. The elemental analyses were collected using a Bruker QUANTAX 70 EDS system (Bruker, Billerica, MA, USA) (percentual data) and a 15 kV SEM FEI Inspect S50 m (FEI Company, Hillsboro, OR, USA) for counts-per-second spectrum data. Measurements of seedling root and shoot lengths were performed manually using a ruler (cm), and their weights (g) were measured with an analytical balance.
Statistical analysis
Correlations between the applied irrigation treatments, which might contribute to crystal formation in a SMX concentration-dependent manner, were performed. Principal Component Analysis (PCA) was implemented using raw data for physicochemical and geochemical variables from soil, as well as for morpho-physiological and biochemical variables from plants [24]. These data were analyzed in R version 4.4.2 [32] with RStudio [33] IDE version 2024.12.0.467 [34] to assess correlations among the irrigation solutions. Therefore, an original R script was specifically designed for data analysis using dplyr [35], ggplot2 [36], nortest [37], tseries [38], car [39], stats [40], FactoMineR [41], and factoextra [33]. Statistical significance was considered for p ≤ 0.05 at 95% confidence. The annotations used in PCA implementation are defined as follows: pH; electrical conductivity (EC); loss on ignition (LOI); moisture (H); dissolved inorganic nitrogen (DIN: N-NH4+, N-NO3−, N-NO2−); bioavailable phosphorus (P-PO43−); mineral elements (Ca, Cu, Fe, K, Mn, Zn, As, Ni, Pb, Rb, As, Sr); percentage of seed germination (X_seed_G); root and stem length (R and S_length); dry biomass of roots and stems (dw_R and dw_S); lipid peroxidation in roots and stems (MDA_R and MDA_S); assimilatory pigments (Chl_a, Chl_b), total chlorophyll (Chl_T), chlorophyll a/b ratio (Chl_a/Chl_b), and carotenoids (Carot); and stomatal conductance (SC). These variables were previously analyzed, interpreted, and reported using Origin® 6.0 statistical and graphical software separately [24].

3. Results

The T20 microphotograph shows a reduction in prismatic crystal formation and their abundance in Solanum lycopersicum L. leaves (var. Zaraza) compared with the SMX treatment. The SMX and AMX antibiotic solutions used to irrigate the Buzau 47 (II) variety both induced prismatic crystal formation, but their shapes were not stellate or acicular, as observed in the first experiment (I). The two tomato varieties (Figure 1—DW for Zaraza and Buzau 47) used in the two phytotoxicity tests responded similarly under SMX treatment (Figure 1—I and II, SMX), regardless of the environmental conditions applied (vegetative chamber versus greenhouse). However, the prismatic crystals induced by SMX, in the Zaraza variety, appeared prismatic–acicular, whereas in the Buzau 47 variety, they had cubic or diamond shapes. A similar crystal shape was obtained in the SMX II treatment induced by the 0.25 mM AMX antibiotic (Figure 1, AMX). The general appearance of the plants suggests an advanced state of calcification, as observed in the SEM microscale images (Figure 1, SEM).
In the subsequent stage of the investigation, attention was directed to analyzing the crystal composition using EDX to identify their elements. The crystal composition of the SMX sample was first analyzed to ascertain whether the concentrations of the detected elements varied across different surface points.
The percentage ratio of each selected element within an SMX-induced crystal was broadly equally distributed from five different measured sample points consisting of C, O, S, Ca, and K. Therefore, we assumed that the mineral assembly did not change between probes or sample points and had stable percentage proportions of elements. As can be seen in Table 1, the percentage ratio of each selected element was broadly equally distributed across the different measured sample points from S1 to S5. Therefore, the percentage proportions of elements within the crystal’s mineral assembly did not change and remained stable in the SMX (I) treatment. It may be noted that the C element was deliberately removed from the determinations and list in Table 1 to ensure better percentage accuracy for the Ca, S, and K elements of interest.
We also carefully investigated whether the crystal morphology varied within the same treatment (SMX I) across the same samples used for EDX. Overall, we observed that acicular needles had a central basal fixing point and a radial distribution on the opposite extremity.
Next, the SMX- and AMX-treated leaf samples were analyzed by EDX to obtain the main element spectrum and compare their scalar levels between the irrigation treatments used in the second experiment (Buzau 47 variety). The automatic detection identified O, C, Ca, K, S and Mg, ranging between 200 k and 300 k counts per second for the DW, SMX and AMX treatments, as shown in Figure 2. In the distilled water control (Figure 2A), the C element ranged around 175 k cps, whereas in the AMX treatment, it was at 200 k cps (Figure 2C), followed by the SMX treatment with C reaching 250 k cps (Figure 2B). Although the C level fluctuated between treatments, its trend remained constantly higher than the O level across all samples. Mg, S, Cl, and Ca exhibited appropriate levels between the DW and SMX treatments. In contrast, K, Ca, and S showed much higher peak concentrations in the AMX treatment compared to DW and SMX (Figure 2D). The Au element was also detected in all samples since all probes were gold-coated.
As EDX element profiling also facilitates the visualization of each element distribution within a sample of interest, a colored map was produced within an SMX-induced crystal using the Buzau 47 leaf variety. In Figure 3, the color intensity of the main constituent elements differs between the vegetal tissue and crystal compositions: Ca (turquoise), K (light violet), and S (dark violet). Mg, O, and C are shared elements between vegetal tissue and crystals, as shown in field EDX analyses, EDX map and in point analyses of two different crystal types.
Next, the SMX solution was subjected to an NTP treatment for 20 min (T20resulting in its complete removal from the initial solution. The resulting sample (T20) was compared with an incomplete removal treatment of the SMX after 5 min of NTP action (T5). Also, the sample was compared with a negative SMX-containing control to assess any plasma potential effects after 20 min of NTP treatment, as well as with plasma-activated water (PAW). SEM high-magnification (HM) microphotographs (between 5 and 20 μm) of the plasma-treated plants (T20) indicated the presence of spherical structures (s) on the leaf surface, similar to those found in the TW solvent control. Such forms were associated with spherical-like crystals and were also visualized in the PAW plasma control and T5 treatments. Also, prismatic crystals (p) were present in the PAW, T20, and T5 irrigation treatments, along with the previously mentioned spherical ones (Figure 4).
In the final phase of the study, the compositions of the two crystal types detected in each treatment sample (Figure 4) were investigated using point analyses (with a resolution of less than 10 μm). These analyses were conducted using the SEM-EDX technique for each treatment in isolation. The automatically detected elements, extracted and displayed as percentages, were analyzed directly for each crystal type in the spherical (s) and prismatic (p) cases. The obtained results for the main elements were confirmed in the EDX spectrum of Solanum lycopersicum L. leaf samples from Buzau 47 variety (Figure 2 and Figure 3), namely C, O, K, Ca, S, and Mg. However, other elements, such as Cl, P, and Mn, although present, appeared in very low amounts and not considered. Therefore, the specific element profiling screening at the microscale level (point analyses) revealed percentage differences between the two types of crystals and between the irrigation treatments for spherical (TWs, PAWs, T20s, and T5s) and prismatic (PAWp, T20p, T5p) crystals, as shown in Figure 5A–D. The significant impact on the classification of the two observed crystals is displayed in Figure 4. The high and low percentages of the displayed minerals indicate that the combination of these elements may play a role (Figure 5) in crystal formation or crystal growth mechanisms. The elemental profile of the DW water control (black line) is used here to represent the physiological status of the plant. It is plotted alongside the spherical crystal profile found in the TW solvent control (red line), which serves as a marker of crystal growth initiation (Figure 5A). Here, the C level in the spherical crystal from TW decreases compared to that in the DW. In contrast, the Ca level increases sharply from 5% in the DW to 25% in the TW. Furthermore, the NTP treatment of TW for 20 min resulted in plasma-activated water (PAW) with properties distinct from those of the other irrigation treatments. At this point, the overall Ca level is midway between the DW and TW controls on the scale in Figure 5A. However, since the percentage level of the S element did not change much, uptake from soil through roots to shoots and bioaccumulation in plants may have occurred. Still, it was present at various percentage concentrations across crystal types. This is clearly displayed in all Figure 5 graphs, as both crystals contained S but at different levels. Based on Figure 5B,C and the patterns observed in Figure 4, NTP has the potential to reduce the SMX effect by alternating prismatic and spherical crystals through specific up-and-down shifts in detected mineral trends.
Regarding the morphometry of the Buzau 47 variety used in the repetition trial with the same SMX initial solution (II), the plant total length (roots and shoots) and the subsequently recorded fresh biomass were significantly smaller than in the control (DW). Small SMX-treated roots were also confirmed in the Buzau 47 (II) variety. In contrast, tomato roots showed no significant differences between the DW and AMX control groups but were larger than in the SMX treatment (Figure 6A). Similarly, the AMX-treated shoots, although significantly higher than those irrigated with the SMX solution, remained below the DW control scale (Figure 6A). The scale pattern of total seedling fresh weight is similar to the trend in plant lengths, even though it was measured in a composite sample (Figure 6A, Biomass). In contrast, the SMX-treated shoots were significantly smaller than the DW control in both experiments (varieties I and II).
The NTP-derived solution comparisons between PAW, T20, and T5 irrigation treatments reveal that root length was significantly reduced only by the PAW treatment compared to T5 (Figure 6B). At the same time, T20 had roots similar to PAW and T5, including the DW and TW controls.
The characterization of the irrigation solutions was performed using soil and plant variables. The raw data set used to illustrate the PCA (Figure 7 and Figure 8) showed a relatively low explained variance between the initial variables and the treatments applied to the plants, with Dim1 at 27.1% and Dim2 at 11.9%. However, the graphical representation of the mean values (n = 5) indicates two distinct but slightly interconnected groups: DW (control) with TW (solvent control), and PAW (plasma control) with T20 (solution without SMX after 20 min NTP treatment). In contrast, the variables obtained with the SMX and T5 solutions appear isolated on the same axis, but at large distances, and are associated with the presence of the SMX compound at different concentrations.
In this context, the first principal component may serve as a primary indicator of physiological vigor, as this dimension is heavily defined by high positive contributions from assimilatory pigments (Chl_a at 6% and Chl_b at 5.2%), total chlorophyll (Chl_T at 5.9%), carotenoids (Carot at 5.8%), and physical growth parameters, such as stem length (S_Length at 5.1%). As illustrated in Figure 7 and Figure 8, the control groups (DW, TW, and PAW) cluster in the positive half of Dim1. In stark contrast, the SMX and T5 groups are isolated in the negative half of Dim1. Interestingly, the graph illustrates a steady positive shift in Dim1-dominant variables from the SMX treatment group to the T5 and T20 groups.
Additionally, the second principal component primarily captures variance related to nitrogen speciation and chemical alteration, with the two dominant contributors to this axis being ammonia nitrogen (N.NH4 at 6.6%) and dissolved inorganic nitrogen (DIN at 6.6%), followed by pH (4.4%). This dimension effectively separates the plasma-treated groups (PAW and T20) from the standard controls. While the T20 group successfully returns to the positive side of Dim1, thus indicating a restoration of physiological vigor comparable to the SMX group, it remains elevated on Dim2 (Figure 7, Figure 8 and Figure 9).

4. Discussion

Observations of crystal formation in tomato leaves under SMX and AMX antibiotic treatments are depicted in Figure 1, Figure 3 and Figure 4, which show induction at a high concentration of 0.25 mM. Two intermediate crystal growth stages were detected after SMX concentration decreased following the 5 min action of NTP treatment of the initial SMX solution (15 mg L−1 SMX, T5)—Figure 4. A further decrease in abundance was observed after SMX was completely removed from the T20 treatment (Figure 1). As a general observation, a low abundance of spherical crystals occurred in the TW control (Figure 4). Despite the crystal shape and size, their general mineral composition did not change much. Thus, the constitutive element profiles detected by EDX, either as cps spectra (Figure 2 and Figure 3) or as percentages, are: O, C, Ca, K, Mg, and S. Moreover, various element concentrations were obtained, depending on the treatment applied or the magnification scale (point versus field analyses). Although the values of certain elements between sampling points varied little, as exemplified in Table 1, statistical differences in means can influence the initial interpretation of the data. For example, differences between Ca and S in PAWs and PAWp crystals were still obvious, as Ca decreased in the true prismatic crystal, whereas S slightly increased (Figure 5B). Similarly, as S was still present in both the T20 and T5 samples, it had the same concentration but was present in different metabolite types or other reaction by-products. Although the SMX-induced prismatic crystal effect remained, the K level likely influenced the plasma effect, reducing spherical crystal abundance and promoting the formation of spherical crystals. Conversely, judging by the limited sampling numbers, as indicated in Figure 10A, it can be concluded that an element (i.e., S) can occupy a stable percentage level in a mature prismatic crystal compared to a spherical one. This affirmation is supported by Table 1, which shows point analyses at different locations within a mature crystal (acicular–prismatic). In contrast, sulfur was not significantly different between a DW tissue control sample and an SMX-intoxicated leaf (Figure 10B), yielding semi-quantitative results. However, other present elements that were not considered may have been present at different concentrations (e.g., P, Mn, Cl, Cu, Au), as partially shown in Figure 2. These and other elements might be actively involved in crystal formation mechanisms under antibiotic stress induced by high SMX or AMX concentrations. Moreover, toxic elements can be present in the environment, ranked as non-essential plant toxic or carcinogenic (e.g., Cd, As, Al, Hg, or Pb), and detected in soil, air, and water, affecting plants [34]. Studies of two cacao tree cultivars with high Cd accumulation revealed that CaOx crystals were involved in Cd accumulation in branches. CaOx was present in all organs of both cultivars. In particular, mature leaves accumulated CaOx content above the 5% dry weight, a limit used to define extreme oxalate accumulation [35].
The herbicidal effect of the initial SMX solution [36] persisted in tomato seedlings (Figure 6A,B), as we observed in both a previous Petri dish experiment for roots [31] and in pots for stems [24]. Also, the AMX treatment reduced seedling size compared to DW (Figure 6B).
Overall, the intuitive mechanism of crystal formation might involve C-O balance, with P increasing and K decreasing, as shown in Figure 10B. However, as both S and K increased and the C-O ratio remained steady, according to Figure 2 (AMX), the contextual data must be carefully and critically tested, documented, and interpreted (Figure 10B).
In our experiments, crystal growth was dependent on the mineral element’s variation in the presence of antibiotic treatment solutions, as determined by the concentrations found in the irrigation treatment (SMX or AMX). The acicular shape or size is likely dependent on other factors such as species variety (Zaraza), antibiotic type (SMX), or experimental growth conditions (climate chamber).
Once Sulfamethoxazole (SMX) enters the soil, it is taken up by plants and enters their cells, where it affects their metabolism through transformation, conjugation, and sequestration reactions. N-glycolysis is the main transformation of SMX in Arabidopsis thaliana L., leading to the formation of S-containing metabolites. The main resulting metabolite is N4-glucosyl-SMX, which is further metabolized into other S-containing compounds [37]. In contrast, in [38], it was stated that the main metabolite of SMX formed in Oryza sativa L. was N4-acetyl-sulfamethoxazole (NASMX). NASMX accumulates mainly in roots, but also in plant tissues. The authors demonstrated that cytochrome P450, as well as acetyltransferases and glycosyltransferases, are involved in metabolite biotransformation, concluding that this antibiotic acts as a phytotoxic stress factor. Also, [39] demonstrated that SMX acts at the rhizosphere level by reducing the abundance of S-oxidizing bacteria (e.g., Sulfuricaulis), thereby influencing S assimilation by plants. Moreover, SMX induces the formation of reactive oxygen species (ROS), which intervene in plant defense mechanisms, thereby activating secondary metabolism and enzymes such as SOD, POD, and CAT [38]. The toxicity mechanisms of SMX also led to decreased photosynthetic efficiency and growth in Hordeum vulgare L. as SMX concentration increased [40]. In this case, the phytotoxicity of SMX manifests as inactivation of the oxygen evolution complex (OEC). This blocking disrupts the flow of electrons to the reaction center of photosystem II (PSII), thereby inhibiting the entire electron transport chain in photosynthesis. Therefore, SMX disrupts the plant’s nutritional and chemical balance, leading to the poor management of S as a macronutrient.
Sulfur crystals formed in plants have rarely been reported, with the literature reporting their occurrence on the surfaces of conifer needles or in the substomatal cavities of longleaf pine following acid rain. The mechanisms of S crystal formation and their role in plant function are unknown [26].
Although S from SMX, once it reaches the plants, is stored as metabolites resulting from detoxification, under the influence of microorganisms in the rhizosphere, the organic compound can also be mineralized. This phenomenon modifies the microbiome and induces the release of S from the organic compound in the form of sulfate ions (SO42−) [41]. It is likely that these ions, once absorbed into the plant cell wall or vacuoles, can precipitate as crystals containing S, similar to crystalluria in medicine.
In human and veterinary medicine, CaOx may act as a toxin or an antinutrient, leading to crystalline nephropathy or kidney stone formation [42,43,44].
Recent research has elucidated key genes involved in the formation of CaOx crystals in plants, which, in addition to their physiological role, have broad ecological implications [23]. The natural occurrence of CaOx crystals in plants has been documented since the 17th century [19] and is observed in most groups of photosynthetic organisms, such as algae, lower vascular plants, gymnosperms and angiosperms [18,45,46,47]. It has been shown that endogenous synthesis of oxalic acid is accompanied by a physicochemical precipitation, in addition to the uptake of Ca derived from the environment [19,47]. The localization of CaOx crystals can be found as intracellular or extracellular deposits. Intracellular crystals are mostly formed in the vacuoles of plant cells and are known as crystal idioblasts or are associated with plant walls as a result of a biomineralization process. It is interesting to note that the crystals have different shapes and sizes and are probably specific to each plant species [18].
Although the presence of CaOx crystals in plants, as well as the mechanisms leading to their formation, are well documented in the literature, the mechanism underlying the formation of CaOx-SMX crystals is still unknown [48].
Generally, the natural phenomenon of CaOx crystal deposition in plants could satisfy the calcium requirements of adjacent cells during their development [49]. However, many plant species contain secondary metabolites in large quantities, such as oxalates, alkaloids, terpenes, and phytotoxins, which can cause diseases or may even be lethal to animal and human populations. Among these metabolites, oxalates are metabolized the slowest or not at all; therefore, free calcium, which is toxic to cells, can induce the formation of calcium oxalate crystals, thus eliminating the excess calcium oxalate [22,50,51,52]. Although it is documented that calcium oxalate crystals can be present in almost all parts of plants, in our studies, we identified calcium oxalate crystals in tomato only in the leaves, induced by sulfamethoxazole and amoxicillin (Figure 1 and Figure 4). We also briefly explored the possibility of crystal formation in Solanum lycopersicum L. roots using SEM, and SMX crystals were not observed. Similarly, in Gomphrena claussenii, crystals were reported in stems and leaves but not in roots, and were associated with Cd [51]. The authors reported heavy metals, such as Cadmium (Cd), that may be involved in the co-localization and ligand coordination of crystals, with a greater abundance of Cd-O-C binding compared to S ligands (Cd-S-C). Such crystals can form independently of Ca supply [51]. Plants have developed intricate defense mechanisms to protect themselves against the toxic effects of certain elements [34], and the formation and function of CaOx may be associated with the elimination of excess pollutants. Plants lack a dedicated excretory system for eliminating solid waste, such as calcium salts [52]. Some defense mechanisms include compartmentalization, pollutant sequestration in cellular organelles, inactivation via complex formation with organic ligands, and exclusion via transporters, ion channels, transcription factors, and signaling molecules [34]. In contrast, it is known that CaOx crystal formation is dynamic and that it can be resorbed at different stages of plant development. It has been found that when idioblasts do not show signs of wall rupture, this phenomenon suggests resorption or internal consumption of CaOx crystals during anther maturation [49].
The PCA results (Figure 7, Figure 8 and Figure 9) for the variables measured in plant and soil suggest that the biological importance of the aboveground parts of plants is greater than that of the roots in this scenario. Analysis of the factors contributing to the variance revealed five measurements that explained most of the variance between treatments and were correlated with each other based on soil physicochemical and geochemical determinations, combined with plant morpho-physiological and biochemical variables. The assimilatory pigments, represented by chlorophylls and carotenoids in plants, together with dissolved inorganic nitrogen from soil, were overcome by physical growth parameters, such as stem length. Plant performance is also shown in Figure 6A,B. As illustrated in Figure 7 and Figure 8, the control groups of DW, TW, and PAW had clusters in the positive half of Dim1. This positioning might correlate with the optimal health of these plants, which is consistent with previous findings where these treatments showed minimal presence of surface crystals [24]. In stark contrast, the SMX and T5 groups were isolated at the negative half of Dim1. This spatial separation could reflect a “stress state” characterized by significantly reduced pigment concentrations and inhibited growth. This inhibition was likely driven by the high abundance of SMX-induced crystals on the tomato leaf surfaces, also identified in previous SEM-EDX analyses [24]. The vertical shift in nitrogenous species suggests that, while 20 minutes of NTP treatment effectively removed the SMX compound and reduced leaf crystal abundance to control levels, it may have simultaneously enriched the system with specific (NH4) slightly, affecting the pH profile of the irrigation solution.
Further research is needed to investigate the factors influencing crystal formation across crops and concentrations to narrow the range of plant stress conditions. The study of interactions between antibiotic residues and other pollutants in the plant–soil–water ecosystem has particular implications for assessing long-term ecological and human health risks.

5. Conclusions

The topic of crystal formation in plants under the SMX and AMX antibiotic treatments is a very new area of research that deserves further exploration. Our results reveal that using non-thermal plasma treatment for SMX-contaminated water (T20) led to a decrease in crystal density in Solanum lycopersicum L. leaves. Both antibiotics suppressed the growth of the two tomato seedlings used. Several main elements were shown to play an important role in the formation of such crystals at different concentration levels. These were O, C, Ca, K, Mg and S, but others, such as Mn, Cl and P, may play a role in CaOx formation under high concentrations of antibiotics. It was shown that NTP removed SMX from the initial solution, reducing the effect of crystal formations in T5 compared to the SMX treatment. Furthermore, crystal abundance was reduced much more than in the SMX and T5 treatments, comparable with PAW (no SMX present). Although the percentage of the main elements in spherical and prismatic crystals vary, the mechanism by which their abundance was reduced might be related to the SMX effect.
To expand the existing knowledge, further investigations are needed to examine the effects of different SMX and AMX concentrations on both tomatoes and other crop species. Furthermore, the temporal dynamics of crystal formation may provide insights into the mechanisms underlying plant stress responses, regardless of whether they manifest in the cotyledons or mature leaves. It is important to determine the metals and/or metalloids that may contribute to the formation of such crystals, followed by the use of PCA based on chemical elements detected in both soil and plants through root uptake. Moreover, fundamental research on the effects of pharmaceuticals could answer questions about crystal formation in edible plants if untreated wastewater is used in agriculture. Applications of NTP can provide key insights, improving this research area within the food industry in order to enhance food safety, sustainability, and public health.

Author Contributions

Conceptualization, S.E.G., A.D.N. and M.C.; methodology, M.C., G.D., A.D.N., A.I. and M.D.; software, M.C. and A.I.; validation, A.D.N., M.D., A.I., M.N., M.C. and S.E.G.; formal analysis, M.C., M.D., A.I., G.D. and A.D.N.; investigation, M.C., A.I., G.D., M.D. and A.D.N.; resources, S.E.G., M.N. and A.D.N.; data curation, S.E.G., M.C., M.D., A.I., G.D. and A.D.N.; writing, M.C. and A.D.N.; original draft preparation, M.C., G.D., M.D. and A.D.N.; review and editing, A.D.N., S.E.G., A.I., M.N., M.D., G.D. and M.C.; visualization, S.E.G., G.D., A.D.N., A.I., M.N., M.D. and M.C.; supervision, S.E.G. and A.D.N.; project administration, A.D.N. and S.E.G.; funding acquisition, A.D.N., M.N., and S.E.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Romanian Ministry of Research, Innovation and Digitalization, through CNCS-UEFISCDI, PNCDI III projects PN-III-P4-ID-PCE-2020-0335, contract PCE 143/2021, and PN-III-P4-ID-PCE-2020-0494, contract PCE 156/2021, the latter exploring the effects of heavy METals and Temperature on the functional Traits space of grassland plants coupled to water-mediated Elemental FLUXes (METTELFLUX), “https://mettelflux.com/ (accessed on 15 March 2026)”.

Data Availability Statement

Data are available from the first author upon request.

Acknowledgments

We are grateful to USAMVB for providing soil, to INFLPR for SEM and EDS training courses and for plasma treatment of contaminated water with antibiotics; to Virgil Iordache from CESEC for laboratory facilities and protocols; and finally, to the Dimitrie Brandza Botanical Garden of the University of Bucharest for hosting and helping with the second experiment in the greenhouse.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. SEM images (50 μm) captured during two different experiments (I and II) using the tomato varieties Zaraza and Buzau 47, respectively, showing abundant crystal formation induced by 0.25 mM SMX and AMX treatments compared to distilled water control (DW) and T20.
Figure 1. SEM images (50 μm) captured during two different experiments (I and II) using the tomato varieties Zaraza and Buzau 47, respectively, showing abundant crystal formation induced by 0.25 mM SMX and AMX treatments compared to distilled water control (DW) and T20.
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Figure 2. The effects of applied irrigation with SMX (B) and AMX (C) treatments on the mineral assemblies of Buzau 47 tomato varieties, as detected by EDX technique (30 μm scale), and also displayed at 2000 and 4000 units distance (D). The DW control level (counts/s) in (A,D) are shown as black lines.
Figure 2. The effects of applied irrigation with SMX (B) and AMX (C) treatments on the mineral assemblies of Buzau 47 tomato varieties, as detected by EDX technique (30 μm scale), and also displayed at 2000 and 4000 units distance (D). The DW control level (counts/s) in (A,D) are shown as black lines.
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Figure 3. EDX map profiling of the SMX-treated tomato seedlings (Buzau 47 variety) showing the distribution of O, C, Ca, K, Mg, and S, and confirming the presence of these mineral elements within an SEM visual field of 100 μm (K, AN series).
Figure 3. EDX map profiling of the SMX-treated tomato seedlings (Buzau 47 variety) showing the distribution of O, C, Ca, K, Mg, and S, and confirming the presence of these mineral elements within an SEM visual field of 100 μm (K, AN series).
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Figure 4. Crystal morphology within control and SMX-NTP-treated leaves (after 30 days). SEM microphotograph captures of DW control with a clean surface, spherical-like crystals found in TWs, PAWs, T20s, and T5s samples (arrows), and prismatic crystals found in PAWp, T20p, T5p, and SMXp samples (arrows). The yellow circles and squares indicate the sampling point for EDX analyses.
Figure 4. Crystal morphology within control and SMX-NTP-treated leaves (after 30 days). SEM microphotograph captures of DW control with a clean surface, spherical-like crystals found in TWs, PAWs, T20s, and T5s samples (arrows), and prismatic crystals found in PAWp, T20p, T5p, and SMXp samples (arrows). The yellow circles and squares indicate the sampling point for EDX analyses.
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Figure 5. EDX percentage spectra of the main elements detected in DW vegetal tissue, spherical crystals (TWs, PAWs, T20s, and T5s), and prismatic crystals (PAWp, T20p, T5p) in each treatment, respectively (A) DW and TW control, (B) PAW, (C) T20, (D) T5.
Figure 5. EDX percentage spectra of the main elements detected in DW vegetal tissue, spherical crystals (TWs, PAWs, T20s, and T5s), and prismatic crystals (PAWp, T20p, T5p) in each treatment, respectively (A) DW and TW control, (B) PAW, (C) T20, (D) T5.
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Figure 6. Seedling morphometry comparisons between irrigation treatments applied to Buzau 47 (A) and Zaraza (B) tomato varieties statistical differences are marked with lowercase letters above the columns (one-way ANOVA, p < 0.05 at 95% confidence).
Figure 6. Seedling morphometry comparisons between irrigation treatments applied to Buzau 47 (A) and Zaraza (B) tomato varieties statistical differences are marked with lowercase letters above the columns (one-way ANOVA, p < 0.05 at 95% confidence).
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Figure 7. PCA implementation of soil–plant variables according to each applied +/− NTP treatment and +/− SMX solution. PCA plot showing spatial distribution of treatment groups along the first two dimensions (Dim1: 27.1%, Dim2: 11.9%).
Figure 7. PCA implementation of soil–plant variables according to each applied +/− NTP treatment and +/− SMX solution. PCA plot showing spatial distribution of treatment groups along the first two dimensions (Dim1: 27.1%, Dim2: 11.9%).
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Figure 8. PCA implementation of soil–plant variables according to each applied +/− NTP treatment and +/− SMX solution. PCA biplot showing the relationship between treatment groups and variable loading vectors.
Figure 8. PCA implementation of soil–plant variables according to each applied +/− NTP treatment and +/− SMX solution. PCA biplot showing the relationship between treatment groups and variable loading vectors.
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Figure 9. PCA implementation of soil–plant variables according to each applied +/− NTP treatment and +/− SMX solution. Variable contributions quantify the percentage contribution of each variable to the total variance.
Figure 9. PCA implementation of soil–plant variables according to each applied +/− NTP treatment and +/− SMX solution. Variable contributions quantify the percentage contribution of each variable to the total variance.
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Figure 10. (A) Sulfur element data extracted from spherical and prismatic crystals based on different numbers (N) of point analyses (5–20 μm). The S column represents the sulfur from the SMX commercial reagent. (B) Mineral composition of Solanum lycopersicum var. Zaraza in leaf surface for DW control and SMX treatment (30 μm field, statistical differences are marked with lowercase letters above the columns, N = 4, one-way ANOVA, p ≤ 0.05 at 95% confidence).
Figure 10. (A) Sulfur element data extracted from spherical and prismatic crystals based on different numbers (N) of point analyses (5–20 μm). The S column represents the sulfur from the SMX commercial reagent. (B) Mineral composition of Solanum lycopersicum var. Zaraza in leaf surface for DW control and SMX treatment (30 μm field, statistical differences are marked with lowercase letters above the columns, N = 4, one-way ANOVA, p ≤ 0.05 at 95% confidence).
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Table 1. Percentage of elements detected in a crystal from the SMX treatment (I) for the Zaraza variety.
Table 1. Percentage of elements detected in a crystal from the SMX treatment (I) for the Zaraza variety.
C. Norm. [wt.%]
K Series ElementsS1SES2SES3SES4SES5SE
O853.544.155.774.957.394.056.144.043.953.3
S1619.530.318.880.417.290.318.340.324.480.4
K1912.960.311.650.313.740.312.760.315.620.3
Ca2013.970.213.710.311.580.212.760.215.950.2
Percentage 100%
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Cicirma, M.; Neagoe, A.D.; Nedelescu, M.; Ionascu, A.; Dumitru, M.; Dinca, G.; Georgescu, S.E. Crystal Formation in Solanum lycopersicum L. Leaves Under Antibiotic Stress Reduced by Non-Thermal Plasma Treated Water. Crops 2026, 6, 35. https://doi.org/10.3390/crops6020035

AMA Style

Cicirma M, Neagoe AD, Nedelescu M, Ionascu A, Dumitru M, Dinca G, Georgescu SE. Crystal Formation in Solanum lycopersicum L. Leaves Under Antibiotic Stress Reduced by Non-Thermal Plasma Treated Water. Crops. 2026; 6(2):35. https://doi.org/10.3390/crops6020035

Chicago/Turabian Style

Cicirma, Marius, Aurora Daniela Neagoe, Mirela Nedelescu, Adrian Ionascu, Marius Dumitru, George Dinca, and Sergiu Emil Georgescu. 2026. "Crystal Formation in Solanum lycopersicum L. Leaves Under Antibiotic Stress Reduced by Non-Thermal Plasma Treated Water" Crops 6, no. 2: 35. https://doi.org/10.3390/crops6020035

APA Style

Cicirma, M., Neagoe, A. D., Nedelescu, M., Ionascu, A., Dumitru, M., Dinca, G., & Georgescu, S. E. (2026). Crystal Formation in Solanum lycopersicum L. Leaves Under Antibiotic Stress Reduced by Non-Thermal Plasma Treated Water. Crops, 6(2), 35. https://doi.org/10.3390/crops6020035

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