Evaluating the Phytotoxic Impact of Pharmaceutical Antibiotics on Spinach: Insights into Growth, Metabolism, and Protein Dynamics

Sanskriti Dutta¹, Pushpa Latha Manda^1, Ramprasath A¹, Abinesh Muthaiyan¹, Saravanabhavan Periyakali², Joen-Rong Sheu³, Jayakumar Thanasekaran^1*

¹Department of Ecology & Environmental Sciences, Pondicherry University, Puducherry India.

²Department of Zoology, Bharathiar University, Coimbatore, Tamil Nadu, India

³Department of Pharmacology, Taipei Medical University, Taipei, Taiwan.

Correspondence to Dr. T. Jayakumar, Email tjayakumar@pondiuni.ac.in; Tel.: 0431 2654325#325” ORCID: https://orcid.org/0000-0002-4516-5955

Abstract

The increasing presence of pharmaceutical contaminants, particularly antibiotics, in agricultural environments poses significant risks to crop health and productivity. This study investigated the phytotoxic effects of Azithromycin and Gentamicin on spinach (Spinacia oleracea) by assessing growth parameters, chlorophyll content, total phenolic content (TPC), total flavonoid content (TFC), and protein profile. Azithromycin at 10 mg/L enhanced seed germination, whereas it inhibited at higher concentrations of 100 and 500 mg/L. In contrast, Gentamicin promoted germination at all-tested concentrations relative to the control. Azithromycin treatment resulted in significant reductions in plant height, root length, chlorophyll a and b contents, total phenolic content (TPC), and total flavonoid content (TFC), indicating impaired growth and metabolic activity. While gentamicin exhibited moderate effects on these parameters, its impact was not statistically significant compared to that of azithromycin. SDS-PAGE analysis revealed qualitative alterations in protein profiles, suggesting disruption of protein synthesis or stability, which was further supported by quantitative protein estimation. Comparatively, azithromycin exerted significant effects on all evaluated parameters. This response may be attributed to its greater ability to penetrate plant tissues, where it can interfere with plastid (chloroplast) and mitochondrial ribosomes, thereby disrupting essential metabolic processes. In contrast, gentamicin likely remains confined to the extracellular spaces, resulting in minimal physiological impact. This study provides novel insights into the concentration-dependent phytotoxicity of Azithromycin in spinach, highlighting its impact on key phytochemicals and protein metabolism. These findings underscore the potential risks of specific antibiotic contamination in agricultural systems and contribute to understanding plant responses to emerging pharmaceutical contaminants.

Keywords:

Antibiotics

Seed germination

Chlorophyll

SDS-PAGE

Spinach plant

Antioxidants

1. Introduction

Antibiotics are widely used in healthcare, veterinary, and agricultural sectors to prevent and treat bacterial infections (Bombaywala et al., 2021). However, their extensive and often unregulated application has led to their recognition as emerging environmental contaminants. A significant proportion of administered antibiotics are excreted unmetabolized by humans and animals, entering soil and aquatic ecosystems through municipal effluents, hospital discharges, and agricultural runoff (Kraemer et al., 2019; Felis et al., 2020). It is estimated that up to 70% of certain antibiotic classes, such as tetracyclines, are released in active form into the environment (Yang et al., 2021). Continuous release of antibiotic residues from wastewater, sewage sludge, and livestock manure leads to their accumulation in agricultural soils (Manyi-Loh et al., 2018). These residues are subsequently taken up by crops through root absorption and can accumulate in plant tissues (Zhao et al., 2022). Such uptake poses dual risks: (i) phytotoxic effects that impair plant growth and metabolism, and (ii) potential entry into the food chain, thereby raising food safety and public health concerns.

Several studies have reported that antibiotics interfere with fundamental physiological processes in plants. They can inhibit photosynthesis, chlorophyll synthesis, and cell division, as demonstrated in green algae and crop plants exposed to various antibiotic classes (Felis et al., 2020; Opriș et al., 2013). For examples, accumulation of antibiotics in edible crops such as carrots and wheat has been shown to disrupt nitrogen metabolism, respiration, and enzymatic activity (Rocha et al., 2021; Han et al., 2019). Similarly, oxytetracycline, doxycycline, and enrofloxacin inhibited root elongation and overall growth in cucumber, rape, and Chinese cabbage (Wang et al., 2019), while barley seeds exposed to enrofloxacin and tylosin exhibited reduced germination rates (Elezz et al., 2019). These findings underscore the potential threat of antibiotic pollution to agricultural productivity and ecosystem health. Antibiotics can also cause oxidative stress by generating reactive oxygen species (ROS), leading to cellular damage and disruption of antioxidant defense systems. Elevated oxidative stress alters the biosynthesis of secondary metabolites such as phenolic compounds and flavonoids, which are key antioxidants involved in plant defence (Dai & Mumper, 2010; Kumar et al., 2023). In addition, antibiotic exposure can interfere with chloroplast ribosomal function and protein synthesis, given the structural similarity between bacterial and plastid ribosomes (Liu et al., 2011; Krupka et al., 2022). Such interference can impair photosynthetic efficiency and protein metabolism, ultimately reducing plant growth and productivity.

Spinach (Spinacia oleracea), an economically important leafy vegetable rich in antioxidants and nutrients, is widely cultivated and consumed globally. Its high-water content and fast growth make it particularly vulnerable to the accumulation of environmental contaminants. Among antibiotics, macrolides (e.g., azithromycin) and aminoglycosides (e.g., gentamicin) are frequently detected in wastewater and agricultural soils due to their extensive clinical and veterinary usage (Skandalis et al., 2021; Coates et al., 2022). The present study aims to evaluate the phytotoxic effects of two widely used antibiotics: Azithromycin and Gentamicin on Spinacia oleracea. Growth parameters (seed germination, plant height, and root length), chlorophyll content, total phenolic content (TPC), total flavonoid content (TFC), and protein profiles (SDS-PAGE) were analyzed to assess the physiological and biochemical responses of spinach in antibiotic stress. This study contributes to understanding how pharmaceutical contaminants influence plant metabolism and provides insights into the potential ecological risks associated with antibiotic residues in agroecosystems.

2. Materials and Methods

2.1. Materials

Seeds of Spinach (Spinacia oleracea) were procured from a local retailer in Pondicherry, India. Azithromycin (tablet formulation) and Gentamicin (injectable solution) were obtained from a nearby licensed pharmacy. All other chemicals and reagents used in the study were of analytical grade.

2.2. Plants Cultivation and Treatments

The experiment setup consisted of eight treatment groups including a control (plants without antibiotic exposure) spinach plants were treated with azithromycin at concentrations of 10 mg/L, 100 mg/L, and 500 mg/L, designated as A10, A100, and A500, respectively. Gentamicin treatments were administered at concentrations of 50 mg/L, 100 mg/L, 200 mg/L represented as G50, G100, G200 respectively An additional group received a combined treatment of azithromycin and gentamicin at 250 mg/L each, designated as A + G (Fig. 1A). Prior to sowing, spinach seeds were rinsed with distilled water. Each pot was filled with same type of soil and seeds were sown under identical conditions. Plants were grown for approximately three months until they reached maturity, characterized by adequate length and healthy green leaves. Antibiotic treatments were administered through irrigation, with each group receiving 100 ml of the respective antibiotic solution daily for 14 consecutive days prior to harvesting. After harvest, a portion of the plant material was used for phytochemical extraction, while the remaining samples were stored at − 20°C for further analyses.

Fig. 1

Effect of antibiotics azithromycin (A) and gentamicin (G) on height (A) and root length (B) of the spinach plants. Spinach plants were treated with different concentrations of azithromycin (10,100 and 500 mg/l), gentamicin (50,100 and 200 mg/l) and both the antibiotics (250mg/l). The height of plants from each antibiotic-treated group was measured from the soil surface to the uppermost part of the shoot. Root length was determined after carefully uprooting the plants, washing off adhering soil, and measuring with a millimeter scale. All measurements were performed in triplicate to ensure accuracy.

The experimental setup consisted of eight treatment groups, including a control (plants without antibiotic exposure). Spinach plants were treated with azithromycin at concentrations of 10 mg/L, 100 mg/L, and 500 mg/L, designated as A10, A100, and A500, respectively. Gentamicin treatments were administered at concentrations of 50 mg/L, 100 mg/L, and 200 mg/L, designated as G50, G100, and G200, respectively. An additional group received a combined treatment of azithromycin and gentamicin at 250 mg/L each, designated as A + G (Fig. 1A). Prior to sowing, spinach seeds were rinsed with distilled water. Each pot was filled with the same type of soil, and seeds were sown under identical conditions. Plants were grown for approximately three months until they reached maturity, characterized by adequate length and healthy green leaves. Antibiotic treatments were administered through irrigation, with each group receiving 100 mL of the respective antibiotic solution daily for 14 consecutive days prior to harvesting. After harvest, a portion of the plant material was used for phytochemical extraction, while the remaining samples were stored at − 20°C for further analyses.

2.3. Measurement of Average Height and Length of Plant Roots

The height of plants from each antibiotic-treated group was measured from the soil surface to the uppermost part of the shoot. Root length was determined after carefully uprooting the plants, washing off adhering soil, and measuring with a millimeter scale. All measurements were performed in triplicate to ensure accuracy.

2.4. Germination Test

Seeds were pre-soaked in distilled water for 1 h, followed by surface sterilization in 1% H₂O₂ for 10 min, and subsequently rinsed with distilled water. For each treatment group (Control, A10, A100, A500, G50, G100, G200 and A + G), 30 seeds were placed in eight sterilized Petri plates lined with filter paper and moistened with 5ml of distilled water for the control group and respective antibiotics solutions for other treatment groups. Seeds were allowed to germinate at room temperature for 7 days. Germination was recorded when the radicle protruded through the seed coat and reached approximately 1 mm in length. The germination percentage was calculated using the formula (Chitwood et al., 2016; Li et al., 2023):

$\:\text{G}\text{E}\text{R}\text{M}\text{I}\text{N}\text{A}\text{T}\text{I}\text{O}\text{N}\:\text{\%}=\:\frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\text{s}\:\text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{e}\text{d}}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\text{s}\:\text{i}\text{n}\:\text{t}\text{h}\text{e}\:\text{p}\text{l}\text{a}\text{t}\text{e}}\times\:100$

2.5. Determination of Chlorophyll

Chlorophyll was extracted from 0.5 g of fresh leaf tissue using 4 ml of 99% acetone and 2 ml ethanol (2:1 v/v). The homogenized mixture was incubated at − 20°C for 30 min and centrifuged at 2000 rpm for 10 min. Cold extraction and centrifugation facilitated pigment preservation and separation of chlorophyll from cellular debris, yielding a clear supernatant. The supernatant was then mixed with 5 ml acetone/ethanol (2:1 v/v). Absorbance was measured at 663 nm and 645 nm using acetone/ethanol (2:1, v/v) as the blank. Total chlorophyll, chlorophyll a, and chlorophyll b contents were calculated using the equations described by Patricio et al. (2018).

Chlorophyll a (mg/g) =

$\:\left(12.7\:\times\:A663\right)-\left(2.59\:\times\:A645\right)$

Chlorophyll b (mg/g) =

$\:\left(22.9\:\times\:A645\right)-\left(4.7\:\times\:A663\right)$

Total chlorophyll (mg/g) =

$\:\left(8.2\:\times\:A663\right)+\left(20.2\:\times\:A645\right)$

2.6. Determination of Total Phenolic Content (TPC)

Dried plant samples were extracted by maceration in 100% ethanol for three days. The extracts were filtered, and the solvent was evaporated to obtain the residue for further analysis. Total phenolic content (TPC) was determined using the Folin–Ciocalteu method, reliable technique for quantifying phenolic compounds that serve as important antioxidants (Zhao et al., 2022).

Gallic acid was used as standard, and a calibration curve was plotted using different concentrations of Gallic acid in ethanol (10–100 µg/mL). For the assay, 1 mL of standard was mixed with 5 mL of Folin – Ciocalteu reagent and 4 mL of 7.5% sodium carbonate (Na₂CO₃). Same procedure was followed for plant extracts. The mixtures were incubated at room temperature for 1 h, and absorbance was recorded at 765 nm. Results were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW).

2.7. Quantification of Total Flavonoids (TF)

Total flavonoid content (TFC) of the ethanolic extract of spinach was determined using the aluminum chloride (AlCl₃) colorimetric method (Kumar et al., 2014). This assay is based on the formation of stable complexes between flavonoids and AlCl₃, allowing accurate quantification. Plant extracts were diluted to a concentration of 1mg/mL. Quercetin was used as the standard, and a calibration curve was prepared with concentrations ranging from (10–100 µg/mL). For the assay, 100 µL of each extract was mixed with 400 µL of methanol and 100 µL of 10% AlCl₃ solution. The mixtures were incubated in the dark for 45 min, and absorbance was measured at 415 nm. Results were expressed as milligrams of quercetin equivalents per gram of dry weight (mg QE/g DW).

2.8. Evaluation of Protein Integrity in Spinach Leaves by SDS-PAGE

Spinach leaves were homogenized in RIPA lysis buffer (Tris–HCl, NaCl, Triton X-100, sodium deoxycholate, SDS, sodium azide) supplemented with protease inhibitors to prevent protein degradation. Lysates were centrifuged at 13,000 rpm for 13 min at 4°C, and supernatants were collected. Protein concentrations were determined using the Bradford assay with BSA standards (0.04–0.2 mg/mL) by measuring absorbance at 595 nm.

SDS-PAGE was performed using a vertical gel electrophoresis apparatus (I Gene Labserve®) on 12% polyacrylamide gels. Protein samples (60–80 µg) were mixed with 4× Laemmli buffer, denatured at 95°C for 5 min, and loaded onto the gels. Electrophoresis was conducted at 80 V in the stacking gel to allow proteins to concentrate into sharp bands, then increased to 100–150 V in the resolving gel for separation based on molecular weight. The run was stopped when the dye front migrated 2–3 mm from the gel bottom. Gels were stained with Coomassie Brilliant Blue for 45–60 min and destained with 20% methanol and 10% acetic acid to visualize protein bands. Protein molecular weights were estimated by comparison with a prestained molecular weight marker (11–245 kDa). Gel images were captured and digitized using an imaging scanner for documentation and further analysis.

2.9. Statistical Analysis

All measurements, including plant height, root length, chlorophyll content, total flavonoid, and total phenolic content, were performed in triplicate and expressed as mean ± standard deviation (SD). One-way ANOVA was used to assess significant differences among treatment groups (p < 0.05). Data analysis and graphical representations were performed using Microsoft Excel 2013.

3. Results

3.1. Effect of Antibiotics on Height and Root Length of plants

Spinach plant exposed to Azithromycin and Gentamicin at varying concentrations exhibited significant differences (p < 0.05) in average height and root length (Figs. 1A and B). Overall, antibiotic-treated plants showed reduced growth compared to the control, indicating inhibitory effects of the antibiotics.The control group had an average plant height of 15.13 cm and root length of 7.57 cm. Plants treated with Azithromycin at 10, 100 and 500 mg/L showed progressively reduced heights of 14.60, 13.03, and 11.77 cm and root length of 6.77, 5.67, and 5.37 cm, respectively. Gentamicin-treated plants at 50, 100, and 200 mg/L exhibited average heights of 13.03, 13.77, and 14.00 cm and root length of 6.03, 5.67, and 6.60 cm respectively. The group treated with both antibiotics at 250 mg/L each had an average height of 14.00 cm and root length of 5.73 cm respectively.

Azithromycin showed a clear dose-dependent inhibitory effect on both plant height and root length, whereas Gentamicin displayed a less consistent pattern. Combined antibiotic treatment also resulted in reduced root length compared to the control, indicating possible additive or synergistic negative effects on root development. These results indicate that antibiotic exposure can adversely affect the growth parameters of spinach plants, with higher concentrations generally causing greater reductions.

3.2. Effect on Antibiotics on Seed Germination

The effect of Azithromycin and Gentamicin on spinach seed germination was concentration and compound-dependent. Seeds treated with Azithromycin at 10, 100 and 500 mg/L showed germination percentages of 46.66%, 23.33% and 20.00% respectively (Fig. 2A), indicating hormetic effect, where low antibiotic levels may stimulate physiological processes and promote germination. In contrast, Gentamicin-treated seeds at 50, 100 and 200 mg/L exhibited germination rates of 50%, 66.66% and 53.33%, respectively. Seeds exposed to both antibiotics at 250 mg/L each showed a germination percentage of 66.66%.

Fig. 2

Effect of antibiotics on seed germination of spinach plants. Seeds were pre-soaked in distilled water for 1 h, followed by surface sterilization in 1% H₂O₂ for 10 min, and subsequently rinsed with distilled water. For each treatment group (Control, A10, A100, A500, G50, G100, G200 and A + G), 30 seeds were placed in eight sterilized petri plates lined with filter paper and moistened with 5ml of distilled water for the control group and respective antibiotics solutions for other treatment groups. Seeds were allowed to germinate at room temperature for 7 days. Germination was recorded when the radicle protruded through the seed coat and reached approximately 1 mm in length.

Compared to the control group 36.66%, the lowest Azithromycin concentration (10 mg/L) promoted germination, while higher concentrations inhibited it (Fig. 2B). Gentamicin consistently supported germination across all tested concentrations, and the combined antibiotic treatment also enhanced germination, suggesting an antagonistic interaction where Gentamicin may partially mitigate the inhibitory effects of high Azithromycin concentrations.(Table 1). These findings indicate that mild stress induced by low Azithromycin levels can activate protective or stimulatory pathways in seeds, enhancing germination, whereas higher concentrations are inhibitory. Gentamicin appears to maintain or promote germination, and the observed antagonistic effect in combined treatments highlights the complexity of multiple antibiotic exposures on plant physiological responses.

Table 1

Germination percentage of each treatment group. (A is Azithromycin and G is Gentamicin)
Sl. No.	Group	Seeds in plate	Number of seeds germinated	Germination percentage (%)
1.	CONTROL	30	11	36.66
2.	A10	30	14	46.66
3.	A100	30	7	23.33
4.	A500	30	6	20.00
5.	G50	30	15	50
6.	G100	30	20	66.66
7.	G200	30	16	53.33
8.	A + G	30	20	66.66

3.3. Effect of Antibiotics on Chlorophyll Content

Effect of azithromycin and gentamicin on chlorophyll a, chlorophyll b and total chlorophyll content are presented in Fig. 3A. Overall, plants exposed to antibiotics exhibited reduced chlorophyll levels compared to the control group.

Fig. 3

Impact of antibiotics on total chlorophyll, chlorophyll a, and chlorophyll b (A), total phenoilc (B) and total flavonoids (C) in spinach plants.

In the control plants, chlorophyll a, b and total chlorophyll values were 37.40, 24.29 and 62.15 mg/g respectively. In Azithromycin-treated plants, chlorophyll a decreased progressively from 36.48 to 24.40 mg/g, chlorophyll b from 19.92 to 8.69 mg/g, and total chlorophyll from 56.86 to 33.42 mg/g across the 10, 100, and 500 mg/L treatments. Gentamicin treatment at 50, 100 and 200 mg/L resulted in chlorophyll a value of 30.25, 31.74 and 31.58 mg/g, chlorophyll b values of 12.07, 12.26 and 12.77 mg/g, and total chlorophyll values of 42.72, 44.43 and 44.47 mg/g respectively. Combined exposure to both antibiotics (250 mg/L each) also led to reductions, with chlorophyll a at 29.47 mg/g, chlorophyll b at 12.59 mg/g, and total chlorophyll at 42.45 mg/g.

Both antibiotics, whether applied individually or in combination, negatively affected chlorophyll biosynthesis or stability in spinach plants. The decline was more pronounced at higher Azithromycin concentrations, indicating a dose-dependent inhibitory effect. Since chlorophyll is essential for photosynthesis, its reduction may contribute to decreased photosynthetic efficiency and overall plant health. Although the combined treatment did not show synergistic inhibition beyond individual effects, chlorophyll content remained lower than in control plants.

3.4. Effect of Antibiotics on Total Phenolic Content (TPC)

Treatment with Azithromycin and Gentamicin led to a marked reduction in TPC values compared to the control group (Fig. 3B). The control plants recorded a TPC of 20.36 mg GAE/g. In Azithromycin-treated plants, TPC values declined to 12.13, 11.21, and 8.09 mg GAE/g at 10, 100, and 500 mg/L, respectively. Gentamicin treatments at 50, 100 and 200 mg/L yielded TPC values of 12.24, 7.44 and 11.83 mg GAE/g respectively. Plants exposed to the combined treatment (250 mg/L each) showed a TPC of 12.07 mg GAE/g.

Both antibiotics caused a significant decline in TPC, with the reduction being more pronounced at higher concentrations, particularly under Azithromycin exposure, indicating a possible dose-dependent effect. Since phenolic compounds are key contributors to plant defense and antioxidant activity, their depletion suggests a weakened stress-response capacity in spinach plants. The combined antibiotic treatment did not exacerbate the reduction beyond the effects of individual antibiotics, indicating the absence of a strong synergistic interaction.

3.5. Effect of Antibiotics on Total Flavonoid Content (TFC)

A reduction in TFC was observed in all antibiotic-treated groups compared to the control (15.78 mg QE/g) (Fig. 3C). In Azithromycin-treated plants, TFC values decreased to 5.91, 4.13 and 4.24 mg QE/g at 10, 100 and 500 mg/L respectively. Gentamicin treatments at 50, 100 and 200 mg/L resulted in TFC value of 9.34, 5.42 and 5.36 mg QE/g, respectively. Plants exposed to the combined treatment (250 mg/L each) recorded a TFC of 6.28 mg QE/g, also lower than the control.

Both antibiotics, individually and in combination, significantly reduced flavonoid biosynthesis or accumulation in spinach. The decline was more pronounced at higher concentration, indicating a dose-dependent inhibitory effect. As flavonoids are vital for antioxidant defense and stress tolerance, their reduction may compromise the plant’s ability to withstand environemnetal stressors. The combined antibiotic treatment did not further decrease TFC beyond the individual treatments, suggesting no strong synergistic interaction.

3.6. Effect of Antibiotics on Protein Expression

SDS-PAGE revealed clear alterations in spinach leaf protein profiles under antibiotic exposure (Fig. 4). The most prominent bands were consistently detected between ~ 35–48 kDa, a range that likely includes photosynthetic proteins such as the RuBisCO large subunit (~ 55 kDa) and other chloroplast-associated enzymes. Additional bands appeared around 63 kDa and within the 75–135 kDa range, possibly representing multiprotein complexes or stress-related proteins, while faint low-molecular-weight bands (< 25 kDa) suggested small peptides or degradation products. Protein band width decreased progressively with higher concentrations of both antibiotics, with the combined treatment (250 mg/L each) showing the narrowest bands, indicative of synergistic inhibition of protein expression.

Fig. 4

Influence of antibiotics on protein profiles (SDS-PAGE) of spinach plants. SDS-PAGE was performed using a vertical gel electrophoresis apparatus (I Gene Labserve®) on 12% polyacrylamide gels. The run was stopped when the dye front migrated 2–3 mm from the gel bottom. Gels were stained with Coomassie Brilliant Blue for 45–60 min and destained with 20% methanol and 10% acetic acid to visualize protein bands. Protein molecular weights were estimated by comparison with a prestained molecular weight marker (11–245 kDa). Gel images were captured and digitized using an imaging scanner for documentation and further analysis.

Quantitative analysis highlighted compound-specific effects. For the ~ 40 kDa band, azithromycin caused reduced intensity at A100 (6,961.52) compared to control (8,499.90), but a marked increase at A500 (12,790.62), suggesting stress-induced upregulation at high doses. The ~ 45 kDa band was consistently suppressed by azithromycin, with partial recovery at A500 (10,126.33). Gentamicin induced milder effects: ~40 kDa band intensity declined at G50 (4,248.86) and G100 (6,680.66) but recovered at G200 (8,002.97), while the ~ 45 kDa band remained relatively stable (G50: 4,159.06; G100: 4,063.20; G200: 6,816.30). In the combined treatment (A + G), ~ 40 kDa (6,904.71) and ~ 45 kDa (5,138.07) bands showed intermediate values, suggesting partial mitigation of azithromycin’s suppressive effect by gentamicin.

These results demonstrate that antibiotics, originally designed to target bacterial ribosomes, disrupt plant protein expression likely through effects on plastid and mitochondrial ribosomes of prokaryotic origin. The observed dose-dependent suppression and occasional induction of proteins highlight the complex phytotoxic responses of spinach to antibiotic stress.

3.6.1. Molecular Impact of Azithromycin

Azithromycin, a macrolide antibiotic, binds to the 50S subunit of bacterial ribosomes, blocking peptide elongation and protein synthesis. Because plastids (e.g., chloroplasts) contain 70S ribosomes of prokaryotic origin, their translation machinery is also sensitive to macrolides. In spinach leaves, low-dose treatment (A10, L2) maintained strong protein bands in the 35–48 kDa range, indicating functional chloroplast protein synthesis. At higher concentrations (A100, L3; A500, L4), band intensity decreased markedly (Fig. 4B), suggesting suppression or degradation of photosynthetic and metabolic proteins.

This decline likely reflects impaired synthesis of plastid-encoded proteins, including the RuBisCO large subunit, ATP synthase subunits, and photosystem components, along with destabilization of the chloroplast proteome. Azithromycin-induced oxidative stress may further contribute by generating reactive oxygen species (ROS), which promote protein oxidation and proteolytic degradation via Clp and FtsH complexes. The loss of high molecular weight complexes (> 100 kDa) supports disruption of photosystem and ATP synthase assembly, ultimately reducing photosynthetic efficiency.

3.6.2. Molecular Impact of Gentamicin

Gentamicin, an aminoglycoside antibiotic, binds to the bacterial 30S ribosomal subunit, leading to mRNA misreading and defective protein synthesis. Although plant chloroplasts contain 70S-type ribosomes, gentamicin exhibits comparatively lower affinity toward plastid ribosomes than azithromycin, resulting in reduced translational disruption. In the present study, gentamicin-treated samples (G50, G100, G200; L5–L7) displayed sustained or enhanced protein band intensity within the 30–50 kDa range, with the highest density observed at the moderate concentration (G100; L6) (Fig. 4B). This pattern suggests that gentamicin induces minimal proteotoxic stress or activates compensatory stress responses that upregulate protective proteins, thereby maintaining overall protein stability.

At higher concentrations (200 mg/L; L7), a slight reduction in band intensity was noted, indicating the onset of mild stress or protein degradation at supra-physiological levels. Additionally, faint to moderate bands between 100–135 kDa were observed, potentially corresponding to multimeric protein complexes or high-molecular-weight stress-responsive proteins such as heat shock proteins (HSPs). The enhanced protein expression profile under gentamicin exposure, along with improved seed germination rates in corresponding assays, suggests that gentamicin may elicit a mild adaptive response involving the upregulation of molecular chaperones, antioxidant enzymes, and other stress-related proteins that stabilize cellular function under antibiotic exposure.

3.6.3. Combined Treatment (A + G)

The combined antibiotic treatment (A + G; L8) exhibited intermediate protein band intensities with individual azithromycin and gentamicin exposures, an indicating an interaction between azithromycin’s inhibitory effects and gentamicin’s comparatively protective influence (Fig. 4B)

Protein bands within the 30–50 kDa and high-molecular-weight regionswere less diminished than those observed under high-dose azithromycin (A500; L4), suggesting partial mitigation of azithromycin’s-induced proteo-toxicity by gentamicin. This antagonistic interaction at the proteomic level corresponds with observations from germination and chlorophyll assays, implying that gentamicin may induce stress-response pathways that stabilize protein expression compromised by azithromycin.

Faint or inconsistent bands below < 25 kDa likely represent degradation products or low-molecular-weight peptides such as signaling molecules or proteolytic fragments, reflecting antibiotic-induced proteolysis. Increased band smearing in high-dose azithromycin lanes further supports enhanced protein degradation or denaturation. In contrast, high-molecular-weight proteins (> 135 kDa) appeared less abundant under azithromycin exposure, suggesting disruption of large multiprotein complexes essential for photosynthetic electron transport and chloroplast membrane integrity.

4. Discussion

This study evaluated the effects of azithromycin and gentamicin on Spinacia oleracea by assessing growth and selected biochemical parameters. Both antibiotics significantly influenced all evaluated traits, with marked differences between treated and control groups. The effects were antibiotic- and concentration-dependent, showing both positive and negative outcomes. Additionally, combined treatments exhibited synergistic or antagonistic interactions, reflecting the complex nature of antibiotic–plant relationships.

Plant height and root length are key indicators of growth performance and are highly sensitive to environmental stresses (Bitella et al., 2024). Antibiotics are known to affect root morphology, reducing elongation, cell accumulation, and water absorption capacity (Minden et al., 2017). In Brassica rapa ssp. chinensis, norfloxacin, tetracycline, and oxytetracycline significantly reduced shoot and root length compared with controls (Khan et al., 2021). Similarly, Triticum aestivum seedlings exposed to enrofloxacin and oxytetracycline exhibited decreased root length at 10–80 mg/L (Li et al., 2023). Tetracycline reduced shoot length in ryegrass at 10 and 100 mg/L (Han et al., 2019), while tomato plants treated with spectinomycin, chloramphenicol, vancomycin, and spiramycin showed inhibited root elongation and development (Bellino et al., 2018). Collectively, these studies demonstrate the broad inhibitory effects of antibiotics on plant growth across various species and antibiotic classes. The present study similarly showed reduced height and root length in spinach under antibiotic stress, aligning with previous findings and underscoring the vulnerability of plant growth to antibiotic contamination.

Antibiotic exposure can elevate oxidative stress, leading to cellular and organelle damage (Narciso et al., 2023; Fiaz et al., 2023). Oxidative stress disrupts metabolic processes and inhibits growth, contributing to stunted development. Moreover, antibiotic stress increases abscisic acid (ABA) levels, which suppress root elongation (Li et al., 2023). Antibiotics also interfere with the soil nitrogen cycle by affecting ureC, nirK, and norB gene expression, thereby impairing urea decomposition and denitrification, which further limit plant growth (Zhai et al., 2023). Alterations in soil microbial communities reduce beneficial plant–microbe interactions, exacerbating growth inhibition (Carballo et al., 2022). For instance, Vigna radiata treated with oxytetracycline and levofloxacin exhibited reduced nitrate reductase activity and increased antioxidant activity (Fiaz et al., 2023). Similarly, β-lactam antibiotics suppressed root elongation in Arabidopsis thaliana due to decreased cell expansion (Gudino et al., 2018).

Antibiotics at higher concentrations also affect seed germination. Delayed germination was observed in Triticum aestivum and Apera spica-venti treated with penicillin G, sulfadiazine, and tetracycline at 5–10 µg/L (Minden et al., 2017). Enrofloxacin, kanamycin, oxytetracycline, penicillin, and tylosin affected germination frequency in several crops, including corn, soybean, and sunflower, even at low concentrations (Eluk et al., 2016). In wheat, oxytetracycline and enrofloxacin increased ABA levels and antioxidant activity, suggesting an adaptive stress response that mitigates antibiotic toxicity (Li et al., 2023). These findings indicate that antibiotic effects on germination can be inhibitory or stimulatory, depending on the compound, concentration, and plant species.

The enhanced germination observed under gentamicin treatment in this study may be attributed to antibiotic-induced modulation of ABA levels, promoting adaptive stress tolerance. Low antibiotic concentrations may also induce mild stress that stimulates defense mechanisms, a phenomenon known as hormesis. Conversely, the inhibitory effects of azithromycin at higher concentrations may result from disrupted ROS signaling, similar to the effects of chlortetracycline in Brassica campestris, where germination was inhibited by interference with H₂O₂ signaling (Cheong et al., 2020). Disruption of ROS-mediated signaling can impair cellular communication and metabolic regulation essential for successful seed germination.

Chlorophyll estimation is a key indicator for assessing the effects of biotic and abiotic stresses on plants, as any reduction in chlorophyll content directly affects photosynthesis and primary productivity (Kalaji et al., 2018). In the present study, chlorophyll a, b, and total chlorophyll levels were higher in control plants than in antibiotic-treated groups. Increasing concentrations of azithromycin resulted in a progressive decline in chlorophyll content, whereas gentamicin treatments showed an increasing trend, indicating differential phytotoxicity between the two antibiotics. Such variability may arise from differences in their chemical nature, mechanisms of action, and plant–antibiotic interactions.

Antibiotics are known to interfere with chlorophyll biosynthesis and photosynthetic machinery (Krupka et al., 2022). δ-Aminolevulinic acid (ALA), a precursor in chlorophyll biosynthesis, was reported to decrease in Selenastrum capricornutum exposed to antibiotics such as erythromycin, ciprofloxacin, and sulfamethoxazole (Liu et al., 2011; Krupka et al., 2022). Conversely, streptomycin exposure increased ALA accumulation, which promoted free radical generation and oxidative damage to cells and organelles (Yaronskaya et al., 2007; Noriega et al., 2007; Krupka et al., 2022). Antibiotic-induced oxidative stress can impair chloroplast function, reduce chlorophyll stability, and disrupt photosystem II (PSII) and photosystem I (PSI) electron flow (Krupka et al., 2022).

Similarly, ofloxacin exposure in tomato plants at 5–20 mg/L caused chloroplast rupture, chlorophyll degradation, and decreased electron transport rate, reducing photosynthetic efficiency (Zhang et al., 2023). Likewise, ciprofloxacin and tetracycline exposure led to a concentration-dependent decline in leaf chlorophyll content in yellow lupin seedlings (Rydzyński et al., 2017). Collectively, these findings, consistent with the present study, indicate that antibiotic contamination adversely affects chlorophyll biosynthesis and photosynthetic performance, thereby impairing overall plant health and productivity.

Phenolic compounds are important secondary metabolites with strong antioxidant properties that protect plants against reactive oxygen species (ROS) and oxidative damage (Dai et al., 2010). They play a crucial role in enhancing cellular defense and overall plant resilience under stress conditions. In the present study, total phenolic content (TPC) decreased under antibiotic stress compared to the control, suggesting inhibition of phenolic biosynthesis. Phenylalanine ammonia-lyase (PAL), a key enzyme in the phenylpropanoid pathway, catalyzes the conversion of phenylalanine to trans-cinnamic acid, a precursor of phenolic compounds (Corso et al., 2020; Hu et al., 2022). Antibiotics may interfere with this pathway by disrupting amino acid metabolism, as observed in Brassica rapa exposed to antibiotics, which showed altered biosynthesis of phenylalanine and other amino acids (Khan et al., 2021). Such disruptions could downregulate the phenylpropanoid pathway and reduce phenolic synthesis.

Interestingly, the highest concentration of gentamicin (200 mg/L) caused a sharp increase in TPC, indicating a dose-dependent response. Elevated oxidative stress at this concentration may have triggered compensatory activation of the phenolic biosynthetic pathway, leading to the accumulation of these protective metabolites (Kumar et al., 2023). Under severe stress, plants often upregulate secondary metabolite production as a defensive mechanism. Overall, the observed changes in TPC reflect the complex and dynamic nature of plant responses to antibiotic-induced oxidative stress, involving both suppression and stimulation of phenolic metabolism depending on the type and intensity of the stressor.

A study on Triticum aestivum L. (wheat) revealed that exposure to textile dyes and antibiotics, including erythromycin, increased total flavonoid content (TFC) at low concentrations but decreased it at higher doses (Copaciu et al., 2016). Flavonoids function as key antioxidants in plants, neutralizing reactive oxygen species (ROS) and protecting cellular components from oxidative damage. Elevated antibiotic concentrations can trigger ROS generation, leading to depletion of flavonoid reserves as they are consumed in oxidative defense (Patil et al., 2024). This mechanism may explain the reduction in TFC observed in antibiotic-treated spinach in the present study, where prolonged exposure likely exhausted the antioxidant defense capacity (Copaciu et al., 2016). Additionally, excessive ROS production under antibiotic stress may directly inhibit flavonoid biosynthesis, further reducing TFC levels (Vinogradova et al., 2023). The observed dose-dependent decline in TFC underscores the sensitivity of the plant antioxidant system to prolonged or high-level antibiotic exposure.

Previous studies have also reported antibiotic-induced alterations in amino acid and protein metabolism consistent with the reductions in protein expression observed here (Khan et al., 2021; Li et al., 2023). Proteins are essential for plant growth, metabolism, and stress adaptation, and their decline can substantially compromise plant health. For instance, florfenicol exposure in wheat seedlings inhibited growth- and photosynthesis-related proteins, accompanied by increased enzymatic activity at higher antibiotic concentrations (Chen et al., 2023). Similarly, pea seedlings exposed to tetracycline exhibited reductions in RuBisCO, OEE1, and OEE2 proteins—key components of the photosynthetic electron transport chain (Margas et al., 2016). In Brassica rapa ssp. chinensis, tetracycline, oxytetracycline, and norfloxacin altered the biosynthesis of several amino acids, including phenylalanine, tyrosine, and tryptophan, and disrupted alanine, aspartate, and glutamate metabolism, indicating impaired amino acid and protein synthesis (Khan et al., 2021).

Oxidative damage to cellular organelles and alterations in metabolic pathways caused by antibiotic toxicity are likely contributors to protein degradation under stress (Krupka et al., 2022; Chen et al., 2023). For example, erythromycin (a macrolide) inhibited protein synthesis by suppressing chloroplast gene expression in Selenastrum capricornutum, whereas streptomycin (an aminoglycoside) increased ALA content and induced structural changes in barley chloroplasts (Yaronskaya et al., 2007; Liu et al., 2011). These findings collectively highlight that antibiotic exposure disrupts antioxidant defenses, protein synthesis, and metabolic stability, ultimately impairing plant growth and productivity.

5. Conclusion

This study demonstrated that azithromycin and gentamicin exert distinct, concentration-dependent phytotoxic effects on Spinacia oleracea. Both antibiotics altered key biochemical and physiological parameters, including chlorophyll content, phenolic and flavonoid levels, and protein expression. Low concentrations stimulated germination, whereas higher doses inhibited it, indicating a hormetic dose–response pattern. Azithromycin impaired plastid ribosome function, leading to reduced photosynthetic efficiency and growth suppression, while gentamicin appeared to induce mild stress responses that enhanced germination and protein stability. SDS-PAGE analysis revealed pronounced alterations in protein profiles and oxidative damage, suggesting disruption of vital biosynthetic pathways and cellular homeostasis. Collectively, these findings indicate that antibiotic contamination compromises plant health and nutritional quality, underscoring the ecological risks associated with the accumulation of pharmaceutical residues in agricultural systems.

Acknowledgements

The Department of Science and Technology-Science and Engineering Research Board (DST-SERB), India is acknowledged for the finacial support through the CRG project (CRG/2023/001796).

Declaration of interest

statement

The authors declare no conflicts of interest in relation to this publication.

Electronic Supplementary Material

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Supplementary Material 1

Additional Files

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References

Abou Elezz A, Easa A, Atia F, Ahmed T (2019) The potential impact data of Tylosin and Enrofloxacin veterinary antibiotics on germination and accumulation in barley seed as a forage crop and good dietary sources using LC/MS-MS. Data Brief 25:104326. https://doi.org/10.1016/j.dib.2019.104326

Bellino A, Lofrano G, Carotenuto M, Libralato G, Baldantoni D (2018) Antibiotic effects on seed germination and root development of tomato (Solanum lycopersicum L). Ecotoxicol Environ Saf 148:135–141. https://doi.org/10.1016/j.ecoenv.2017.10.006

Bitella G, Bochicchio R, Castronuovo D, Lovelli S, Mercurio G, Rivelli AR, Rossi R (2024) Monitoring plant height and spatial distribution of biometrics with a low-cost proximal platform. Plants 13(8):1085. https://doi.org/10.3390/plants13081085

Bombaywala S, Mandpe A, Paliya S, Kumar S (2021) Antibiotic resistance in the environment: a critical insight on its occurrence, fate, and eco-toxicity. Environ Sci Pollut Res 28(20):24889–24916. https://doi.org/10.1007/s11356-021-13143-x

Carballo M, Rodríguez A, de la Torre A (2022) Phytotoxic effects of antibiotics on terrestrial crop plants and wild plants: a systematic review. Arch Environ Contam Toxicol 82(1):48–61. https://doi.org/10.1007/s00244-021-00893-5

Chen H, Jin J, Hu S, Shen L, Zhang P, Li Z, Liu H (2023) Metabolomics and proteomics reveal the toxicological mechanisms of florfenicol stress on wheat (Triticum aestivum L.) seedlings. J Hazard Mater 443:130264. https://doi.org/10.1016/j.jhazmat.2022.130264

Cheong MS, Yoon YE, Kim JW, Hong YK, Kim SC, Lee YB (2020) Chlortetracycline inhibits seed germination and seedling growth in Brassica campestris by disrupting H₂O₂ signaling. Appl Biol Chem 63. https://doi.org/10.1186/s13765-019-0484-7

Chitwood J, Shi A, Evans M, Rom C, Gbur EE, Motes D, Hensley D (2016) Effect of temperature on seed germination in spinach (Spinacia oleracea). HortScience 51(12):1475–1478. https://doi.org/10.21273/HORTSCI10977-16

Coates J, Bostick KJ, Jones BA, Caston N, Ayalew M (2022) What is the impact of aminoglycoside exposure on soil and plant root-associated microbiota? A systematic review protocol. Environ Evid 11(1):18. https://doi.org/10.1186/s13750-022-00270-9

Copaciu F, Opriş O, Niinemets Ü, Copolovici L (2016) Toxic influence of key organic soil pollutants on the total flavonoid content in wheat leaves. Water, Air, & Soil Pollution, 227, Article number. https://doi.org/10.1007/s11270-016-2873-6

Corso M, Perreau F, Mouille G, Lepiniec L (2020) Specialized phenolic compounds in seeds: structures, functions, and regulations. Plant Sci 296:110471. https://doi.org/10.1016/j.plantsci.2020.110471

Dai J, Mumper RJ (2010) Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 15(10):7313–7352. https://doi.org/10.3390/molecules15107313

Eluk D, Nagel OG, Zimmermann J, Molina MP, Althaus RL (2016) Effect of antibiotics on the germination and root elongation of argentine intensive crops. Int J Environ Res 10(4):471–480. https://doi.org/10.22059/ijer.2016.59521

Felis E, Kalka J, Sochacki A, Kowalska K, Bajkacz S, Harnisz M, Korzeniewska E (2020) Antimicrobial pharmaceuticals in the aquatic environment—occurrence and environmental implications. Eur J Pharmacol 866:172813. https://doi.org/10.1016/j.ejphar.2019.172813

Fiaz M, Ahmed I, Hassan SMU, Niazi AK, Khokhar MF, Farooq MA, Arshad M (2023) Antibiotics-induced changes in nitrogen metabolism and antioxidative enzymes in mung bean (Vigna radiata). Sci Total Environ 873:162449. https://doi.org/10.1016/j.scitotenv.2023.162449

Gudiño ME, Blanco-Touriñán N, Arbona V, Gómez-Cadenas A, Blázquez MA, Navarro-García F (2018) β-Lactam antibiotics modify root architecture and indole glucosinolate metabolism in Arabidopsis thaliana. Plant Cell Physiol 59(10):2086–2098. https://doi.org/10.1093/pcp/pcy172

Han T, Liang Y, Wu Z, Zhang L, Liu Z, Li Q, Wu M (2019) Effects of tetracycline on growth, oxidative stress response, and metabolite pattern of ryegrass. J Hazard Mater 380:120885. https://doi.org/10.1016/j.jhazmat.2019.120885

Hu W, Sarengaowa, Guan Y, Feng K (2022) Biosynthesis of phenolic compounds and antioxidant activity in fresh-cut fruits and vegetables. Front Microbiol 13:906069. https://doi.org/10.3389/fmicb.2022.906069

Kalaji HM, Bąba W, Gediga K, Goltsev V, Samborska IA, Cetner MD, Kompała-Bąba A (2018) Chlorophyll fluorescence as a tool for nutrient status identification in rapeseed plants. Photosynth Res 136:329–343. https://doi.org/10.1007/s11120-017-0503-z

Khan KY, Ali B, Zhang S, Stoffella PJ, Yuan S, Xia Q, Guo Y (2021) Effects of antibiotics stress on growth variables, ultrastructure, and metabolite pattern of Brassica rapa ssp. chinensis. Sci Total Environ 778:146333. https://doi.org/10.1016/j.scitotenv.2021.146333

Kraemer SA, Ramachandran A, Perron GG (2019) Antibiotic pollution in the environment: from microbial ecology to public policy. Microorganisms 7(6):180. https://doi.org/10.3390/microorganisms7060180

Krupka M, Piotrowicz-Cieślak AI, Michalczyk DJ (2022) Effects of antibiotics on the photosynthetic apparatus of plants. J Plant Interact 17(1):96–104. https://doi.org/10.1080/17429145.2022.2048261

Kumar D, Jamwal A, Madaan R, Kumar S (2014) Estimation of total phenols and flavonoids in selected Indian traditional plants. J Pharmacognosy Phytochemistry Res Methods 2(1):42–46. https://doi.org/10.15415/jptrm.2014.21006

Kumar K, Debnath P, Singh S, Kumar N (2023) An overview of plant phenolics and their involvement in abiotic stress tolerance. Stresses 3(3):570–585. https://doi.org/10.3390/stresses3030033

Li L, Li T, Liu Y, Li L, Huang X, Xie J (2023) Effects of antibiotics stress on root development, seedling growth, antioxidant status and abscisic acid level in wheat (Triticum aestivum L). Ecotoxicol Environ Saf 252:114621. https://doi.org/10.1016/j.ecoenv.2023.114621

Liu B, Liu W, Nie X, Guan C, Yang Y, Wang Z, Liao W (2011) Growth response and toxic effects of three antibiotics on Selenastrum capricornutum evaluated by photosynthetic rate and chlorophyll biosynthesis. J Environ Sci 23(9):1558–1563. https://doi.org/10.1016/S1001-0742(10)60539-7

Loh C, Mamphweli S, Meyer E, Okoh A (2018) Antibiotic use in agriculture and its consequential resistance in environmental sources: potential public health implications. Molecules 23(4):795. https://doi.org/10.3390/molecules23040795

Margas M, Piotrowicz-Cieślak AI, Ziółkowska A, Adomas B (2016) Tetracycline accumulation in pea seedlings and its effects on proteome and enzyme activities. Int J Agric Biology 18(4). https://doi.org/10.17957/IJAB/15.0166

Minden V, Deloy A, Volkert AM, Leonhardt SD, Pufal G (2017) Antibiotics impact plant traits, even at small concentrations. AoB Plants 9(2):plx010. https://doi.org/10.1093/aobpla/plx010

Narciso A, Barra Caracciolo A, De Carolis C (2023) Overview of direct and indirect effects of antibiotics on terrestrial organisms. Antibiotics 12(9):1471. https://doi.org/10.3390/antibiotics12091471

Noriega GO, Balestrasse KB, Batlle A, Tomaro ML (2007) Cadmium induced oxidative stress in soybean plants also by the accumulation of δ-aminolevulinic acid. Biometals 20(6):841–851. https://doi.org/10.1007/s10534-006-9077-0

Opriş O, Copaciu F, Soran ML, Ristoiu D, Niinemets Ü, Copolovici L (2013) Influence of nine antibiotics on key secondary metabolites and physiological characteristics in Triticum aestivum: leaf volatiles as a promising new tool to assess toxicity. Ecotoxicol Environ Saf 87:70–79. https://doi.org/10.1016/j.ecoenv.2012.09.006

Patil JR, Mhatre KJ, Yadav K, Yadav LS, Srivastava S, Nikalje GC (2024) Flavonoids in plant–environment interactions and stress responses. Discover Plants 1(1):1–19. https://doi.org/10.1007/s44154-023-00056-3

Pérez-Patricio M, Camas-Anzueto JL, Sanchez-Alegría A, Aguilar-González A, Gutiérrez-Miceli F, Escobar-Gómez E, Grajales-Coutiño R (2018) Optical method for estimating the chlorophyll contents in plant leaves. Sensors 18(2):650. https://doi.org/10.3390/s18020650

Rocha DC, da Silva Rocha C, Tavares DS, de Morais Calado SL, Gomes MP (2021) Veterinary antibiotics and plant physiology: An overview. Sci Total Environ 767:144902. https://doi.org/10.1016/j.scitotenv.2021.144902

Rydzyński D, Piotrowicz-Cieślak AI, Grajek H, Michalczyk DJ (2019) Chlorophyll degradation by tetracycline and cadmium in spinach (Spinacia oleracea L.) leaves. Int J Environ Sci Technol 16:6301–6314. https://doi.org/10.1007/s13762-018-2070-9

Rydzyński D, Piotrowicz-Cieślak AI, Grajek H, Michalczyk DJ (2017) Instability of chlorophyll in yellow lupin seedlings grown in soil contaminated with ciprofloxacin and tetracycline. Chemosphere 184:62–73. https://doi.org/10.1016/j.chemosphere.2017.05.007

Skandalis N, Maeusli M, Papafotis D, Miller S, Lee B, Theologidis I, Luna B (2021) Environmental spread of antibiotic resistance. Antibiotics 10(6):640. https://doi.org/10.3390/antibiotics10060640

Vinogradova N, Vinogradova E, Chaplygin V, Mandzhieva S, Kumar P, Rajput VD, Singh RK (2023) Phenolic compounds of the medicinal plants in an anthropogenically transformed environment. Molecules 28(17):6322. https://doi.org/10.3390/molecules28176322

Wang R, Wang J, Wang J, Zhu L, Zhang W, Zhao X, Ahmad Z (2019) Growth-inhibiting effects of four antibiotics on cucumber, rape and Chinese cabbage. Bull Environ Contam Toxicol 103:187–192. https://doi.org/10.1007/s00128-019-02578-9

Yang Q, Gao Y, Ke J, Show PL, Ge Y, Liu Y, Chen J (2021) Antibiotics: an overview on the environmental occurrence, toxicity, degradation, and removal methods. Bioengineered 12(1):7376–7416. https://doi.org/10.1080/21655979.2021.1983280

Yaronskaya EB, Gritskevich ER, Trukhanov NL, Averina NG (2007) Effect of kinetin on early stages of chlorophyll biosynthesis in streptomycin-treated barley seedlings. Russ J Plant Physiol 54(3):388–395. https://doi.org/10.1134/S1021443707030144

Zhai W, Jiang W, Guo Q, Wang Z, Liu D, Zhou Z, Wang P (2023) Existence of antibiotic pollutant in agricultural soil: exploring the correlation between microbiome and pea yield. Sci Total Environ 871:162152. https://doi.org/10.1016/j.scitotenv.2023.162152

Zhang Z, Liu X, Li N, Cao B, Huang T, Li P, Xu K (2023) Effect of ofloxacin levels on growth, photosynthesis and chlorophyll fluorescence kinetics in tomato. Plant Physiol Biochem 194:374–382. https://doi.org/10.1016/j.plaphy.2023.05.016

Zhao M, Li J, Zhou S, Rao G, Xu D (2022) Effects of tetracycline on the secondary metabolites and nutritional value of oilseed rape (Brassica napus L). Environ Sci Pollut Res 29(54):81222–81233. https://doi.org/10.1007/s11356-022-19679-y

Abbreviations

A, Azithromycin; G, Gentamicin; v/v, volume/volume; µg/mL, micrograms per milliliter; mL, milliliter; µL, microliter; mg/mL, milligrams per milliliter; RIPA, Radioimmuno Precipitation Assay; SDS, Sodium Dodecyl Sulfate; SDS-PAGE, Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis; BSA, Bovine Serum Albumin; ANOVA, Analysis of Variance; TPC, Total Phenolic Content; GAE/g, Gallic Acid Equivalents per gram; TFC, Total Flavonoid Content; QE/g, Quercetin Equivalents per gram; kDa, kilodalton; C, Control; ssp, subspecies; ABA, Abscisic acid; N, Nitrogen; ureC, alpha subunit of urease; norB, nitric oxide reductase subunit B; nirK, copper-containing nitrite reductase enzyme; ENR, Enrofloxacin; OTC, Oxytetracycline; ROS, Reactive Oxygen Species; ALA, Aminolevulinic acid; PS, Photosystem; OFL, ofloxacin; PAL, Phenylalanine ammonia-lyase; RuBisCo, Ribulose-1,5-bisphosphate carboxylase/oxygenase; OEE, Oxygen Evolving Enhancer proteins.

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Author Contributions:

Sanskriti Dutta (SD): Conceptualization; Methodology; Investigation; Data curation; Formal analysis; Visualization; Writing original draft. Pushpa Latha Manda (PLM): Visualization; Resources; Validation; Writing – review & editing. Ramprasath A (RA): Methodology; Investigation; Data curation; Laboratory support. Abinesh Muthaiyan (AM): Software; Data processing; Assistance in biochemical analysis; Review & editing. Saravanabhavan Periyakali (SP): Supervision; Guidance on experimental design; Resources. Joen-Rong Sheu (JRS): Critical review of manuscript. Jayakumar Thanasekaran (JT): Supervision; Conceptualization; Methodological oversight; Writing – review & editing; Project administration.