Impact of synthetic and herbal dyes on photosynthesis and ROS scavenging enzyme activities in Spirodela polyrhiza

Abstract

Synthetic colors (SCs) are widely used in various industries, yet their discharge into aquatic ecosystems poses a significant environmental threat. This study investigates the impact of SCs and herbal colors (HCs), used during the ‘festival of colors- Holi in India’ on the physiological and photosynthetic performance of Spirodela polyrhiza, an aquatic macrophyte known for its bioindicator potential. The effects were assessed through chlorophyll a fluorescence transient analysis (O-J-I-P), biochemical assays, and energy flux parameters. The results indicate that exposure to SCs severely disrupted PSII functionality, as evidenced by a drastic decline in the quantum yield of primary photochemistry (φPo), electron transport efficiency (φEo), and performance index (PIcs). At 2.5 g/L SC, a 94.18% reduction in trapped energy flux (TR₀/CSm) and a 96.1% decline in electron transport rate (ET₀/CSm) were observed, along with a significant decrease in chlorophyll content. Furthermore, SCs markedly reduced the plastoquinone pool size, leading to impaired photosynthetic electron transport. Antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT), were significantly elevated in SC-treated fronds, indicating oxidative stress. Conversely, no major alterations were observed in plants grown on HCs, which maintained stable photosynthetic parameters and even exhibited enhanced PIcs and φEo, likely due to nutrient enrichment from organic flower-derived pigments. The findings establish S. polyrhiza as an effective bioindicator for monitoring SC pollution in aquatic environments. This study underscores the urgent need to replace synthetic dyes with eco-friendly alternatives, as HCs not only mitigate environmental risks but also enhance growth and productivity of aquatic plants. The research provides novel insights into the differential physiological responses of aquatic plants to SCs and HCs, emphasizing the importance of promoting sustainable, non-toxic coloring agents to safeguard aquatic ecosystems.

Introduction

Dyes and pigments have played a significant role in enhancing the visual appeal of the world and have been utilized since ancient times in various industries, including food, textiles, artifacts, and paper production¹. The increasing demand for colorants has led to extensive research on the development of natural dyes from renewable sources. Natural colorants offer several advantages over synthetic dyes, as they are eco-friendly, safe, renewable, and aesthetically pleasing. Unlike synthetic dyes, natural dyes pose no significant disposal issues and are non-toxic to human health and the environment. India, with its rich biodiversity, serves as a significant reservoir of natural colorants, boasting approximately 500 plant species known to produce colorants that can be extracted easily².

Historical evidence suggests that the use of dyes and pigments dates back to the dawn of human civilization, with ancient Indian, Egyptian, Roman, and Greek societies utilizing natural colorants³. However, the widespread use of natural dyes continued uninterrupted until the mid-nineteenth century, when William Henry Perkin synthesized the first synthetic dye, mauve or aniline purple, in 1856^4,5. The advent of synthetic dyes revolutionized the dye industry due to their high durability, broad color range, cost-effectiveness, and ease of application⁶. Today, thousands of synthetic dyes are produced, with the textile industry consuming nearly 800,000 tons annually. Despite these advantages, synthetic dyes present significant environmental and health concerns. The global consumption of natural dyes remains limited to only 1% of total dye usage due to their high production costs, poor binding capacity with fabrics, and limited availability of dye content in plant materials⁷.

Synthetic dyes contribute substantially to environmental pollution, particularly in industrial sectors such as textiles, food, pharmaceuticals, cosmetics, and paper manufacturing⁸. These industries release large volumes of colored wastewater into water bodies, which significantly impacts aquatic ecosystems by reducing sunlight penetration, increasing biochemical oxygen demand (BOD) and chemical oxygen demand (COD), and inhibiting photosynthesis⁹. Synthetic dyes are often recalcitrant, bio-accumulative, toxic, mutagenic, and carcinogenic¹⁰. Due to their hazardous nature, several synthetic dyes, especially azo-based compounds, have been banned in many countries, prompting industries to seek safer natural dye alternatives^11–13.

The textile industry is one of the largest generators of liquid effluent pollutants due to the excessive water consumption in dyeing processes. It is estimated that approximately 280,000 tons of textile dyes are discharged into industrial effluents annually¹⁴. The traditional textile finishing process requires nearly 100 L of water per kilogram of textile material, making wastewater treatment a major concern. The presence of synthetic dyes in wastewater is visually identifiable and serves as an early indicator of water pollution. The high levels of colored effluents can severely damage aquatic ecosystems and pose serious risks to human health^15,16. Many synthetic dyes contain known carcinogens, such as benzidine and other aromatic compounds, further elevating their toxicity concerns^1,6.

In India, the celebration of the Holi festival involves the application of colored powders and liquids. Historically, these colors were derived from natural sources such as flowers and leaves. However, modern Holi colors are predominantly synthetic and contain hazardous dyes like rhodamine B, malachite green, methyl violet, orange II, and auramine O¹⁷. These synthetic colors often include toxic adulterants like lead oxide, copper sulfate, mercury sulfite, chromium iodide, silica, and mica dust¹⁸. Since these synthetic colors are openly sold without regulatory oversight, they pose severe environmental and health risks. The eventual discharge of these synthetic colors into water bodies exacerbates water pollution, leading to reduced light transmission, inhibition of photosynthesis in aquatic plants, and toxicity to aquatic organisms⁸.

Duckweeds (Lemnaceae) are small, floating aquatic plants that thrive in various water bodies and are often associated with nutrient enrichment¹⁹. Common species include Lemna minor, L. gibba, L. valdiviana, L. aequinoctialis, Landoltia punctata, and Spirodela polyrhiza²⁰. Duckweeds, particularly S. polyrhiza, are recognized for their rapid growth and potential as bioindicators in aquatic ecotoxicology^21,22. The impact of synthetic colors on photosynthesis in aquatic plants remains largely unexplored. The polyphasic OJIP chlorophyll a fluorescence technique provides an effective means to assess the photosynthetic efficiency of plants under environmental stress^23–31. By analyzing polyphasic OJIP Chl a fluorescence transients, phenomenological energy fluxes (ABS/CSm, TR/CSm, ET/CSm, DI/CSm), RC/CSm, φPo, φEo, and PIcsm, researchers can quantify the effects of synthetic and natural dyes on photosynthetic performance³².

In addition to fluorescence-based assessments, enzymatic antioxidant parameters such as superoxide dismutase (SOD) and catalase (CAT) provide insights into oxidative stress responses in plants exposed to synthetic and natural dyes. The chlorophyll content of plants serves as another vital indicator of photosynthetic health and potential toxicity effects³³. The current study aims to evaluate the effects of synthetic and natural dyes on the growth and photosynthetic efficiency of S. polyrhiza by analyzing OJIP Chl a fluorescence kinetics, antioxidant enzyme activities (SOD and CAT), and chlorophyll content. This research will contribute to understanding how synthetic dyes impact aquatic ecosystems and whether natural dyes can serve as eco-friendly alternatives. However, limited studies have evaluated the comparative toxicity of synthetic and natural dyes using photosynthetic parameters and antioxidant enzyme markers in aquatic plants. Notably, the impact of commonly used Holi colors on the photosynthetic machinery of S. polyrhiza, a potential bioindicator species, remains unexplored. Hence, efforts were done to fulfil the study critical gap of knowledge by systematically assessing the physiological and biochemical responses of S. polyrhiza to both synthetic and natural Holi colors.

Materials and methods

Plant selection, culture and experimental design

Spirodela polyrhiza L. was collected from the freshwater lake Fateh Sagar, located in Udaipur, India (24.5854° N, 73.7125° E). The collected plants were surface-sterilized by immersion in 0.5% sodium hypochlorite (NaOCl) for 3 min, followed by rinsing with double-distilled water for 1 min. The sterilized plants were then aseptically cultured in sterile 10% Hoagland nutrient solution³⁴ and maintained under controlled conditions at 25 ± 2 °C with 65–70% relative humidity. A photoperiod of 16 h light and 8 h dark was provided at an intensity of 45 µmol photons m⁻² s⁻¹. The pure culture of S. polyrhiza was acclimatized in a controlled growth chamber for one week before initiating the experiment. Following acclimation, healthy plants were exposed to Hoagland solution supplemented with varying concentrations (0.5–2.5 g L⁻¹) of red synthetic colorants (SCs) and hydrocolloids (HCs). Plants grown in Hoagland solution without the addition of SCs and HCs served as the experimental control (Fig. 1a).

Fig. 1.

Control plants of Spirodela polyrhiza (a) were maintained under standard growth conditions without the addition of SCs or HCs. The UV–visible spectroscopic analysis confirmed the presence of Rhodamine B in the SCs, as indicated by its characteristic absorption spectrum (b).

The SCs were purchase from the local market of Udaipur, India. The red SC, commonly used during the Holi festival, was analyzed using UV-spectroscopy, confirming the presence of rhodamine B as a key constituent. Purchased Holi color contain 2 mg/g of rhodamine B. Commercial Holi color were used in this study without detailed chemical characterization. These mixtures may contain fillers, and unknown contaminants along with rhodamine B, which could influence biological responses. The commercial SC preparation used in this study may contain unidentified compounds in varying concentrations, which could influence the observed physiological responses. Therefore, caution must be exercised in generalizing the results, and future studies employing purified or chemically characterized compounds are warranted to confirm the specific effects of rhodamine or related constituents. In contrast, the red HC was prepared from dried petals of Butea monosperma, known for its natural pigments and bioactive compounds. Fresh petals of B. monosperma were collected, washed thoroughly with distilled water to remove surface impurities, and shade-dried for 7–10 days until crisp. The dried petals were then ground into a fine powder using a mechanical grinder and sieved to obtain a uniform particle size. The resulting powder was stored in airtight containers at room temperature in a dark, dry place until use. Stock solutions of SC and HC were prepared in distilled water and diluted to various concentrations (0.5–2.5 g/L) for experimental treatments.

Growth inhibition rate

The growth inhibition test was conducted following the standard protocol (OECD, 2006), with modifications to assess the impact of synthetic color and herbal color on S. polyrhiza. All treatments, including controls, were conducted in triplicates and incubated for 24 h under photoautotrophic conditions at a temperature of 24 ± 1 °C, with a light intensity of 30 watts and continuous illumination in a plant growth incubator. The average specific growth rate (µ) and growth inhibition (%I) were calculated using the following equations ³⁵:

where N1 and N2 represent the initial and final frond count after 24 h, respectively, while t₁ denotes the start of the test and t₂ denotes the endpoint. and indicate the growth rates of the control and treatment groups, respectively.

Estimation of chlorophyll content

The chlorophyll content was estimated following Arnon’s method. S. polyrhiza plants were treated with control, synthetic, and herbal colors at different concentrations. After the treatment, chlorophyll was extracted using acetone as a solvent. The absorbance of the extract was measured at 663 nm and 645 nm using a Shimadzu spectrophotometer. The total chlorophyll content was then calculated using the following formula³⁶:

Estimation of antioxidant enzyme activity

The activities of superoxide dismutase (SOD) and catalase (CAT) were measured to assess the antioxidant response of S. polyrhiza under control, synthetic, and herbal color treatments.

Superoxide dismutase (SOD) activity

SOD activity was measured based on its capacity to inhibit the photoreduction of nitroblue tetrazolium (NBT), as described by Kono (1978)³⁷. Plant tissues from each treatment group were homogenized in 50 mM chilled phosphate buffer (pH 7.0) and centrifuged at 10,000 rpm for 10 min at 4 °C. The resulting supernatant was used as the enzyme source. The 2.0 mL reaction mixture contained 500 µL of 96 µM NBT, 100 µL of 0.6% Triton X-100, and 1.3 mL of 50 mM Na–K buffer (pH 10.0). The reaction was initiated by the addition of 100 µL of 20 mM hydroxylamine hydrochloride (pH 6.0), and after 2 min, 70 µL of enzyme extract was added. The change in absorbance was recorded at 540 nm. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of NBT reduction under assay conditions. Results were expressed as units per mg protein (U/mg protein).

Catalase (CAT) activity

CAT activity was determined by the method of Teranishi et al. (1974)³⁸, based on the decomposition of hydrogen peroxide (H₂O₂). Plant tissues were homogenized in 50 mM chilled phosphate buffer (pH 7.0) and centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was used as the enzyme extract. The 3.0 mL reaction mixture consisted of 0.1 mL enzyme extract, 20 mM H₂O₂, and 50 mM phosphate buffer (pH 7.0). The reaction was allowed to proceed for 5 min, then terminated by adding 2.0 mL of titanium reagent. This reagent was prepared by mixing 1 g of titanium dioxide and 10 g of potassium sulfate in 150 mL concentrated H₂SO₄, digesting for 2 h, and diluting the cooled mixture to 1.5 L with distilled water. The mixture was centrifuged at 10,000 rpm for 10 min at 4 °C, and the absorbance of the supernatant was measured at 410 nm. One unit of CAT activity was defined as the amount of enzyme that decomposes 1 µmol of H₂O₂ per minute under assay conditions. Results were expressed as U/mg protein.

Fast chl a fluorescence kinetics

Chl a fluorescence O-J-I-P transients were recorded by Plant Efficiency Analyzer (Hansatech Instruments, U.K.). Prior to the measurements, control and experimental plants were adapted to darkness for 1 h. Fluorescence transients were induced over a frond area of 4 mm diameter by a red light (peak at 650 nm) of 3000 µmol m⁻² s⁻¹ (sufficient excitation intensity to ensure closure of all PSII RCs to obtain a true fluorescence intensity of F_m) provided by a high intensity LED array of three light emitting diodes. A total measuring time of one second was used thought out the experiments. Thereafter, primary fluorescence data were used to calculate area between fluorescence induction curve and F_m, phenomenological energy fluxes, RC/CSm, φP_o and φE_o and PI_cs using equations of JIP test^23,39,40 (Table 1).

Table 1.

Glossary and formulae of terms used for the analysis of the fluorescence transients O-J-I-P induced in response of different concentrations of SCs and HCs in S. polyrhiza.

Extracted and technical fluorescence parameters
F0 or F50µs	Fluorescence intensity at 50 µs when all PSII RCs are open
FM or FP	Maximal fluorescence intensity when all PSII RCs are closed
Area	Area above fluorescence curve between F0 and Fm
VJ, relative variable fluorescence at the J-step
M0, approximated initial slope (in ms−1)
Quantum efficiencies or flux ratios (at t = 0)
Po (TR0/ABS), maximum quantum yield of PSII
Eo (ET0/ABS), quantum yield of electron transport
Density of reaction centers
RC/CSm, concentration of active PSII
Phenomenological fluxes (at t = 0)
ABS/CSm, absorption flux per cross section	Fluorescence intensity at 50 µs
TR0/CSm, trapped energy flux per cross section
ET0/CSm, electron transport flux per cross section
DI0/CSm, dissipated energy flux per cross section
Performance index
PIcs, performance index on cross section basis

Statistical analysis

The results represent the mean values derived from three independent measurements for each treatment. Statistical analyses were conducted using GraphPad Prism 8, with significance determined at p < 0.05. To assess the impact of HCs and SCs, a one-way analysis of variance (ANOVA) was performed, followed by Tukey’s Honestly Significant Difference (HSD) test (p = 0.05) to identify significant differences among means. These statistical approaches ensured a comprehensive evaluation of dataset variations, with the Tukey HSD test applied for post hoc comparisons at a significance threshold of α ≤ 0.05. Additionally, graphical representations, including various plots and radar charts, were generated using OriginPro 18.0 to effectively visualize data trends.

Results

Effects on morphology, frond-specific growth rate, and growth inhibition percentage

The present study evaluates the impact of SCs and HCs on the physiological and biochemical responses of S. polyrhiza. UV-spectroscopic analysis confirmed the presence of Rhodamine B in red SCs (Fig. 1b). Exposure to SCs resulted in a significant reduction in chlorophyll concentration (Fig. 2a–c) and increased mortality in S. polyrhiza. In contrast, plants displayed healthy growth in all concentrations of HC (Fig. 2d–f). The effects of SCs and HCs on average growth rate and percentage growth inhibition of S. polyrhiza after 24 h of exposure are summarized in Table 2. The average growth rate was reduced up to 175, 366.6 and 750% in 0.5,1.5 and 2.5 SCs treatments respectively, while the growth rate was exponentially enhanced under HCs treatment 66.66, 83.33 and 200% in 0.5, 1.5 and 2.5 HCs exposures. The growth inhibition also revealed the same results %I was reduced in HCs up to 58, 102 and 184% under 0.5, 1.5 and 2.5 SCs concentrations. In contrary to this, the %I enhanced up to 176, 370 and 748% under 0.5, 1.5 and 2.5 HCs concentrations.

Fig. 2.

S. polyrhiza exposed to different concentrations (0.5–2.5 g/L) of SC (a–c) and HC (d–f). The red coloration observed in the SC-treated plants is attributed to the presence of Rhodamine B dye.

Table 2.

Changes in average specific growth rate (µ) and growth inhibition (%I) in S. polyrhiza subjected to various concentrations of SCs and HCs. Different letters indicate significant differences between treatments (P ≤ 0.05).

Treatments	µ (Average specific growth rate)	%I (Growth inhibition)
Control	0.12 ± 0.02a	0.00 ± 0.00ab
0.5HC	0.20 ± 0.05a	− 58.90 ± 13.86a
1.5HC	0.25 ± 0.03ab	− 102.49 ± 27.46a
2.5HC	0.36 ± 0.08b	− 184.09 ± 15.39a
0.5SC	-0.09 ± 0.04c	176.30 ± 34.66b
1.5SC	-0.32 ± 0.06d	370.65 ± 94.27b
2.5SC	-0.78 ± 0.12e	748.13 ± 216.01c

Values represent the mean of 30 measurements ± standard error (SE).

Chlorophyll content, SOD and CAT activity

The chlorophyll content of the control sample was approximately 2.5 mg g⁻¹ FW. Treatment with HCs at concentrations of 0.5 g/L, 1.5 g/L, and 2.5 g/L resulted in negligible changes in chlorophyll levels, with only a slight, statistically insignificant fluctuation of 5% compared to the control. This suggests that herbal color did not adversely affect the photosynthetic pigment concentration. In contrast, SCs exhibited a clear negative impact on chlorophyll content in a dose-dependent manner. The exposure of 0.5 g/L SCs reduced chlorophyll content by approximately 44%, while 1.5 g/L SC caused a 72% decline. The most severe effect was observed with 2.5 g/L SC, which led to a drastic 78% reduction in chlorophyll levels compared to the untreated control (Fig. 3a).

Fig. 3.

Variations in (a) chlorophyll content, (b) superoxide dismutase (SOD) activity, and (c) catalase (CAT) activity in S. polyrhiza subjected to different concentrations (0.5–2.5 g/L) of SCs and HCs, compared to the control.

The SOD activity in the control and HCs treated samples remained relatively stable, with activity levels ranging from approximately 8 to 10 U mL⁻¹ g⁻¹ FW. In contrast, plants treated with SCs showed a significant increase in SOD activity. Specifically, 0.5 g/L SC led to a 50% rise in SOD activity compared to the control. At 1.5 g/L SC, SOD levels surged by approximately 135%, and at 2.5 g/L SC, there was a pronounced increase of around 275% (Fig. 3b).

Catalase activity in the control plants and those treated with HCs remained consistent, averaging around 6 U mL⁻¹ g⁻¹ FW across all HC concentrations tested. In contrast, catalase activity was significantly elevated in the presence of SCs, indicating oxidative stress. Treatment with 0.5 g/L SCs increased catalase levels by approximately 50%. A further increase was observed at 1.5 g/L SC, with catalase activity nearly doubling—showing a 90% rise compared to the control (Fig. 3c). The most substantial effect occurred at 2.5 g/L SC, which caused catalase activity to rise by about 100%.

Chlorophyll a fluorescence, F_o and F_m

A complete OJIP fluorescence transient curve was observed in control plants, characterized by distinct intermediate J and I phases. In all treatments with HCs, no noteworthy changes were observed in the OJIP curves, which closely resembled that of the control. In contrast, the OJIP curves were significantly altered in plants treated with SCs. At 0.5 g/L SC, a notable reduction in fluorescence intensity was observed throughout the curve. At higher concentrations (1.5 and 2.5 g/L), the OJIP curves appeared markedly flattened, indicating severe disruption and near-complete loss of the characteristic OJIP phases (Fig. 4b). The minimum fluorescence (F₀) represents the basal level of fluorescence when all PSII reaction centers are open. In S. polyrhiza fronds treated with HCs, no significant changes were observed in F_o compared to the control. However, a slight increase of approximately 18% in F_o was noted in plants treated with 0.5 g/L SC (Fig. 4a). At higher SC concentrations of 1.5 and 2.5 g/L, F_o showed a marked increase of 32% and 41%, respectively. In contrast, the maximum fluorescence (F_m), which reflects the fluorescence when all PSII reaction centers are closed, was significantly reduced in SC-treated plants by 20%, 35%, and 43% at 0.5, 1.5, and 2.5 g/L, respectively. Meanwhile, plants treated with HCs showed only minor variations in F_m, with an insignificant increase of about 1–3% across all concentrations (Fig. 4a).

Fig. 4.

Radar plot representing the effect of SCs and HCs on key photosynthetic parameters of S. polyrhiza, including area, reaction centers per cross-section (RC/CS), maximum quantum yield of primary photochemistry (φPo), quantum yield of electron transport (φEo), and performance index (PIcs), along with phenomenological fluxes (a). Chlorophyll fluorescence OJIP induction curve demonstrating variations in photosynthetic activity under SC and HC treatments (b).

Area and density of active reaction centers per cross-section (RC/CS)

The “Area” under the chlorophyll fluorescence induction curve represents the size of the plastoquinone pool and is a proxy for the total electron acceptor capacity on the acceptor side of PSII. In S. polyrhiza treated with HCs, the area values remained close to the control level, with negligible variations (± 4%). However, under SC treatment, there was a significant decline in area. A reduction of approximately 27% was observed at 0.5 g/L SC, which became more severe at higher concentrations of 53% at 1.5 g/L and 66% at 2.5 g/L (Fig. 4a). The density of active reaction centers per cross-section (RC/CS) is an indicator of the functional integrity of PSII complexes in the photosynthetic apparatus. In S. polyrhiza fronds treated with HCs, RC/CS values remained unchanged, showing only minor fluctuations within ± 5% of control levels. However, plants exposed to SCs exhibited a marked, concentration-dependent decline in RC/CS values. At 0.5 g/L SC, there was 25% decrease, which intensified further with a 45% reduction at 1.5 g/L and a dramatic 60% decline at 2.5 g/L. These results indicated that SCs significantly deactivate PSII reaction centers (Fig. 4a).

Phenomenological fluxes

The absorption per cross-section (ABS/CS) parameter indicates the total light energy absorbed by the antenna system per unit cross-section of leaf tissue. In S. polyrhiza fronds treated with HCs, ABS/CS values remained comparable to the control, showing minimal fluctuation within ± 3%. However, a clear decline was recorded in SC-treated samples. ABS/CS dropped by approximately 18% at 0.5 g/L, and this reduction intensified at higher concentrations 33% at 1.5 g/L and 45% at 2.5 g/L (Fig. 4a). Similarly, trapping per cross-section (TR/CS), which reflects the energy trapped in PSII reaction centers for photochemistry, showed a slight increase (~ 3%) under HC treatments. Conversely, SC treatments caused a progressive decrease in TR/CS, with reductions of 22%, 38%, and 50% at 0.5, 1.5, and 2.5 g/L concentrations, respectively. The electron transport per cross-section (ET/CS), which quantifies the portion of trapped energy used for electron transport, also remained stable or slightly improved (3–5%) under HCs, whereas SCs triggered a substantial reduction of 28%, 52%, and 63% at increasing concentrations. In contrast, energy dissipation per cross-section (DI/CS), representing the proportion of energy lost as heat, showed only a slight increase (5–8%) under HC treatments. However, DI/CS rose sharply in SC-treated plants, increasing by 40% at 0.5 g/L and increasing to 72% and 90% at 1.5 and 2.5 g/L, respectively. The change in overall phenomenological fluxes (ABS/CS, TR/CS, ET/CS and DI/CS) were also represented by leaf model (Fig. 5).

Fig. 5.

Representation of the effect of SCs and HCs at concentrations of 0.5–2.5 g/L on the phenomenological energy fluxes in S. polyrhiza. Leaf models illustrate variations in energy flux parameters under different treatments compared to the control leaf model, providing a visual representation of changes in photosynthetic efficiency.

Quantum efficiencies

The maximum quantum yield of PSII (ΦPo) is a critical indicator of the photochemical efficiency of PSII under optimal light conditions. In S. polyrhiza plants treated with HCs, ΦPo remained comparable to the control group, with only minimal variations ranging between 0–2%, suggesting that HCs did not impair the primary photochemistry of PSII. In contrast, SC treatments led to a dose-dependent decline in ΦPo. At 0.5 g/L SC, ΦPo decreased by approximately 25%, indicating moderate inhibition of PSII efficiency (Fig. 4a). This reduction became more pronounced at higher SC concentrations, with declines of around 42% and 48% observed at 1.5 g/L and 2.5 g/L, respectively. The quantum yield for electron transport (ΦEo) reflects the efficiency with which absorbed energy is used to drive electron transport beyond Q_A⁻ in PSII. In plants treated with HCs, a slight improvement in ΦEo was observed, ranging between 2–4% across concentrations, suggesting a mildly favourable effect on electron transport (Fig. 4a). However, SC treatments resulted in a sharp and progressive decline in ΦEo. A reduction of approximately 29% was recorded at 0.5 g/L SC, indicating early signs of stress on the electron transport chain. This inhibition intensified at higher concentrations, with ΦEo decreasing by nearly 53% and 60% at 1.5 g/L and 2.5 g/L SC, respectively. These results clearly demonstrate that SC exposure severely compromises the efficiency of photosynthetic electron transport in S. polyrhiza. The quantum yield of energy dissipation (ΦDo) represents the proportion of absorbed light energy that is not used in photochemistry but instead dissipated as heat. In HC-treated plants, a slight increase in ΦDo (about 3–5%) was observed, indicating minimal stress or enhanced photoprotective energy dissipation. In stark contrast, SC treatments led to a substantial rise in ΦDo, reflecting heightened energy loss and photoinhibition. At 0.5 g/L SC, ΦDo increased by approximately 35%, while higher concentrations of 1.5 and 2.5 g/L caused drastic increases of 70% and 100%, respectively (Fig. 4a).

Performance index (PIcs)

PIcs is a comprehensive parameter that integrates the overall functional activity of PSII, making it a sensitive indicator of photosynthetic status of plant. In fronds treated with HCs, PIcs showed no significant alteration compared to control (fluctuation within ± 5%). In contrast, SC treatments caused a drastic decline in PIcs values. A decrease of about 35% was noted at 0.5 g/L SC, which steeply dropped to 68% at 1.5 g/L and further to 82% at 2.5 g/L (Fig. 4a).

Discussion

SCs widely used during the Holi festival in India and globally, eventually enter natural aquatic ecosystems, altering the physico-chemical properties of potable water, and affecting aquatic flora. The present study evaluated the physiological and biochemical effects of SCs and HCs on Spirodela polyrhiza. SCs caused marked declines in chlorophyll concentration and significantly increased mortality in S. polyrhiza followed a concentration-dependent response⁴¹, suggesting a severe inhibitory effect on photosynthesis and plant vitality. The chlorophyll content was significantly reduced in fronds treated with SCs, indicating potential photosynthetic impairment, whereas no notable alterations were observed in plants exposed to HCs^42,43. Chlorophyll degradation is often associated with oxidative stress, which is commonly induced by environmental pollutants^44,45. In contrast, all concentrations of HCs supported healthy growth and even stimulated biomass accumulation. The positive response under HC treatments may indicate a fertilizer-like effect, likely due to bioavailable nutrients or other beneficial phytochemicals present in the herbal formulations.

Reactive oxygen species (ROS) are key indicators of plant stress and play a crucial role in determining cellular damage^46,47. Various studies have demonstrated that ROS production increases proportionally with stress severity in plants. Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), serve as essential defense mechanisms against oxidative stress by mitigating ROS accumulation^48,49. SOD catalyzes the dismutation of superoxide radicals (O₂⁻) into hydrogen peroxide (H₂O₂), while CAT further detoxifies H₂O₂ by converting it into water and oxygen, thereby preventing cellular damage^50,51. In the present study, no significant changes in SOD and CAT activities were observed in plants treated with HCs, suggesting that HC do not induce oxidative stress and any other types of toxicity in S. polyrhiza. The growth promoting effects of HC may be due to the presence of various natural antioxidants, flavonoids, or osmoprotectants. However, a substantial increase in the activities of both enzymes was recorded in SC-treated plants, with the highest enzymatic activity detected at 2.5 g/L SC concentration. This recommends that exposure to SCs led to excessive ROS accumulation, triggering an enhanced antioxidant response as a protective mechanism. The elevated enzyme activity at higher SC concentrations indicates severe oxidative stress, potentially leading to cellular damage and reduced plant vitality.

The analysis of the fluorescence transient O-J-I-P provided valuable insights into the functionality of PSII under the influence of SCs and HCs. In healthy and HC-treated plants, the typical OJIP curve was maintained, indicating intact photosynthetic electron transport. In contrast, SC-treated plants exhibited an incomplete OJIP curve with significant reductions in F_o and F_m, suggesting disruption at both the antenna level and PSII reaction centers^23,52,53. This SC-induced reduction is likely attributed to decreased chlorophyll content and enhanced non-radiative energy dissipation^54,55. Similar fluorescence responses have been reported under drought stress conditions⁵⁶. Previous studies^57,58 demonstrated that the loss of the IP phase is indicative of PSI impairment, further supporting the SC-induced stress effect. Conversely, an increase in F_o has been observed in several plant species exposed to heat^59,60 and water stress^29,30,61.

The complementary area between the fluorescence induction curve and Fm, representing the plastoquinone (PQ) pool size, showed a significant decline with increasing SC concentration (Fig. 4a). A reduction in the PQ pool size may suggests degradation of the D1 protein within PSII, a crucial component for maintaining PSII functionality^32,62. Phenomenological fluxes such as ABS/CSm, TR₀/CSm, and ET₀/CSm showed significant impairment under SC treatments, with up to 94% reduction in energy trapping and transport efficiency at 2.5 g/L SC. This marked decline indicates that the SC treatment likely interferes with the light-harvesting antenna complex (LHCII), impairing the efficient absorption and transfer of excitation energy to the PSII reaction center⁶³. Since LHCII plays a crucial role in directing light energy to PSII, its disruption lead to insufficient excitation of active reaction centers. Consequently, this reduces the number of effectively operating PSII RCs, as the excitation energy required for photochemistry becomes limited. The observed decline in RC/CS thus reflects the SC-induced impairment of both energy harvesting and functional reaction centers. These effects also arise from decline in antenna size, pigment bleaching, thylakoid disorganization, or direct chemical interaction of SC constituents with the photosynthetic electron transport chain possibly at the Q_A, Q_B binding sites or PQ pool^25,64. In contrast, HC exposure either maintained or slightly enhanced these parameters, especially at lower concentrations. The marginal increase in RC/CS and φEo suggests improved PSII stability and electron transport, possibly linked to micronutrient enrichment or secondary metabolites acting as electron donors or stabilizers.

The PIcs, an integrative indicator of PSII functionality, showed a dramatic reduction in SC-treated plants and enhancement under HC treatments. The decline in φPo, and φEo under SC exposure reflects impaired primary photochemistry and reduced photochemical efficiency. In HC-treated plants, the improvement in these parameters may result from enhanced chlorophyll content, better electron transfers kinetics, or stress-mitigating effects of bioactive herbal compounds. While the beneficial impact of HCs may be attributed to the presence of essential macro- and micronutrients, further chemical characterization is warranted to confirm this. Additionally, exploring the binding affinity of SCs to specific photosynthetic proteins or redox carriers may shed light on their toxic mode of action.

Conclusion

The present study demonstrates that SCs significantly impair the photosynthetic performance of S. polyrhiza, leading to increased mortality and severe physiological stress. In contrast, HCs, derived from dried flower petals, serve as a natural organic amendment, enriching water bodies with essential nutrients and supporting plant growth. The findings highlight that S. polyrhiza undergoes distinct physiological alterations upon SC exposure, making it a promising bioindicator for monitoring water contamination by SC-containing effluents. Given the detrimental effects of SCs, promoting the use of herbal-based, eco-friendly dyes should be prioritized, while stringent regulations should be imposed to restrict the use of synthetic colorants with hazardous components.

Abbreviations

SC

Synthetic Holi color

HC

Herbal Holi color

Chl

Chlorophyll

PS

Photosystem

RC

Reaction center

F₀

Fluorescence at 50 µs

F_m

Maximal fluorescence intensity

ABS/CSm

Absorbed photon flux per excited cross section of PSII

ET₀/CSm

Electron transport flux from Q_A⁻ to PQ per cross section

DI/CS

Dissipated energy flux per cross section

RC/CSm

Density of active PSII RCs

φP_o

Maximum quantum yield of primary PSII photochemistry (TR/ABS or Fv/Fm)

φE_o

Quantum yield of electron transport from Q_A⁻ to PQ (ET/ABS)

PI_CS

Performance index on cross section basis

Author contributions

U.B. and P.T. conducted the experiments, while V.S. conceived the research idea. V.S. supervised the entire study. U.B. drafted the manuscript and prepared the figures. V.S. and R.J.S. reviewed the manuscript, and finalized it.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Consent for publication

All authors are agreeing for publication.